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Sample records for chimeric antibodies expressed

  1. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    PubMed

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.

  2. Expression and secretion of aequorin as a chimeric antibody by means of a mammalian expression vector.

    PubMed Central

    Casadei, J; Powell, M J; Kenten, J H

    1990-01-01

    A fusion protein has been expressed from the relevant genes in mammalian cells consisting of the photoprotein aequorin and an anti-4-hydroxy-3-nitrophenacetyl antibody gene. This chimeric antibody has allowed the development of a sensitive luminescent immunoassay. Initially the cDNA of the photoprotein aequorin from Aequorea victoria was cloned and expressed in Escherichia coli. The gene was expressed as apoaequorin and, by using luciferin isolated from Renilla reniformis, its activity was found essentially identical to native aequorin. The aequorin gene was subcloned into a mammalian expression vector to produce a fusion protein directing secretion of apoaequorin; the aequorin gene was fused to the 3' terminus of an immunoglobulin heavy-chain gene that directed expression of an anti-4-hydroxy-3-nitrophenacetyl antibody. The gene fusion contained the variable region, the constant region domain 1, and part of domain 2 for the IgG2b mouse immunoglobulin, followed by the aequorin gene. Transfection of the chimeric gene into a cell line expressing the complementary lambda 1 light chain, J558L, allowed recovery of a chimeric antibody with binding specificity for the 4-hydroxy-3-nitrophenacetyl group and the related 4-hydroxy-3-iodo-5-nitrophenacetyl hapten. The Ca2(+)-dependent bioluminescent activity of aequorin was also recovered. Images PMID:2315301

  3. Immunotherapy for cancer: construction, expression and functional characterization of chimeric antibodies.

    PubMed

    Motmans, K; Thirion, S; Heyligen, H; Janssens, J; Raus, J; Vandevyver, C

    1996-12-01

    Monoclonal antibodies (Mabs) are a potential key component for the treatment of cancer, because of their specificity and multiple effector functions. Hybridoma technology and progress in genetic engineering made it possible to customize antibody molecules, rendering them more suitable for selective application. A widely used technique is the construction of mouse-human hybrid molecules by recombinant DNA techniques. These so-called chimeric antibodies contain the murine variable (V) regions fused to the human constant (C) regions. In this report, a general approach is described for the production of chimeric antibodies. The gene segments encoding the murine variable heavy and light chain are isolated by the polymerase chain reaction and cloned into expression vectors containing the human gamma 1 heavy chain gene and the human K light chain gene, respectively. Subsequently, these constructs are transfected into a non-Ig-producing murine hybridoma, eg SP2/0 cells. The in vitro study of the functional characteristics and biological properties of the thus obtained chimeric antibodies are discussed.

  4. Reconstruction and expression of chimeric anti-HBx antibody in Escherichia coli.

    PubMed

    Zhou, G; Liu, K D; Tang, Z Y; Chen, Y H; Wu, X F; Schroeder, C H

    1997-01-01

    The variable regions of murine monoclonal anti-HBx immunoglobulin and the constant region of human antibody were cloned by reverse transcript-polymerase chain reaction (RT-PCR). The heavy-chain and light-chain variable regions were connected and coexpressed with human constant region C-r3 and C-k3 in the reconstructed vector of E. coli. The products showed high specificity and binding ability with HBx. Which is closely associated with hepatocarcinogenesis. This makes it possible to humanize the mouse monoclonal antibodies and express the fusion protein in E.coli for potential radioimmunotherapy in patients with hepatocellular carcinoma.

  5. Chimeric human/murine monoclonal IgM antibodies to HIV-1 Nef antigen expressed on chronically infected cells.

    PubMed

    Kawai, Masahiro; He, Lianying; Kawamura, Takeshi; Omoto, Shinya; Fujii, Yoichi R; Okada, Noriko

    2003-01-01

    Human IgM antibody (Ab) to gangliosides induced cytolysis of HIV-1-infected cells by homologous human complement. We expected that any human IgM Ab reactive with HIV-1 infected cells could cause complement-mediated cytolysis. The trans-chromosome mouse (TC mouse) contains human chromosomes harboring genes responsible for immunoglobulin production. Spleen cells from TC mice immunized with recombinant Nef were fused with mouse myeloma cells to generate hybridomas, and we selected those that produced human mu-chain-positive Abs reactive with Nef fixed on an ELISA plate. However, the L-chain of the monoclonal Abs (mAbs) were murine lambda in type and were chimeric, and we could not succeed in obtaining mAb with human mu- and human kappa-chains. The chimeric mAbs reacted with the HIV-1 infected cells as seen with flow cytometric analysis, and the surface expression of Nef was also detectable on chronically infected OM10.1 cells which had no detectable gp120. However, although the reaction of the chimeric IgM mAb with HIV-1-infected MOLT4 cells induced C3 deposition on cell surfaces on incubation with fresh human serum, the cells remained unlysed, as determined by 51Cr release assay. The amount of Nef antigen on the cells might not have been high enough to overcome the function of HRF20 (CD59) that restricts formation of membrane attack complexes of homologous complement. However, combination of anti-Nef IgM mAb with other IgM mAbs reactive with the surface of HIV-1-infected cells may induce a synergistic effect in complement mediated cytolysis.

  6. A Bivalent, Chimeric Rabies Virus Expressing Simian Immunodeficiency Virus Envelope Induces Multifunctional Antibody Responses

    PubMed Central

    Dunkel, Amber; Shen, Shixue; LaBranche, Celia C.; Montefiori, David

    2015-01-01

    Abstract We previously showed that a matrix (M) gene-deleted rabies virus (RABV)-based vaccine (RABV-ΔM) is highly immunogenic and induces potent B cell responses in the context of RABV infection. We speculated that RABV-ΔM expressing HIV proteins would also induce potent B cell responses against HIV antigens. As a prerequisite to future studies in nonhuman primates, we completed immunogenicity studies in mice to confirm the ability of RABV-ΔM to induce polyfunctional B cell responses in the context of HIV. To that end, the envelope protein from the mac239 strain of SIV (SIVmac239Env) was cloned into RABV-ΔM, resulting in RABV-ΔM-Env. Infectious virus was recovered following standard methods and propagated on baby hamster kidney cells stably expressing RABV M [>107 focus forming units (ffu)/ml]. Western blot analysis of cell lysates or of purified virions confirmed Env expression on the surface of infected cells and within virus particles, respectively. Positive neutralization activity against a neutralization-sensitive SIV strain and to a lesser extent against a neutralization-resistant SIV strain was detected in mice after a single intramuscular inoculation with RABV-ΔM-Env. The quality, but not quantity, of the antibody response was enhanced via boosting with recombinant gp130 or RABV-ΔM-Env as measured by an increase in antibody avidity and a skewing toward a Th1-type antibody response. We also show that an intradermal inoculation induces higher antibodies than an intramuscular or intranasal inoculation. An intradermal inoculation of RABV-ΔM-Env followed by a boost inoculation with recombinant gp130 produced anti-SIV antibodies with neutralizing and nonneutralizing antibody (nNAb) effector functions. Together, RABV-ΔM-Env induces B cells to secrete antibodies against SIV with the potential to clear both “free” and cell-associated virus. Strategies capable of eliciting both NAbs as well as nNAbs might help to improve the efficacy of HIV-1 vaccines

  7. [Construction of a recombinant baculovirus transfer vector with two promoters expressing the anti-human CD28 chimeric antibody by using TP-PCR method].

    PubMed

    Zhu, Yan; Chen, Yong-Jing; Qiu, Yu-Hua; Zheng, Feng-Feng; Zhu, Jiang

    2005-09-01

    CD28, a cell surface glycoprotein, predominantly expressed on T cells, belongs to the Ig superfamily and provides critical co-stimulatory signals. The data which have published indicate that the monoclonal antibody against CD28 can decrease curative effects when it was applied in vivo for a long time. In order to avoid the human-anti-mouse action, anti-CD28 mAb must be humanized before it can be used in clinical study. Chimeric antibody, consisting of variable regions of mouse antibody and the constant regions of human IgG1, is often chosen by designers in generating humanized antibody. In this study, to prepare the anti-human CD28 chimeric antibody, the genes coding variable regions of anti-CD28 mAb and the constant regions of human IgG1 were cloned by PCR method. Then, the target genes were assembled by TP-PCR, a novel method developed for fusing genes without designing endonuclease sites at the both end of the target genes, and inserted into the baculovirus transfer vector pAcUW3 respectively. Thus, the recombinant baculovirus transfer vector with two strong promoters, ph and p10 was successfully constructed, which can express two different foreign genes at the same time. The recombinant vector was identified by the methods of restriction digesting, electrophoresis, PCR amplification and further verified by DNA sequence analysis. This work will contribute to expressing the chimeric CD28 antibody in insect cells.

  8. Chimeric antibodies with extended half-life in ferrets

    PubMed Central

    Nesspor, Thomas C; Scallon, Bernard

    2014-01-01

    Background Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long-term studies. Objective Our aim was to identify an antibody design with reduced immunogenicity and longer circulating half-life in ferrets. Methods The constant region coding sequences for ferret immunoglobulin G were cloned, and chimeric human/ferret antibodies were expressed and purified. Some of the chimeric antibodies included substitutions that have been shown to extend the half-life of human IgG antibodies. These chimeric antibodies were tested for binding to recombinant ferret FcRn receptor and then evaluated in pharmacokinetic studies in ferrets. Results A one-residue substitution in the ferret Fc domain, S252Y, was identified that increased binding affinity to the ferret neonatal receptor by 24-fold and extended half-life from 65 ± 27 to 206 ± 28 hours or ∼9 days. Ferrets dosed twice with this surrogate antibody showed no indications of an immune response. Conclusion Expressing the variable region of a candidate human therapeutic antibody with ferret constant regions containing the S252Y substitution can offer long half-life and limit immunogenicity. PMID:25074755

  9. Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions.

    PubMed

    Ma, Biao; Osborn, Michael J; Avis, Suzanne; Ouisse, Laure-Hélène; Ménoret, Séverine; Anegon, Ignacio; Buelow, Roland; Brüggemann, Marianne

    2013-12-31

    Expression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human VH, D and JH genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human VH-region (comprising 22 VHs, all Ds and all JHs in natural configuration) but linked to different rat CH-genes and regulatory sequences. The endogenous IgH locus was silenced by zinc-finger nucleases. After breeding, all lines produced exclusively chimeric human H-chain with near normal IgM levels. However, in two lines poor IgG expression and inefficient immune responses were observed, implying that high expression, class-switching and hypermutation are linked to optimal enhancer function provided by the large regulatory region at the 3' end of the IgH locus. Furthermore, exclusion of Cδ and its downstream interval region may assist recombination. Highly diverse IgG and immune responses similar to normal rats were identified in two strains carrying diverse and differently spaced C-genes.

  10. Chimeric antibodies with extended half-life in ferrets.

    PubMed

    Nesspor, Thomas C; Scallon, Bernard

    2014-09-01

    Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long-term studies. Our aim was to identify an antibody design with reduced immunogenicity and longer circulating half-life in ferrets. The constant region coding sequences for ferret immunoglobulin G were cloned, and chimeric human/ferret antibodies were expressed and purified. Some of the chimeric antibodies included substitutions that have been shown to extend the half-life of human IgG antibodies. These chimeric antibodies were tested for binding to recombinant ferret FcRn receptor and then evaluated in pharmacokinetic studies in ferrets. A one-residue substitution in the ferret Fc domain, S252Y, was identified that increased binding affinity to the ferret neonatal receptor by 24-fold and extended half-life from 65 ± 27 to 206 ± 28 hours or ~9 days. Ferrets dosed twice with this surrogate antibody showed no indications of an immune response. Expressing the variable region of a candidate human therapeutic antibody with ferret constant regions containing the S252Y substitution can offer long half-life and limit immunogenicity. © 2014 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  11. Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans.

    PubMed

    Boado, Ruben J; Zhang, Yufeng; Zhang, Yun; Xia, Chun-fang; Wang, Yuntao; Pardridge, William M

    2008-03-01

    The genetic engineering, expression, and validation of a fusion protein of avidin (AV) and a chimeric monoclonal antibody (mAb) to the human insulin receptor (HIR) is described. The 15 kDa avidin monomer was fused to the carboxyl terminus of the heavy chain of the HIRMAb. The fusion protein heavy chain reacted with antibodies specific for human IgG and avidin, and had the same affinity for binding to the HIR extracellular domain as the original chimeric HIRMAb. The fusion protein qualitatively bound biotinylated ligands, but was secreted fully saturated with biotin by COS cells, owing to the high level of biotin in tissue culture medium. Chinese hamster ovary (CHO) cells were permanently transfected with a tandem vector expressing the fusion protein genes, and high expressing cell lines were isolated by methotrexate amplification and dilutional cloning. The product expressed by CHO cells had high binding to the HIR, and migrated as a homogeneous species in size exclusion HPLC and native polyacrylamide gel electrophoresis. The CHO cells were adapted to a 4 week culture in biotin depleted medium, and the HIRMAb-AV fusion protein expressed under these conditions had 1 unoccupied biotin binding site per molecule, based on a [3H]-biotin ultrafiltration assay. The HIRMAb-AV increased biotin uptake by human cells >15-fold, and mediated the endocytosis of fluorescein-biotin, as demonstrated by confocal microscopy. In summary, the HIRMAb-AV fusion protein is a new drug targeting system for humans that can be adapted to monobiotinylated drugs or nucleic acids.

  12. [Expression and antiviral activity of a chimeric porcinized monoclonal antibody (cHQ06) against E2 protein of classical swine fever virus].

    PubMed

    Chen, Shucheng; Sun, Huimin; Li, Su; Liu, Pinghuang; Ma, Jifei; Qiu, Huaji

    2017-08-25

    Classical swine fever (CSF), one of OIE-listed diseases, is a highly contagious and economically important disease of pigs. Classical swine fever virus (CSFV) is the causative agent of CSF. The capsid (C) protein and the glycoproteins Erns, E1 and E2, are structural components of the virus. E2 is the most immunogenic protein of the CSFV glycoproteins, inducing neutralizing antibodies that provide protection against lethal CSFV challenge. In a previous study, we developed a murine MAb HQ06 against the E2 protein of CSFV. In this study, the variable region genes from HQ06 and constant regions gene of swine antibody are fused and cloned into the eukaryotic expression vectors to establish a cell line which can stably express a chimeric porcinized MAb (cHQ06) against E2 in CHO cell. The purified cHQ06 antibody protein was determined to be successfully generated, which exhibited high reactivity between cHQ06 and the E2 protein of CSFV by enzyme-linked immunosorbent assay (ELISA) and Western blotting. More importantly, we investigated the neutralizing activity of cHQ06 against CSFV. In conclusion, this study generated cHQ06 for efficient and stable production which can be used against to develop novel diagnostic assays, investigate the structure and function of the E2 protein and generate novel preparations of diagnosis and treatment.

  13. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  14. Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies to Toxoplasma gondii

    PubMed Central

    Hackett, John; Hoff-Velk, Jane; Golden, Alan; Brashear, Jeff; Robinson, John; Rapp, Margaret; Klass, Michael; Ostrow, David H.; Mandecki, Wlodek

    1998-01-01

    In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays. PMID:9574691

  15. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells.

    PubMed Central

    Liu, A Y; Robinson, R R; Hellström, K E; Murray, E D; Chang, C P; Hellström, I

    1987-01-01

    A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man. PMID:3106970

  16. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

    PubMed

    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  17. Engineering and characterization of a chimeric anti-platelet glycoprotein Ibalpha monoclonal antibody and preparation of its Fab fragment.

    PubMed

    Yang, Jianfeng; Ji, Shundong; Dong, Ningzheng; Zhao, Yiming; Ruan, Changgeng

    2010-04-01

    Glycoprotein Ibalpha (GPIbalpha) is a platelet-specific membrane protein. It mediates platelet adhesion to collagen exposed at the vascular injury site by binding to von Willebrand factor (VWF) in plasma. This process is crucial for arterial thrombus formation. Blocking interaction between GPIbalpha and VWF may prevent platelet adhesion and thrombus formation. We previously generated a high affinity monoclonal antibody against human platelet GPIbalpha, SZ2, which inhibits both ristocetin- and botrocetin-induced platelet aggregation in vitro. To convert SZ2 into mouse/human chimeric antibody for anti-platelet therapy in humans, in this study, we constructed a mouse/human chimeric antibody derived from the hybridoma cells producing murine antibody against platelet glycoprotein Ibalpha, conducted its expression in dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells, and prepared its chimeric Fab fragment. Results from ELISA and Western blot analysis showed that the chimeric antibody was secreted from the cells and that the heavy and light chains were assembled correctly. Flow cytometry analysis confirmed specific binding of the chimeric antibody to the GPIb-expressing CHO cells. In vitro functional studies revealed that the chimeric antibody and its Fab fragment prevented platelet adhesion to VWF under high shear stress and inhibited ristocetin-induced platelet aggregation in a dose-dependent manner. These results demonstrated that the chimeric antibody was successfully engineered and suggested that the Fab fragment of chimeric antibody against GPIbalpha is a promising therapeutic antibody more suitable for prevention and treatment of human arterial thrombosis.

  18. Enhanced antigen detection in immunohistochemical staining using a 'digitized' chimeric antibody.

    PubMed

    Eng, Hui-Yan; Wang, Cheng-I; Xue, Yuezhen; Lee, Chia-Yin; Zulkifli, Sarah Binte; Chiam, Poh-Cheang; Ghadessy, Farid J; Lane, David P

    2016-01-01

    The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma.

    PubMed Central

    Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma. Images Figure 7 PMID:8795576

  20. Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration

    PubMed Central

    Ansari, Sahar; Moshaverinia, Alireza; Pi, Sung Hee; Han, Alexander; Abdelhamid, Alaa I.; Zadeh, Homayoun H.

    2013-01-01

    Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti- BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis. PMID:24055525

  1. Expression studies of catalytic antibodies

    SciTech Connect

    Ulrich, H.D.; Patten, P.A.; Yang, P.L.

    1995-12-05

    We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived form a number of catalytic antibodies. Expression yields of eight hybridoma-and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high-density fermentation. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies. 41 refs., 4 figs., 1 tab.

  2. Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

    PubMed

    Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

    2011-04-01

    We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs.

  3. [Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein].

    PubMed

    Zhou, Rongping; Sun, Lina; Liu, Yang; Wu, Wei; Li, Chuan; Liang, Mifang; Qiu, Peihong

    2016-01-01

    The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

  4. Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E.

    PubMed

    Mukamoto, Masafumi; Maeda, Hiroaki; Kohda, Tomoko; Nozaki, Chikateru; Takahashi, Motohide; Kozaki, Shunji

    2013-01-01

    A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

  5. Plant-based production of two chimeric monoclonal IgG antibodies directed against immunodominant epitopes of Vibrio cholerae lipopolysaccharide.

    PubMed

    Levinson, Kara J; Giffen, Samantha R; Pauly, Michael H; Kim, Do H; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J; Zeitlin, Larry; Mantis, Nicholas J

    2015-07-01

    We have produced and characterized two chimeric human IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest.

  6. Plant-based Production of Two Chimeric Monoclonal IgG Antibodies Directed against Immunodominant Epitopes of Vibrio cholerae Lipopolysaccharide

    PubMed Central

    Levinson, Kara J.; Giffen, Samantha R.; Pauly, Michael H.; Kim, Do H.; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J.; Zeitlin, Larry; Mantis, Nicholas J.

    2015-01-01

    We have produced and characterized two chimeric IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest. PMID:25865265

  7. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

    SciTech Connect

    Zhang Fanglin; Wu Xingan; Luo Wen; Bai Wentao; Liu Yong; Yan Yan; Wang Haitao; Xu Zhikai . E-mail: zhikaixu@fmmu.edu.cn

    2007-03-23

    Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice.

  8. A human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro.

    PubMed

    Ding, Guipeng; Chen, Ximin; Zhu, Jin; Duesbery, Nicholas S; Cheng, Xunjia; Cao, Brian

    2013-01-01

    Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46 × 10(7) L/mol and could neutralize LeTx with an EC50 of 85  μ g/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.

  9. Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor.

    PubMed

    Zhang, Mei-Yun; Feng, Yang; Wang, Yanping; Dimitrov, Dimiter S

    2009-01-01

    The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool.

  10. Characterization of a chimeric monoclonal antibody against the insulin-like growth factor-I receptor

    PubMed Central

    Feng, Yang; Wang, Yanping; Dimitrov, Dimiter S

    2009-01-01

    The insulin-like growth factors (IGFs) signaling system has been shown to play important roles in neoplasia. The IGF receptor type 1 (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and there is substantial experimental and clinical evidence that targeting IGF-IR is a promising therapeutic strategy against cancer. It has been previously reported that a mouse monoclonal antibody (mAb), 4G11, blocked IGF-I binding to IGF-IR and downregulated the IGF-IR in MCF-7 cells. We cloned this antibody, constructed a human-mouse chimeric antibody, designated m590, and characterized it. The chimeric IgG1 m590 bound to cell-associated IGF-IR on NWT c43 stably transfected cells and MCF-7 breast cancer cells as efficiently as the parental murine antibody. Using purified IGF-IR extracellular domains, we found that both the chimeric m590 and the parental 4G11 antibodies bind to conformational epitopes on IGF-IR. Neither of these antibodies bound to the insulin receptor (IR) ectodomain. Furthermore, IgG1 m590 blocked the binding of IGF-I and IGF-II to IGF-IR, and inhibited both IGF-I and IGF-II induced phosphorylation of IGF-IR in MCF-7 cells. These results suggest that m590 could be an useful antibody in diagnosis and treatment of cancer, as well as a research tool. PMID:20065647

  11. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha.

    PubMed

    Doki, Tomoyoshi; Takano, Tomomi; Hohdatsu, Tsutomu

    2016-10-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2-4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2-4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2-4) by fusing the variable region of mouse mAb 2-4 to the constant region of feline antibody. The chimeric mAb 2-4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2-4 and chimeric mAb 2-4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2-4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2-4 was reduced. In contrast, in cats treated with chimeric mAb 2-4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2-4-treated cats.

  12. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

    PubMed Central

    DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

    2016-01-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2–4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2–4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2–4) by fusing the variable region of mouse mAb 2–4 to the constant region of feline antibody. The chimeric mAb 2–4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2–4 and chimeric mAb 2–4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2–4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2–4 was reduced. In contrast, in cats treated with chimeric mAb 2–4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2–4-treated cats. PMID:27264736

  13. Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A

    PubMed Central

    Prigent, Julie; Mazuet, Christelle; Boquet, Didier; Lamourette, Patricia; Volland, Hervé; Popoff, Michel R.; Créminon, Christophe; Simon, Stéphanie

    2010-01-01

    Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species. PMID:20967241

  14. A Novel Chimeric Anti-PA Neutralizing Antibody for Postexposure Prophylaxis and Treatment of Anthrax.

    PubMed

    Xiong, Siping; Tang, Qi; Liang, Xudong; Zhou, Tingting; Yang, Jin; Liu, Peng; Chen, Ya; Wang, Changjun; Feng, Zhenqing; Zhu, Jin

    2015-07-02

    Anthrax is a highly lethal infectious disease caused by the bacterium Bacillus anthracis, and the associated shock is closely related to the lethal toxin (LeTx) produced by the bacterium. The central role played by the 63 kDa protective antigen (PA63) region of LeTx in the pathophysiology of anthrax makes it an excellent therapeutic target. In the present study, a human/murine chimeric IgG mAb, hmPA6, was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmPA6 expressed in 293F cells could neutralize LeTx both in vitro and in vivo. At a dose of 0.3 mg/kg, it could protect all tested rats from a lethal dose of LeTx. Even administration of 0.6 mg/kg hmPA6 48 h before LeTx challenge protected all tested rats. The results indicate that hmPA6 is a potential candidate for clinical application in anthrax treatment.

  15. High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG

    PubMed Central

    Miyazaki‐Komine, Kaori; Takai, Yoshiki; Huang, Ping; Kusano‐Arai, Osamu; Iwanari, Hiroko; Misu, Tatsuro; Koda, Katsushi; Mitomo, Katsuyuki; Sakihama, Toshiko; Toyama, Yoshiaki; Fujihara, Kazuo; Hamakubo, Takao; Yasui, Masato

    2015-01-01

    Background and Purpose Most of the cases of neuromyelitis optica (NMO) are characterized by the presence of an autoantibody, NMO‐IgG, which recognizes the extracellular domains of the water channel, aquaporin‐4. Binding of NMO‐IgG to aquaporin‐4 expressed in end‐feet of astrocytes leads to complement‐dependent disruption of astrocytes followed by demyelination. One therapeutic option for NMO is to prevent the binding of NMO‐IgG to aquaporin‐4, using high‐avidity, non‐pathogenic–chimeric, monoclonal antibodies to this water channel. We describe here the development of such antibodies. Experimental Approach cDNAs encoding variable regions of heavy and light chains of monoclonal antibodies against the extracellular domains of human aquaporin‐4 were cloned from hybridoma total RNA and fused to those encoding constant regions of human IgG1 and Igκ respectively. Then mammalian expression vectors were constructed to establish stable cell lines secreting mature chimeric antibodies. Key Results Original monoclonal antibodies showed high avidity binding to human aquaporin‐4, as determined by ELISA. Live imaging using Alexa‐Fluor‐555‐labelled antibodies revealed that the antibody D15107 more rapidly bound to cells expressing human aquaporin‐4 than others and strongly enhanced endocytosis of this water channel, while D12092 also bound rapidly to human aquaporin‐4 but enhanced endocytosis to a lesser degree. Chimeric D15107 prevented complement‐dependent cytotoxicity induced by NMO‐IgG from patient sera in vitro. Conclusions and Implications We have established non‐pathogenic, high‐avidity, chimeric antibodies against the extracellular domains of human aquaporin‐4, which provide a novel therapeutic option for preventing the progress and recurrence of NMO/NMO spectrum disorders. PMID:26398585

  16. Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines.

    PubMed

    Ye, Ling; Wen, Zhiyuan; Dong, Ke; Wang, Xi; Bu, Zhigao; Zhang, Huizhong; Compans, Richard W; Yang, Chinglai

    2011-01-01

    Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.

  17. Chimeric antigen receptors and bispecific antibodies to retarget T cells in pediatric oncology

    PubMed Central

    Suzuki, Maya; Curran, Kevin J.; Cheung, Nai-Kong V.

    2016-01-01

    Cancer immunotherapy using antigen-specific T cells has broad therapeutic potential. Chimeric antigen receptors and bispecific antibodies can redirect T cells to kill tumors without human leukocyte antigens (HLA) restriction. Key determinants of clinical potential include the choice of target antigen, antibody specificity, antibody affinity, tumor accessibility, T cell persistence, and tumor immune evasion. For pediatric cancers, additional constraints include their propensity for bulky metastatic disease and the concern for late toxicities from treatment. Nonetheless, the recent preclinical and clinical developments of these T cell based therapies are highly encouraging. PMID:25832831

  18. Production of Hybrid Chimeric PVX Particles Using a Combination of TMV and PVX-Based Expression Vectors

    PubMed Central

    Dickmeis, Christina; Honickel, Mareike Michaela Antonia; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    We have generated hybrid chimeric potato virus X (PVX) particles by coexpression of different PVX coat protein fusions utilizing tobacco mosaic virus (TMV) and PVX-based expression vectors. Coinfection was achieved with a modified PVX overcoat vector displaying a fluorescent protein and a TMV vector expressing another PVX fluorescent overcoat fusion protein. Coexpression of the PVX-CP fusions in the same cells was confirmed by epifluorescence microscopy. Labeling with specific antibodies and transmission electron microscopy revealed chimeric particles displaying green fluorescent protein and mCherry on the surface. These data were corroborated by bimolecular fluorescence complementation. We used split-mCherry fragments as PVX coat fusions and confirmed an interaction between the split-mCherry fragments in coinfected cells. The presence of assembled split-mCherry on the surface confirmed the hybrid character of the chimeric particles. PMID:26636076

  19. Production of Hybrid Chimeric PVX Particles Using a Combination of TMV and PVX-Based Expression Vectors.

    PubMed

    Dickmeis, Christina; Honickel, Mareike Michaela Antonia; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    We have generated hybrid chimeric potato virus X (PVX) particles by coexpression of different PVX coat protein fusions utilizing tobacco mosaic virus (TMV) and PVX-based expression vectors. Coinfection was achieved with a modified PVX overcoat vector displaying a fluorescent protein and a TMV vector expressing another PVX fluorescent overcoat fusion protein. Coexpression of the PVX-CP fusions in the same cells was confirmed by epifluorescence microscopy. Labeling with specific antibodies and transmission electron microscopy revealed chimeric particles displaying green fluorescent protein and mCherry on the surface. These data were corroborated by bimolecular fluorescence complementation. We used split-mCherry fragments as PVX coat fusions and confirmed an interaction between the split-mCherry fragments in coinfected cells. The presence of assembled split-mCherry on the surface confirmed the hybrid character of the chimeric particles.

  20. Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein

    PubMed Central

    Soleimani, Meysam; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    Background: Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein. Materials and Methods: In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains. Results: Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins. Conclusions: The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains. PMID:27169101

  1. Anti-Bovine Programmed Death-1 Rat–Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

    PubMed Central

    Okagawa, Tomohiro; Konnai, Satoru; Nishimori, Asami; Maekawa, Naoya; Ikebuchi, Ryoyo; Goto, Shinya; Nakajima, Chie; Kohara, Junko; Ogasawara, Satoshi; Kato, Yukinari; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2017-01-01

    Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat–bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals. PMID:28638381

  2. Anti-Bovine Programmed Death-1 Rat-Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle.

    PubMed

    Okagawa, Tomohiro; Konnai, Satoru; Nishimori, Asami; Maekawa, Naoya; Ikebuchi, Ryoyo; Goto, Shinya; Nakajima, Chie; Kohara, Junko; Ogasawara, Satoshi; Kato, Yukinari; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2017-01-01

    Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat-bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4(+) T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.

  3. Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination.

    PubMed

    Wenthur, Cody J; Cai, Xiaoqing; Ellis, Beverly A; Janda, Kim D

    2017-08-15

    Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  5. A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate.

    PubMed

    Kalbina, Irina; Wallin, Anita; Lindh, Ingrid; Engström, Peter; Andersson, Sören; Strid, Ke

    2011-12-01

    The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP - up to 3% of TSP. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

    PubMed Central

    Zvirbliene, Aurelija; Kucinskaite-Kodze, Indre; Razanskiene, Ausra; Petraityte-Burneikiene, Rasa; Klempa, Boris; Ulrich, Rainer G.; Gedvilaite, Alma

    2014-01-01

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications. PMID:24513568

  7. The use of chimeric virus-like particles harbouring a segment of hantavirus Gc glycoprotein to generate a broadly-reactive hantavirus-specific monoclonal antibody.

    PubMed

    Zvirbliene, Aurelija; Kucinskaite-Kodze, Indre; Razanskiene, Ausra; Petraityte-Burneikiene, Rasa; Klempa, Boris; Ulrich, Rainer G; Gedvilaite, Alma

    2014-02-07

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80-89) or site #4 (aa 280-289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.

  8. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

    PubMed

    Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

    2017-08-08

    Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

  9. Eliciting neutralizing antibodies against the membrane proximal external region of HIV-1 Env by chimeric live attenuated influenza A virus vaccines.

    PubMed

    Zang, Yang; Du, Dongchuan; Li, Na; Su, Weiheng; Liu, Xintao; Zhang, Yan; Nie, Jianhui; Wang, Youchun; Kong, Wei; Jiang, Chunlai

    2015-07-31

    Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Treatment of vitiligo with a chimeric monoclonal antibody to CD20: a pilot study

    PubMed Central

    Ruiz-Argüelles, A; García-Carrasco, M; Jimenez-Brito, G; Sánchez-Sosa, S; Pérez-Romano, B; Garcés-Eisele, J; Camacho-Alarcón, C; Reyes-Núñez, V; Sandoval-Cruz, M; Mendoza-Pinto, C; López-Colombo, A

    2013-01-01

    Five patients with active disseminated vitiligo were given 1 g of a chimeric (murine/human) monoclonal antibody to CD20 in a single intravenous infusion and followed-up for 6 months. Three of the patients showed an overt clinical and histological improvement of the disease, one presented slight improvement and the remaining patient showed no changes. Improvement was neither associated with changes in laboratory parameters nor to a specific human leucocyte antigen D-related (HLA-DR) phenotype. We believe that these preliminary results are encouraging, and further clinical trials should be undertaken. An important aim should be the finding of a marker with a good response to this therapeutic approach. PMID:23815517

  11. Treatment of vitiligo with a chimeric monoclonal antibody to CD20: a pilot study.

    PubMed

    Ruiz-Argüelles, A; García-Carrasco, M; Jimenez-Brito, G; Sánchez-Sosa, S; Pérez-Romano, B; Garcés-Eisele, J; Camacho-Alarcón, C; Reyes-Núñez, V; Sandoval-Cruz, M; Mendoza-Pinto, C; López-Colombo, A

    2013-11-01

    Five patients with active disseminated vitiligo were given 1g of a chimeric (murine/human) monoclonal antibody to CD20 in a single intravenous infusion and followed-up for 6 months. Three of the patients showed an overt clinical and histological improvement of the disease, one presented slight improvement and the remaining patient showed no changes. Improvement was neither associated with changes in laboratory parameters nor to a specific human leucocyte antigen D-related (HLA-DR) phenotype. We believe that these preliminary results are encouraging, and further clinical trials should be undertaken. An important aim should be the finding of a marker with a good response to this therapeutic approach. © 2013 British Society for Immunology.

  12. First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers.

    PubMed

    Stevens, Misty W; Henry, Ralph L; Owens, S Michael; Schutz, Ralph; Gentry, W Brooks

    2014-01-01

    This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17-19 d in the 3 highest dose groups and volume of distribution of 5-6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.

  13. Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

    PubMed

    Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

    2014-01-01

    Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.

  14. Evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue functional antibody assays.

    PubMed

    Yamanaka, Atsushi; Suzuki, Ryosuke; Konishi, Eiji

    2014-07-23

    Dengue fever and dengue hemorrhagic fever are endemic throughout tropical and subtropical countries. Four serotypes of dengue viruses (DENV-1 to DENV-4), each with several genotypes including various subclades, are co-distributed in most endemic areas. Infection-neutralizing and -enhancing antibodies are believed to play protective and pathogenic roles, respectively. Measurement of these functional antibodies against a variety of viral strains is thus important for evaluating coverage and safety of dengue vaccine candidates. Although transportation of live virus materials beyond national borders is increasingly limited, this difficulty may be overcome using biotechnology that enables generation of an antibody-assay antigen equivalent to authentic virus based on viral sequence information. A rapid system to produce flavivirus single-round infectious particles (SRIPs) was recently developed using a Japanese encephalitis virus (JEV) subgenomic replicon plasmid. This system allows production of chimeric SRIPs that have surface proteins of other flaviviruses. In the present study, SRIPs of DENV-1 (D1-SRIPs) were evaluated as an antigen for functional antibody assays. Inclusion of the whole mature capsid gene of JEV into the replicon plasmid provided higher D1-SRIP yields than did its exclusion in cases where a DENV-1 surface-protein-expressing plasmid was used for co-transfection of 293T cells with the replicon plasmid. In an assay to measure the balance between neutralizing and enhancing activities, dose (antibody dilution)-dependent activity curves in dengue-immune human sera or mouse monoclonal antibodies obtained using D1-SRIP antigen were equivalent to those obtained using DENV-1 antigen. Similar results were obtained using additional DENV-2 and DENV-3 systems. In a conventional Vero-cell neutralization test, a significant correlation was shown between antibody titers obtained using D1-SRIP and DENV-1 antigens. These results demonstrate the utility of D1-SRIPs as

  15. Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease

    PubMed Central

    Zanchin, Nilson Ivo Tonin; Brasil, Tatiana de Arruda Campos; Foti, Leonardo; de Souza, Wayner Vieira; Silva, Edmilson Domingos; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2016-01-01

    The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies. PMID:27517281

  16. Production and Characterization of a Set of Mouse-Human Chimeric Immunoglobulin G (IgG) Subclass and IgA Monoclonal Antibodies with Identical Variable Regions Specific for Pseudomonas aeruginosa Serogroup O6 Lipopolysaccharide

    PubMed Central

    Preston, Michael J.; Gerçeker, A. Alev; Reff, Mitchell E.; Pier, Gerald B.

    1998-01-01

    The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy. PMID

  17. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  18. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  19. Functional analysis of aldehyde oxidase using expressed chimeric enzyme between monkey and rat.

    PubMed

    Itoh, Kunio; Asakawa, Tasuku; Hoshino, Kouichi; Adachi, Mayuko; Fukiya, Kensuke; Watanabe, Nobuaki; Tanaka, Yorihisa

    2009-01-01

    Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie-Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater V(max) values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively.

  20. Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells

    PubMed Central

    Dotti, Gianpietro; Gottschalk, Stephen; Savoldo, Barbara; Brenner, Malcolm K

    2013-01-01

    Summary Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking and effector functions of a T-cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CART cells. Our review then describes our own and other investigators’ work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR-T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained. PMID:24329793

  1. Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

    PubMed Central

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

  2. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    PubMed

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR(+) T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+) tumor targets. This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  3. Engineering, expression and purification of a chimeric fibrin-specific streptokinase.

    PubMed

    Taheri, Mohammad Naser; Behzad-Behbahani, Abbas; Rafiei Dehbidi, Gholamreza; Salehi, Saeede; Sharifzadeh, Sedigheh

    2016-12-01

    Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric streptokinase by fusing the fibrin binding Kringle 2 domain of tissue plasminogen activator (TPA) to the streptokinase N-terminal end. The chimeric SK construct (KSK) with inserted Kringle 2 domain was cloned into pET28a expression vector. The expression of recombinant protein was carried out in Escherichia coli origami (DE3) and confirmed by SDS-PAGE and Western blotting analyses. We used the chromogenic substrate S-2251 method to assess the specific activities of the chimeric and control wild-type proteins. Then, the two proteins were added in amounts with equal activity to fibrin clots of identical size. Finally, the supernatant above the fibrin clots was collected and subjected to the chromogenic assay to analyze the specificity of the chimeric protein. The specific activities of the chimeric and wild-type proteins were found to be 0.06 U/mg and 0.07 U/mg, respectively. Because of the binding of the chimeric protein to fibrin, the mean specific activity was significantly lower in the KSK supernatant (0.01) compared with the control (approximately 0.06) (p < 0.05). Our in vitro results indicate that the chimeric streptokinase protein has strong fibrin-specific activity compared to the wild-type protein. However, further in vivo studies are needed to evaluate its potential fibrinolytic effects. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. A nontoxic chimeric enterotoxin adjuvant induces protective immunity in both mucosal and systemic compartments with reduced IgE antibodies.

    PubMed

    Kweon, Mi-Na; Yamamoto, Masafumi; Watanabe, Fumiko; Tamura, Shinichi; Van Ginkel, Frederik W; Miyauchi, Akira; Takagi, Hiroaki; Takeda, Yoshifumi; Hamabata, Takashi; Fujihashi, Kohtaro; McGhee, Jerry R; Kiyono, Hiroshi

    2002-11-01

    A novel nontoxic form of chimeric mucosal adjuvant that combines the A subunit of mutant cholera toxin E112K with the pentameric B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli was constructed by use of the Brevibacillus choshinensis expression system (mCTA/LTB). Nasal immunization of mice with tetanus toxoid (TT) plus mCTA/LTB elicited significant TT-specific immunoglobulin A responses in mucosal compartments and induced high serum immunoglobulin G and immunoglobulin A anti-TT antibody responses. Although TT plus native CT induced high total and TT-specific immunoglobulin E responses, use of the chimera molecule as mucosal adjuvant did not. Furthermore, all mice immunized with TT plus mCTA/LTB were protected from lethal systemic challenge with tetanus toxin. Importantly, the mice were completely protected from influenza virus infection after nasal immunization with inactivated influenza vaccine together with mCTA/LTB. These results show that B. choshinensis-derived mCTA/LTB is an effective and safe mucosal adjuvant for the induction of protective immunity against potent bacterial exotoxin and influenza virus infection.

  5. Expression and assembly of a fully active antibody in algae

    NASA Astrophysics Data System (ADS)

    Mayfield, Stephen P.; Franklin, Scott E.; Lerner, Richard A.

    2003-01-01

    Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

  6. Equivalent perceptual asymmetries for free viewing of positive and negative emotional expressions in chimeric faces.

    PubMed

    Christman, S D; Hackworth, M D

    1993-06-01

    Research employing chimeric stimuli (in which smiling and neutral half-faces are paired) has demonstrated greater influence of the left half-face in determining perceived intensity of expression. To date, no studies have examined how emotional expressions other than happiness are perceived in this format. Right-handed subjects viewed chimeric faces depicting both positive (happiness, pleasant surprise) and negative (sadness, anger) emotions in a free vision task. Results indicated a left half-face bias for all four emotions, supporting the hypothesis of a greater right hemisphere role in emotional perception. The lack of differences in strength of left half-face bias as a function of the specific emotion depicted suggests that results obtained with typical chimeric half-face paradigms can be generalized to emotions other than happiness.

  7. Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

    SciTech Connect

    Hajra, A.; Liu, P.; Collins, E.S.

    1994-09-01

    A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicated by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.

  8. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    PubMed

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG1) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  9. Effect of VK framework-1 glycosylation on the binding affinity of lymphoma-specific murine and chimeric LL2 antibodies and its potential use as a novel conjugation site.

    PubMed

    Leung, S O; Dion, A S; Pellegrini, M C; Losman, M J; Grebenau, R C; Goldenberg, D M; Hansen, H J

    1995-02-08

    A potential asparagine (Asn)-linked glycosylation site was identified in the VK FRI sequence of an anti-B lymphoma monoclonal antibody (MAb), LL2.SDS-PAGE analysis and endo-F treatment of both murine and chimeric LL2 antibodies indicated that this site was glycosylated; however, no differences in the binding affinity to Raji cells were observed between the native murine LL2 and the endo-F-deglycosylated murine LL2 antibodies. Elimination of the glycosylation site from the chimeric LL2 antibody was accomplished by an Asn to Gln mutation in the tri-acceptor site found in the light chain. The resultant aglycosylated chimeric LL2 exhibited a similar Raji cell binding affinity to that of the glycosylated form. The results are in agreement with computer modeling studies which suggested the lack of interactions between the oligosaccharide moiety and the CDRs. The finding is interesting because it enables a wider choice of human framework sequences, which in most cases do not have a corresponding glycosylation site, for the humanization of the LL2 VK domain, as well as a greater latitude of host expression systems. Most importantly, the LL2 VK carbohydrate moiety might be used as a novel conjugation site for drugs and radionuclides without compromising the immunoreactivity of the antibody.

  10. Exploiting chimeric human antibodies to characterize a protective epitope of Neisseria adhesin A, one of the Bexsero vaccine components.

    PubMed

    Bertoldi, Isabella; Faleri, Agnese; Galli, Barbara; Lo Surdo, Paola; Liguori, Alessia; Norais, Nathalie; Santini, Laura; Masignani, Vega; Pizza, Mariagrazia; Giuliani, Marzia Monica

    2016-01-01

    Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation. © FASEB.

  11. Immunizations with chimeric hepatitis B virus-like particles to induce potential anti-hepatitis C virus neutralizing antibodies.

    PubMed

    Vietheer, Patricia T K; Boo, Irene; Drummer, Heidi E; Netter, Hans-Jürgen

    2007-01-01

    Virus-like particles (VLPs) are highly immunogenic and proven to induce protective immunity. The small surface antigen (HBsAg-S) of hepatitis B virus (HBV) self-assembles into VLPs and its use as a vaccine results in protective antiviral immunity against HBV infections. Chimeric HBsAg-S proteins carrying foreign epitopes allow particle formation and have the ability to induce anti-foreign humoral and cellular immune responses. The insertion of the hypervariable region 1 (HVR1) sequence derived from the envelope protein 2 (E2) of hepatitis C virus (HCV) into the major antigenic site of HBsAg-S ('a'-determinant) resulted in the formation of highly immunogenic VLPs that retained the antigenicity of the inserted HVR1 sequence. BALB/c mice were immunized with chimeric VLPs, which resulted in antisera with anti-HCV activity. The antisera were able to immunoprecipitate native HCV envelope complexes (E1E2) containing homologous or heterologous HVR1 sequences. HCV E1E2 pseudotyped HIV-1 particles (HCVpp) were used to measure entry into HuH-7 target cells in the presence or absence of antisera that were raised against chimeric VLPs. Anti-HVR1 VLP sera interfered with entry of entry-competent HCVpps containing either homologous or heterologous HVR1 sequences. Also, immunizations with chimeric VLPs induced antisurface antigen (HBsAg) antibodies, indicating that HBV-specific antigenicity and immunogenicity of the 'a'-determinant region is retained. A multivalent vaccine against different pathogens based on the HBsAg delivery platform should be possible. We hypothesize that custom design of VLPs with an appropriate set of HCV-neutralizing epitopes will induce antibodies that would serve to decrease the viral load at the initial infecting inoculum.

  12. Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.

    PubMed

    Rueda, P; Hurtado, A; del Barrio, M; Martínez-Torrecuadrada, J L; Kamstrup, S; Leclerc, C; Casal, J I

    1999-10-10

    An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses. Copyright 1999 Academic Press.

  13. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    PubMed

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system.

  14. Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

    PubMed Central

    2011-01-01

    Background To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. Methods A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. Results The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38) and a clinical specificity of 100% (n = 24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Conclusions The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria. PMID:21529346

  15. Transient Expression of Chimeric Genes Delivered into Pollen by Microprojectile Bombardment 1

    PubMed Central

    Twell, David; Klein, Theodore M.; Fromm, Michael E.; McCormick, Sheila

    1989-01-01

    Chimeric genes containing a pollen-specific promoter from tomato (Lycopersicon esculentum) or the CaMV35S promoter were transiently expressed following their introduction into tobacco (Nicotiana tabacum) pollen using high velocity microprojectiles. Transient expression of the microprojectile-introduced genes in leaves and pollen was similar to that observed for these genes in stably transformed tobacco plants. Images Figure 2 PMID:16667175

  16. Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies

    PubMed Central

    Wang, Joshua W.; Jagu, Subhashini; Wu, Wai-Hong; Viscidi, Raphael P.; Macgregor-Das, Anne; Fogel, Jessica M.; Kwak, Kihyuck; Daayana, Sai; Kitchener, Henry; Stern, Peter L.; Gravitt, Patti E.; Trimble, Cornelia L.

    2015-01-01

    Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies. PMID:25972404

  17. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

    PubMed Central

    2012-01-01

    Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized

  18. Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry.

    PubMed

    Zheng, Zhili; Chinnasamy, Nachimuthu; Morgan, Richard A

    2012-02-13

    There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Currently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments. We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized mouse antibody based CARs

  19. Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

    PubMed

    Marciello, Marzia; Filice, Marco; Olea, David; Velez, Marisela; Guisan, José M; Mateo, Cesar

    2014-12-16

    The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

  20. [Novel therapy for malignant lymphoma: adoptive immuno-gene therapy using chimeric antigen receptor(CAR)-expressing T lymphocytes].

    PubMed

    Ozawa, Keiya

    2014-03-01

    Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

  1. Mouse/human chimeric IgG1 and IgG4 antibodies directed to the house dust mite allergen Der p 2: use in quantification of allergen specific IgG.

    PubMed

    Schuurman, J; Perdok, G J; Mueller, G A; Benjamin, D C; Yong Tan, K; Chapman, M D; Aalberse, R C

    1997-09-01

    Chimeric mouse/human monoclonal IgG1 and IgG4 antibodies were developed against the house dust mite allergen Der p 2. These chimeric IgG antibodies, hIgG1-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains of the original murine monoclonal antibody 2B12, whereas the heavy chain constant domains have been replaced by the human IgG1 or IgG4 heavy chain. The expression level of hIgG1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 microg/mL, respectively. Since all IgG in these culture supernatants is allergen-specific, they are useful reference reagents and enable the calculation of the amount of allergen specific IgG1 and IgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgG1 and IgG4 RAST and with reversed IgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found. With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.

  2. Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

    PubMed Central

    Ye, Xiaohua; Ku, Zhiqiang; Liu, Qingwei; Wang, Xiaoli; Shi, Jinping; Zhang, Yunfang; Kong, Liangliang; Cong, Yao

    2014-01-01

    Enterovirus 71 (EV71) is a major causative agent of hand, food, and mouth disease, which frequently occurs in young children. Since there are 11 subgenotypes (A, B1 to B5, and C1 to C5) within EV71, an EV71 vaccine capable of protecting against all of these subgenotypes is desirable. We report here the vaccine potential and protective mechanism of two chimeric virus-like particles (VLPs) presenting conserved neutralizing epitopes of EV71. We show that fusions of hepatitis B core antigen (HBc) with the SP55 or SP70 epitope of EV71, designated HBcSP55 and HBcSP70, respectively, can be rapidly generated and self-assembled into VLPs with the epitopes displayed on the surface. Immunization with the chimeric VLPs induced carrier- and epitope-specific antibody responses in mice. Anti-HBcSP55 and anti-HBcSP70 sera, but not anti-HBc sera, were able to neutralize in vitro multiple genotypes and strains of EV71. Importantly, passive immunization with anti-HBcSP55 or anti-HBcSP70 sera protected neonatal mice against lethal EV71 infections. Interestingly, anti-HBcSP70 sera could inhibit EV71 attachment to susceptible cells, whereas anti-HBcSP55 sera could not. However, both antisera were able to neutralize EV71 infection in vitro at the postattachment stage. The divergent mechanism of neutralization and protection conferred by anti-SP70 and anti-SP55 sera is in part attributed to their respective ability to bind authentic viral particles. Collectively, our study not only demonstrates that chimeric VLPs displaying the SP55 and SP70 epitopes are promising candidates for a broad-spectrum EV71 vaccine but also reveals distinct mechanisms of neutralization by the SP55- and SP70-targeted antibodies. PMID:24131712

  3. Exchanging Murine and Human Immunoglobulin Constant Chains Affects the Kinetics and Thermodynamics of Antigen Binding and Chimeric Antibody Autoreactivity

    PubMed Central

    Torres, Marcela; Fernandez-Fuentes, Narcis; Fiser, András; Casadevall, Arturo

    2007-01-01

    Mouse-human chimeric antibodies composed of murine variable (V) and human (C) chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of CH glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb) 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch) and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by CH region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the CH domains through an allosteric effect involving networks of highly connected amino acids. PMID:18074033

  4. Chimeric Anti-Staphylococcal Enterotoxin B Antibodies and Lovastatin Act Synergistically to Provide In Vivo Protection against Lethal Doses of SEB

    PubMed Central

    Tilahun, Mulualem E.; Kwan, Alan; Natarajan, Kannan; Quinn, Megan; Tilahun, Ashenafi Y.; Xie, Chen; Margulies, David H.; Osborne, Barbara A.; Goldsby, Richard A.; Rajagopalan, Govindarajan

    2011-01-01

    Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts. PMID:22102880

  5. Functional expression and properties of a nicotinic alpha9/5-HT3A chimeric receptor.

    PubMed

    Verbitsky, Miguel; Plazas, Paola V; Elgoyhen, A Belén

    2003-10-27

    We describe the functional properties of a nicotinic alpha9/serotonin subtype 3A (5HT3A) chimeric receptor expressed in Xenopus laevis oocytes. The chimera preserved ligand-binding properties of alpha9 and channel properties of 5HT3A. Thus, it responded to acetylcholine in a concentration-dependent manner with an EC50 of 70 microM but not to serotonin. It was blocked by methyllycaconitine, strychnine, atropine and nicotine, with the same rank order of potency as alpha9 receptors. The current-voltage relationship of currents through the alpha9/5HT3A chimera was similar to that of the 5HT3A receptors. These results are an evidence of functional coupling between the ligand-binding and the channel domains of the chimeric receptor.

  6. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model

    PubMed Central

    Suarez, Eloah Rabello; Chang, De-Kuan; Sun, Jiusong; Sui, Jianhua; Freeman, Gordon J.; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A.

    2016-01-01

    Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50–80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen. PMID:27145284

  7. Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma

    PubMed Central

    Eger, Christin; Siebert, Nikolai; Seidel, Diana; Zumpe, Maxi; Jüttner, Madlen; Brandt, Sven; Müller, Hans-Peter; Lode, Holger N.

    2016-01-01

    Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2 overexpressed in NB

  8. A recombinant chimeric La Crosse virus expressing the surface glycoproteins of Jamestown Canyon virus is immunogenic and protective against challenge with either parental virus in mice or monkeys.

    PubMed

    Bennett, R S; Gresko, A K; Nelson, J T; Murphy, B R; Whitehead, S S

    2012-01-01

    La Crosse virus (LACV) and Jamestown Canyon virus (JCV), family Bunyaviridae, are mosquito-borne viruses that are endemic in North America and recognized as etiologic agents of encephalitis in humans. Both viruses belong to the California encephalitis virus serogroup, which causes 70 to 100 cases of encephalitis a year. As a first step in creating live attenuated viral vaccine candidates for this serogroup, we have generated a recombinant LACV expressing the attachment/fusion glycoproteins of JCV. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. For the M segment, the open reading frame (ORF) of LACV is replaced with that derived from JCV and is flanked by the untranslated regions of LACV. The resulting chimeric virus retained the same robust growth kinetics in tissue culture as observed for either parent virus, and the virus remains highly infectious and immunogenic in mice. Although both LACV and JCV are highly neurovirulent in 21 day-old mice, with 50% lethal dose (LD₅₀) values of 0.1 and 0.5 log₁₀ PFU, respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 10³ PFU. Parenteral vaccination of mice with 10¹ or 10³ PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and induced neutralizing antibodies to JCV, LACV, and TAHV. When vaccinated monkeys were challenged with JCV, they were protected against the development of viremia. Generation of highly attenuated yet immunogenic chimeric bunyaviruses could be an efficient general method for development of vaccines effective against these pathogenic viruses.

  9. Constant domains influence binding of mouse–human chimeric antibodies to the capsular polypeptide of Bacillus anthracis

    PubMed Central

    Hubbard, Mark A; Thorkildson, Peter; Kozel, Thomas R; AuCoin, David P

    2013-01-01

    Our laboratory previously described the binding characteristics of the murine IgG3 monoclonal antibody (MuAb) F26G3. This antibody binds the poly-glutamic acid capsule (PGA) of Bacillus anthracis, an essential virulence factor in the progression of anthrax. F26G3 IgG3 MuAb binds PGA with a relatively high functional affinity (10 nM), produces a distinct “rim” quellung reaction, and is protective in a murine model of pulmonary anthrax. This study engineered an IgG subclass family of F26G3 mouse–human chimeric antibodies (ChAb). The F26G3 ChAbs displayed 9- to 20-fold decreases in functional affinity, as compared with the parent IgG3 MuAb. Additionally, the quellung reactions that were produced by the ChAbs all differed from the parent IgG3 MuAb in that they appeared “puffy” in nature. This study demonstrates that human constant domains may influence multiple facets of antibody binding to microbial capsular antigens despite their spatial separation from the traditional antigen-binding site. PMID:23863605

  10. Constant domains influence binding of mouse-human chimeric antibodies to the capsular polypeptide of Bacillus anthracis.

    PubMed

    Hubbard, Mark A; Thorkildson, Peter; Kozel, Thomas R; AuCoin, David P

    2013-08-15

    Our laboratory previously described the binding characteristics of the murine IgG3 monoclonal antibody (MuAb) F26G3. This antibody binds the poly-glutamic acid capsule (PGA) of Bacillus anthracis, an essential virulence factor in the progression of anthrax. F26G3 IgG3 MuAb binds PGA with a relatively high functional affinity (10 nM), produces a distinct "rim" quellung reaction, and is protective in a murine model of pulmonary anthrax. This study engineered an IgG subclass family of F26G3 mouse-human chimeric antibodies (ChAb). The F26G3 ChAbs displayed 9- to 20-fold decreases in functional affinity, as compared with the parent IgG3 MuAb. Additionally, the quellung reactions that were produced by the ChAbs all differed from the parent IgG3 MuAb in that they appeared "puffy" in nature. This study demonstrates that human constant domains may influence multiple facets of antibody binding to microbial capsular antigens despite their spatial separation from the traditional antigen-binding site.

  11. Multi-petal cyclamen flowers produced by AGAMOUS chimeric repressor expression.

    PubMed

    Tanaka, Yuri; Oshima, Yoshimi; Yamamura, Tomomichi; Sugiyama, Masao; Mitsuda, Nobutaka; Ohtsubo, Norihiro; Ohme-Takagi, Masaru; Terakawa, Teruhiko

    2013-01-01

    Cyclamen persicum (cyclamen) is a commercially valuable, winter-blooming perennial plant. We cloned two cyclamen orthologues of AGAMOUS (AG), CpAG1 and CpAG2, which are mainly expressed in the stamen and carpel, respectively. Cyclamen flowers have 5 petals, but expression of a chimeric repressor of CpAG1 (CpAG1-SRDX) caused stamens to convert into petals, resulting in a flower with 10 petals. By contrast, CpAG2-SRDX only caused incomplete formation of stamens and carpels. Expression in Arabidopsis thaliana showed similar effects on flower organ specification. Simultaneous expression of CpAG1-SRDX and CpAG2-SRDX in cyclamen induced rose-like, multi-petal flowers, a potentially valuable trait in commercial ornamental varieties. Expression of CpAG2-SRDX in a cyclamen mutant lacking expression of CpAG1 more effectively produced multi-petal flowers. Here, we controlled the number of petals in cyclamen by simple genetic engineering with a chimeric repressor. This strategy may be applicable useful for other ornamental plants with two distinct AG orthologues.

  12. Successful treatment with a chimeric anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab) for a patient with relapsed mantle cell lymphoma who developed a human anti-chimeric antibody.

    PubMed

    Maeda, T; Yamada, Y; Tawara, M; Yamasaki, R; Yakata, Y; Tsutsumi, C; Onimaru, Y; Kamihira, S; Tomonaga, M

    2001-07-01

    Mantle cell lymphoma (MCL) has a poor prognosis without cure; the median overall survival ranges only from 3 to 4 years irrespective of conventional therapeutic regimens. IDEC-C2B8 (rituximab), a chimeric monoclonal antibody against the B-cell-specific antigen CD20, induces an evaluable clinical response in patients with MCL with mild toxicities. However, the single agent rituximab cannot cure MCL. Due to its low immunogenicity, an antibody against IDEC-C2B8 (human antichimeric antibody [HACA]) has rarely been produced in vivo. We report a patient with relapsed MCL who was successfully treated with IDEC-C2B8 for over a year although she developed HACA 6 months after the initial administration of IDEC-C2B8 in the phase II clinical trial conducted by Zenyaku Kogyo Co. Ltd. We followed the pharmacokinetics of IDEC-C2B8, the serum HACA titer, and the number of B lymphocytes in the peripheral blood in relation to clinical response. The HACA became undetectable soon after subsequent administrations of IDEC-C2B8. When the serum level of IDEC-C2B8 was kept elevated, clinical responses were apparently observed and HACA disappeared during this response period. There were no significant clinical toxicities related to the appearance of HACA. The present findings suggested that IDEC-C2B8 is effective and safe even in patients who have developed HACA.

  13. Differential effect of isotype on efficacy of anti-tumor necrosis factor alpha chimeric antibodies in experimental septic shock

    PubMed Central

    1994-01-01

    Immune complexes containing human gamma (g)1 or murine g2a antibodies generate secondary effector mechanisms via Fc receptor binding or complement activation, whereas those containing human g4 or murine g1 antibodies generally do not. Therefore, isotype selection of therapeutic antibodies may have important clinical consequences. In a rabbit model of human tumor necrosis factor (rhuTNF)-induced pyrexia, a murine/human chimeric g4 anti-human TNF-alpha monoclonal antibody (mAb) (cCB0011) showed a dose-dependent inhibition of pyrexia, whereas a g1 isotype variant of the same mAb gave a marked pyrexia that was seen at all doses indicative of an immune complex-mediated response. To investigate whether isotype difference could influence mAb efficacy in pathological disease states, hamster/murine chimeric g1 and g2a anti- murine TNF-alpha mAbs (TN3g1, TN3g2a) were studied in experimental shock in mice and rats. In lipopolysaccharide-induced shock in mice, treatment with TN3g1 mAb at 30 and 3 mg/kg resulted in 90% survival by 72 h (p < or = 0.004), and prolonged survival to 45 h (p < or = 0.05), respectively, compared with 100% mortality by 27 h in controls. In contrast, a g2a isotype variant of the same mAb (30 mg/kg) resulted in only 10% survival by 72 h (p < or = 0.05). In a neutropenic sepsis model in rats there was greater survival in animals receiving the g1 isotype of TN3 compared with g2a isotype variant (70 vs. 27%; p < or = 0.005) with 100% mortality in the controls. These differences were not due to the pharmacokinetic profiles of the mAbs. In models of experimental shock antibody isotype can affect outcome with inactive isotypes (human g4 and murine g1) being more efficacious than active isotypes (human g1 and murine g2a). PMID:8113678

  14. Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum.

    PubMed

    Dubois, Mathieu; Fenaille, François; Clement, Gilles; Lechmann, Martin; Tabet, Jean-Claude; Ezan, Eric; Becher, François

    2008-03-01

    In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Following digestion with trypsin, specific peptides from light and heavy chains were monitored in the selected reaction monitoring (SRM) mode. Assay variability below 20% was provided through optimization of the digestion step and rigorous monitoring of the whole analytical process using an appropriate internal standard. The 20 ng/mL lower limit of quantification was similar to that of ELISA methods. These results show that this mass spectrometric approach is a potential alternative for pharmacokinetic evaluation of mAbs during clinical development.

  15. Emotion processing in chimeric faces: hemispheric asymmetries in expression and recognition of emotions.

    PubMed

    Indersmitten, Tim; Gur, Ruben C

    2003-05-01

    Since the discovery of facial asymmetries in emotional expressions of humans and other primates, hypotheses have related the greater left-hemiface intensity to right-hemispheric dominance in emotion processing. However, the difficulty of creating true frontal views of facial expressions in two-dimensional photographs has confounded efforts to better understand the phenomenon. We have recently described a method for obtaining three-dimensional photographs of posed and evoked emotional expressions and used these stimuli to investigate both intensity of expression and accuracy of recognizing emotion in chimeric faces constructed from only left- or right-side composites. The participant population included 38 (19 male, 19 female) African-American, Caucasian, and Asian adults. They were presented with chimeric composites generated from faces of eight actors and eight actresses showing four emotions: happiness, sadness, anger, and fear, each in posed and evoked conditions. We replicated the finding that emotions are expressed more intensely in the left hemiface for all emotions and conditions, with the exception of evoked anger, which was expressed more intensely in the right hemiface. In contrast, the results indicated that emotional expressions are recognized more efficiently in the right hemiface, indicating that the right hemiface expresses emotions more accurately. The double dissociation between the laterality of expression intensity and that of recognition efficiency supports the notion that the two kinds of processes may have distinct neural substrates. Evoked anger is uniquely expressed more intensely and accurately on the side of the face that projects to the viewer's right hemisphere, dominant in emotion recognition.

  16. Isolation and characterization of chimeric human Fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow.

    PubMed

    Burdick, Monica M; Reynolds, Nathan M; Martin, Eric W; Hawes, Jacquelyn V; Carlson, Grady E; Cuckler, Chaz M; Bates, Michael C; Barthel, Steven R; Dimitroff, Charles J

    2014-01-08

    Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization

  17. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle

    PubMed Central

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  18. Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

    PubMed

    Knipping, Karen; Simons, Peter J; Buelens-Sleumer, Laura S; Cox, Linda; den Hartog, Marcel; de Jong, Niels; Teshima, Reiko; Garssen, Johan; Boon, Louis; Knippels, Léon M J

    2014-01-01

    Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

  19. Development of β-Lactoglobulin-Specific Chimeric Human IgEκ Monoclonal Antibodies for In Vitro Safety Assessment of Whey Hydrolysates

    PubMed Central

    Buelens-Sleumer, Laura S.; Cox, Linda; den Hartog, Marcel; de Jong, Niels; Teshima, Reiko; Garssen, Johan; Boon, Louis; Knippels, Léon M. J.

    2014-01-01

    Background Cow’s milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow’s milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. Objective An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Methods Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. Results After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow’s milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow’s milk allergic children. Conclusion Usage of our ‘unlimited’ source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow’s milk allergic patients to assess safety

  20. Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific IgE.

    PubMed

    Schuurman, J; Perdok, G J; Lourens, T E; Parren, P W; Chapman, M D; Aalberse, R C

    1997-04-01

    A chimeric human IgE monoclonal antibody was developed against the house dust mite allergen Der p 2. This chimeric antibody (hIgE-Dp2A) was composed of the heavy-chain variable domains and light chains of the original murine monoclonal antibody retaining its binding characteristics, whereas the heavy-chain constant domains were exchanged with the human IgE heavy chain. The chimeric IgE expression level was IgE 600 IU/ml (1 IU = 2.4 ng/ml). The binding of the chimeric hIgE-Dp2A to mite extract was indistinguishable from that of the original mouse monoclonal antibody. Parallel dose-response curves were found when the binding of hIgE-Dp2A to mite extract and anti-IgE coupled to sepharose were compared. Binding levels were not identical; however, hIgE-Dp2A bound significantly better to the mite-extract sepharose. This result indicates that the commonly used anti-IgE on solid phase calibration systems may lead to an overestimation of the amount of allergen-specific IgE present in the serum sample. The less efficient binding of the detector anti-IgE in case of the anti-IgE sepharose is likely to be because of the occupation of epitopes of the IgE by the sepharose-bound anti-IgE. Dose-response curves of serial dilutions of patient samples were parallel with the hIgE-Dp2A dose-response curve, which indicates that hIgE-Dp2A behaves like natural IgE antibodies in binding to allergen coupled to solid phase. This antibody is well suited for use as a reference reagent in the RAST and enables the expression of the amount of allergen-specific IgE present in a patient sample in absolute amounts.

  1. The B-cell tumor–associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor

    PubMed Central

    Schmitt, Thomas M.; Baskar, Sivasubramanian; Lupo-Stanghellini, Maria Teresa; Nishida, Tetsuya; Yamamoto, Tori N.; Bleakley, Marie; Turtle, Cameron J.; Chang, Wen-Chung; Greisman, Harvey A.; Wood, Brent; Maloney, David G.; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2010-01-01

    Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells. PMID:20702778

  2. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor.

    PubMed

    Hudecek, Michael; Schmitt, Thomas M; Baskar, Sivasubramanian; Lupo-Stanghellini, Maria Teresa; Nishida, Tetsuya; Yamamoto, Tori N; Bleakley, Marie; Turtle, Cameron J; Chang, Wen-Chung; Greisman, Harvey A; Wood, Brent; Maloney, David G; Jensen, Michael C; Rader, Christoph; Riddell, Stanley R

    2010-11-25

    Monoclonal antibodies and T cells modified to express chimeric antigen receptors specific for B-cell lineage surface molecules such as CD20 exert antitumor activity in B-cell malignancies, but deplete normal B cells. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) was identified as a highly expressed gene in B-cell chronic lymphocytic leukemia (B-CLL), but not normal B cells, suggesting it may serve as a tumor-specific target for therapy. We analyzed ROR1-expression in normal nonhematopoietic and hematopoietic cells including B-cell precursors, and in hematopoietic malignancies. ROR1 has characteristics of an oncofetal gene and is expressed in undifferentiated embryonic stem cells, B-CLL and mantle cell lymphoma, but not in major adult tissues apart from low levels in adipose tissue and at an early stage of B-cell development. We constructed a ROR1-specific chimeric antigen receptor that when expressed in T cells from healthy donors or CLL patients conferred specific recognition of primary B-CLL and mantle cell lymphoma, including rare drug effluxing chemotherapy resistant tumor cells that have been implicated in maintaining the malignancy, but not mature normal B cells. T-cell therapies targeting ROR1 may be effective in B-CLL and other ROR1-positive tumors. However, the expression of ROR1 on some normal tissues suggests the potential for toxi-city to subsets of normal cells.

  3. The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity

    PubMed Central

    Ogasawara, Satoshi; Fujii, Yuki; Oki, Hiroharu; Fukayama, Masashi; Nishioka, Yasuhiko; Kaneko, Mika K.

    2015-01-01

    Podoplanin (PDPN/Aggrus/T1α) binds to C-type lectin-like receptor-2 (CLEC-2) and induces platelet aggregation. PDPN is associated with malignant progression, tumor metastasis, and poor prognosis in several types of cancer. Although many anti-human PDPN (hPDPN) monoclonal antibodies (mAbs), such as D2-40 and NZ-1, have been established, these epitopes are limited to the platelet aggregation-stimulating (PLAG) domain (amino acids 29-54) of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-7, which is more sensitive than D2-40 and NZ-1, using the Cancer-specific mAb (CasMab) method. The epitope of LpMab-7 was shown to be entirely different from that of NZ-1, a neutralizing mAb against the PLAG domain according to an inhibition assay and lectin microarray analysis. In the present study, we produced a mouse-human chimeric anti-hPDPN mAb, chLpMab-7. ChLpMab-7 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, chLpMab-7 inhibited the growth of hPDPN-expressing tumors in vivo. Although chLpMab-7 recognizes a non-PLAG domain of hPDPN, it suppressed the hematogenous metastasis of hPDPN-expressing tumors. These results indicated that chLpMab-7 suppressed tumor development and hematogenous metastasis in a neutralization-independent manner. In conclusion, hPDPN shows promise as a target in the development of a novel antibody-based therapy. PMID:26416352

  4. Analytical characterization of ch14.18: a mouse-human chimeric disialoganglioside-specific therapeutic antibody.

    PubMed

    Soman, Gopalan; Kallarakal, Abraham T; Michiel, Dennis; Yang, Xiaoyi; Saptharish, Nirmala; Jiang, Hengguang; Giardina, Steve; Gilly, John; Mitra, George

    2012-01-01

    Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2-8°C is stable for more than five years.

  5. Expression of an immunogenic LTB-based chimeric protein targeting Zaire ebolavirus epitopes from GP1 in plant cells.

    PubMed

    Ríos-Huerta, Regina; Monreal-Escalante, Elizabeth; Govea-Alonso, Dania O; Angulo, Carlos; Rosales-Mendoza, Sergio

    2017-02-01

    An antigenic protein targeting two epitopes from the Zaire ebolavirus GP1 protein was expressed in plant cells rendering an antigen capable of inducing humoral responses in mouse when administered subcutaneously or orally. The 2014 Ebola outbreak made clear that new treatments and prophylactic strategies to fight this disease are needed. Since vaccination is an intervention that could achieve the control of this epidemic disease, exploring the production of new low-cost vaccines is a key path to consider; especially in developing countries. In this context, plants are attractive organisms for the synthesis and delivery of subunit vaccines. This study aimed at producing a chimeric protein named LTB-EBOV, based on the B subunit of the Escherichia coli heat-labile enterotoxin as an immunogenic carrier and two epitopes from the Zaire ebolavirus GP1 protein recognized by neutralizing antibodies. The LTB-EBOV protein was expressed in plant tissues at levels up to 14.7 µg/g fresh leaf tissue and proven to be immunogenic in BALB/c mice when administered by either subcutaneous or oral routes. Importantly, IgA and IgG responses were induced following the oral immunization. The potential use of the plant-made LTB-EBOV protein against EBOV is discussed.

  6. Expression of a new chimeric protein with a highly repeated sequence in tobacco cells.

    PubMed

    Saumonneau, Amélie; Rottier, Karine; Conrad, Udo; Popineau, Yves; Guéguen, Jacques; Francin-Allami, Mathilde

    2011-07-01

    In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS(W)) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS(M)-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.

  7. Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: a case study.

    PubMed

    Dorvignit, Denise; Palacios, Julio L; Merino, Maylin; Hernández, Tays; Sosa, Katya; Casaco, Angel; López-Requena, Alejandro; Mateo de Acosta, Cristina

    2012-01-01

    The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

  8. Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles.

    PubMed

    Abraham, Ambily; Natraj, Usha; Karande, Anjali A; Gulati, Ashutosh; Murthy, Mathur R N; Murugesan, Sathyabalan; Mukunda, Pavithra; Savithri, Handanahal S

    2016-02-24

    The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

  9. Optimizing antibody expression: The nuts and bolts.

    PubMed

    Ayyar, B Vijayalakshmi; Arora, Sushrut; Ravi, Shiva Shankar

    2017-03-01

    Antibodies are extensively utilized entities in biomedical research, and in the development of diagnostics and therapeutics. Many of these applications require high amounts of antibodies. However, meeting this ever-increasing demand of antibodies in the global market is one of the outstanding challenges. The need to maintain a balance between demand and supply of antibodies has led the researchers to discover better means and methods for optimizing their expression. These strategies aim to increase the volumetric productivity of the antibodies along with the reduction of associated manufacturing costs. Recent years have witnessed major advances in recombinant protein technology, owing to the introduction of novel cloning strategies, gene manipulation techniques, and an array of cell and vector engineering techniques, together with the progress in fermentation technologies. These innovations were also highly beneficial for antibody expression. Antibody expression depends upon the complex interplay of multiple factors that may require fine tuning at diverse levels to achieve maximum yields. However, each antibody is unique and requires individual consideration and customization for optimizing the associated expression parameters. This review provides a comprehensive overview of several state-of-the-art approaches, such as host selection, strain engineering, codon optimization, gene optimization, vector modification and process optimization that are deemed suitable for enhancing antibody expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins.

    PubMed

    Li, A; Baba, T W; Sodroski, J; Zolla-Pazner, S; Gorny, M K; Robinson, J; Posner, M R; Katinger, H; Barbas, C F; Burton, D R; Chou, T C; Ruprecht, R M

    1997-05-20

    A panel of 14 human IgG monoclonal antibodies (MAbs) specific for envelope antigens of the human immunodeficiency virus type 1 (HIV-1), 2 high-titer human anti-HIV-1 immunoglobulin (HIVIG) preparations, and 15 combinations of MAbs or MAb/HIVIG were tested for their ability to neutralize infection of cultured human T cells (MT-2) with a chimeric simian immunodeficiency virus (SHIV-vpu+), which expressed HIV-1 IIIB envelope antigens. Eleven MAbs and both HIVIGs were neutralizing. When used alone, the anti-CD4-binding site MAb b12, the anti-gp41 MAb 2F5, and the anti-gp120 MAb 2G12 were the most potent. When combination regimens involving two MAbs targeting different epitopes were tested, synergy was seen in all paired MAbs, except for one combination that revealed additive effects. The lowest effective antibody concentration for 50% viral neutralization (EC50) and EC90 were achieved with combinations of MAbs b12, 2F5, 2G12, and the anti-V3 MAb 694/98D. Depending on the combination regimen, the concentration of MAbs required to reach 90% virus neutralization was reduced approximately 2- to 25-fold as compared to the dose requirement of individual MAbs to produce the same effect. Synergy of the combination regimens implies that combinations of antibodies may have a role in passive immunoprophylaxis against HIV-1. The ability of SHIV to replicate in rhesus macaques will allow us to test such approaches in vivo.

  11. Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

    PubMed Central

    Abraham, Ambily; Natraj, Usha; Karande, Anjali A.; Gulati, Ashutosh; Murthy, Mathur R. N.; Murugesan, Sathyabalan; Mukunda, Pavithra; Savithri, Handanahal S.

    2016-01-01

    The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies–D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies. PMID:26905902

  12. Engineered platform for bioethylene production by a cyanobacterium expressing a chimeric complex of plant enzymes.

    PubMed

    Jindou, Sadanari; Ito, Yuki; Mito, Natsumi; Uematsu, Keiji; Hosoda, Akifumi; Tamura, Hiroto

    2014-07-18

    Ethylene is an industrially important compound, but more sustainable production methods are desirable. Since cellulosomes increase the ability of cellulolytic enzymes by physically linking the relevant enzymes via dockerin-cohesin interactions, in this study, we genetically engineered a chimeric cellulosome-like complex of two ethylene-generating enzymes from tomato using cohesin-dockerins from the bacteria Clostridium thermocellum and Acetivibrio cellulolyticus. This complex was transformed into Escherichia coli to analyze kinetic parameters and enzyme complex formation and into the cyanobacterium Synechococcus elongatus PCC 7942, which was then grown with and without 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Only at minimal protein expression levels (without IPTG), the chimeric complex produced 3.7 times more ethylene in vivo than did uncomplexed enzymes. Thus, cyanobacteria can be used to sustainably generate ethylene, and the synthetic enzyme complex greatly enhanced production efficiency. Artificial synthetic enzyme complexes hold great promise for improving the production efficiency of other industrial compounds.

  13. Clinical effects of a chimeric anti-EpCAM monoclonal antibody in combination with granulocyte-macrophage colony-stimulating factor in patients with metastatic colorectal carcinoma.

    PubMed

    Liljefors, Maria; Nilsson, Bo; Fagerberg, Jan; Ragnhammar, Peter; Mellstedt, Håkan; Frödin, Jan-Erik

    2005-06-01

    The EpCAM antigen is highly expressed on colorectal carcinoma (CRC) cells. Murine anti-EpCAM MAb (anti-EpCAM mMAb) alone or in combination with cytokines may induce clinical responses including long-lasting complete remissions (CR) in patients with metastatic disease. The chimeric variant of anti-EpCAM MAb (anti-EpCAM cMAb) interacts more efficiently with human effector cells (ADCC) than the murine counterpart in the killing of colorectal carcinoma cells in vitro, an important mechanism of action for antibody in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) augments immune effector cell functions in vivo and may enhance the therapeutic effect of MAbs. In this study, the therapeutic efficacy of the combination of anti-EpCAM cMAb and GM-CSF was evaluated in 24 patients with metastatic CRC. GM-CSF was given s.c. once daily for 10 consecutive days and on day 3, anti-EpCAM cMAb was given i.v. A treatment cycle was repeated every 4th week. Five patients achieved stable disease > 3 months (overall response rate 21%). Responding patients survived significantly longer than non-responding patients (p = 0.030). The frequency of patients with an immediate-type allergic reaction (ITAR) against anti-EpCAM cMAb at the 1st, 2nd, 3rd and 4th treatment cycles was as 13%, 29%, 25% and 19% respectively. Compared to a previous study where anti-EpCAM mMAb was used in a similar treatment regimen, the present protocol did not augment the overall or progression-free survival. The overall response rate was also similar to anti-EpCAM mMAb treated patients (6/22, 27%), but the anti-EpCAM mMAb treatment protocol induced two CR, one MR and three SD. Further studies are warranted to establish the role of EpCAM as a target for antibody therapy, specifically the significance of chimeric or humanized anti-EpCAM MAbs.

  14. The Chimeric Protein Domain III-Capsid of Dengue Virus Serotype 2 (DEN-2) Successfully Boosts Neutralizing Antibodies Generated in Monkeys upon Infection with DEN-2▿

    PubMed Central

    Valdés, Iris; Gil, Lázaro; Romero, Yaremis; Castro, Jorge; Puente, Pedro; Lazo, Laura; Marcos, Ernesto; Guzmán, María G.; Guillén, Gerardo; Hermida, Lisset

    2011-01-01

    Use of a heterologous prime-boost strategy based on a combination of nonreplicative immunogens and candidate attenuated virus vaccines against dengue virus in the same schedule is an attractive approach. These combinations may result in a condensed immunization regime for humans, thus reducing the number of doses with attenuated virus and the time spacing. The present work deals with the evaluation of the heterologous prime-boost strategy combining a novel chimeric protein (domain III-capsid) of dengue virus serotype 2 (DEN-2) and the infective homologous virus in the same immunization schedule in monkeys. Primed monkeys received one dose of infective DEN-2 and were then vaccinated with the recombinant protein. We found that animals developed a neutralizing antibody response after the infective dose and were notably boosted with a second dose of the chimeric protein 3 months later. The neutralizing antibodies induced were long lasting, and animals also showed the ability to induce a specific cellular response 6 months after the booster dose. As a conclusion, we can state that the domain III region, when it is properly presented as a fusion protein to the immune system, is able to recall the neutralizing antibody response elicited following homologous virus infection in monkeys. Further prime-boost approaches can be performed in a condensed regime combining the chimeric domain III-capsid protein and candidate live attenuated vaccines against DEN-2. PMID:21209159

  15. Design, expression and characterization of a single chain anti-CD20 antibody; a germline humanized antibody derived from Rituximab.

    PubMed

    Ahmadzadeh, Vahideh; Farajnia, Safar; Hosseinpour Feizi, Mohammad Ali; Khavarinejad, Ramazan Ali

    2014-10-01

    CD20 is a B cell lineage specific surface antigen involved in various B cell malignancies. So far, several murine and chimeric antibodies have been produced against this antigen among which Rituximab is a commercially approved antibody widely used in treatment of cancers associated with CD20 overexpression. The current study reports the production and characterization of a humanized single chain version of Rituximab through CDR grafting method. For either heavy or light chain variable domains, a human antibody with the highest sequence homology to Rituximab was selected from human germline sequences and used as framework donors. Vernier zone residues in framework regions were replaced with those of Rituximab to retain the antigen binding affinity of parental antibody. The reactivity of humanized single chain antibody with CD20 was examined by ELISA and dot blot assays. The ability of antibody to suppress the growth of CD20 overexpressing Raji cells was tested by MTT assay. Analysis of reactivity with CD20 antigen revealed that the humanized single chain antibody reacted to the target antigen with high affinity. Proliferation inhibition assay showed that humanized scFv could suppress the proliferation of Raji cells efficiently in a dose-dependent manner. This successful production of a humanized scFv with the ability to inhibit growth of CD20-expressing cancer cell may provide a promising alternative strategy for CD20 targeted therapy.

  16. Radioimmunotherapy with astatine-211 using chimeric monoclonal antibody U36 in head and neck squamous cell carcinoma.

    PubMed

    Cheng, Junping; Ekberg, Tomas; Engström, Mats; Nestor, Marika; Jensen, Holger J; Tolmachev, Vladimir; Anniko, Matti

    2007-06-01

    In advanced head and neck squamous cell carcinoma (HNSCC), there is a need for an adjuvant treatment. We aim to evaluate the biodistribution and therapeutic effect of radioimmunotherapy using the alpha emitting, astatine-211-labeled, chimeric monoclonal antibody U36 (U36) on the HNSCC cell line UT-SCC7 in vivo. Xenograft tumors were inoculated subcutaneously in nude mice. Astatine-211-labeled U36 was injected intravenously with or without blocking of target with nonlabeled U36. In the biodistribution experiments, radioactivity was measured in tumors and various organs at set time points. In the therapeutic experiments, two groups (with or without blocking) received therapy, and the tumor growth was compared with that of controls. In addition, one group received nonlabeled U36 only. The biodistribution experiments demonstrated that astatine-211-labeled U36 could target UT-SCC7 xenografts in nude mice. With time, uptake increased in tumors and decreased in normal organs. Nonlabeled U36 did not influence tumor growth. In the two therapy groups, 18 of 20 tumors responded to therapy by decreasing or stabilizing their volumes. Significant difference was seen between the treated groups and the controls (P < .05). The study illustrates the specific binding of astatine-211-labeled U36 to HNSCC and suggests radioimmunotherapy with the alpha emitting radionuclide to be a useful treatment modality.

  17. From therapeutic antibodies to chimeric antigen receptors (CARs): making better CARs based on antigen-binding domain.

    PubMed

    Wu, Yanling; Jiang, Shibo; Ying, Tianlei

    2016-12-01

    A variety of approaches are being pursued to improve the safety and antitumor potency of chimeric antigen receptor (CAR) T-cell therapy. However, most engineering efforts have thus far been focused on its intracellular signaling domain, while its extracellular antigen-binding domain has received less attention. Areas covered: Herein, the authors summarize the current knowledge of CAR T-cell therapy. Accordingly, they focus on its antigen-binding domain, discuss key considerations for selecting an optimal single-chain variable fragment (scFv) when designing a CAR, and suggest potential directions aimed at developing the next-generation CARs. Expert opinion: The extracellular region of CARs can play a decisive role in their safety and efficacy. Instead of directly translating an available therapeutic mAb to a scFv-based CAR construct, the authors suggest that various CAR-displayed scFvs with different affinity, specificity and binding epitopes against an individual target molecule should be generated and evaluated side-by-side. Incorporating new antibody formats that possess characteristics superior to those of scFvs may be one way to engineer safer and more effective CARs. The authors expect that further CAR engineering will enable us to target more antigens involved in hematological and solid malignancies with minimal side effects to serve unmet clinical needs.

  18. Immunogenic targeting of recombinant peptide vaccines to human antigen-presenting cells by chimeric anti-HLA-DR and anti-surface immunoglobulin D antibody Fab fragments in vitro.

    PubMed Central

    Baier, G; Baier-Bitterlich, G; Looney, D J; Altman, A

    1995-01-01

    To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence-activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV-1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV-1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system. PMID:7533857

  19. Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

    PubMed Central

    Calvanese, Vincenzo; Mallya, Meera; Campbell, R Duncan; Aguado, Begoña

    2008-01-01

    Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F) undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C) and not on their own. PMID:18817541

  20. Chimeric anti-IL-17 full-length monoclonal antibody is a novel potential candidate for the treatment of rheumatoid arthritis.

    PubMed

    Bai, Fuliang; Tian, Hui; Niu, Zeshan; Liu, Mingyao; Ren, Guiping; Yu, Yinhang; Sun, Tian; Li, Siming; Li, Deshan

    2014-03-01

    Rheumatoid arthritis (RA) is an autoimmune disease, primarily manifesting as inflammatory arthritis. It is associated with chronic inflammation of the synovial joints, mostly in the hands and feet, as well as with systemic extra-articular inflammation. The chimeric anti-interleukin (IL)-17 full-length monoclonal antibody (CMa17Aab) targets IL-17A, which is an important cytokine in the pathogenesis of RA and other inflammatory disorders. In this study, we investigated whether CMa17Aab exerts therapeutic effects in a mouse model of type II collagen-induced arthritis (CIA). Mice with CIA were subcutaneously injected with the humanized CMa17Aab antibody. The effects of treatment were assessed by estimating the arthritis severity score, the extent of histological damage and bone destruction, the autoreactive humoral and cellular immune responses and the production of cytokines. Treatment with CMa17Aab exerted beneficial effects in the mice with CIA as regards clinical and histological parameters. Compared with the controls, treatment with CMa17Aab resulted in a significant alleviation of the severity of the symptoms of arthritis, by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and downregulating the expression of IL-6, IL-8, matrix metalloproteinase (MMP)-3, IL-17, IL-1β, tumor necrosis factor (TNF)-α, receptor activator for nuclear factor-κB ligand (RANKL) and interferon (IFN)-γ in inflamed tissues. In conclusion, our study demonstrates that treatment with CMa17Aab exerts beneficial effects in mice with CIA, by preventing joint inflammation, cartilage destruction and bone damage. These preliminary results suggest that CMa17Aab is an important regulator in RA, and that it may represent a novel therapeutic agent that may prove useful in the treatment of this disease.

  1. A tool kit for rapid cloning and expression of recombinant antibodies.

    PubMed

    Dodev, Tihomir S; Karagiannis, Panagiotis; Gilbert, Amy E; Josephs, Debra H; Bowen, Holly; James, Louisa K; Bax, Heather J; Beavil, Rebecca; Pang, Marie O; Gould, Hannah J; Karagiannis, Sophia N; Beavil, Andrew J

    2014-07-30

    Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

  2. A tool kit for rapid cloning and expression of recombinant antibodies

    PubMed Central

    Dodev, Tihomir S.; Karagiannis, Panagiotis; Gilbert, Amy E.; Josephs, Debra H.; Bowen, Holly; James, Louisa K.; Bax, Heather J.; Beavil, Rebecca; Pang, Marie O.; Gould, Hannah J.; Karagiannis, Sophia N.; Beavil, Andrew J.

    2014-01-01

    Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions. PMID:25073855

  3. Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C-staphylococcus A protein.

    PubMed

    Cao, Haiyan; Zhang, Guo-Rong; Geller, Alfred I

    2010-09-10

    Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC-glial cell line-derived neurotrophic factor or gC-brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC-ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2 months) expression.

  4. Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody.

    PubMed

    Soto, Yosdel; Mesa, Niurka; Alfonso, Yumisley; Pérez, Arlenis; Batlle, Fernando; Griñán, Tania; Pino, Adonis; Viera, Justo; Frómeta, Milagros; Brito, Victor; Olivera, Armando; Zayas, Francisco; Vázquez, Ana M

    2014-01-01

    The progression of atherosclerosis is favored by increasing amounts of chondroitin sulfate proteoglycans in the artery wall. We previously reported the reactivity of chP3R99 monoclonal antibody (mAb) with sulfated glycosaminoglycans and its association with the anti-atherogenic properties displayed. Now, we evaluated the accumulation of this mAb in atherosclerotic lesions and its potential use as a probe for specific in vivo detection of the disease. Atherosclerosis was induced in NZW rabbits (n = 14) by the administration of Lipofundin 20% using PBS-receiving animals as control (n = 8). Accumulation of chP3R99 mAb in atherosclerotic lesions was assessed either by immunofluorescence detection of human IgG in fresh-frozen sections of aorta, or by immunoscintigraphy followed by biodistribution of the radiotracer upon administration of (99m)Tc-chP3R99 mAb. Immunofluorescence studies revealed the presence of chP3R99 mAb in atherosclerotic lesions 24 h after intravenous administration, whereas planar images showed an evident accumulation of (99m)Tc-chP3R99 mAb in atherosclerotic rabbit carotids. Accordingly, (99m)Tc-chP3R99 mAb uptake by lesioned aortic arch and thoracic segment was increased 5.6-fold over controls and it was 3.9-folds higher in carotids, in agreement with immunoscintigrams. Moreover, the deposition of (99m)Tc-chP3R99 mAb in the artery wall was associated both with the presence and size of the lesions in the different portions of evaluated arteries and was greater than in non-targeted organs. In conclusion, chP3R99 mAb preferentially accumulates in arterial atherosclerotic lesions supporting the potential use of this anti-glycosaminoglycans antibody for diagnosis and treatment of atherosclerosis.

  5. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    PubMed

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  6. Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response.

    PubMed

    Sjatha, Fithriyah; Kuwahara, Miwa; Sudiro, T Mirawati; Kameoka, Masanori; Konishi, Eiji

    2014-02-01

    Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) gene-based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  7. 78 FR 13691 - Prospective Grant of Exclusive License: The Development of m971 and m972 Chimeric Antigen...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-28

    ... m971 and m972 Chimeric Antigen Receptors (CARs) for the Treatment of B Cell Malignancies AGENCY... worldwide, and the field of use may be limited to: Treatment of B cell malignancies that express CD22 on their cell surface using chimeric antigen receptors which contain the m971 or m972 antibody...

  8. A Chimeric HIV-1 Envelope Glycoprotein Trimer with an Embedded Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Domain Induces Enhanced Antibody and T Cell Responses*

    PubMed Central

    van Montfort, Thijs; Melchers, Mark; Isik, Gözde; Menis, Sergey; Huang, Po-Ssu; Matthews, Katie; Michael, Elizabeth; Berkhout, Ben; Schief, William R.; Moore, John P.; Sanders, Rogier W.

    2011-01-01

    An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric EnvGM-CSF enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines. PMID:21515681

  9. A chimeric HIV-1 envelope glycoprotein trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain induces enhanced antibody and T cell responses.

    PubMed

    van Montfort, Thijs; Melchers, Mark; Isik, Gözde; Menis, Sergey; Huang, Po-Ssu; Matthews, Katie; Michael, Elizabeth; Berkhout, Ben; Schief, William R; Moore, John P; Sanders, Rogier W

    2011-06-24

    An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric Env(GM-CSF) enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines.

  10. Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

    SciTech Connect

    Pancera, Marie; McLellan, Jason S.; Wu, Xueling; Zhu, Jiang; Changela, Anita; Schmidt, Stephen D.; Yang, Yongping; Zhou, Tongqing; Phogat, Sanjay; Mascola, John R.; Kwong, Peter D.

    2010-11-03

    HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 {angstrom}, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming {approx}20 {angstrom} above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P = 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.

  11. Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

    PubMed Central

    Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

    2015-01-01

    Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

  12. Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen.

    PubMed

    Chen, Tsung-Hsien; Chen, Ten-Hong; Hu, Chung-Chi; Liao, Jia-Teh; Lee, Chin-Wei; Liao, Jiunn-Wang; Lin, Maw-Yeong; Liu, Hung-Jen; Wang, Min-Ying; Lin, Na-Sheng; Hsu, Yau-Heiu

    2012-06-01

    Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  14. Antibody-Independent Protection against Rotavirus Infection of Mice Stimulated by Intranasal Immunization with Chimeric VP4 or VP6 Protein

    PubMed Central

    Choi, Anthony H.-C.; Basu, Mitali; McNeal, Monica M.; Clements, John D.; Ward, Richard L.

    1999-01-01

    This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-μg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-μg doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient μMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody. PMID:10438847

  15. Evidence for Anger Saliency during the Recognition of Chimeric Facial Expressions of Emotions in Underage Ebola Survivors.

    PubMed

    Ardizzi, Martina; Evangelista, Valentina; Ferroni, Francesca; Umiltà, Maria A; Ravera, Roberto; Gallese, Vittorio

    2017-01-01

    One of the crucial features defining basic emotions and their prototypical facial expressions is their value for survival. Childhood traumatic experiences affect the effective recognition of facial expressions of negative emotions, normally allowing the recruitment of adequate behavioral responses to environmental threats. Specifically, anger becomes an extraordinarily salient stimulus unbalancing victims' recognition of negative emotions. Despite the plethora of studies on this topic, to date, it is not clear whether this phenomenon reflects an overall response tendency toward anger recognition or a selective proneness to the salience of specific facial expressive cues of anger after trauma exposure. To address this issue, a group of underage Sierra Leonean Ebola virus disease survivors (mean age 15.40 years, SE 0.35; years of schooling 8.8 years, SE 0.46; 14 males) and a control group (mean age 14.55, SE 0.30; years of schooling 8.07 years, SE 0.30, 15 males) performed a forced-choice chimeric facial expressions recognition task. The chimeric facial expressions were obtained pairing upper and lower half faces of two different negative emotions (selected from anger, fear and sadness for a total of six different combinations). Overall, results showed that upper facial expressive cues were more salient than lower facial expressive cues. This priority was lost among Ebola virus disease survivors for the chimeric facial expressions of anger. In this case, differently from controls, Ebola virus disease survivors recognized anger regardless of the upper or lower position of the facial expressive cues of this emotion. The present results demonstrate that victims' performance in the recognition of the facial expression of anger does not reflect an overall response tendency toward anger recognition, but rather the specific greater salience of facial expressive cues of anger. Furthermore, the present results show that traumatic experiences deeply modify the perceptual

  16. Evidence for Anger Saliency during the Recognition of Chimeric Facial Expressions of Emotions in Underage Ebola Survivors

    PubMed Central

    Ardizzi, Martina; Evangelista, Valentina; Ferroni, Francesca; Umiltà, Maria A.; Ravera, Roberto; Gallese, Vittorio

    2017-01-01

    One of the crucial features defining basic emotions and their prototypical facial expressions is their value for survival. Childhood traumatic experiences affect the effective recognition of facial expressions of negative emotions, normally allowing the recruitment of adequate behavioral responses to environmental threats. Specifically, anger becomes an extraordinarily salient stimulus unbalancing victims’ recognition of negative emotions. Despite the plethora of studies on this topic, to date, it is not clear whether this phenomenon reflects an overall response tendency toward anger recognition or a selective proneness to the salience of specific facial expressive cues of anger after trauma exposure. To address this issue, a group of underage Sierra Leonean Ebola virus disease survivors (mean age 15.40 years, SE 0.35; years of schooling 8.8 years, SE 0.46; 14 males) and a control group (mean age 14.55, SE 0.30; years of schooling 8.07 years, SE 0.30, 15 males) performed a forced-choice chimeric facial expressions recognition task. The chimeric facial expressions were obtained pairing upper and lower half faces of two different negative emotions (selected from anger, fear and sadness for a total of six different combinations). Overall, results showed that upper facial expressive cues were more salient than lower facial expressive cues. This priority was lost among Ebola virus disease survivors for the chimeric facial expressions of anger. In this case, differently from controls, Ebola virus disease survivors recognized anger regardless of the upper or lower position of the facial expressive cues of this emotion. The present results demonstrate that victims’ performance in the recognition of the facial expression of anger does not reflect an overall response tendency toward anger recognition, but rather the specific greater salience of facial expressive cues of anger. Furthermore, the present results show that traumatic experiences deeply modify the perceptual

  17. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.

  18. Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

    PubMed

    Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W; Kaufman, Joshua D; Dearborn, Altaira D; Trenbeath, Joni L; Eren, Elif; Steven, Alasdair C; Rader, Christoph; Wingfield, Paul T

    2017-10-06

    Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ∼10(-12) m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

  19. The cytotoxicity and microdosimetry of astatine-211-labeled chimeric monoclonal antibodies in human glioma and melanoma cells in vitro.

    PubMed

    Larsen, R H; Akabani, G; Welsh, P; Zalutsky, M R

    1998-02-01

    The cytotoxicity of alpha-particle-emitting endoradiotherapeutic compounds is of increasing interest because clinical evaluation of these potential therapeutic agents is commencing. Astatine-211 is a radionuclide with a 7.2-h half-life that emits 5.87 and 7.45 MeV alpha particles. In the present work, we have investigated the in vitro cytotoxicity of 211At-labeled chimeric monoclonal antibodies (mAbs) in monolayers of D-247 MG human glioma cells and SK-MEL-28 human melanoma cells. The mAbs studied were 81C6, reactive with the extracellular matrix antigen tenascin, Mel-14, directed against the cell membrane antigen proteoglycan chondroitin sulfate, and a nonspecific control mAb, TPS3.2. Cell uptake increased as a function of activity concentration after a 1-h exposure to the 211At-labeled mAbs. The retention of activity was also measured to calculate cumulative activity associated with the cells and the medium. The clonogenic survival as a function of activity concentration was linear in all cases with no detectable shoulder. Microdosimetric analyses were performed based on measured cell geometry, cumulative activity and Monte Carlo transport of alpha particles. Using 18 kBq/ml activity concentration and 1 h of incubation, a two to five times higher activity bound to the microcolonies was found for the specific mAbs compared to the nonspecific mAb. These calculations indicated that a survival fraction of 0.37 was achieved with 0.24-0.28 Gy for D-247 MG cells and 0.27-0.29 Gy for SK-MEL-28 cells. The microdosimetric cell sensitivity, z0, for D-247 MG cells was significantly lower than for SK-MEL-28 cells (0.08 compared to 0.15 Gy). For both cell lines, reduction in survival to 0.37 required an average of only 1-2 alpha-particle hits to the cell nucleus.

  20. Dissecting Antibodies Induced by a Chimeric Yellow Fever-Dengue, Live-Attenuated, Tetravalent Dengue Vaccine (CYD-TDV) in Naive and Dengue-Exposed Individuals.

    PubMed

    Henein, Sandra; Swanstrom, Jesica; Byers, Anthony M; Moser, Janice M; Shaik, S Farzana; Bonaparte, Matthew; Jackson, Nicholas; Guy, Bruno; Baric, Ralph; de Silva, Aravinda M

    2017-02-01

    Sanofi Pasteur has developed a chimeric yellow fever-dengue, live-attenuated, tetravalent dengue vaccine (CYD-TDV) that is currently approved for use in several countries. In clinical trials, CYD-TDV was efficacious at reducing laboratory-confirmed cases of dengue disease. Efficacy varied by dengue virus (DENV) serotype and prevaccination dengue immune status. We compared the properties of antibodies in naive and DENV-exposed individuals who received CYD-TDV. We depleted specific populations of DENV-reactive antibodies from immune serum samples to estimate the contribution of serotype-cross-reactive and type-specific antibodies to neutralization. Subjects with no preexisting immunity to DENV developed neutralizing antibodies to all 4 serotypes of DENV. Further analysis demonstrated that DENV4 was mainly neutralized by type-specific antibodies whereas DENV1, DENV2, and DENV3 were mainly neutralized by serotype cross-reactive antibodies. When subjects with preexisting immunity to DENV were vaccinated, they developed higher levels of neutralizing antibodies than naive subjects who were vaccinated. In preimmune subjects, CYD-TDV boosted cross-reactive neutralizing antibodies while maintaining type-specific neutralizing antibodies acquired before vaccination. Our results demonstrate that the quality of neutralizing antibodies induced by CYD-TDV varies depending on DENV serotype and previous immune status. We discuss the implications of these results for understanding vaccine efficacy. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  1. Characterization of NoV P particle-based chimeric protein vaccines developed from two different expression systems.

    PubMed

    Fu, Lu; Jin, Hao; Yu, Yongjiao; Yu, Bin; Zhang, Haihong; Wu, Jiaxin; Yin, Yuhe; Yu, Xianghui; Wu, Hui; Kong, Wei

    2017-02-01

    The Norovirus (NoV) P domain, with three surface loops for foreign antigen insertion, has been demonstrated as an excellent platform for antigen presentation and novel vaccine development. The P domain alone can self-assemble into a P dimer, 12-mer small particle or 24-mer P particle, and vaccines based on those particles may elicit different levels of immunogenicity. Currently, P particles are generally produced in soluble expression systems in Escherichia coli, mainly in the 24-mer form. However, the low yield of the soluble protein has hindered further clinical applications of P particle-based protein vaccines. In this study, we inserted the Alzheimer's disease (AD) immunogen Aβ1-6 into the three loops of the P particle to generate an AD protein vaccine. To increase the yield of this chimeric protein, we tested the generation of proteins in a soluble expression system and an inclusion body expression system separately in E. coli. The result showed that the inclusion body expression system could greatly enhance the product yield of the chimeric protein compared with the soluble expression system. The refolded protein from the inclusion bodies was mainly in the 12-mer form, while the protein generated from the soluble supernatant was mainly in the 24-mer form. Moreover, the immunogenicity of soluble proteins was significantly stronger than that of the refolded proteins. Thus, comparisons between the two expression methods suggested that the soluble expression system generated chimeric P particles with better immunogenicity, while inclusion body expression system yielded more P particle proteins.

  2. Transplantation of GFP-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos.

    PubMed

    Zou, Jian; Wei, Xiangyun

    2010-07-19

    Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed.

  3. Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9

    SciTech Connect

    Wathen, M.W.; Aeed, P.A.; Elhammer, A.P. )

    1991-03-19

    The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with ({sup 3}H)glucosamine and infected wit a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal{beta}1-3GalNAc constituting 17 and 66% of the total number of structures respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked {alpha}1-6 to the asparagine-linked N-acetylglucosamine.

  4. Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models

    NASA Astrophysics Data System (ADS)

    Maawy, Ali A.; Hiroshima, Yukihiko; Kaushal, Sharmeela; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2013-12-01

    The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.

  5. Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin.

    PubMed

    Huang, Nai-Jia; Pishesha, Novalia; Mukherjee, Jean; Zhang, Sicai; Deshycka, Rhogerry; Sudaryo, Valentino; Dong, Min; Shoemaker, Charles B; Lodish, Harvey F

    2017-09-04

    A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications.The therapeutic use of single-chain antibodies (VHHs) is limited by their short half-life in the circulation. Here the authors engineer mouse and human red blood cells to express VHHs against botulinum neurotoxin A (BoNT/A) on their surface and show that an infusion of these cells into mice confers long lasting protection against a high dose of BoNT/A.

  6. Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

    DOE PAGES

    Xu, Qi; Knoshaug, Eric P.; Wang, Wei; ...

    2017-07-24

    Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for

  7. Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi.

    PubMed

    Xu, Qi; Knoshaug, Eric P; Wang, Wei; Alahuhta, Markus; Baker, John O; Yang, Shihui; Vander Wall, Todd; Decker, Stephen R; Himmel, Michael E; Zhang, Min; Wei, Hui

    2017-07-24

    Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel

  8. In Vitro Generation of Human NK cells Expressing Chimeric Antigen Receptor through Differentiation of Gene-Modified Hematopoietic Stem Cells

    PubMed Central

    Lowe, Emily; Truscott, Laurel C.; De Oliveira, Satiro N.

    2016-01-01

    Summary NK cells represent a very promising source for adoptive cellular approaches for cancer immunotherapy, and extensive research has been conducted, including clinical trials. Gene modification of NK cells can direct their specificity and enhance their function, but the efficiency of gene transfer techniques is very limited. Here we describe two protocols designed to generate mature human NK cells from gene-modified hematopoietic stem cells. These protocols use chimeric antigen receptor as the transgene, but could potentially be modified for the expression any particular transgene in human NK cells. PMID:27177671

  9. Expression of a chimeric CSF1R-LTK mediates ligand-dependent neurite outgrowth.

    PubMed

    Yamada, Shigeru; Nomura, Takashi; Takano, Kota; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2008-11-19

    In an earlier screening, we identified several genes for kinases that might control the extension of neurites. One of these genes encoded a leukocyte tyrosine kinase (LTK), which is a receptor tyrosine kinase whose ligands remain to be identified. To examine the possible role of this LTK in neurite outgrowth, we constructed a chimeric receptor, in which the extracellular domain of the receptor for colony-stimulating factor-1 was fused to the cytoplasmic domain of LTK, which allowed the selective activation of LTK by colony-stimulating factor-1. Our studies using this chimeric receptor suggest that activation of the tyrosine kinase activity of LTK is sufficient to promote neurite outgrowth through pathways that include reactions catalyzed by phosphatidylinositol 3-kinase and MAPK.

  10. Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

    PubMed

    Shen, Chan-Juan; Yang, Yu-Xiu; Han, Ethan Q; Cao, Na; Wang, Yun-Fei; Wang, Yi; Zhao, Ying-Ying; Zhao, Li-Ming; Cui, Jian; Gupta, Puja; Wong, Albert J; Han, Shuang-Yin

    2013-05-09

    Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRv

  11. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    SciTech Connect

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku Okada, Naoki

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  12. Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy.

    PubMed

    Espinosa, Diego A; Yadava, Anjali; Angov, Evelina; Maurizio, Paul L; Ockenhouse, Christian F; Zavala, Fidel

    2013-08-01

    The development of vaccine candidates against Plasmodium vivax-the most geographically widespread human malaria species-is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax-infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP.

  13. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

  14. Porcine circovirus type 2 protective epitope densely carried by chimeric papaya ringspot virus-like particles expressed in Escherichia coli as a cost-effective vaccine manufacture alternative.

    PubMed

    Aguilera, Brenda Eugenia; Chávez-Calvillo, Gabriela; Elizondo-Quiroga, Darwin; Jimenez-García, Mónica Noemí; Carrillo-Tripp, Mauricio; Silva-Rosales, Laura; Hernández-Gutiérrez, Rodolfo; Gutiérrez-Ortega, Abel

    2017-05-01

    Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus-like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self-assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N- and C-termini and a predicted antigenic region located near the N-terminus. Wild-type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope-presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small-scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  15. Expression level of a chimeric kinase governs entry into sporulation in Bacillus subtilis.

    PubMed

    Eswaramoorthy, Prahathees; Dravis, Ashlee; Devi, Seram Nganbiton; Vishnoi, Monika; Dao, Hoang-Anh; Fujita, Masaya

    2011-11-01

    Upon starvation, Bacillus subtilis cells switch from growth to sporulation. It is believed that the N-terminal sensor domain of the cytoplasmic histidine kinase KinA is responsible for detection of the sporulation-specific signal(s) that appears to be produced only under starvation conditions. Following the sensing of the signal, KinA triggers autophosphorylation of the catalytic histidine residue in the C-terminal domain to transmit the phosphate moiety, via phosphorelay, to the master regulator for sporulation, Spo0A. However, there is no direct evidence to support the function of the sensor domain, because the specific signal(s) has never been found. To investigate the role of the N-terminal sensor domain, we replaced the endogenous three-PAS repeat in the N-terminal domain of KinA with a two-PAS repeat derived from Escherichia coli and examined the function of the resulting chimeric protein. Despite the introduction of a foreign domain, we found that the resulting chimeric protein, in a concentration-dependent manner, triggered sporulation by activating Spo0A through phosphorelay, irrespective of nutrient availability. Further, by using chemical cross-linking, we showed that the chimeric protein exists predominantly as a tetramer, mediated by the N-terminal domain, as was found for KinA. These results suggest that tetramer formation mediated by the N-terminal domain, regardless of the origin of the protein, is important and sufficient for the kinase activity catalyzed by the C-terminal domain. Taken together with our previous observations, we propose that the primary role of the N-terminal domain of KinA is to form a functional tetramer, but not for sensing an unknown signal.

  16. Dystrophic Muscle in Mice Chimeric for Expression of α5 Integrin

    PubMed Central

    Taverna, Daniela; Disatnik, Marie-Helene; Rayburn, Helen; Bronson, Roderick T.; Yang, Joy; Rando, Thomas A.; Hynes, Richard O.

    1998-01-01

    α5-deficient mice die early in embryogenesis (Yang et al., 1993). To study the functions of α5 integrin later in mouse embryogenesis and during adult life we generated α5 −/−;+/+ chimeric mice. These animals contain α5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that α5 −/− cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (Taverna et al., 1998). Histological analysis of α5 −/−;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration–regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of α5 −/− ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is α5-integrin-dependent. PMID:9813102

  17. The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen.

    PubMed

    Boutaud, A; Castellino, F J

    1993-06-01

    A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees

  18. Enhanced resistance to Sclerotinia sclerotiorum in Brassica napus by co-expression of defensin and chimeric chitinase genes.

    PubMed

    Zarinpanjeh, Nasim; Motallebi, Mostafa; Zamani, Mohammad Reza; Ziaei, Mahboobeh

    2016-11-01

    Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the major fungal diseases of Brassica napus L. To develop resistance against this fungal disease, the defensin gene from Raphanus sativus and chimeric chit42 from Trichoderma atroviride with a C-terminal fused chitin-binding domain from Serratia marcescens were co-expressed in canola via Agrobacterium-mediated transformation. Twenty transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR), with 4.8 % transformation efficiency. The chitinase activity of PCR-positive transgenic plants were measured in the presence of colloidal chitin, and five transgenic lines showing the highest chitinase activity were selected for checking the copy number of the transgenes through Southern blot hybridisation. Two plants carried a single copy of the transgenes, while the remainder carried either two or three copies of the transgenes. The antifungal activity of two transgenic lines that carried a single copy of the transgenes (T4 and T10) was studied by a radial diffusion assay. It was observed that the constitutive expression of these transgenes in the T4 and T10 transgenic lines suppressed the growth of S. sclerotiorum by 49 % and 47 %, respectively. The two transgenic lines were then let to self-pollinate to produce the T2 generation. Greenhouse bioassays were performed on the transgenic T2 young leaves by challenging with S. sclerotiorum and the results revealed that the expression of defensin and chimeric chitinase from a heterologous source in canola demonstrated enhanced resistance against sclerotinia stem rot disease.

  19. Collagen Sponge Functionalized with Chimeric Anti-BMP-2 Monoclonal Antibody Mediates Repair of Critical-Size Mandibular Continuity Defects in a Nonhuman Primate Model

    PubMed Central

    Xie, Yilin; Su, Yingying; Tang, Jianxia; Goh, Bee Tin; Saigo, Leonardo; Zhang, Chunmei; Wang, Jinsong; Khojasteh, Arash; Wang, Songlin

    2017-01-01

    Antibody-mediated osseous regeneration (AMOR) has been introduced by our research group as a tissue engineering approach to capture of endogenous growth factors through the application of specific monoclonal antibodies (mAbs) immobilized on a scaffold. Specifically, anti-Bone Morphogenetic Protein- (BMP-) 2 mAbs have been demonstrated to be efficacious in mediating bone repair in a number of bone defects. The present study sought to investigate the application of AMOR for repair of mandibular continuity defect in nonhuman primates. Critical-sized mandibular continuity defects were created in Macaca fascicularis locally implanted with absorbable collagen sponges (ACS) functionalized with chimeric anti-BMP-2 mAb or isotype control mAb. 2D and 3D analysis of cone beam computed tomography (CBCT) imaging demonstrated increased bone density and volume observed within mandibular continuity defects implanted with collagen scaffolds functionalized with anti-BMP-2 mAb, compared with isotype-matched control mAb. Both CBCT imaging and histologic examination demonstrated de novo bone formation that was in direct apposition to the margins of the resected bone. It is hypothesized that bone injury may be necessary for AMOR. This is evidenced by de novo bone formation adjacent to resected bone margins, which may be the source of endogenous BMPs captured by anti-BMP-2 mAb, in turn mediating bone repair. PMID:28401163

  20. Enhanced Tumor Trafficking of GD2 Chimeric Antigen Receptor T Cells by Expression of the Chemokine Receptor CCR2b

    PubMed Central

    Craddock, John A; Lu, An; Bear, Adham; Pule, Martin; Brenner, Malcolm K; Rooney, Cliona M; Foster, Aaron E

    2010-01-01

    For adoptive T cell therapy to be effective against solid tumors, tumor-specific T cells must be able to migrate to the tumor site. One requirement for efficient migration is that the effector cells express chemokine receptors that match the chemokines produced either by tumor or tumor-associated cells. In this study, we investigated whether the tumor trafficking of activated T cells (ATCs) bearing a chimeric antigen receptor specific for the tumor antigen GD2 (GD2-CAR) could be enhanced by forced co-expression of the chemokine receptor CCR2b, since this receptor directs migration towards CCL2, a chemokine produced by many tumors, including neuroblastoma. Neuroblastoma cell lines (SK-N-SH and SK-N-AS) and primary tumor cells isolated from six patients all secreted high levels of CCL2, but GD2-CAR transduced ATCs lacked expression of CCR2 (<5%) and migrated poorly to recombinant CCL2 or tumor supernatants. Following retroviral transduction, however, ATCs expressed high levels of CCR2b (>60%) and migrated well in vitro. We expressed firefly luciferase in CCR2b-expressing ATCs and observed improved homing (>10-fold) to CCL2-secreting neuroblastoma compared to CCR2 negative ATCs. As a result, ATCs co-modified with both CCR2b and GD2-CAR had greater anti-tumor activity in vivo. PMID:20842059

  1. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors

    PubMed Central

    Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E.; Laskowski, Tamara; McNamara, George; Cooper, Laurence J. N.

    2016-01-01

    Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR’s in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies. PMID:27548616

  2. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

    PubMed

    Thokala, Radhika; Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E; Laskowski, Tamara; McNamara, George; Cooper, Laurence J N

    2016-01-01

    Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

  3. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.).

    PubMed

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants.

  4. In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection

    PubMed Central

    Nishimori, Asami; Okagawa, Tomohiro; Maekawa, Naoya; Ikebuchi, Ryoyo; Goto, Shinya; Sajiki, Yamato; Suzuki, Yasuhiko; Kohara, Junko; Ogasawara, Satoshi; Kato, Yukinari; Murata, Shiro; Ohashi, Kazuhiko

    2017-01-01

    Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM+ B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4+ T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application. PMID:28445479

  5. In vitro and in vivo antivirus activity of an anti-programmed death-ligand 1 (PD-L1) rat-bovine chimeric antibody against bovine leukemia virus infection.

    PubMed

    Nishimori, Asami; Konnai, Satoru; Okagawa, Tomohiro; Maekawa, Naoya; Ikebuchi, Ryoyo; Goto, Shinya; Sajiki, Yamato; Suzuki, Yasuhiko; Kohara, Junko; Ogasawara, Satoshi; Kato, Yukinari; Murata, Shiro; Ohashi, Kazuhiko

    2017-01-01

    Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM+ B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4+ T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.

  6. Chimeric, divalent and tetravalent anti-CD19 monoclonal antibodies with potent in vitro and in vivo antitumor activity against human B-cell lymphoma and pre-B acute lymphoblastic leukemia cell lines.

    PubMed

    Liu, Xiao-Yun; Pop, Laurentiu M; Tsai, Lydia; Pop, Iliodora V; Vitetta, Ellen S

    2011-07-15

    CD19 is an attractive therapeutic target for treating human B-cell tumors. In our study, chimeric (c) divalent (cHD37) and tetravalent (cHD37-DcVV) anti-CD19 monoclonal antibodies (MAbs) were constructed, expressed and evaluated for their binding to human 19-positive (CD19(+)) tumor cell lines. They were also tested for proapoptotic activity and the ability to mediate effector functions. The antitumor activity of these MAbs was further tested in mice xenografted with the CD19(+) Burkitt's lymphoma cell line, Daudi or the pre-B acute lymphoblastic leukemia (ALL) cell line, NALM-6. The cHD37 and cHD37-DcVV MAbs exhibited specific binding and comparable proapoptotic activity on CD19(+) tumor cell lines in vitro. In addition, the cHD37 and cHD37-DcVV MAbs were similar in their ability to mediate antibody-dependent cell-mediated phagocytosis (ADCP). However, the tetravalent cHD37-DcVV MAb bound more avidly, had a slower dissociation rate, and did not internalize as well. It also had enhanced antibody-dependent cellular cytotoxicity (ADCC) with human but not murine effector cells. The cHD37 and cHD37-DcVV MAbs exhibited comparable affinity for the human neonatal Fc receptor (FcRn) and similar pharmacokinetics (PKs) in mice. Moreover, all the HD37 constructs were similar in extending the survival of mice xenografted with Daudi or NALM-6 tumor cells. Therefore, the cHD37 and cHD37-DcVV MAbs have potent antitumor activity and should be further developed for use in humans. Although not evident in mice, due to its increased ability to mediate ADCC with human but not mouse effector cells, the cHD37-DcVV MAb should have superior therapeutic efficacy in humans.

  7. Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

    PubMed

    Nagelkerke, S Q; Tacke, C E; Breunis, W B; Geissler, J; Sins, J W R; Appelhof, B; van den Berg, T K; de Boer, M; Kuijpers, T W

    2015-09-01

    The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

  8. Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

    PubMed

    Qin, Haiying; Cho, Monica; Haso, Waleed; Zhang, Ling; Tasian, Sarah K; Oo, Htoo Zarni; Negri, Gian Luca; Lin, Yongshun; Zou, Jizhong; Mallon, Barbara S; Maude, Shannon; Teachey, David T; Barrett, David M; Orentas, Rimas J; Daugaard, Mads; Sorensen, Poul H B; Grupp, Stephan A; Fry, Terry J

    2015-07-30

    Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.

  9. Regulated expression of the feline panleukopenia virus P38 promoter on extrachromosomal FPV/EBV chimeric plasmids.

    PubMed Central

    Clemens, D L; Carlson, J O

    1989-01-01

    Feline panleukopenia virus/Epstein-Barr virus (FPV/EBV) chimeric expression plasmids were constructed to study regulation of the structural protein gene of the parvovirus, FPV, in a homologous cell culture system. Detection and quantitation of activity from the native FPV promoter, P38, was facilitated by fusing the Escherichia coli lacZ gene with the FPV structural protein gene. Feline cell lines which stably maintained these plasmids extrachromosomally were established. Constitutive beta-galactosidase activity was low but increased up to 40-fold after infection with FPV. Expression of beta-galactosidase was only detected when the FPV/lacZ gene was oriented in the same transcriptional direction as the Epstein-Barr virus gene coding for EBNA-1. When a small open reading frame upstream of the FPV/lacZ initiation codon was deleted, beta-galactosidase expression increased another 4.7- to 26-fold. These changes in beta-galactosidase activity indicate that expression of the FPV structural protein gene is regulated both transcriptionally and posttranscriptionally. Images PMID:2542586

  10. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor–expressing T cells

    PubMed Central

    Lynn, Rachel C.; Poussin, Mathilde; Kalota, Anna; Feng, Yang; Low, Philip S.; Dimitrov, Dimiter S.

    2015-01-01

    T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ+ AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ+ THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34+ HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. PMID:25887778

  11. Eradication of B-ALL using chimeric antigen receptor–expressing T cells targeting the TSLPR oncoprotein

    PubMed Central

    Qin, Haiying; Cho, Monica; Haso, Waleed; Zhang, Ling; Tasian, Sarah K.; Oo, Htoo Zarni; Negri, Gian Luca; Lin, Yongshun; Zou, Jizhong; Mallon, Barbara S.; Maude, Shannon; Teachey, David T.; Barrett, David M.; Orentas, Rimas J.; Daugaard, Mads; Sorensen, Poul H. B.; Grupp, Stephan A.

    2015-01-01

    Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell–associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL. PMID:26041741

  12. Reverse Genetics System for Uukuniemi Virus (Bunyaviridae): RNA Polymerase I-Catalyzed Expression of Chimeric Viral RNAs

    PubMed Central

    Flick, Ramon; Pettersson, Ralf F.

    2001-01-01

    We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of the Bunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5′- and 3′-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm. PMID:11160662

  13. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  14. Translational Implications for Off-the-shelf Immune Cells Expressing Chimeric Antigen Receptors.

    PubMed

    Torikai, Hiroki; Cooper, Laurence Jn

    2016-08-01

    Chimeric antigen receptor (CAR) endows specificity to T-cells independent of human leukocyte antigen (HLA). This enables one immunoreceptor to directly target the same surface antigen on different subsets of tumor cells from multiple HLA-disparate recipients. Most approaches manufacture individualized CAR(+)T-cells from the recipient or HLA-compatible donor, which are revealing promising clinical results. This is the impetus to broaden the number of patients eligible to benefit from adoptive immunotherapy such as to infuse third-party donor derived CAR(+)T-cells. This will overcome issues associated with (i) time to manufacture T-cells, (ii) cost to generate one product for one patient, (iii) inability to generate a product from lymphopenic patients or patient's immune cells fail to complete the manufacturing process, and (iv) heterogeneity of T-cell products produced for or from individual recipients. Establishing a biobank of allogeneic genetically modified immune cells from healthy third-party donors, which are cryopreserved and validated in advance of administration, will facilitate the centralizing manufacturing and widespread distribution of CAR(+)T-cells to multiple points-of-care in a timely manner. To achieve this, it is necessary to engineer an effective strategy to avoid deleterious allogeneic immune responses leading to toxicity and rejection. We review the strategies to establish "off-the-shelf" donor-derived biobanks for human application of CAR(+)T-cells as a drug.

  15. Armed Oncolytic Adenovirus-Expressing PD-L1 Mini-Body Enhances Antitumor Effects of Chimeric Antigen Receptor T Cells in Solid Tumors.

    PubMed

    Tanoue, Kiyonori; Rosewell Shaw, Amanda; Watanabe, Norihiro; Porter, Caroline; Rana, Bhakti; Gottschalk, Stephen; Brenner, Malcolm; Suzuki, Masataka

    2017-04-15

    Chimeric antigen receptor-modified T cells (CAR T cells) produce proinflammatory cytokines that increase expression of T-cell checkpoint signals such as PD-L1, which may inhibit their functionality against solid tumors. In this study, we evaluated in human tumor xenograft models the proinflammatory properties of an oncolytic adenovirus (Onc.Ad) with a helper-dependent Ad (HDAd) that expresses a PD-L1 blocking mini-antibody (mini-body; HDPDL1) as a strategy to enhance CAR T-cell killing. Coadministration of these agents (CAd-VECPDL1) exhibited oncolytic effects with production of PD-L1 mini-body locally at the tumor site. On their own, HDPDL1 exhibited no antitumor effect and CAd-VECPDL1 alone reduced tumors only to volumes comparable to Onc.Ad treatment. However, combining CAd-VECPDL1 with HER2.CAR T cells enhanced antitumor activity compared with treatment with either HER2.CAR T cells alone or HER2.CAR T cells plus Onc.Ad. The benefits of locally produced PD-L1 mini-body by CAd-VECPDL1 could not be replicated by infusion of anti-PD-L1 IgG plus HER2.CAR T cells and coadministration of Onc.Ad in an HER2(+) prostate cancer xenograft model. Overall, our data document the superiority of local production of PD-L1 mini-body by CAd-VECPDL1 combined with administration of tumor-directed CAR T cells to control the growth of solid tumors. Cancer Res; 77(8); 2040-51. ©2017 AACR. ©2017 American Association for Cancer Research.

  16. Antitumor activity of chimeric immunoreceptor gene-modified Tc1 and Th1 cells against autologous carcinoembryonic antigen-expressing colon cancer cells.

    PubMed

    Sasaki, Takeshi; Ikeda, Hiroaki; Sato, Masayoshi; Ohkuri, Takayuki; Abe, Hiroyuki; Kuroki, Masahide; Onodera, Masafumi; Miyamoto, Masaki; Kondo, Satoshi; Nishimura, Takashi

    2006-09-01

    To generate tumor-specific and interferon (IFN)-gamma-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3zeta. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2-/- mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-gamma in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer.

  17. Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

    PubMed Central

    Klessig, D F; Brough, D E; Cleghon, V

    1984-01-01

    The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells. Images PMID:6542172

  18. Pharmacokinetics and tolerability of human mouse chimeric anti-CD22 monoclonal antibody in Chinese patients with CD22-positive non-Hodgkin lymphoma

    PubMed Central

    Li, Su; Zhang, Dongsheng; Sun, Jian; Li, Zhinming; Deng, Liting; Zou, Benyan; Zhan, Jing

    2012-01-01

    The safety and pharmacokinetics assessment of antibodies targeting CD22 (e.g., epratuzumab) have been established in western Caucasian populations, but there are no reports of the effects in Chinese populations. This dose-escalation study examines the safety, pharmacokinetics and biologic effects of multiple doses of anti-CD22 human-murine chimeric monoclonal antibody SM03 in 21 Chinese patients with CD22-positive non-Hodgkin lymphoma. Most of drug-related adverse events (AEs) were mild and reversible. Two patients experienced serious AEs (hemorrhage); one patient had grade 4 neutropenia; one patient had asymptomatic grade III prolongation of activated partial thromboplastin time (APTT). Major AEs included fever (71%), prolongation of APTT (42.8%), leukocytopenia (44.4%), alanine transaminase elevation (28.6%), elevated serum creatinine (23.8%) and injection site skin redness (14.3%). Circulating B cells transiently decreased without significant effects on T cells or immunoglobulin levels. Pharmacokinetic data revealed that mean maximum observed SM03 concentration and mean AUC from time zero to infinity increased in a dose-dependent manner up to 360 mg/m2 SM03. Mean clearance was similar at doses ≤360 mg/m2 and decreased significantly at dose 480 mg/m2, supporting saturation of B-cell binding at 360 mg/m2. Across all dose levels and histologies, one patient achieved partial response at 480 mg/m2 dose; 14 patients had stable disease as best response and four patients progressed. Overall, SM03 was tolerated at doses ranging from 60–480 mg/m2 and had potential efficacy in Chinese patients with follicular lymphoma. PMID:22453099

  19. Repeated infusions of infliximab, a chimeric anti-TNFα monoclonal antibody, in patients with active spondyloarthropathy: one year follow up

    PubMed Central

    Kruithof, E; Van den Bosch, F; Baeten, D; Herssens, A; De Keyser, F; Mielants, H; Veys, E

    2002-01-01

    Background: In a pilot study, the anti-tumour necrosis factor α monoclonal antibody, infliximab, induced a rapid and significant improvement in global, peripheral, and axial disease manifestations of patients with active spondyloarthropathy. Objective: To determine whether repeated infusions of infliximab would effectively and safely maintain the observed effect. Methods: Safety and efficacy of a maintenance regimen (5 mg/kg infliximab every 14 weeks) was evaluated using the measurements reported in the pilot study. Of the 21 patients, 19 completed the one year follow up for efficacy; two patients changed to another dosing regimen after week 12 owing to partial lack of efficacy. However, they are still being followed up for safety analysis. Results: After each re-treatment a sustained significant decrease of all disease manifestations was observed. Before re-treatment, symptoms recurred in 3/19 (16%) at week 20, in 13/19 (68%) at week 34, and in 15/19 (79%) at week 48. No withdrawals due to adverse events occurred. Twelve minor infectious episodes were observed. Twelve patients (57%) developed antinuclear antibodies; in four of them (19%) anti-dsDNA antibodies were detected. However, no lupus-like symptoms occurred. Conclusion: In this open study of infliximab in patients with active spondyloarthropathy, the significant improvement of all disease manifestations was maintained over a one year follow up period without major adverse events. Although recurrence of symptoms was noted in a rising number of patients before each re-treatment, no loss of efficacy was observed after re-treatment. PMID:11830424

  20. T cells expressing CD19/CD20 bi-specific chimeric antigen receptors prevent antigen escape by malignant B cells

    PubMed Central

    Zah, Eugenia; Lin, Meng-Yin; Silva-Benedict, Anne; Jensen, Michael C.; Chen, Yvonne Y.

    2016-01-01

    The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, but multiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bi-specific CARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies. We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate that optimized bi-specific CARs can control both wild-type B-cell lymphoma and CD19− mutants with equal efficiency in vivo. To our knowledge, this is the first bi-specific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in the development of receptors with higher-level complexity. PMID:27059623

  1. Fast track antibody V-gene rescue, recombinant expression in plants and characterization of a PfMSP4-specific antibody.

    PubMed

    Kapelski, Stephanie; Boes, Alexander; Spiegel, Holger; de Almeida, Melanie; Klockenbring, Torsten; Reimann, Andreas; Fischer, Rainer; Barth, Stefan; Fendel, Rolf

    2015-02-05

    Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequences enables not only the recombinant expression of the original antibody for further applications, but opens the road for antibody engineering towards innovative diagnostic or therapeutic applications. A time- and cost-efficient production system enabling the detailed analysis of the antibodies is an essential requirement in this context. Sequences were rescued from three hybridoma cell lines, subjected to sequence analysis, subcloned into binary expression vectors and recombinantly expressed as chimeric mAb (constant regions of human IgG1:k1) in Nicotiana benthamiana plants. The properties of the recombinant and the murine mAbs were compared using competition enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. The recognition of native PfMSP4 by the recombinant mAb was analysed by immunofluorescence staining of Pf 3D7A schizonts and by western blot analysis of merozoite extract. The rescued sequences of all three hybridoma cell lines were identical. The recombinant mAb was successfully expressed as IgG in plants at moderate levels (45 mg/kg fresh leaf weight). Preservation of the original epitope was demonstrated in a competition ELISA, using recombinant mAb and the three murine mAbs. EGF_PfMSP4-specific affinities were determined by SPR spectroscopy to 8 nM and 10 nM for the murine or recombinant mAb, respectively. Binding to parasite PfMSP4 was confirmed in an immunofluorescence assay showing a characteristic staining pattern and by western blot

  2. Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

    PubMed Central

    Srinivasan, J; Singh, O; Chakrabarti, S; Talwar, G P

    1995-01-01

    We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response. PMID:7591154

  3. Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses.

    PubMed

    Brandler, Samantha; Ruffie, Claude; Najburg, Valérie; Frenkiel, Marie-Pascale; Bedouelle, Hughes; Desprès, Philippe; Tangy, Frédéric

    2010-09-24

    Dengue disease is an increasing global health problem that threatens one-third of the world's population. To control this emerging arbovirus, an efficient preventive vaccine is still needed. Because four serotypes of dengue virus (DV) coexist and antibody-dependent enhanced infection may occur, most strategies developed so far rely on the administration of tetravalent formulations of four live attenuated or chimeric viruses. Here, we evaluated a new strategy based on the expression of a single minimal tetravalent DV antigen by a single replicating viral vector derived from pediatric live-attenuated measles vaccine (MV). We generated a recombinant MV vector expressing a DV construct composed of the four envelope domain III (EDIII) from the four DV serotypes fused with the ectodomain of the membrane protein (ectoM). After two injections in mice susceptible to MV infection, the recombinant vector induced neutralizing antibodies against the four serotypes of dengue virus. When immunized mice were further inoculated with live DV from each serotype, a strong memory neutralizing response was raised against all four serotypes. A combined measles-dengue vaccine might be attractive to immunize infants against both diseases where they co-exist.

  4. A new microcolumn-type microchip for examining the expression of chimeric fusion genes using a nucleic acid sandwich hybridization technique.

    PubMed

    Ohnishi, Michihiro; Sasaki, Naoyuki; Kishimoto, Takuya; Watanabe, Hidetoshi; Takagi, Masatoshi; Mizutani, Shuki; Kishii, Noriyuki; Yasuda, Akio

    2014-11-01

    We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Chimeric hepatitis B virus (HBV)/hepatitis C virus (HCV) subviral envelope particles induce efficient anti-HCV antibody production in animals pre-immunized with HBV vaccine.

    PubMed

    Beaumont, Elodie; Roingeard, Philippe

    2015-02-18

    The development of an effective, affordable prophylactic vaccine against hepatitis C virus (HCV) remains a medical priority. The recently described chimeric HBV-HCV subviral envelope particles could potentially be used for this purpose, as they could be produced by industrial procedures adapted from those established for the hepatitis B virus (HBV) vaccine. We show here, in an animal model, that pre-existing immunity acquired through HBV vaccination does not influence the immunogenicity of the HCV E2 protein presented by these chimeric particles. Thus, these chimeric HBV-HCV subviral envelope particles could potentially be used as a booster in individuals previously vaccinated against HBV, to induce protective immunity to HCV.

  6. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.)

    PubMed Central

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet’s salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na+/H+ antiporter and H+-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na+ and K+ in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na+-toxicity for plants. PMID:26284097

  7. Preserved Activity of CD20-Specific Chimeric Antigen Receptor-Expressing T Cells in the Presence of Rituximab.

    PubMed

    Rufener, Gregory A; Press, Oliver W; Olsen, Philip; Lee, Sang Yun; Jensen, Michael C; Gopal, Ajay K; Pender, Barbara; Budde, Lihua E; Rossow, Jeffrey K; Green, Damian J; Maloney, David G; Riddell, Stanley R; Till, Brian G

    2016-06-01

    CD20 is an attractive immunotherapy target for B-cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation is that residual serum rituximab might block CAR binding to CD20 and thereby impede T cell-mediated anti-lymphoma responses. The activity of CD20 CAR-modified T cells in the presence of various concentrations of rituximab was tested in vitro and in vivo CAR-binding sites on CD20(+) tumor cells were blocked by rituximab in a dose-dependent fashion, although at 37°C blockade was incomplete at concentrations up to 200 μg/mL. T cells with CD20 CARs also exhibited modest dose-dependent reductions in cytokine secretion and cytotoxicity, but not proliferation, against lymphoma cell lines. At rituximab concentrations of 100 μg/mL, CAR T cells retained ≥50% of baseline activity against targets with high CD20 expression, but were more strongly inhibited when target cells expressed low CD20. In a murine xenograft model using a rituximab-refractory lymphoma cell line, rituximab did not impair CAR T-cell activity, and tumors were eradicated in >85% of mice. Clinical residual rituximab serum concentrations were measured in 103 lymphoma patients after rituximab therapy, with the median level found to be only 38 μg/mL (interquartile range, 19-72 μg/mL). Thus, despite modest functional impairment in vitro, the in vivo activity of CD20-targeted CAR T cells remains intact at clinically relevant levels of rituximab, making use of these T cells clinically feasible. Cancer Immunol Res; 4(6); 509-19. ©2016 AACR

  8. In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

    PubMed

    Dudczig, Stefanie; Currie, Peter D; Poggi, Lucia; Jusuf, Patricia R

    2017-03-22

    The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

  9. T cells expressing an anti–B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma

    PubMed Central

    Ali, Syed Abbas; Shi, Victoria; Maric, Irina; Wang, Michael; Stroncek, David F.; Rose, Jeremy J.; Brudno, Jennifer N.; Stetler-Stevenson, Maryalice; Feldman, Steven A.; Hansen, Brenna G.; Fellowes, Vicki S.; Hakim, Frances T.; Gress, Ronald E.

    2016-01-01

    Therapies with novel mechanisms of action are needed for multiple myeloma (MM). B-cell maturation antigen (BCMA) is expressed in most cases of MM. We conducted the first-in-humans clinical trial of chimeric antigen receptor (CAR) T cells targeting BCMA. T cells expressing the CAR used in this work (CAR-BCMA) specifically recognized BCMA-expressing cells. Twelve patients received CAR-BCMA T cells in this dose-escalation trial. Among the 6 patients treated on the lowest 2 dose levels, limited antimyeloma activity and mild toxicity occurred. On the third dose level, 1 patient obtained a very good partial remission. Two patients were treated on the fourth dose level of 9 × 106 CAR+ T cells/kg body weight. Before treatment, the first patient on the fourth dose level had chemotherapy-resistant MM, making up 90% of bone marrow cells. After treatment, bone marrow plasma cells became undetectable by flow cytometry, and the patient’s MM entered a stringent complete remission that lasted for 17 weeks before relapse. The second patient on the fourth dose level had chemotherapy-resistant MM making up 80% of bone marrow cells before treatment. Twenty-eight weeks after this patient received CAR-BCMA T cells, bone marrow plasma cells were undetectable by flow cytometry, and the serum monoclonal protein had decreased by >95%. This patient is in an ongoing very good partial remission. Both patients treated on the fourth dose level had toxicity consistent with cytokine-release syndrome including fever, hypotension, and dyspnea. Both patients had prolonged cytopenias. Our findings demonstrate antimyeloma activity of CAR-BCMA T cells. This trial was registered to www.clinicaltrials.gov as #NCT02215967. PMID:27412889

  10. Detection of thromboembolism with ⁹⁹mTc-labeled F(ab)₂ fragment of anti-glycoprotein IIIa chimeric monoclonal antibody in beagle canines.

    PubMed

    Ji, Shundong; Fang, Wei; Dong, Ningzheng; He, Zuoxiang; Ruan, Changgeng

    2012-11-01

    Rapid and timely diagnosis of pulmonary embolism (PE) and deep venous thrombosis (DVT) is important to improve patient outcome. The goal of this study was using (99m)Tc-chSZ21-F(ab)(2), F(ab)(2) fragment of anti-glycoprotein IIIa chimeric monoclonal antibody, to image experimental thromboembolism (DVT and PE) in dogs. Flow cytometry assay and adenosine diphosphate (ADP) stimulated platelet aggregation was performed to determine the specificity and affinity of chSZ21-F(ab)(2) to the GPIIb/IIIa receptor on human or canine platelets. Both PE and DVT were induced in 12 beagle canines by catheter under X-ray direction. After (99m)Tc-chSZ21-F(ab)(2) injection,animals were imaged for up to 3 hours then heparinized and sacrificed. Specific binding of chSZ21-F (ab)(2) to GPIIb/IIIa on human or canine platelets was verified by flow cytometry assay. chSZ21-F (ab)(2) inhibited ADP induced platelet aggregation with a dose-dependent manner, the concentration required to inhibit 50% (IC(50)) of platelet aggregation was 11.6 ± 7.9 nM and 24.9 ± 18.8 nM for human and canine, respectively. In vivo, focal uptake was observed in planar images as early as 30 min (DVT) and 60 min (PE), and became clearer within 3 hours after injection. Lesion-to-background ratio averaged 12.8 (PE-to-lung), 7.2 (DVT-to-blood), and 117.0(DVT-to-muscle), respectively. These results suggested that (99m)Tc-chSZ21-F(ab)(2) with high DVT and PE uptake is a promising agent for imaging vascular thrombosis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    SciTech Connect

    Xu, Yu; Liu, Zhengchun; Kong, Haiyan; Sun, Wenjie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  12. Flow cytometric evaluation of red blood cell chimerism after bone marrow transplantation in Iranian patients: a preliminary study.

    PubMed

    Shaiegan, Mojgan; Hadjati, Esmerdis; Aghaiipour, Mahnaz; Iravani, Masoud; David, Gaelle; Bernard, Daniel

    2006-10-01

    The aim of this study was to evaluate mixed red cells population and red blood cell chimerism after hematopoietic stem cell transplantation. Red blood cell chimerism after hematopoietic stem cell transplantation was analyzed using a series of fluorescein isothiocyanate-conjugated monoclonal antibodies (BioAtlantic, France) directed against ABH, Rh (D, C, E, c, e), Kell, Duffy, Kidd, and Ss antigens on blood samples of 14 patients with hematologic disorders undergoing hematopoietic stem cell transplantation, by flow cytometric method on days 15, 30, and 60 after transplantation. All patients showed expression of donor red cell antigens within days 15 - 30 after hematopoietic stem cell transplantation. Graft versus host disease and ABO incompatibility did not affect the expression of chimerism. Flow cytometric analysis is a simple, accurate, and valuable test which is of significant help in monitoring chimerism in allogeneic hematopoietic stem cell transplantation.

  13. Phase I study of chimeric anti-CD20 monoclonal antibody in Chinese patients with CD20-positive non-Hodgkin's lymphoma

    PubMed Central

    Gui, Lin; Han, Xiaohong; He, Xiaohui; Song, Yuanyuan; Yao, Jiarui; Yang, Jianliang; Liu, Peng; Qin, Yan; Zhang, Shuxiang; Zhang, Weijing; Gai, Wenlin; Xie, Liangzhi

    2016-01-01

    Objective: This study was designed to determine the safety, pharmacokinetics and biologic effects of a human-mouse chimeric anti-CD20 monoclonal antibody (SCT400) in Chinese patients with CD20-positive B-cell non-Hodgkin's lymphoma (CD20+ B-cell NHL). SCT400 has an identical amino acid sequence as rituximab, with the exception of one amino acid in the CH1 domain of the heavy chain, which is common in Asians. Methods: Fifteen patients with CD20+ B-cell NHL received dose-escalating SCT400 infusions (250 mg/m2: n=3; 375 mg/m2: n=9; 500 mg/m2: n=3) once weekly for 4 consecutive weeks with a 24-week follow-up period. The data of all patients were collected for pharmacokinetics and pharmacodynamics analyses. Results: No dose-limiting toxicities were observed. Most drug-related adverse events were grade 1 or 2. Two patients had grade 3 or 4 neutropenia. Under premedication, the drug-related infusion reaction was mild. A rapid, profound and durable depletion of circulating B cells was observed in all dose groups without significant effects on T cell count, natural killer (NK) cell count or immunoglobulin levels. No patient developed anti-SCT400 antibodies during the course of the study. SCT400 serum half-life (T1/2), maximum concentration (Cmax) and area under the curve (AUC) generally increased between the first and fourth infusions (P<0.05). At the 375 mg/m2 dose, the T1/2 was 122.5±46.7 h vs. 197.0±75.0 h, respectively, and the Cmax was 200.6±20.2 g/mL vs. 339.1±71.0 g/mL, respectively. From 250 mg/m2 to 500 mg/m2, the Cmax and AUC increased significantly in a dose-dependent manner (P<0.05). Patients with a high tumor burden had markedly lower serum SCT400 concentrations compared with those without or with a low tumor burden. Of the 9 assessable patients, 1 achieved complete response and 2 achieved partial responses. Conclusions: SCT400 is well-tolerated and has encouraging preliminary efficacy in Chinese patients with CD20+ B-cell NHL. PMID:27199517

  14. A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma.

    PubMed

    Maekawa, Naoya; Konnai, Satoru; Takagi, Satoshi; Kagawa, Yumiko; Okagawa, Tomohiro; Nishimori, Asami; Ikebuchi, Ryoyo; Izumi, Yusuke; Deguchi, Tatsuya; Nakajima, Chie; Kato, Yukinari; Yamamoto, Keiichi; Uemura, Hidetoshi; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2017-08-21

    Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.

  15. Chimeric NKG2D CAR-Expressing T Cell-Mediated Attack of Human Ovarian Cancer Is Enhanced by Histone Deacetylase Inhibition

    PubMed Central

    Song, De-Gang; Ye, Qunrui; Santoro, Stephen; Fang, Chongyun; Best, Andrew

    2013-01-01

    Abstract NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4+ and CD8+ NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer. PMID:23297870

  16. Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition.

    PubMed

    Song, De-Gang; Ye, Qunrui; Santoro, Stephen; Fang, Chongyun; Best, Andrew; Powell, Daniel J

    2013-03-01

    NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4(+) and CD8(+) NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer.

  17. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

    PubMed

    Sainsbury, Frank; Jutras, Philippe V; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.

  18. Construction, expression and characterization of a chimeric multi-domain protein mediating specific DNA transfer.

    PubMed

    Gao, Peng; Li, Xiao; Liu, Yanjing; Liu, Yan; Kan, Shifu; Jin, Jing; Wang, Shuqi; Yuan, Changji; Jin, Ningyi

    2010-12-01

    The delivery of plasmid DNA to target cells using a simple, defined, non-viral system is an area of intense research in gene therapy. Here, we describe a novel DNA carrier protein termed TG, consisting of the DNA-binding domain of the yeast transcriptional activator GAL4 and human immunodeficiency virus type 1 Tat protein, which can transfer modified naked plasmid DNA into target cells to express foreign genes of interest. The TG protein was expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni(2+) affinity chromatography column. SDS-PAGE and Western blotting revealed that the fusion protein was highly expressed with a yield of approximately 275 mg/L. We also constructed the pIRES-UAS-EGFP DNA vector, consisting of upstream activating sequences (UASs) for the specific binding of the DNA-binding protein and the enhanced green fluorescent protein (EGFP) gene. The TG protein could bind specifically to pIRES-UAS-EGFP, forming a complex which could efficiently transfect target cells and result in detectable EGFP protein expression. Thus, these results provide a basis for development of efficient non-viral DNA transfer vectors for further improvements of gene therapy strategies.

  19. Codon engineering for improved antibody expression in mammalian cells.

    PubMed

    Carton, Jill M; Sauerwald, Tina; Hawley-Nelson, Pam; Morse, Barry; Peffer, Nancy; Beck, Heena; Lu, Jin; Cotty, Adam; Amegadzie, Bernard; Sweet, Ray

    2007-10-01

    While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in research and manufacturing of protein biotherapeutics. We are studying the effect of codon engineering on expression of recombinant antibodies with an emphasis on developing manufacturing cell lines. CNTO 888, a human mAb specific for human MCP-1, was obtained by antibody phage display in collaboration with MorphoSys AG. The isolated DNA sequence of the antibody was biased towards bacterial codons, reflecting the engineering of the Fab library for phage display expression in Escherichia coli. We compared the expression of CNTO 888 containing the parental V-region sequences with two engineered coding variants. In the native codon exchanged (NCE) variant, the V-region codons were replaced with those used in naturally derived human antibody genes. In the human codon optimized (HCO) variant the V-region codons were those used at the highest frequency based on a human codon usage table. The antibody expression levels from stable transfections in mammalian host cells were measured. The HCO codon variant of CNTO 888 yielded the highest expressing cell lines and the highest average expression for the screened populations. This had a significant positive effect on the process to generate a CNTO 888 production cell line and indicates the potential to improve antibody expression in mammalian expression systems by codon engineering.

  20. Complex chimerism

    PubMed Central

    Ma, Kimberly K.; Petroff, Margaret G.; Coscia, Lisa A.; Armenti, Vincent T.; Adams Waldorf, Kristina M.

    2013-01-01

    Thousands of women with organ transplantation have undergone successful pregnancies, however little is known about how the profound immunologic changes associated with pregnancy might influence tolerance or rejection of the allograft. Pregnant women with a solid organ transplant are complex chimeras with multiple foreign cell populations from the donor organ, fetus, and mother of the pregnant woman. We consider the impact of complex chimerism and pregnancy-associated immunologic changes on tolerance of the allograft both during pregnancy and the postpartum period. Mechanisms of allograft tolerance are likely dynamic during pregnancy and affected by the influx of fetal microchimeric cells, HLA relationships (between the fetus, pregnant woman and/or donor), peripheral T cell tolerance to fetal cells, and fetal minor histocompatibility antigens. Further research is necessary to understand the complex immunology during pregnancy and the postpartum period of women with a solid organ transplant. PMID:23974274

  1. Production and characterisation of a monoclonal antibody that recognises the chicken CSF1 receptor and confirms that expression is restricted to macrophage-lineage cells.

    PubMed

    Garcia-Morales, Carla; Rothwell, Lisa; Moffat, Lindsey; Garceau, Valerie; Balic, Adam; Sang, Helen M; Kaiser, Pete; Hume, David A

    2014-02-01

    Macrophages contribute to innate and acquired immunity as well as many aspects of homeostasis and development. Studies of macrophage biology and function in birds have been hampered by a lack of definitive cell surface markers. As in mammals, avian macrophages proliferate and differentiate in response to CSF1 and IL34, acting through the shared receptor, CSF1R. CSF1R mRNA expression in the chicken is restricted to macrophages and their progenitors. To expedite studies of avian macrophage biology, we produced an avian CSF1R-Fc chimeric protein and generated a monoclonal antibody (designated ROS-AV170) against the chicken CSF1R using the chimeric protein as immunogen. Specific binding of ROS-AV170 to CSF1R was confirmed by FACS, ELISA and immunohistochemistry on tissue sections. CSF1 down-regulated cell surface expression of the CSF1R detected with ROS-AV170, but the antibody did not block CSF1 signalling. Expression of CSF1R was detected on the surface of bone marrow progenitors only after culture in the absence of CSF1, and was induced during macrophage differentiation. Constitutive surface expression of CSF1R distinguished monocytes from other myeloid cells, including heterophils and thrombocytes. This antibody will therefore be of considerable utility for the study of chicken macrophage biology. Copyright © 2013. Published by Elsevier Ltd.

  2. Leaf proteome analysis of transgenic plants expressing antiviral antibodies.

    PubMed

    Di Carli, Mariasole; Villani, Maria Elena; Renzone, Giovanni; Nardi, Luca; Pasquo, Alessandra; Franconi, Rosella; Scaloni, Andrea; Benvenuto, Eugenio; Desiderio, Angiola

    2009-02-01

    The expression of exogenous antibodies in plant is an effective strategy to confer protection against viral infection or to produce molecules with pharmaceutical interest. However, the acceptance of the transgenic technology to obtain self-protecting plants depends on the assessment of their substantial equivalence compared to non-modified crops with an established history of safe use. In fact, the possibility exists that the introduction of transgenes in plants may alter expression of endogenous genes and/or normal production of metabolites. In this study, we investigated whether the expression in plant of recombinant antibodies directed against viral proteins may influence the host leaf proteome. Two transgenic plant models, generated by Agrobacterium tumefaciens-mediated transformation, were analyzed for this purpose, namely, Lycopersicon esculentum cv. MicroTom and Nicotiana benthamiana, expressing recombinant antibodies against cucumber mosaic virus and tomato spotted wilt virus, respectively. To obtain a significant representation of plant proteomes, optimized extraction procedures have been devised for each plant species. The proteome repertoire of antibody-expressing and control plants was compared by 2-DE associated to DIGE technology. Among the 2000 spots detected within the gels, about 10 resulted differentially expressed in each transgenic model and were identified by MALDI-TOF PMF and muLC-ESI-IT-MS/MS procedures. Protein variations were restricted to a limited number of defined differences with an average ratio below 2.4. Most of the differentially expressed proteins were related to photosynthesis or defense function. The overall results suggest that the expression of recombinant antibodies in both systems does not significantly alter the leaf proteomic profile, contributing to assess the biosafety of resistant plants expressing antiviral antibodies.

  3. Neurophysiological evidence (ERPs) for hemispheric processing of facial expressions of emotions: Evidence from whole face and chimeric face stimuli.

    PubMed

    Damaskinou, Nikoleta; Watling, Dawn

    2017-08-31

    This study was designed to investigate the patterns of electrophysiological responses of early emotional processing at frontocentral sites in adults and to explore whether adults' activation patterns show hemispheric lateralization for facial emotion processing. Thirty-five adults viewed full face and chimeric face stimuli. After viewing two faces, sequentially, participants were asked to decide which of the two faces was more emotive. The findings from the standard faces and the chimeric faces suggest that emotion processing is present during the early phases of face processing in the frontocentral sites. In particular, sad emotional faces are processed differently than neutral and happy (including happy chimeras) faces in these early phases of processing. Further, there were differences in the electrode amplitudes over the left and right hemisphere, particularly in the early temporal window. This research provides supporting evidence that the chimeric face test is a test of emotion processing that elicits right hemispheric processing.

  4. Expression of human antibodies in eukaryotic micro-algae.

    PubMed

    Mayfield, Stephen P; Franklin, Scott E

    2005-03-07

    Protein based therapeutics have enjoyed great success over the past decade. Unfortunately, with this clinical success comes a heavy price tag, owing to the inherently high costs of capitalization and production using mammalian cell fermentation. To address this problem, we have begun developing a system for the expression of recombinant proteins in the unicellular eukaryotic green algae, Chlamydomonas reinhardtii, leading to the production of human IgA single chain antibodies. The expression of human monoclonal antibodies in C. reinhardtii offers an attractive alternative to traditional mammalian based expression systems for several reasons, including an ability to rapidly obtain stable plastid and nuclear transformants, coupled with inherently low costs of capitalization and production.

  5. Clinical Experience with α-Particle–Emitting 211At: Treatment of Recurrent Brain Tumor Patients with 211At-Labeled Chimeric Antitenascin Monoclonal Antibody 81C6

    PubMed Central

    Zalutsky, Michael R.; Reardon, David A.; Akabani, Gamal; Coleman, R. Edward; Friedman, Allan H.; Friedman, Henry S.; McLendon, Roger E.; Wong, Terence Z.; Bigner, Darell D.

    2010-01-01

    α-Particle–emitting radionuclides, such as 211At, with a 7.2-h half-life, may be optimally suited for the molecularly targeted radiotherapy of strategically sensitive tumor sites, such as those in the central nervous system. Because of the much shorter range and more potent cytotoxicity of α-particles than of β-particles, 211At-labeled agents may be ideal for the eradication of tumor cells remaining after surgical debulking of malignant brain tumors. The main goal of this study was to investigate the feasibility and safety of this approach in patients with recurrent malignant brain tumors. Methods Chimeric antitenascin monoclonal antibody 81C6 (ch81C6) (10 mg) was labeled with 71–347 MBq of 211At by use of N-succinimidyl 3-[211At]astatobenzoate. Eighteen patients were treated with 211At-labeled ch81C6 (211At-ch81C6) administered into a surgically created resection cavity (SCRC) and then with salvage chemotherapy. Serial γ-camera imaging and blood sampling over 24 h were performed. Results A total of 96.7% ± 3.6% (mean ± SD) of 211At decays occurred in the SCRC, and the mean blood-pool percentage injected dose was ≤0.3. No patient experienced dose-limiting toxicity, and the maximum tolerated dose was not identified. Six patients experienced grade 2 neurotoxicity within 6 wk of 211At-ch81C6 administration; this neurotoxicity resolved fully in all but 1 patient. No toxicities of grade 3 or higher were attributable to the treatment. No patient required repeat surgery for radionecrosis. The median survival times for all patients, those with glioblastoma multiforme, and those with anaplastic astrocytoma or oligodendroglioma were 54, 52, and 116 wk, respectively. Conclusion This study provides proof of concept for regional targeted radiotherapy with 211At-labeled molecules in oncology. Specifically, the regional administration of 211At-ch81C6 is feasible, safe, and associated with a promising antitumor benefit in patients with malignant central nervous system

  6. Designing a recombinant chimeric construct contain MUC1 and HER2 extracellular domain for prediagnostic breast cancer.

    PubMed

    Gheybi, Elaheh; Amani, Jafar; Salmanian, Ali Hatef; Mashayekhi, Farhad; Khodi, Samaneh

    2014-11-01

    Breast cancer is the most common cancer among women in the world. One of the approaches for diagnosis of breast cancer is detection of its tumor-associated markers. Mucin 1 (MUC1), a tumor-associated antigen, is a transmembrane glycoprotein expressed by normal epithelial cells and overexpressed by carcinomas of epithelial origin. Also, human epidermal growth factor receptor-2 (HER2/erbB-2) belongs to the one of four members of tyrosin kinase type 1 family in which overexpression of HER2 is associated with malignancy in breast cancer. This study was aimed to bioinformatics analysis and designing a recombinant chimeric protein containing MUC1 and HER2 antigens to express in prokaryotic host (Escherichia coli) as breast cancer diagnosis tools. The immunogenic sequences of MUC1 and HER2 were extracted and fused together by a linker. The chimeric construct was analyzed by bioinformatics softwares. The optimization and purification, evaluation of the expression of chimeric protein was performed using Western blotting, ELISA, and immunohistochemistry. The results showed that the chimeric construct was stable and immunogenic domains were exposed. The pET-28a vector containing chimeric gene had high level of protein expression. The recombinant chimeric protein was confirmed using Western blotting, and it was investigated using ELISA and IHC. Then, the MUC1 and HER2 combined peptides can be used as coating antigens in ELISA for detection of antibodies against MUC1 or HER2 in human serum.

  7. Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody-based chimeric antigen receptors indicates affinity/avidity thresholds.

    PubMed

    Oren, Ravit; Hod-Marco, Moran; Haus-Cohen, Maya; Thomas, Sharyn; Blat, Dan; Duvshani, Nerri; Denkberg, Galit; Elbaz, Yael; Benchetrit, Fabrice; Eshhar, Zelig; Stauss, Hans; Reiter, Yoram

    2014-12-01

    Adoptive transfer of Ag-specific T lymphocytes is an attractive form of immunotherapy for cancers. However, acquiring sufficient numbers of host-derived tumor-specific T lymphocytes by selection and expansion is challenging, as these cells may be rare or anergic. Using engineered T cells can overcome this difficulty. Such engineered cells can be generated using a chimeric Ag receptor based on common formats composed from Ag-recognition elements such as αβ-TCR genes with the desired specificity, or Ab variable domain fragments fused with T cell-signaling moieties. Combining these recognition elements are Abs that recognize peptide-MHC. Such TCR-like Abs mimic the fine specificity of TCRs and exhibit both the binding properties and kinetics of high-affinity Abs. In this study, we compared the functional properties of engineered T cells expressing a native low affinity αβ-TCR chains or high affinity TCR-like Ab-based CAR targeting the same specificity. We isolated high-affinity TCR-like Abs recognizing HLA-A2-WT1Db126 complexes and constructed CAR that was transduced into T cells. Comparative analysis revealed major differences in function and specificity of such CAR-T cells or native TCR toward the same antigenic complex. Whereas the native low-affinity αβ-TCR maintained potent cytotoxic activity and specificity, the high-affinity TCR-like Ab CAR exhibited reduced activity and loss of specificity. These results suggest an upper affinity threshold for TCR-based recognition to mediate effective functional outcomes of engineered T cells. The rational design of TCRs and TCR-based constructs may need to be optimized up to a given affinity threshold to achieve optimal T cell function.

  8. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    PubMed

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  9. Optimizing antibody expression by using the naturally occurring framework diversity in a live bacterial antibody display system.

    PubMed

    Lombana, T Noelle; Dillon, Michael; Bevers, Jack; Spiess, Christoph

    2015-12-03

    Rapid identification of residues that influence antibody expression and thermostability is often needed to move promising therapeutics into the clinic. To establish a method that can assess small expression differences, we developed a Bacterial Antibody Display (BAD) system that overcomes previous limitations, enabling the use of full-length formats for antibody and antigen in a live cell setting. We designed a unique library of individual framework variants using natural diversity introduced by somatic hypermutation, and screened half-antibodies for increased expression using BAD. We successfully identify variants that dramatically improve expression yields and in vitro thermostability of two therapeutically relevant antibodies in E. coli and mammalian cells. While we study antibody expression, bacterial display can now be expanded to examine the processes of protein folding and translocation. Additionally, our natural library design strategy could be applied during antibody humanization and library design for in vitro display methods to maintain expression and formulation stability.

  10. PreC and C Regions of Woodchuck Hepatitis Virus Facilitate Persistent Expression of Surface Antigen of Chimeric WHV-HBV Virus in the Hydrodynamic Injection BALB/c Mouse Model

    PubMed Central

    Wu, Weimin; Liu, Yan; Lin, Yong; Pan, Danzhen; Yang, Dongliang; Lu, Mengji; Xu, Yang

    2017-01-01

    In the hydrodynamic injection (HI) BALB/c mouse model with the overlength viral genome, we have found that woodchuck hepatitis virus (WHV) could persist for a prolonged period of time (up to 45 weeks), while hepatitis B virus (HBV) was mostly cleared at week four. In this study, we constructed a series of chimeric genomes based on HBV and WHV, in which the individual sequences of a 1.3-fold overlength HBV genome in pBS-HBV1.3 were replaced by their counterparts from WHV. After HI with the WHV-HBV chimeric constructs in BALB/c mice, serum viral antigen, viral DNA (vDNA), and intrahepatic viral antigen expression were analyzed to evaluate the persistence of the chimeric genomes. Interestingly, we found that HI with three chimeric WHV-HBV genomes resulted in persistent antigenemia in mice. All of the persistent chimeric genomes contained the preC region and the part of the C region encoding the N-terminal 1–145 amino acids of the WHV genome. These results indicated that the preC region and the N-terminal part of the C region of the WHV genome may play a role in the persistent antigenemia. The chimeric WHV-HBV genomes were able to stably express viral antigens in the liver and could be further used to express hepadnaviral antigens to study their pathogenic potential. PMID:28230775

  11. Augmented anti-tumor activity of NK-92 cells expressing chimeric receptors of TGF-βR II and NKG2D.

    PubMed

    Wang, Zhongjuan; Guo, Linghua; Song, Yuan; Zhang, Yinsheng; Lin, Dandan; Hu, Bo; Mei, Yu; Sandikin, Dedy; Liu, Haiyan

    2017-04-01

    The capacity of natural killer (NK) cells to kill tumor cells without specific antigen recognition provides an advantage over T cells and makes them potential effectors for tumor immunotherapy. However, the efficacy of NK cell adoptive therapy can be limited by the immunosuppressive tumor microenvironment. Transforming growth factor-β (TGF-β) is a potent immunosuppressive cytokine that can suppress NK cell function. To convert the suppressive signal induced by TGF-β to an activating signal, we genetically modified NK-92 cells to express a chimeric receptor with TGF-β type II receptor extracellular and transmembrane domains and the intracellular domain of NK cell-activating receptor NKG2D (TN chimeric receptor). NK-92 cells expressing TN receptors were resistant to TGF-β-induced suppressive signaling and did not down-regulate NKG2D. These modified NK-92 cells had higher killing capacity and interferon γ (IFN-γ) production against tumor cells compared with the control cells and their cytotoxicity could be further enhanced by TGF-β. More interestingly, the NK-92 cells expressing TN receptors were better chemo-attracted to the tumor cells expressing TGF-β. The presence of these modified NK-92 cells significantly inhibited the differentiation of human naïve CD4(+) T cells to regulatory T cells. NK-92-TN cells could also inhibit tumor growth in vivo in a hepatocellular carcinoma xenograft tumor model. Therefore, TN chimeric receptors can be a novel strategy to augment anti-tumor efficacy in NK cell adoptive therapy.

  12. Immunization with soluble BDC 2.5 T cell receptor-immunoglobulin chimeric protein:antibody specificity and protection of nonobese diabetic mice against adoptive transfer of diabetes by maternal immunization

    PubMed Central

    1996-01-01

    The BDC 2.5 T cell clone is specific for pancreatic beta-cell antigen presented by I-Ag7, and greatly accelerates diabetes when injected into 10-21-d-old nonobese diabetic (NOD) mice. The BDC 2.5 T cell receptor (TCR) has been solubilized as a TCR-IgG1 chimeric protein. All NOD mice immunized against BDC 2.5 TCR-IgG1 produced antibodies recognizing TCR C alpha/C beta epitopes that were inaccessible on the T cell surface. 56% of the mice produced antibodies against the BDC 2.5 clonotype that specifically blocked antigen activation of BDC 2.5 cells. We have used the adoptive transfer model of diabetes to demonstrate that maternal immunization with soluble TCR protects young mice from diabetes induced by the BDC 2.5 T cell clone. PMID:8920864

  13. Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

    PubMed Central

    Powilleit, Frank; Breinig, Tanja; Schmitt, Manfred J.

    2007-01-01

    A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 α-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production. PMID:17476337

  14. Invasiveness, chimerism and genetic diversity.

    PubMed

    Ben-Shlomo, Rachel

    2017-09-26

    Adaptation for invasiveness should comprise the capability to exploit and prosper in a wide range of ecological conditions, and is therefore expected to be associated with a certain level of genetic diversity. Paradoxically, however, invasive populations are established by only a few founders, resulting in low genetic diversity. As a conceivable way of attaining high genetic diversity and high variance of gene expression even when a small number of founders is involved in invasiveness, I suggest here chimerism, a fusion between different individuals-a common phenomenon found in numerous phyla. The composite entity offers the chimeric organism genetic flexibility and higher inclusive fitness that depends on the joint genomic fitness of the original partners. The ability to form a chimeric entity is also applied to subsequent generations and, consequently, the level of genetic diversity does not decline over generations of population establishment following invasion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  15. Chimeric Bovine/Human Parainfluenza Virus Type 3 Expressing Respiratory Syncytial Virus (RSV) F Glycoprotein: Effect of Insert Position on Expression, Replication, Immunogenicity, Stability, and Protection against RSV Infection

    PubMed Central

    Munir, Shirin; Amaro-Carambot, Emerito; Surman, Sonja; Mackow, Natalie; Yang, Lijuan; Buchholz, Ursula J.; Collins, Peter L.; Schaap-Nutt, Anne

    2014-01-01

    ABSTRACT A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109–114, 2012; C.-F. Yang et al., Vaccine 31:2822–2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity. IMPORTANCE The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young

  16. In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli

    PubMed Central

    Novinrooz, Aytak; Zahraei Salehi, Taghi; Firouzi, Roya; Arabshahi, Sina; Derakhshandeh, Abdollah

    2017-01-01

    E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2–4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression

  17. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.

    PubMed

    Ellebrecht, Christoph T; Bhoj, Vijay G; Nace, Arben; Choi, Eun Jung; Mao, Xuming; Cho, Michael Jeffrey; Di Zenzo, Giovanni; Lanzavecchia, Antonio; Seykora, John T; Cotsarelis, George; Milone, Michael C; Payne, Aimee S

    2016-07-08

    Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease.

  18. Tissue-specific expressed antibody variable gene repertoires.

    PubMed

    Briney, Bryan S; Willis, Jordan R; Finn, Jessica A; McKinney, Brett A; Crowe, James E

    2014-01-01

    Recent developments in genetic technologies allow deep analysis of the sequence diversity of immune repertoires, but little work has been reported on the architecture of immune repertoires in mucosal tissues. Antibodies are the key to prevention of infections at the mucosal surface, but it is currently unclear whether the B cell repertoire at mucosal surfaces reflects the dominant antibodies found in the systemic compartment or whether mucosal tissues harbor unique repertoires. We examined the expressed antibody variable gene repertoires from 10 different human tissues using RNA samples derived from a large number of individuals. The results revealed that mucosal tissues such as stomach, intestine and lung possess unique antibody gene repertoires that differed substantially from those found in lymphoid tissues or peripheral blood. Mutation frequency analysis of mucosal tissue repertoires revealed that they were highly mutated, with little evidence for the presence of naïve B cells, in contrast to blood. Mucosal tissue repertoires possessed longer heavy chain complementarity determining region 3 loops than lymphoid tissue repertoires. We also noted a large increase in frequency of both insertions and deletions in the small intestine antibody repertoire. These data suggest that mucosal immune repertoires are distinct in many ways from the systemic compartment.

  19. Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses

    PubMed Central

    Wykes, Michelle N; Beattie, Lynette; MacPherson, Gordon G; Hart, Derek N

    2004-01-01

    CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b− DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs. PMID:15500618

  20. Chimeric bacteriophage fr virus-like particles harboring the immunodominant C-terminal region of hamster polyomavirus VP1 induce a strong VP1-specific antibody response in rabbits and mice.

    PubMed

    Voronkova, Tatyana; Grosch, Adrian; Kazaks, Andris; Ose, Velta; Skrastina, Dace; Sasnauskas, Kestutis; Jandrig, Burkhard; Arnold, Wolfgang; Scherneck, Siegfried; Pumpens, Paul; Ulrich, Rainer

    2002-01-01

    The late region of the hamster polyomavirus (HaPyV, former HaPV) genome encodes three structural proteins VP1, VP2, and VP3, where VP1 represents the major capsid protein of 384 amino acids. Screening of sera from HaPyV-infected papilloma-bearing and papilloma-free hamsters demonstrated the immunodominant features of all three capsid proteins. For both groups of hamsters in the C-terminal region of VP1 immunodominant B-cell epitopes were identified in the regions between amino acids 305 and 351 and amino acids 351 and 384. The high flexibility of the C-terminal region of VP1 was confirmed by the formation of chimeric virus-like particles based on the coat protein of the RNA bacteriophage fr which was previously found to tolerate only very short-sized foreign insertions. Phage fr coat protein-derived virus-like particles tolerated the N-terminal fusion of amino acids 333-384, 351-384, 351-374, and 364-384, respectively, of VP1. The induction of VP1-specific antibodies in rabbits and mice by immunization with chimeric virus-like particles harboring amino acids 333-384, 351-384, and 364-384, respectively, of VP1 suggested the immunodominant nature of the C-terminal region of VP1.

  1. Expression of recombinant vaccines and antibodies in plants.

    PubMed

    Ko, Kisung

    2014-06-01

    Plants are able to perform post-translational maturations of therapeutic proteins required for their functional biological activity and suitable in vivo pharmacokinetics. Plants can be a low-cost, large-scale production platform of recombinant biopharmaceutical proteins such as vaccines and antibodies. Plants, however, lack mechanisms of processing authentic human N-glycosylation, which imposes a major limitation in their use as an expression system for therapeutic glycoproducts. Efforts have been made to circumvent plant-specific N-glycosylation, as well as to supplement the plant's endogenous system with human glycosyltransferases for non-immunogenic and humanized N-glycan production. Herein we review studies on the potential of plants to serve as production systems for therapeutic and prophylactic biopharmaceuticals. We have especially focused on recombinant vaccines and antibodies and new expression strategies to overcome the existing problems associated with their production in plants.

  2. Downregulation of transferrin receptor surface expression by intracellular antibody

    SciTech Connect

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin . E-mail: guanxin_shen@yahoo.com.cn

    2007-03-23

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.

  3. Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo.

    PubMed

    Chen, Joy; Peterson, Kenneth R; Iancu-Rubin, Camelia; Bieker, James J

    2010-09-28

    Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNA-binding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.

  4. Induction of Pluripotent Protective Immunity Following Immunisation with a Chimeric Vaccine against Human Cytomegalovirus

    PubMed Central

    Zhong, Jie; Rist, Michael; Cooper, Leanne; Smith, Corey; Khanna, Rajiv

    2008-01-01

    Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8+ and CD4+ T-cells which co-expressed IFN-γ and TNF-α, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8+ and CD4+ T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings. PMID:18806877

  5. EASE vectors for rapid stable expression of recombinant antibodies.

    PubMed

    Aldrich, Teri L; Viaje, Aurora; Morris, Arvia E

    2003-01-01

    Over the past 10 years, monoclonal antibodies and antibody fragments have become an increasingly important source of therapeutic molecules in the biotechnology industry. Drug development strategies rely on screening large numbers of candidate molecules in search of an optimized drug candidate. This strategy requires efficient production of ten to a few hundred milligrams of candidate molecules for screening in bioassays and animal models. Typically, this amount of recombinant protein expression involves large numbers of transient transfections or cloning of a recombinant cell line. Both of these approaches are time-consuming and labor-intensive. In this report, we describe the application of an EASE vector system that is capable of generating stable pools of transfected Chinese hamster ovary cells. These pooled populations of cells produce high quantities of antibody candidates without labor-intensive cloning in a 3-5 week time frame. When an optimal drug candidate has been selected, pools generated with EASE-containing vectors can also be used in subsequent cloning steps to make cell lines with improved expression levels. We demonstrate that EASE increases expression in nonamplified pools in addition to increasing amplification and viability of clonal cell lines generated with the EASE-containing vectors compared with pools and cell lines generated without EASE.

  6. Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

    PubMed

    Hernández-Guzmán, K; Sahagún-Ruiz, A; Vallecillo, A J; Cruz-Mendoza, I; Quiroz-Romero, H

    2016-01-01

    Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.

  7. Development of a Novel Antibody-Drug Conjugate for the Potential Treatment of Ovarian, Lung, and Renal Cell Carcinoma Expressing TIM-1.

    PubMed

    Thomas, Lawrence J; Vitale, Laura; O'Neill, Thomas; Dolnick, Ree Y; Wallace, Paul K; Minderman, Hans; Gergel, Lauren E; Forsberg, Eric M; Boyer, James M; Storey, James R; Pilsmaker, Catherine D; Hammond, Russell A; Widger, Jenifer; Sundarapandiyan, Karuna; Crocker, Andrea; Marsh, Henry C; Keler, Tibor

    2016-12-01

    T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I transmembrane protein that was originally described as kidney injury molecule 1 (KIM-1) due to its elevated expression in kidney and urine after renal injury. TIM-1 expression is also upregulated in several human cancers, most notably in renal and ovarian carcinomas, but has very restricted expression in healthy tissues, thus representing a promising target for antibody-mediated therapy. To this end, we have developed a fully human monoclonal IgG1 antibody specific for the extracellular domain of TIM-1. This antibody was shown to bind purified recombinant chimeric TIM-1-Fc protein and TIM-1 expressed on a variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma), IGROV-1 (human ovarian adenocarcinoma), and A549 (human lung carcinoma). Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by cells in vitro, and internalization was confirmed by quantitative imaging flow cytometry. An antibody-drug conjugate (ADC) was produced with the anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E (MMAE), and designated CDX-014. The ADC was shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1-expressing cell lines, but not on TIM-1-negative cell lines. Using the Caki-1, IGROV-1, and A549 xenograft mouse models, CDX-014 showed significant antitumor activity in a clinically relevant dose range. Safety evaluation in nonhuman primates has demonstrated a good profile and led to the initiation of clinical studies of CDX-014 in renal cell carcinoma and potentially other TIM-1-expressing tumors. Mol Cancer Ther; 15(12); 2946-54. ©2016 AACR.

  8. Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development

    PubMed Central

    1995-01-01

    The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis- regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse alpha 1(II) collagen gene. Chondrocyte densities and levels of alpha 1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form alpha 1(IX) collagen, alpha 2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of alpha 1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components. PMID:7822417

  9. The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules.

    PubMed

    Carsolio, C; Campos, F; Sánchez, F; Rocha-Sosa, M

    1994-12-01

    In Phaseolus vulgaris there is a nodulin family, Npv30, of ca. 30 kDa, as detected in an in vitro translation assay [2]. We isolated a gene (npv30-1) for one of the members of this family. The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes. The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation. GUS activity was only detected in the infected cells of the nodules of transgenic plants. By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.

  10. In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

    PubMed

    Caprodossi, Sara; Galluzzi, Luca; Biagetti, Simona; Della Chiara, Giulia; Pelicci, Pier Giuseppe; Magnani, Mauro; Fanelli, Mirco

    2005-01-01

    Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

  11. Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia

    PubMed Central

    Barrett, David; Aplenc, Richard; Porter, David L.; Rheingold, Susan R.; Teachey, David T.; Chew, Anne; Hauck, Bernd; Wright, J. Fraser; Milone, Michael C.; Levine, Bruce L.; June, Carl H.

    2014-01-01

    Summary Chimeric antigen receptor–modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre–B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×106 to 1.2×107 CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce anti-leukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor–modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL. PMID:23527958

  12. Human liver catalase: cloning, expression and characterization of monoclonal antibodies.

    PubMed

    Jin, Li Hua; Kim, Dae Won; Eum, Won Sik; Yoon, Chang Sik; Jang, Sang Ho; Choi, Hee Soon; Choi, Soo Hyun; Kim, Young Hoon; Kim, So Young; Jung, Mi Ryoung; Kang, Tae-Cheon; Won, Moo Ho; Lee, Hyeon Yong; Kang, Jung Hoon; Kwon, Oh-Shin; Cho, Sung-Woo; Lee, Kil Soo; Park, Jinseu; Choi, Soo Young

    2003-06-30

    We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.

  13. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

    PubMed

    Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

    2016-05-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Copyright© Ferrata Storti Foundation.

  14. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma

    PubMed Central

    Drent, Esther; Groen, Richard W.J.; Noort, Willy A.; Themeli, Maria; Lammerts van Bueren, Jeroen J.; Parren, Paul W.H.I.; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W.C.J.; Martens, Anton C.M.; Lokhorst, Henk M.; Mutis, Tuna

    2016-01-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38+ fractions of CD34+ hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38+ malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. PMID:26858358

  15. Half molecular exchange of IgGs in the blood of healthy humans: chimeric lambda-kappa-immunoglobulins containing HL fragments of antibodies of different subclasses (IgG1-IgG4).

    PubMed

    Sedykh, Sergey E; Lekchnov, Evgenii A; Prince, Viktor V; Buneva, Valentina N; Nevinsky, Georgy A

    2016-10-20

    In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies recognizing a single antigen (monospecific). There is a common belief that IgGs in mammalian biological fluids are monospecific molecules having stable structures and two identical antigen-binding sites. But the issue concerning the possibility of exchange by HL-fragments between the antibody molecules in human blood is still unexplored. Different physico-chemical and immunological methods for analysis of half-molecule exchange between human blood IgGs were used. Using eighteen blood samples of healthy humans we have shown unexpected results for the first time: blood antibodies undergo extensive post-transcriptional half-molecule exchange and IgG pools on average consist of 62.4 ± 6.5% IgGs containing kappa light chains (kappa-kappa-IgGs), 29.8.6 ± 5.4% lambda light chains (lambda-lambda-IgGs), and 8.8 ± 2.7% (range 2.6-16.8%) IgGs containing both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained on average (%): IgG1 (36.0 and 32.3), IgG2 (50.9 and 51.4), IgG3 (9.7 and 9.9), and IgG4 (6.5 and 5.7), while chimeric kappa-lambda-IgGs consisted of (%): 25.5 ± 4.2 IgG1, 50.8 ± 3.9 IgG2, 9.1 ± 2.1 IgG3, and 14.5 ± 2.2 IgG4. Our unexpected data are indicative of the possibility of half-molecule exchange between blood IgGs of various subclasses, raised against different antigens. The existence of blood chimeric bifunctional IgGs with different binding sites destroys the classic paradigm. Due to the phenomenon of polyspecificity and cross-reactivity of bifunctional IgGs containing HL-fragments of different types to different antigens, such IgGs may be important in human blood for widening their different biological functions.

  16. Secretion of a chimeric T-cell receptor-immunoglobulin protein.

    PubMed Central

    Gascoigne, N R; Goodnow, C C; Dudzik, K I; Oi, V T; Davis, M M

    1987-01-01

    To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable. Images PMID:3472243

  17. Chimeric Antigen Receptor T Cells Guided by the Single-Chain Fv of a Broadly Neutralizing Antibody Specifically and Effectively Eradicate Virus Reactivated from Latency in CD4+ T Lymphocytes Isolated from HIV-1-Infected Individuals Receiving Suppressive Combined Antiretroviral Therapy.

    PubMed

    Liu, Bingfeng; Zou, Fan; Lu, Lijuan; Chen, Cancan; He, Dalian; Zhang, Xu; Tang, Xiaoping; Liu, Chao; Li, Linghua; Zhang, Hui

    2016-11-01

    Despite the advent of combined antiretroviral therapy (cART), the persistence of viral reservoirs remains a major barrier to curing human immunodeficiency virus type 1 (HIV-1) infection. Recently, the shock and kill strategy, by which such reservoirs are eradicated following reactivation of latent HIV-1 by latency-reversing agents (LRAs), has been extensively practiced. It is important to reestablish virus-specific and reliable immune surveillance to eradicate the reactivated virus-harboring cells. In this report, we attempted to reach this goal by using newly developed chimeric antigen receptor (CAR)-T cell technology. To generate anti-HIV-1 CAR-T cells, we connected the single-chain variable fragment of the broadly neutralizing HIV-1-specific antibody VRC01 to a third-generation CAR moiety as the extracellular and intracellular domains and subsequently transduced this into primary CD8(+) T lymphocytes. We demonstrated that the resulting VC-CAR-T cells induced T cell-mediated cytolysis of cells expressing HIV-1 Env proteins and significantly inhibited HIV-1 rebound after removal of antiviral inhibitors in a viral infectivity model in cell culture that mimics the termination of the cART in the clinic. Importantly, the VC-CAR-T cells also effectively induced the cytolysis of LRA-reactivated HIV-1-infected CD4(+) T lymphocytes isolated from infected individuals receiving suppressive cART. Our data demonstrate that the special features of genetically engineered CAR-T cells make them a particularly suitable candidate for therapeutic application in efforts to reach a functional HIV cure. The presence of latently infected cells remains a key obstacle to the development of a functional HIV-1 cure. Reactivation of dormant viruses is possible with latency-reversing agents, but the effectiveness of these compounds and the subsequent immune response require optimization if the eradication of HIV-1-infected cells is to be achieved. Here, we describe the use of a chimeric

  18. Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

    PubMed

    Sun, Hengyi; Zang, Xiaonan; Liu, Yuantao; Cao, Xiaofei; Wu, Fei; Huang, Xiaoyun; Jiang, Minjie; Zhang, Xuecheng

    2015-12-01

    Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

  19. Co-expression of chimeric chitinase and a polygalacturonase-inhibiting protein in transgenic canola (Brassica napus) confers enhanced resistance to Sclerotinia sclerotiorum.

    PubMed

    Ziaei, Mahboobeh; Motallebi, Mostafa; Zamani, Mohammad Reza; Panjeh, Nasim Zarin

    2016-06-01

    Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is one of the major fungal diseases of canola. To develop resistance against this fungal disease, the chit42 from Trichoderma atroviride with chitin-binding domain and polygalacturonase-inhibiting protein 2 (PG1P2) of Phaseolus vulgaris were co-expressed in canola via Agrobacterium-mediated transformation. Stable integration and expression of transgenes in T0 and T2 plants was confirmed by PCR, Southern blot and RT-PCR analyses. Chitinase activity and PGIP2 inhibition were detected by colorimetric and agarose diffusion assay in transgenic lines but not in untransformed plants. The crude proteins from single copy transformant leaves having high chitinase and PGIP2 activity (T16, T8 and T3), showed up to 44 % inhibition of S. sclerotiorum hyphal growth. The homozygous T2 plants, showing inheritance in Mendelian fashion (3:1), were further evaluated under greenhouse conditions for resistance to S. sclerotiorum. Intact plants contaminated with mycelia showed resistance through delayed onset of the disease and restricted size and expansion of lesions as compared to wild type plants. Combined expression of chimeric chit42 and pgip2 in Brassica napus L. provide subsequent protection against SSR disease and can be helpful in increasing the canola production in Iran.

  20. The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor1

    PubMed Central

    Jin, Zeming; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Olivares, Simon; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J.N.

    2014-01-01

    Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with a SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells. PMID:21451576

  1. Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

    PubMed Central

    Singh, Harjeet; Figliola, Matthew J.; Dawson, Margaret J.; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E.; Huls, Helen; Cooper, Laurence J. N.

    2013-01-01

    Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

  2. Expression of a Chimeric Gene Encoding Insecticidal Crystal Protein Cry1Aabc of Bacillus thuringiensis in Chickpea (Cicer arietinum L.) Confers Resistance to Gram Pod Borer (Helicoverpa armigera Hubner.).

    PubMed

    Das, Alok; Datta, Subhojit; Thakur, Shallu; Shukla, Alok; Ansari, Jamal; Sujayanand, G K; Chaturvedi, Sushil K; Kumar, P A; Singh, N P

    2017-01-01

    Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer (Helicoverpa armigera H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by Bt (cryI) genes. We designed a plant codon optimized chimeric Bt gene (cry1Aabc) using three domains from three different cry1A genes (domains I, II, and III from cry1Aa, cry1Ab, and cry1Ac, respectively) and expressed it under the control of a constitutive promoter in chickpea (cv. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The in vitro regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric cry1Aabc expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program.

  3. Expression of a Chimeric Gene Encoding Insecticidal Crystal Protein Cry1Aabc of Bacillus thuringiensis in Chickpea (Cicer arietinum L.) Confers Resistance to Gram Pod Borer (Helicoverpa armigera Hubner.)

    PubMed Central

    Das, Alok; Datta, Subhojit; Thakur, Shallu; Shukla, Alok; Ansari, Jamal; Sujayanand, G. K.; Chaturvedi, Sushil K.; Kumar, P. A.; Singh, N. P.

    2017-01-01

    Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer (Helicoverpa armigera H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by Bt (cryI) genes. We designed a plant codon optimized chimeric Bt gene (cry1Aabc) using three domains from three different cry1A genes (domains I, II, and III from cry1Aa, cry1Ab, and cry1Ac, respectively) and expressed it under the control of a constitutive promoter in chickpea (cv. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The in vitro regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric cry1Aabc expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program. PMID:28871265

  4. Specific tumor labeling enhanced by polyethylene glycol linkage of near infrared dyes conjugated to a chimeric anti-carcinoembryonic antigen antibody in a nude mouse model of human pancreatic cancer

    NASA Astrophysics Data System (ADS)

    Maawy, Ali A.; Hiroshima, Yukihiko; Zhang, Yong; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2014-10-01

    Labeling of metastatic tumors can aid in their staging and resection of cancer. Near infrared (NIR) dyes have been used in the clinic for tumor labeling. However, there can be a nonspecific uptake of dye by the liver, lungs, and lymph nodes, which hinders detection of metastasis. In order to overcome these problems, we have used two NIR dyes (DyLight 650 and 750) conjugated to a chimeric anti-carcinoembryonic antigen antibody to evaluate how polyethylene glycol linkage (PEGylation) can improve specific tumor labeling in a nude mouse model of human pancreatic cancer. The conjugated PEGylated and non-PEGylated DyLight 650 and 750 dyes were injected intravenously into non-tumor-bearing nude mice. Serum samples were collected at various time points in order to determine serum concentrations and elimination kinetics. Conjugated PEGylated dyes had significantly higher serum dye concentrations than non-PEGylated dyes (p=0.005 for the 650 dyes and p<0.001 for the 750 dyes). Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with conjugated PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (p<0.001 for the 650 dyes; p=0.01 for 750 dyes). PEGylation of the NIR dyes also decreased their accumulation in lymph nodes, liver, and lung. These results demonstrate enhanced selective tumor labeling by PEGylation of dyes conjugated to a tumor-specific antibody, suggesting their future clinical use in fluorescence-guided surgery.

  5. Effect of hepatitis C virus infection on the mRNA expression of drug transporters and cytochrome p450 enzymes in chimeric mice with humanized liver.

    PubMed

    Kikuchi, Ryota; McCown, Matthew; Olson, Pamela; Tateno, Chise; Morikawa, Yoshio; Katoh, Yumiko; Bourdet, David L; Monshouwer, Mario; Fretland, Adrian J

    2010-11-01

    The expression of drug transporters and metabolizing enzymes is a primary determinant of drug disposition. Chimeric mice with humanized liver, including PXB mice, are an available model that is permissive to the in vivo infection of hepatitis C virus (HCV), thus being a promising tool for investigational studies in development of new antiviral molecules. To investigate the potential of HCV infection to alter the pharmacokinetics of small molecule antiviral therapeutic agents in PXB mice, we have comprehensively determined the mRNA expression profiles of human ATP-binding cassette (ABC) transporters, solute carrier (SLC) transporters, and cytochrome P450 (P450) enzymes in the livers of these mice under noninfected and HCV-infected conditions. Infection of PXB mice with HCV resulted in an increase in the mRNA expression levels of a series of interferon-stimulated genes in the liver. For the majority of genes involved in drug disposition, minor differences in the mRNA expression of ABC and SLC transporters as well as P450s between the noninfected and HCV-infected groups were observed. The exceptions were statistically significantly higher expression of multidrug resistance-associated protein 4 and organic anion-transporting polypeptide 2B1 and lower expression of organic cation transporter 1 and CYP2D6 in HCV-infected mice. Furthermore, the enzymatic activities of the major human P450s were, in general, comparable in the two experimental groups. These data suggest that the pharmacokinetic properties of small molecule antiviral therapies in HCV-infected PXB mice are likely to be similar to those in noninfected PXB mice. However, caution is needed in the translation of this relationship to HCV-infected patients as the PXB mouse model does not accurately reflect the pathology of patients with chronic HCV infection.

  6. Structural defect linked to nonrandom mutations in the matrix gene of Biden strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes

    SciTech Connect

    Ayata, M.; Hirano, A.; Wong, T.C.

    1989-03-01

    Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 M/sub r/ protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general.

  7. Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection.

    PubMed

    Boardman, D A; Philippeos, C; Fruhwirth, G O; Ibrahim, M A A; Hannen, R F; Cooper, D; Marelli-Berg, F M; Watt, F M; Lechler, R I; Maher, J; Smyth, L A; Lombardi, G

    2017-04-01

    Regulatory T cell (Treg) therapy using recipient-derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor-MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune-mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  8. The Nuclear Gene Rf3 Affects the Expression of the Mitochondrial Chimeric Sequence R Implicated in S-Type Male Sterility in Maize

    PubMed Central

    Zabala, G.; Gabay-Laughnan, S.; Laughnan, J. R.

    1997-01-01

    The mitochondrial genomes of maize plants exhibiting S-type cytoplasmic male sterility (cms-S) contain a repeated DNA region designated R. This region was found to be rearranged in the mitochondria of all cms-S cytoplasmically revertant fertile plants in all nuclear backgrounds analyzed. A 1.6-kb mRNA transcribed from the R region in mitochondria of sterile plants was absent from all cytoplasmic revertants examined. The nuclear gene Rf3, which suppresses the cms-S phenotype, was found to have a specific effect on the expression of the R sequence; the abundance of the major R transcripts, including the cms-S-specific 1.6-kb mRNA, is decreased in mitochondria of restored plants. Nucleotide sequence analysis of R has revealed similarities to the R1 plasmid found in some South American maize races with RU cytoplasm, to the M1 plasmid found in one source of Zea luxurians teosinte, to the atp9 mitochondrial gene and its 3' flanking sequence, and also to a region 3' to the orf221 gene. The derived amino acid sequence of the R region predicts two open reading frames (ORFs). These ORFs contain the similarities to R1, M1, atp9 and orf221. The present report reveals the chimeric nature of the R region, describes the complex effect of Rf3 on the expression of the R sequence and implicates R in the sterile phenotype of cms-S maize. PMID:9335619

  9. Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

    PubMed Central

    Proff, Julia; Schaft, Niels; Dörrie, Jan; Full, Florian; Ensser, Armin; Muller, Yves A.; Cerwenka, Adelheid; Abken, Hinrich; Parolini, Ornella; Ambros, Peter F.; Kovar, Heinrich; Holter, Wolfgang

    2012-01-01

    We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection. PMID:22355347

  10. A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells

    PubMed Central

    Gutierrez-Guerrero, Alejandra; Cobo, Marién; Muñoz, Pilar

    2014-01-01

    Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells. PMID:24400083

  11. Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

    PubMed

    Schmueck-Henneresse, Michael; Omer, Bilal; Shum, Thomas; Tashiro, Haruko; Mamonkin, Maksim; Lapteva, Natalia; Sharma, Sandhya; Rollins, Lisa; Dotti, Gianpietro; Reinke, Petra; Volk, Hans-Dieter; Rooney, Cliona M

    2017-07-01

    The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO(+)CCR7(-) effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  12. Males without apparent alloimmunization could have HLA antibodies that recognize target HLA specificities expressed on cells.

    PubMed

    Nakamura, J; Nakajima, F; Kamada, H; Tadokoro, K; Nagai, T; Satake, M

    2017-05-01

    Human leukocyte antigen (HLA) antibodies, which are involved in the development of transfusion-related side effects such as transfusion-related lung injury, are sometimes found in males without a history of alloimmunization (eg, transplantation and transfusion). Whether HLA antibodies in male donors can interact with their target HLA specificities expressed on cells have not been completely investigated. The HLA antibodies detected in 7 male donors were characterized. Flow cytometry and immunocomplex capture fluorescence analysis were performed to evaluate the ability of these antibodies to bind with target HLA specificities expressed on cells. The association of these antibodies with complement was examined using anti-C1q antibody. Sustainability of HLA antibodies over time was compared in 26 male vs 57 female donors. The antibodies from all 7 donors recognized intact HLA molecules coated onto microbeads. The antibodies in 2 of 7 donors also recognized their target HLA specificities expressed on cells. Furthermore, the antibodies in one of these 2 donors showed HLA specificities that involved complement binding. Twenty-one of 26 initially positive male donors had turned negative for HLA antibody at least 1 year after their initial positive screening, whereas HLA antibody positivity was maintained for a long time in most female donors. Males without apparent alloimmunization could have HLA antibodies that recognize their target HLA specificities on cells and that could potentially modify molecular events in affected cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Transcriptome Sequencing for the Detection of Chimeric Transcripts.

    PubMed

    Chu, Hsueh-Ting

    2016-01-01

    The occurrence of chimeric transcripts has been reported in many cancer cells and seen as potential biomarkers and therapeutic targets. Modern high-throughput sequencing technologies offer a way to investigate individual chimeric transcripts and the systematic information of associated gene expressions about underlying genome structural variations and genomic interactions. The detection methods of finding chimeric transcripts from massive amount of short read sequence data are discussed here. Both assembly-based and alignment-based methods are used for the investigation of chimeric transcripts.

  14. Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy

    DOE PAGES

    DebRoy, Swati; Hiraga, Nobuhiko; Imamura, Michio; ...

    2016-06-08

    Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. In this paper, to elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albuminmore » (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. Finally, the results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes.« less

  15. Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy

    SciTech Connect

    DebRoy, Swati; Hiraga, Nobuhiko; Imamura, Michio; Hayes, C. Nelson; Akamatsu, Sakura; Canini, Laetitia; Perelson, Alan S.; Pohl, Ralf T.; Persiani, Stefano; Uprichard, Susan L.; Tateno, Chise; Dahari, Harel; Chayama, Kazuaki

    2016-06-08

    Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. In this paper, to elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. Finally, the results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes.

  16. Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy

    SciTech Connect

    DebRoy, Swati; Hiraga, Nobuhiko; Imamura, Michio; Hayes, C. Nelson; Akamatsu, Sakura; Canini, Laetitia; Perelson, Alan S.; Pohl, Ralf T.; Persiani, Stefano; Uprichard, Susan L.; Tateno, Chise; Dahari, Harel; Chayama, Kazuaki

    2016-06-08

    Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. In this paper, to elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. Finally, the results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes.

  17. Humanization of excretory pathway in chimeric mice with humanized liver.

    PubMed

    Okumura, Hirotoshi; Katoh, Miki; Sawada, Toshiro; Nakajima, Miki; Soeno, Yoshinori; Yabuuchi, Hikaru; Ikeda, Toshihiko; Tateno, Chise; Yoshizato, Katsutoshi; Yokoi, Tsuyoshi

    2007-06-01

    The liver of a chimeric urokinase-type plasminogen activator (uPA)(+/+)/severe combined immunodeficient (SCID) mouse line recently established in Japan could be replaced by more than 80% with human hepatocytes. We previously reported that the chimeric mice with humanized liver could be useful as a human model in studies on drug metabolism and pharmacokinetics. In the present study, the humanization of an excretory pathway was investigated in the chimeric mice. Cefmetazole (CMZ) was used as a probe drug. The CMZ excretions in urine and feces were 81.0 and 5.9% of the dose, respectively, in chimeric mice and were 23.7 and 59.4% of the dose, respectively, in control uPA(-/-)/SCID mice. Because CMZ is mainly excreted in urine in humans, the excretory profile of chimeric mice was demonstrated to be similar to that of humans. In the chimeric mice, the hepatic mRNA expression of human drug transporters could be quantified. On the other hand, the hepatic mRNA expression of mouse drug transporters in the chimeric mice was significantly lower than in the control uPA(-/-)/SCID mice. In conclusion, chimeric mice exhibited a humanized profile of drug excretion, suggesting that this chimeric mouse line would be a useful animal model in excretory studies.

  18. Multimodal imaging and detection strategy with 124 I-labeled chimeric monoclonal antibody cG250 for accurate localization and confirmation of extent of disease during laparoscopic and open surgical resection of clear cell renal cell carcinoma.

    PubMed

    Povoski, Stephen P; Hall, Nathan C; Murrey, Douglas A; Sharp, David S; Hitchcock, Charles L; Mojzisik, Cathy M; Bahnson, Eamonn E; Knopp, Michael V; Martin, Edward W; Bahnson, Robert R

    2013-02-01

    Renal cell carcinoma (RCC) accounts for approximately 85% to 90% of all primary kidney malignancies, with clear cell RCC (ccRCC) constituting approximately 70% to 85% of all RCCs. This study describes an innovative multimodal imaging and detection strategy that uses (124)I-labeled chimeric monoclonal antibody G250 ((124)I-cG250) for accurate preoperative and intraoperative localization and confirmation of extent of disease for both laparoscopic and open surgical resection of ccRCC. Two cases presented herein highlight how this technology can potentially guide complete surgical resection and confirm complete removal of all diseased tissues. This innovative (124)I-cG250 (ie, (124)I-girentuximab) multimodal imaging and detection approach, which would be clinically very useful to urologic surgeons, urologic medical oncologists, nuclear medicine physicians, radiologists, and pathologists who are involved in the care of ccRCC patients, holds great potential for improving the diagnostic accuracy, operative planning and approach, verification of disease resection, and monitoring for evidence of disease recurrence in ccRCC patients.

  19. Definition and application of good manufacturing process-compliant production of CEA-specific chimeric antigen receptor expressing T-cells for phase I/II clinical trial.

    PubMed

    Guest, Ryan D; Kirillova, Natalia; Mowbray, Sam; Gornall, Hannah; Rothwell, Dominic G; Cheadle, Eleanor J; Austin, Eric; Smith, Keith; Watt, Suzanne M; Kühlcke, Klaus; Westwood, Nigel; Thistlethwaite, Fiona; Hawkins, Robert E; Gilham, David E

    2014-02-01

    Adoptive cell therapy employing gene-modified T-cells expressing chimeric antigen receptors (CARs) has shown promising preclinical activity in a range of model systems and is now being tested in the clinical setting. The manufacture of CAR T-cells requires compliance with national and European regulations for the production of medicinal products. We established such a compliant process to produce T-cells armed with a first-generation CAR specific for carcinoembryonic antigen (CEA). CAR T-cells were successfully generated for 14 patients with advanced CEA(+) malignancy. Of note, in the majority of patients, the defined procedure generated predominantly CD4(+) CAR T-cells with the general T-cell population bearing an effector-memory phenotype and high in vitro effector function. Thus, improving the process to generate less-differentiated T-cells would be more desirable in the future for effective adoptive gene-modified T-cell therapy. However, these results confirm that CAR T-cells can be generated in a manner compliant with regulations governing medicinal products in the European Union.

  20. Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172.

    PubMed

    Zimmerman, Carl-Ulrich R; Rosengarten, Renate; Spergser, Joachim

    2011-02-01

    Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable 'UU172 element' from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses. © 2010 Blackwell Publishing Ltd.

  1. Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

    PubMed

    Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2015-06-01

    The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs.

  2. Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B.

    PubMed

    Makvandi-Nejad, Shokouh; McLean, Michael D; Hirama, Tomoko; Almquist, Kurt C; Mackenzie, C Roger; Hall, J Christopher

    2005-10-01

    Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T(0)) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 mug of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T(1) plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T(0) plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T(1) generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.

  3. Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.

    PubMed

    Doering, Christopher B; Denning, Gabriela; Dooriss, Kerry; Gangadharan, Bagirath; Johnston, Jennifer M; Kerstann, Keith W; McCarty, David A; Spencer, H Trent

    2009-07-01

    Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

  4. Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

    PubMed Central

    Doering, Christopher B; Denning, Gabriela; Dooriss, Kerry; Gangadharan, Bagirath; Johnston, Jennifer M; Kerstann, Keith W; McCarty, David A; Spencer, H Trent

    2009-01-01

    Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus–based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved. PMID:19259064

  5. Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

    PubMed Central

    Deniger, Drew C; Switzer, Kirsten; Mi, Tiejuan; Maiti, Sourindra; Hurton, Lenka; Singh, Harjeet; Huls, Helen; Olivares, Simon; Lee, Dean A; Champlin, Richard E; Cooper, Laurence JN

    2013-01-01

    Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR+γδ T cells were expanded on CD19+ artificial antigen-presenting cells (aAPC), which resulted in >109 CAR+γδ T cells from <106 total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR+γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19+ tumor cell lines compared with CARnegγδ T cells. CD19+ leukemia xenografts in mice were reduced with CAR+γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells. PMID:23295945

  6. Immune response and protective profile elicited by a multi-epitope chimeric protein derived from Leptospira interrogans.

    PubMed

    Fernandes, Luis G V; Teixeira, Aline F; Filho, Antonio F S; Souza, Gisele O; Vasconcellos, Silvio A; Heinemann, Marcos B; Romero, Eliete C; Nascimento, Ana L T O

    2017-04-01

    Pathogenic Leptospira is the causative agent of leptospirosis, a widely disseminated disease of human and veterinary concern. The development of vaccines that elicit cross-protective immunity through multiple leptospiral serovars has long been pursued. The aim of this study was to develop a novel chimeric multi-epitope fusion antigen, containing sequences of previously studied outer membrane proteins (OMPs) of Leptospira. The chimeric protein was designed based on the amino acid sequences of the LigA, Mce, Lsa45, OmpL1, and LipL41 proteins, cloned into pAE vector, the protein expressed in Escherichia coli, and its immune response evaluated in the hamster infection model. The recombinant chimeric protein (rChi) was recognized by antibodies present in serum samples of confirmed cases of human leptospirosis and experimentally infected hamsters, demonstrating that the rChi protein participates in the immune response activation during infection. However, despite high antibody titers achieved when the rChi protein was administered with either Alhydrogel or Bordetella pertussis monophosphoryl lipid A (MPLA), only 50% of the hamsters were protected against infection. Although a complete characterization of the immune response elicited by rChi/adjuvant in hamsters is required, it is believed that the construction of chimeric genes is an important attempt towards the generation of an effective vaccine against leptospirosis. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  7. Chimeric human papilloma virus-simian/human immunodeficiency virus virus-like-particle vaccines: immunogenicity and protective efficacy in macaques.

    PubMed

    Dale, C Jane; Liu, Xiaosong Song; De Rose, Robert; Purcell, Damian F J; Anderson, Jenny; Xu, Yan; Leggatt, Graham R; Frazer, Ian H; Kent, Stephen J

    2002-09-15

    Vaccines to efficiently block or limit sexual transmission of both HIV and human papilloma virus (HPV) are urgently needed. Chimeric virus-like-particle (VLP) vaccines consisting of both multimerized HPV L1 proteins and fragments of SIV gag p27, HIV-1 tat, and HIV-1 rev proteins (HPV-SHIV VLPs) were constructed and administered to macaques both systemically and mucosally. An additional group of macaques first received a priming vaccination with DNA vaccines expressing the same SIV and HIV-1 antigens prior to chimeric HPV-SHIV VLP boosting vaccinations. Although HPV L1 antibodies were induced in all immunized macaques, weak antibody or T cell responses to the chimeric SHIV antigens were detected only in animals receiving the DNA prime/HPV-SHIV VLP boost vaccine regimen. Significant but partial protection from a virulent mucosal SHIV challenge was also detected only in the prime/boosted macaques and not in animals receiving the HPV-SHIV VLP vaccines alone, with three of five prime/boosted animals retaining some CD4+ T cells following challenge. Thus, although some immunogenicity and partial protection was observed in non-human primates receiving both DNA and chimeric HPV-SHIV VLP vaccines, significant improvements in vaccine design are required before we can confidently proceed with this approach to clinical trials.

  8. Study of a chimeric foot-and-mouth disease virus DNA vaccine containing structural genes of serotype O in a genome backbone of serotype Asia 1 in guinea pigs.

    PubMed

    Chockalingam, A K; Thiyagarajan, S; Govindasamy, N; Patnaikuni, R; Garlapati, S; Golla, R R; Joyappa, D H; Krishnamshetty, P; Veluvarti, V V S; Veluvati, V V S

    2010-01-01

    Since foot-and-mouth disease virus (FMDV) serotypes display a great genetic and antigenic diversity, there is a constant requirement to monitor the performance of FMDV vaccines in the field with respect to their antigenic coverage. To avoid possible antigenic changes in field FMDV isolates during their adaptation to BHK-21 cells, a standard step used in production of conventional FMDV vaccines, the custom-made chimeric conventional or DNA vaccines, in which antigenic determinants are replaced with those of appropriate field strains, should be constructed. Using this approach, we made a plasmid-based chimeric FMDV DNA vaccine containing structural genes of serotype O in the genome backbone of serotype Asia 1, all under the control of Human cytomegalovirus (HCMV) immediate early gene promoter. BHK-21 cells transfected with the chimeric DNA vaccine did not show cytopathic effect (CPE), but expressed virus-specific proteins as demonstrated by 35S-methionine labeling and immunoprecipitation. Guinea pigs immunized with the chimeric DNA vaccine produced virus-specific antibodies assayed by ELISA and virus neutralization test (VNT), respectively. The chimeric DNA vaccine showed a partial protection of guinea pigs challenged with the virulent FMDV. Although the chimeric DNA vaccine, in general, was not as effective as a conventional one, this study encourages further work towards the development of genetically engineered custom-made chimeric vaccines against FMDV.

  9. A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug‐induced liver injury and postinjury inflammation in mice

    PubMed Central

    Lea, Jonathan D.; Sowinska, Agnieszka; Ottosson, Lars; Fürst, Camilla Melin; Steen, Johanna; Aulin, Cecilia; Clarke, Joanna I.; Kipar, Anja; Klevenvall, Lena; Yang, Huan; Palmblad, Karin; Park, B. Kevin; Tracey, Kevin J.; Blom, Anna M.; Andersson, Ulf

    2016-01-01

    Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP‐induced acute liver injury (APAP‐ALI) and justifies development of anti‐inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N‐acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP‐ALI. The anti‐HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti‐HMGB1 polyclonal antibody treatment improves survival in a model of APAP‐ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti‐HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP‐ALI. The mouse anti‐HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody‐Fc backbones. Effector function‐deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP‐ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP‐induced serum elevations of alanine aminotransferase and microRNA‐122 and completely abrogated markers of APAP‐induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C‐X‐C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. Conclusion: This is the first report describing the generation of a partly humanized HMGB1‐neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP‐ALI. The therapeutic effect was mediated by HMGB1 neutralization and

  10. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  11. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    PubMed Central

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  12. Expression, purification and characterization of the recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO), an active immunotherapeutic vaccine component.

    PubMed

    Xu, Bingze; Lundgren, Mats; Magnusson, Ann-Christine; Fuentes, Alexis

    2010-11-01

    The active vaccine component recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO) has been expressed in CHO-K1 cells. It contains two identical polypeptide chains with 338 amino acid residues in each chain connected by two disulfide bridges. The cell lines were adapted to suspension culture in a serum-free medium. An expression level of 60 mg/L was obtained after 8 days in a shaking flask at a temperature of 31.5 degrees C. The OSO protein has been purified to homogeneity by a combination of three chromatographic steps. Virus inactivation and reduction by solvent detergent treatment and nano-filtration were included in the process. The residual host cell protein content was less than 50 ng/mg OSO as analyzed by ELISA. Purity was analyzed by SDS-PAGE under reducing and non-reducing conditions and was estimated by densitometry to be above 99.0%. The dimer content was less than 0.1% as estimated by analytical size exclusion chromatography. The molecular mass, as estimated by SDS-PAGE, is 90 kDa. A value of around 74 kDa was calculated from its amino acid composition. This indicates that the protein is heavily glycosylated containing around 18% carbohydrate. Isoelectric focusing in polyacrylamide gel disclosed a ladder type band pattern with pI values in the pH-range 7.0-8.3, indicating a variation in the sialic acid content. The OSO protein is not stable at temperatures above 40 degrees C and at pH values below 4 indicating that virus inactivation by incubating the protein solution at higher temperature or at lower pH is not possible. Copyright 2010 Elsevier Inc. All rights reserved.

  13. Structural defect linked to nonrandom mutations in the matrix gene of biken strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes.

    PubMed Central

    Ayata, M; Hirano, A; Wong, T C

    1989-01-01

    Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 Mr protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations in both the amino and carboxy regions. A surprising number of mutations in both the coding and noncoding regions of the Biken M gene were identical to those in an independently isolated SSPE virus strain with a similar defect. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general. Images PMID:2915379

  14. Immunization of N terminus of enterovirus 71 VP4 elicits cross-protective antibody responses

    PubMed Central

    2013-01-01

    Background Enterovirus 71 (EV71) is major cause of hand, foot and mouth disease. Large epidemics of EV71 infection have been recently reported in the Asian-Pacific region. Currently, no vaccine is available to prevent EV71 infection. Results The peptide (VP4N20) consisting of the first 20 amino acids at the N-terminal of VP4 of EV71 genotype C4 were fused to hepatitis B core (HBcAg) protein. Expression of fusion proteins in E. coli resulted in the formation of chimeric virus-like particles (VLPs). Mice immunized with the chimeric VLPs elicited anti-VP4N20 antibody response. In vitro microneutralization experiments showed that anti-chimeric VLPs sera were able to neutralize not only EV71 of genotype C4 but also EV71 of genotype A. Neonatal mice model confirmed the neutralizing ability of anti-chimeric VLPs sera. Eiptope mapping led to the identification of a “core sequence” responsible for antibody recognition within the peptide. Conclusions Immunization of chimeric VLPs is able to elicit antibodies displaying a broad neutralizing activity against different genotypes of EV71 in vitro. The “core sequence” of EV71-VP4 is highly conserved across EV71 genotypes. The chimeric VLPs have a great potential to be a novel vaccine candidate with a broad cross-protection against different EV71 genotypes. PMID:24320792

  15. A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

    PubMed

    Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

    2013-08-20

    Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior

  16. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. © The Author(s).

  17. Expression of antibodies using single open reading frame (sORF) vector design

    PubMed Central

    Gion, Wendy R.; Davis-Taber, Rachel A.; Regier, Dean A.; Fung, Emma; Medina, Limary; Santora, Ling C.; Bose, Sahana; Ivanov, Alexander V.; Perilli-Palmer, Barbara A.; Chumsae, Chris M.; Matuck, Joseph G.; Kunes, Yune Z.; Carson, Gerald R.

    2013-01-01

    Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chains are included within a single open reading frame (sORF) separated by an in-frame intein gene. A single mRNA and subsequent polypeptide are produced upon transient and stable transfection into HEK293 and CHO cells, respectively. Heavy and light chains are separated by the autocatalytic action of the intein and antibody processing proceeds to produce active, secreted antibody. Here, we report advances in sORF technology toward establishment of a viable manufacturing platform for therapeutic antibodies in CHO cells. Increasing expression levels and improving antibody processing by intein and signal peptide selection are discussed. PMID:23774760

  18. Antibodies against HLA-DP recognize broadly expressed epitopes.

    PubMed

    Simmons, Daimon P; Kafetzi, Maria L; Wood, Isabelle; Macaskill, Peter C; Milford, Edgar L; Guleria, Indira

    2016-12-01

    HLA matching and avoidance of pre-transplant donor-specific antibodies are important in selection of donors for solid organ transplant. Solid phase testing with single antigen beads allows resolution of antibody reactivity to the level of the allele. Single antigen bead testing results at a large transplant center were reviewed to identify selective reactivity patterns of anti-HLA antibodies. Many HLA-DP antibodies were identified in the context of other HLA antibodies, but some sera had antibodies against only HLA-DP. B cell flow crossmatch testing was positive for 2 out of 9 sera with HLA-DP antibodies. Many patterns of reactivity corresponded to epitopes in hypervariable regions C and F of DPB1, but some matched epitopes in other regions or DPA1. Through analysis of single antigen bead testing from a large number of patients, we report that anti-HLA-DP antibodies predominantly recognize broadly cross-reactive epitopes. The United Network for Organ Sharing has mandated HLA-DP typing on all deceased kidney donors, and HLA-DP epitopes should be considered as the major antigens for avoidance of pre-transplant donor-specific antibodies.

  19. An improved bioluminescence-based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor.

    PubMed

    Fukutani, Yosuke; Ishii, Jun; Noguchi, Keiichi; Kondo, Akihiko; Yohda, Masafumi

    2012-12-01

    The goal of this work was to improve the bioluminescence-based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast-expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G-protein α-subunit (Gpa1p) with the olfactory-specific Gα(olf), the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence-based odor-sensing system using OR-expressing yeast.

  20. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

    PubMed

    Zhao, Bingxin; Pan, Xiaoxia; Teng, Yumei; Xia, Wenyue; Wang, Jing; Wen, Yuling; Chen, Yuanding

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  1. A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

    PubMed Central

    2013-01-01

    Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome

  2. Antigenicity of a Bacterially Expressed Triple Chimeric Antigen of Plasmodium falciparum AARP, MSP-311 and MSP-119: PfAMSP-Fu35

    PubMed Central

    Pandey, Alok Kumar; Chauhan, Virander S.

    2016-01-01

    Development of fusion chimeras as potential vaccine candidates is considered as an attractive strategy to generate effective immune responses to more than one antigen using a single construct. Here, we described the design, production, purification and antigenicity of a fusion chimera (PfAMSP-Fu35), comprised of immunologically relevant regions of three vaccine target malaria antigens, PfAARP, PfMSP-3 and PfMSP-1. The recombinant PfAMSP-Fu35 is expressed as a soluble protein and purified to homogeneity with ease at a yield of ~ 7 mg L-1. Conformational integrity of the C-terminal fragment of PfMSP-1, PfMSP-119 was retained in the fusion chimera as shown by ELISA with conformation sensitive monoclonal antibodies. High titre antibodies were raised to the fusion protein and to all the three individual components in mice and rabbits upon immunization with fusion chimera in two different adjuvant formulations. The sera against PfAMSP-Fu35 recognized native parasite proteins corresponding to the three components of the fusion chimera. As shown by invasion inhibition assay and antibody mediated cellular inhibition assay, antibodies purified from the PfAMSP-Fu35 immunized serum successfully and efficiently inhibited parasite invasion in P. falciparum 3D7 in vitro both directly and in monocyte dependent manner. However, the invasion inhibitory activity of anti-AMSP-Fu35 antibody is not significantly enhanced as expected as compared to a previously described two component fusion chimera, MSP-Fu24. Therefore, it may not be of much merit to consider AMSP-Fu35 as a vaccine candidate for preclinical development. PMID:27798691

  3. Expression of the C-terminal domain of human apolipoprotein A-I using a chimeric apolipoprotein.

    PubMed

    Sallee, Daniel E; Horn, James V C; Fuentes, Lukas A; Weers, Paul M M

    2017-09-01

    Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179-243 of apoA-I, with a unique methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification. The chimera was expressed in E. coli, purified by Ni-affinity chromatography, and cleaved by cyanogen bromide. SDS-PAGE revealed the presence of three proteins with masses of 7 kDa (CT-apoA-I), 18 kDa (apoLp-III), and a minor 26 kDa band of uncleaved chimera. The digest was reloaded on the Ni-affinity column to bind apoLp-III and uncleaved chimera, while CT-apoA-I was washed from the column and collected. Alternatively, CT-apoA-I was isolated from the digest by reversed-phase HPLC. CT-apoA-I was α-helical, highly effective in solubilizing phospholipid vesicles and disaggregating LPS micelles. However, CT-apoA-I was less active compared to full-length apoA-I in protecting lipolyzed low density lipoproteins from aggregating, and disrupting phosphatidylglycerol bilayer vesicles. Thus the novel expression system produced mg quantities of functional CT-apoA-I, facilitating structural and functional studies of this critical domain of apoA-I. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Expression of chimeric P450 genes encoding flavonoid-3', 5'-hydroxylase in transgenic tobacco and petunia plants(1).

    PubMed

    Shimada, Y; Nakano-Shimada, R; Ohbayashi, M; Okinaka, Y; Kiyokawa, S; Kikuchi, Y

    1999-11-19

    Flavonoid-3',5'-hydroxylase (F3'5'H), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3', 5'-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3'5'H. To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme. Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3',5'-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3',5'-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3'5'H activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3'5'H activity between tobacco and petunia is discussed.

  5. Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer.

    PubMed

    Feng, Kaichao; Guo, Yelei; Dai, Hanren; Wang, Yao; Li, Xiang; Jia, Hejin; Han, Weidong

    2016-05-01

    The successes achieved by chimeric antigen receptor-modified T (CAR-T) cells in hematological malignancies raised the possibility of their use in non-small lung cancer (NSCLC). In this phase I clinical study (NCT01869166), patients with epidermal growth factor receptor (EGFR)-positive (>50% expression), relapsed/refractory NSCLC received escalating doses of EGFR-targeted CAR-T cell infusions. The EGFR-targeted CAR-T cells were generated from peripheral blood after a 10 to 13-day in vitro expansion. Serum cytokines in peripheral blood and copy numbers of CAR-EGFR transgene in peripheral blood and in tissue biopsy were monitored periodically. Clinical responses were evaluated with RECIST1.1 and immune- related response criteria, and adverse events were graded with CTCAE 4.0. The EGFR-targeted CAR-T cell infusions were well-tolerated without severe toxicity. Of 11 evaluable patients, two patients obtained partial response and five had stable disease for two to eight months. The median dose of transfused CAR(+) T cells was 0.97×10(7) cells kg(-1) (interquartile range (IQR), 0.45 to 1.09×10(7) cells kg(-1)). Pathological eradication of EGFR positive tumor cells after EGFR-targeted CAR-T cell treatment can be observed in tumor biopsies, along with the CAR-EGFR gene detected in tumor-infiltrating T cells in all four biopsied patients. The EGFR-targeted CAR-T cell therapy is safe and feasible for EGFR-positive advanced relapsed/refractory NSCLC.

  6. Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors.

    PubMed

    Ren, Fangli; Wang, Ruoke; Zhang, Yanquan; Liu, Chunxiao; Wang, Yinyin; Hu, Jim; Zhang, Linqi; Chang, Zhijie

    2014-12-01

    CREPT (cell-cycle related and expression-elevated protein in tumor), a novel gene also called RPRD1B and C20ORF77, was recently identified to promote tumorigenesis through up-regulation of the expression of genes related to cell cycle. The previous study demonstrated that CREPT is highly expressed in a variety of tumors and enhances the expression of Cyclin D1 by promoting the formation of a chromatin loop. To study the correlation of CREPT expression with clinical factors in different tumors, we generated a monoclonal antibody (3E10) using purified recombinant human GST-CREPT protein as an antigen. In this study, we characterized the specificity of the monoclonal antibody and cloned the gene encoding the antibody for preparation of industrial production. Our results showed that the monoclonal antibody 3E10 was sensitive and specific to recognize human endogenous CREPT protein. We have mapped the epitope of the antibody and cloned the variable region sequence of the gene encoding the antibody. We confirmed that the cloned gene produced an equivalent antibody as that produced by the original hybridoma. This study provided a basis for large-scale production of the CREPT antibody, which will be useful for the study of the role of CREPT in different tumors.

  7. Characterization of a Monoclonal Antibody Against CREPT, a Novel Protein Highly Expressed in Tumors

    PubMed Central

    Ren, Fangli; Wang, Ruoke; Zhang, Yanquan; Liu, Chunxiao; Wang, Yinyin; Hu, Jim; Zhang, Linqi

    2014-01-01

    CREPT (cell-cycle related and expression-elevated protein in tumor), a novel gene also called RPRD1B and C20ORF77, was recently identified to promote tumorigenesis through up-regulation of the expression of genes related to cell cycle. The previous study demonstrated that CREPT is highly expressed in a variety of tumors and enhances the expression of Cyclin D1 by promoting the formation of a chromatin loop. To study the correlation of CREPT expression with clinical factors in different tumors, we generated a monoclonal antibody (3E10) using purified recombinant human GST-CREPT protein as an antigen. In this study, we characterized the specificity of the monoclonal antibody and cloned the gene encoding the antibody for preparation of industrial production. Our results showed that the monoclonal antibody 3E10 was sensitive and specific to recognize human endogenous CREPT protein. We have mapped the epitope of the antibody and cloned the variable region sequence of the gene encoding the antibody. We confirmed that the cloned gene produced an equivalent antibody as that produced by the original hybridoma. This study provided a basis for large-scale production of the CREPT antibody, which will be useful for the study of the role of CREPT in different tumors. PMID:25545209

  8. Expression of a functional single-chain antibody via Corynebacterium pseudodiphtheriticum.

    PubMed

    Sundaram, R K; Hurwitz, I; Matthews, S; Hoy, E; Kurapati, S; Crawford, C; Sundaram, P; Durvasula, R V

    2008-07-01

    Antibody-based therapeutics are effective against conditions ranging from acute infections to malignancy. They may prove crucial in combating bioterrorism and responding to drug-resistant and emerging pathogens. At present the cost of producing therapeutic monoclonal antibodies is between $1,000 to $6,000 per gram. The need to administer antibodies parenterally at frequent intervals further drives the cost of this treatment. Here we present an antibody delivery system, termed paratransgenesis, with the potential to overcome these limitations. The paratransgenic approach involves genetically transforming a commensal or symbiont bacterium to express foreign molecules that target pathogens. We describe transformation of Corynebacterium pseudodiptheriticum, a commensal bacterium found in the human respiratory tract, to express a murine single-chain antibody binding progesterone. The antibody was functional and bound specifically to progesterone in a concentration-dependent manner. This marker antibody system is the precursor to development of expression systems producing recombinant humanized single-chain antibodies. Studies are in progress evaluating fitness, transgene stablility, and pathogenecity of the genetically engineered C. pseudodiptheriticum. We anticipate developing a repertoire of expressed molecules targeting infectious agents and surface epitopes of pulmonary mass lesions. If expression systems for anti-pathogen molecules in C. pseudodiptheriticum and other respiratory commensal bacteria can be optimized, these bacteria have the potential for a range of therapeutic and prophylactic applications.

  9. Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

    PubMed

    Kim, Se Mi; Kim, Young-Il; Park, Su-Jin; Kim, Eun-Ha; Kwon, Hyeok-Il; Si, Young-Jae; Lee, In-Won; Song, Min-Suk; Choi, Young Ki

    2017-03-15

    In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian

  10. Chimeric Pestivirus Experimental Vaccines.

    PubMed

    Reimann, Ilona; Blome, Sandra; Beer, Martin

    2016-01-01

    Chimeric pestiviruses have shown great potential as marker vaccine candidates against pestiviral infections. Exemplarily, we describe here the construction and testing of the most promising classical swine fever vaccine candidate "CP7_E2alf" in detail. The description is focused on classical cloning technologies in combination with reverse genetics.

  11. Role of plant expression systems in antibody production for passive immunization.

    PubMed

    Virdi, Vikram; Depicker, Ann

    2013-01-01

    Passive immunization is a method to achieve immediate protection against infectious agents by administering pathogen-specific antibodies. It has proven to be lifesaving for many acute infections, and it is now also used for cancer treatment. Passive immunization therapies, however, are extremely expensive because they require large amounts of specific antibodies that are produced predominantly in mammalian expression systems. The cost for manufacturing plant-made antibodies is estimated to be comparatively low since plant production systems require relatively less capital investments. In addition, they are not prone to mammalian pathogens, which also eases downstream processing along with making it a safe expression system. Moreover, some of the recent developments in transient expression have enabled rapid, cGMP (current Good Manufacturing Practices) compliant manufacturing of antibodies. Whether lower production costs will be reflected in a lower market price for purified antibodies will be known when more plant-produced antibodies come to the market. Promisingly, the current molecular techniques in the field of in planta expression have enabled high-level production of a variety of antibodies in different plant organs, like roots/tubers/fruits, leaves and seeds, of a variety of plants, like potato, tobacco, maize, rice, tomato and pea, providing a very wide range of possible plant-based passive immunization therapies. For instance, the production of antibodies in edible tissues would allow for a unique, convenient, needle-less, oral passive immunization at the gastric mucosal surface. The technological advances, together with the innate capacity of plant tissues to assemble complex antibodies, will enable carving a niche in the antibody market. This non-exhaustive review aims to shed light on the role of plants as a flexible expression system for passive immunotherapy, which we envisage to progress alongside the conventional production platforms to manufacture

  12. Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II.

    PubMed

    Ferrara, Claudia; Brünker, Peter; Suter, Tobias; Moser, Samuel; Püntener, Ursula; Umaña, Pablo

    2006-04-05

    The effector functions elicited by IgG antibodies strongly depend on the carbohydrate moiety linked to the Fc region of the protein. Therefore several approaches have been developed to rationally manipulate these glycans and improve the biological functions of the antibody. Overexpression of recombinant beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in production cell lines leads to antibodies enriched in bisected oligosaccharides. Moreover, GnT-III overexpression leads to increases in non-fucosylated and hybrid oligosaccharides. Such antibody glycovariants have increased antibody-dependent cellular cytotoxicity (ADCC). To explore a further variable besides overexpression of GnT-III, we exchanged the localization domain of GnT-III with that of other Golgi-resident enzymes. Our results indicate that chimeric GnT-III can compete even more efficiently against the endogenous core alpha1,6-fucosyltransferase (alpha1,6-FucT) and Golgi alpha-mannosidase II (ManII) leading to higher proportions of bisected non-fucosylated hybrid glycans ("Glyco-1" antibody). The co-expression of GnT-III and ManII led to a similar degree of non-fucosylation as that obtained for Glyco-1, but the majority of the oligosaccharides linked to this antibody ("Glyco-2") are of the complex type. These glycovariants feature strongly increased ADCC activity compared to the unmodified antibody, while Glyco-1 (hybrid-rich) features reduced complement-dependent cytotoxicity (CDC) compared to Glyco-2 or unmodified antibody. We show that apart from GnT-III overexpression, engineering of GnT-III localization is a versatile tool to modulate the biological activities of antibodies relevant for their therapeutic application. (c) 2006 Wiley Periodicals, Inc.

  13. Antibodies Expressed by Intratumoral B Cells as the Basis for a Diagnostic Test for Lung Cancer

    DTIC Science & Technology

    2015-06-01

    expression, purification, and characterization of monoclonal antibodies (mAbs) cloned from intratumoral B lymphocytes (ITLs). While this work was...successful in identifying a mAb with preferential binding to tropomyosin 4, all cloned mAbs exhibited polyreactivity. This suggested that the isolated ITLs...our most recent efforts have focused on methods to permit the cloning and expression of recombinant antibodies specifically from oligoclonal ITLs. We

  14. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

    PubMed Central

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-01-01

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

  15. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

    PubMed

    Mohiuddin, Muhammad M; Singh, Avneesh K; Corcoran, Philip C; Thomas, Marvin L; Clark, Tannia; Lewis, Billeta G; Hoyt, Robert F; Eckhaus, Michael; Pierson, Richard N; Belli, Aaron J; Wolf, Eckhard; Klymiuk, Nikolai; Phelps, Carol; Reimann, Keith A; Ayares, David; Horvath, Keith A

    2016-04-05

    Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

  16. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft

    PubMed Central

    Mohiuddin, Muhammad M.; Singh, Avneesh K.; Corcoran, Philip C.; Thomas III, Marvin L.; Clark, Tannia; Lewis, Billeta G.; Hoyt, Robert F.; Eckhaus, Michael; Pierson III, Richard N.; Belli, Aaron J.; Wolf, Eckhard; Klymiuk, Nikolai; Phelps, Carol; Reimann, Keith A.; Ayares, David; Horvath, Keith A.

    2016-01-01

    Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days. PMID:27045379

  17. Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

    PubMed Central

    Zimmermann, Jana; Saalbach, Isolde; Jahn, Doreen; Giersberg, Martin; Haehnel, Sigrun; Wedel, Julia; Macek, Jeanette; Zoufal, Karen; Glünder, Gerhard; Falkenburg, Dieter; Kipriyanov, Sergey M

    2009-01-01

    Background Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Results Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. Conclusion The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market. PMID:19747368

  18. Expression of crossreactive idiotypes by human antibodies specific for the capsular polysaccharide of Hemophilus influenzae B.

    PubMed Central

    Lucas, A H

    1988-01-01

    Human antibodies specific, for polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hemophilus influenzae b, were studied using idiotypic analysis. Antisera were prepared against purified F(ab')2 anti-PRP from two unrelated adults, H.H. and P.T. After repeated absorption with IgG myeloma proteins and with PRP-absorbed normal human Ig and donor Ig, anti-idiotypic (anti-Id) sera were obtained that specifically reacted with anti-PRP antibodies. Anti-IdHH and anti-IdPT reciprocally crossreacted with H.H. and P.T. anti-PRP antibodies and F(ab')2 fragments, and also reacted with the serum anti-PRP antibodies from three additional adults unrelated to P.T. and H.H. Both anti-Id sera partially inhibited anti-PRP paratopes but not anti-tetanus toxoid paratopes. PRP did not inhibit anti-Id recognition of shared or crossreactive idiotypic (CRI) determinants. Naturally occurring and PRP immunization-induced anti-PRP antibodies expressed CRI. While CRI titer increased after immunization, the increase was usually less than the rise in total anti-PRP antibody. Quantitative differences in CRI expression were also apparent between natural and immunization-induced H.H. and P.T. anti-PRP antibodies as shown by their differential inhibitability by anti-Id. Our data demonstrate that anti-PRP antibodies from five unrelated adults express CRI determinants that are probably distant from the PRP combining site. Naturally occurring and immunization-induced anti-PRP antibodies share CRI and therefore appear to be clonally related, although immunization apparently induces the expression CRI-negative antibodies as well. These results, taken with previous studies showing restricted and identical anti-PRP isoelectric focusing spectrotypes in unrelated adults, suggest that some PRP-specific V domains are structurally conserved and probably germ-line encoded. PMID:3257499

  19. Immunoreactivity evaluation of a new recombinant chimeric protein against Brucella in the murine model

    PubMed Central

    Abdollahi, Abbas; Mansouri, Shahla; Amani, Jafar; Fasihi-Ramandi, Mahdi; Moradi, Mohammad

    2016-01-01

    Background and Objectives: Brucellosis is an important health problem in developing countries and no vaccine is available for the prevention of infection in humans. Because of clinically infectious diseases and their economic consequences in human and animals, designing a proper vaccine against Brucella is desirable. In this study, we evaluated the immune responses induced by a designed recombinant chimera protein in murine model. Materials and Methods: Three immunodominant antigens of Brucella have been characterized as potential immunogenic and protective antigens including: trigger factor (TF), Omp31 and Bp26 were fused together by EAAAK linkers to produce a chimera (structure were designed in silico), which was synthesized, cloned, and expressed in E. coli BL21 (DE3). The purification of recombinant protein was performed using Ni-NTA agarose. SDS-PAGE and anti-His antibody was used for confirmation purified protein (Western blot). BALB/c immunization was performed by purified protein and adjuvant, and sera antibody levels were measured by ELISA. otted. Results: SDS-PAGE and Western blotting results indicated the similarity of in silico designing and in vitro experiments. ELISA result proved that the immunized sera of mice contain high levels of antibodies (IgG) against recombinant chimeric protein. Conclusion: The recombinant chimeric protein could be a potential antigen candidate for the development of a subunit vaccine against Brucella. PMID:27928487

  20. Envelope-chimeric Entry-targeted Measles Virus Escapes Neutralization and Achieves Oncolysis

    PubMed Central

    Miest, Tanner S; Yaiw, Koon-Chu; Frenzke, Marie; Lampe, Johanna; Hudacek, Andrew W; Springfeld, Christoph; von Messling, Veronika; Ungerechts, Guy; Cattaneo, Roberto

    2011-01-01

    Measles virus (MV) is a promising vector for cancer therapy and multivalent vaccination, but high prevalence of pre-existing neutralizing antibodies may reduce therapeutic efficacy, particularly following systemic administration. MV has only one serotype, but here we show that its envelope glycoproteins can be exchanged with those of the closely related canine distemper virus (CDV), generating a chimeric virus capable of escaping neutralization. To target its entry, we displayed on the CDV attachment protein a single-chain antibody specific for a designated receptor. To enhance oncolytic efficacy we armed the virus with a prodrug convertase gene capable of locally activating chemotherapeutic prodrugs. The new virus achieved high titers, was genetically stable, and was resistant to neutralization by sera from both MV-immunized mice and MV-immune humans. The new virus targeted syngeneic murine tumor cells expressing the designated receptor implanted in immunocompetent mice, and synergized with a chemotherapeutic prodrug in a model of oncolysis. Importantly, the chimeric MV remained oncolytic when administered systemically even in the presence of anti-MV antibodies capable of abrogating the therapeutic efficacy of the parental, nonshielded MV. This work shows that targeting, arming, and shielding can be combined to generate a tumor-specific, neutralization-resistant virus that can synergize with chemotherapeutics. PMID:21610701

  1. Chimeric Antigen Receptor (CAR)-Engineered Lymphocytes for Cancer Therapy

    PubMed Central

    Ramos, Carlos A.; Dotti, Gianpietro

    2011-01-01

    Introduction Chimeric antigen receptors (CARs) usually combine the antigen binding site of a monoclonal antibody with the signal activating machinery of a T cell, freeing antigen recognition from major histocompatibility complex restriction and thus breaking one of the barriers to more widespread application of cellular therapy. Similar to treatment strategies employing monoclonal antibodies, T cells expressing CARs are highly targeted, but additionally offer the potential benefits of active trafficking to tumor sites, in vivo expansion and long term persistence. Furthermore, gene transfer allows the introduction of countermeasures to tumor immune evasion and of safety mechanisms. Areas covered The authors review the basic structure of so-called first and later generation CARs and their potential advantages over other immune therapy systems. It is described how these molecules can be grafted into immune cells (including retroviral and non-retroviral transduction methods) and strategies to improve the in vivo persistence and function of immune cells expressing CARs are discussed. Examples of tumor associated antigens that have been targeted in preclinical models are presented and clinical experience with these modified cells is summarized. Finally, a discussion on safety issues surrounding CAR gene transfer into T cells and potential solutions to them, are presented. Expert opinion Because of recent advances in immunology, genetics and cell processing, CAR-modified T cells will likely play an increasing role in the cellular therapy of cancer, chronic infections and autoimmune disorders. PMID:21463133

  2. Effect of antigen turnover rate and expression level on antibody penetration into tumor spheroids.

    PubMed

    Ackerman, Margaret E; Pawlowski, David; Wittrup, K Dane

    2008-07-01

    Poor tissue penetration is a significant obstacle to the development of successful antibody drugs for immunotherapy of solid tumors, and diverse alterations to the properties of antibody drugs have been made to improve penetration and homogeneity of exposure. However, in addition to properties of the antibody drug, mathematical models of antibody transport predict that the antigen expression level and turnover rate significantly influence penetration. As intrinsic antigen properties are likely to be difficult to modify, they may set inherent limits to penetration. Accordingly, in this study, we assess their contribution by evaluating the distance to which antibodies penetrate spheroids when these antigen properties are systematically varied. Additionally, the penetration profiles of antibodies against carcinoembryonic antigen and A33, two targets of clinical interest, are compared. The results agree well with the quantitative predictions of the model and show that localizing antibody to distal regions of tumors is best achieved by selecting slowly internalized targets that are not expressed above the level necessary for recruiting a toxic dose of therapeutic. Each antibody-bound antigen molecule that is turned over or present in excess incurs a real cost in terms of penetration depth-a limiting factor in the development of effective therapies for treating solid tumors.

  3. [Expression and purification of GST-CML28 fusion protein and preparation of its polyclonal antibody].

    PubMed

    Mao, Xia; Zhang, Bing; Bai, Xue-Ling; Liu, Long-Long; Zhang, Dong-Hua

    2012-12-01

    This study was aimed to investigate the expression of GST-CML28 in Escherichia Coli and to prepare its antibody. The constructed recombinant expression vectors CML28-pGEX-3X were transformed into Escherichia Coli BL21 under IPTG induction. The protein was abstracted from the transformers, and purified by a GSTrap FF column. The rabbits were immunized by the purified fusion protein to produce serum with anti-CML28 antibody. The serum was purified by chromatographic column stuffed with glutathione Sephamse 4B to get the antibody. The specific antibody against CML28 was further identified by ELISA, Western blot, immunohistochemistry and quantum dot luminescence. The results indicated that GST-CML28 fusion protein was expressed in Escherichia coli and its specific polyclonal antibody was obtained. It is concluded that the anti-CML28 polyclonal antibodies with high titer and specificity are successfully prepared. These antibodies provide an useful experimental tool to profoundly research the physiological significance and biological function of the CML28 gene.

  4. The full-length clone of cucumber green mottle mosaic virus and its application as an expression system for Hepatitis B surface antigen.

    PubMed

    Ooi, Aikseng; Tan, Sianghee; Mohamed, Rosmawati; Rahman, Noorsaadah Abdul; Othman, Rofina Yasmin

    2006-02-24

    A cucumber green mosaic mottle virus (CGMMV) full-length clone was developed for the expression of Hepatitis B surface antigen (HBsAg). The expression of the surface displayed HBsAg by the chimeric virus was confirmed through a double antibody sandwich ELISA. Assessment of the coat protein composition of the chimeric virus particles by SDS-PAGE analysis showed that 50% of the coat proteins were fused to the HBsAg. Biological activity of the expressed HBsAg was assessed through the stimulation of in vitro antibody production by cultured peripheral blood mononuclear cells (PBMC). PBMC that were cultured in the presence of the chimeric virus showed up to an approximately three-fold increase in the level of anti HBsAg immunoglobulin thus suggesting the possible use of this new chimeric virus as an effective Hepatitis B vaccine.

  5. Chimeric plantibody passively protects mice against aerosolized ricin challenge.

    PubMed

    Sully, Erin K; Whaley, Kevin J; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do H; Pauly, Michael H; Velasco, Jesus; Hiatt, Ernie; Morton, Josh; Swope, Kelsi; Roy, Chad J; Zeitlin, Larry; Mantis, Nicholas J

    2014-05-01

    Recent incidents in the United States and abroad have heightened concerns about the use of ricin toxin as a bioterrorism agent. In this study, we produced, using a robust plant-based platform, four chimeric toxin-neutralizing monoclonal antibodies that were then evaluated for the ability to passively protect mice from a lethal-dose ricin challenge. The most effective antibody, c-PB10, was further evaluated in mice as a therapeutic following ricin exposure by injection and inhalation.

  6. Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

    PubMed

    Choudhary, Nandlal; Roy, Avijit; Guillermo, Leon M; Picton, D D; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2013-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.

  7. AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization

    PubMed Central

    Rada, Cristina; Jarvis, John M.; Milstein, César

    2002-01-01

    Somatic hypermutation generates variants of antibody genes and underpins the affinity maturation of antibodies. It is restricted to the V-gene segments, and although it decays exponentially toward the 3′end, it includes recognizable hot spots. Although the detailed mechanism of hypermutation remains elusive, the process may take place in two separate stages, preferentially targeting G/Cs in the first and A/Ts in the second stage. It seems that MSH2 is involved in the second stage, and that activation induced deaminase (AID) is implicated in the control of hypermutation. The constitutively hypermutating cell line Ramos expresses AID, and we have prepared transfectants that express a chimeric AID-green fluorescent protein. The fluorescence is strongly detected in the cytoplasm but not in the nucleus. Yet, the chimeric protein increases the hypermutation rate either directly or, more likely, indirectly, by favoring the transport of AID into the nucleus. Thus, in Ramos, AID seems to be rate limiting. Unexpectedly, the proportion of deletions also is increased. The increase in mutation rate detected by a fast cytofluorimetric method based on the accumulation of sIgM-loss mutants correlates with the increase measured by mutations defined by sequence analysis. The higher mutation rate is largely explained by the higher proportion of mutated clones, indicating that AID controls the number of cells that undergo hypermutation but not the number of mutations that are incorporated in each mutation round. PMID:12011459

  8. Engineering Chimeric Antigen Receptors

    PubMed Central

    Kulemzin, S. V.; Kuznetsova, V. V.; Mamonkin, M.; Taranin, A. V.; Gorchakov, A. A.

    2017-01-01

    Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering. PMID:28461969

  9. Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation.

    PubMed

    Kettleborough, C A; Saldanha, J; Heath, V J; Morrison, C J; Bendig, M M

    1991-10-01

    A mouse monoclonal antibody (mAb 425) with therapeutic potential was 'humanized' in two ways. Firstly the mouse variable regions from mAb 425 were spliced onto human constant regions to create a chimeric 425 antibody. Secondly, the mouse complementarity-determining regions (CDRs) from mAb 425 were grafted into human variable regions, which were then joined to human constant regions, to create a reshaped human 425 antibody. Using a molecular model of the mouse mAb 425 variable regions, framework residues (FRs) that might be critical for antigen-binding were identified. To test the importance of these residues, nine versions of the reshaped human 425 heavy chain variable (VH) regions and two versions of the reshaped human 425 light chain variable (VL) regions were designed and constructed. The recombinant DNAs coding for the chimeric and reshaped human light and heavy chains were co-expressed transiently in COS cells. In antigen-binding assays and competition-binding assays, the reshaped human antibodies were compared with mouse 425 antibody and to chimeric 425 antibody. The different versions of 425-reshaped human antibody showed a wide range of avidities for antigen, indicating that substitutions at certain positions in the human FRs significantly influenced binding to antigen. Why certain individual FR residues influence antigen-binding is discussed. One version of reshaped human 425 antibody bound to antigen with an avidity approaching that of the mouse 425 antibody.

  10. Chimeric antigen receptor-modified T cells strike back

    PubMed Central

    Frigault, Matthew J.

    2016-01-01

    Chimeric antigen receptors (CARs) are engineered molecules designed to endow a polyclonal T-cell population with the ability to recognize tumor-associated surface antigens. In their simplest form, CARs comprise a targeting moiety in the form of a single-chain variable fragment from an antibody connected to various intracellular signaling domains allowing for T-cell activation. This powerful approach combines the specificity of an antibody with the cytotoxic ability of a T cell. There has been much excitement since early phase trials of CAR-T cells targeting CD19 expressed on B-cell malignancies demonstrated remarkable efficacy in inducing long-term, stable remissions in otherwise relapsed/refractory disease. Despite these successes, we have just begun to understand the intricacies of CAR biology with efforts underway to utilize this platform in the treatment of other, previously refractory malignancies. Challenges currently include identification of viable cancer targets, management strategies for potentially severe and irreversible toxicities and overcoming the immunosuppressive nature of the tumor microenvironment. This review will focus on basic CAR structure and function, previous success and new approaches aimed at the broader application of CAR-T-cell therapy. PMID:27021308

  11. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

    PubMed

    Drake, Pascal M W; Barbi, Tommaso; Drever, Matthew R; van Dolleweerd, Craig J; Porter, Andrew J R; Ma, Julian K-C

    2010-03-01

    We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen-binding regions of the microcystin-specific single-chain antibody, 3A8, with constant regions from the murine IgG1kappa, Guy's 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC-LR. Antibody yields in transgenic plant leaves reached a maximum of 64 microg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 microg/g root dry weight/24 h. Rhizosecreted antibody bound to MC-LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC-LR compared to wild-type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin-LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC-LR.-Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.-C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

  12. Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

    SciTech Connect

    Didier, P.; Weiss, E.; Sibler, A.-P.; Philibert, P.; Martineau, P.; Bigot, J.-Y.; Guidoni, L.

    2008-02-22

    Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

  13. AAV-directed persistent expression of a gene encoding anti-nicotine antibody for smoking cessation.

    PubMed

    Hicks, Martin J; Rosenberg, Jonathan B; De, Bishnu P; Pagovich, Odelya E; Young, Colin N; Qiu, Jian-ping; Kaminsky, Stephen M; Hackett, Neil R; Worgall, Stefan; Janda, Kim D; Davisson, Robin L; Crystal, Ronald G

    2012-06-27

    Current strategies to help tobacco smokers quit have limited success as a result of the addictive properties of the nicotine in cigarette smoke. We hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector expressing high levels of an anti-nicotine antibody would persistently prevent nicotine from reaching its receptors in the brain. To test this hypothesis, we constructed an AAVrh.10 vector that expressed a full-length, high-affinity, anti-nicotine antibody derived from the Fab fragment of the anti-nicotine monoclonal antibody NIC9D9 (AAVantiNic). In mice treated with this vector, blood concentrations of the anti-nicotine antibody were dose-dependent, and the antibody showed high specificity and affinity for nicotine. The antibody shielded the brain from systemically administered nicotine, reducing brain nicotine concentrations to 15% of those in naïve mice. The amount of nicotine sequestered in the serum of vector-treated mice was more than seven times greater than that in untreated mice, with 83% of serum nicotine bound to immunoglobulin G. Treatment with the AAVantiNic vector blocked nicotine-mediated alterations in arterial blood pressure, heart rate, and locomotor activity. In summary, a single administration of a gene transfer vector expressing a high-affinity anti-nicotine monoclonal antibody elicited persistent (18 weeks), high titers of an anti-nicotine antibody that obviated the physiologic effects of nicotine. If this degree of efficacy translates to humans, AAVantiNic could be an effective preventative therapy for nicotine addiction.

  14. Cloning, expression and polyclonal antibody preparation of the asialoglycoprotein receptor of Marmota himalayan.

    PubMed

    Yang, Yan; Huang, Huang; Zhang, Zhenghua; Wang, Baoju; Tian, Yongjun; Lu, Mengji; Yang, Dongliang

    2007-08-01

    The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota Himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

  15. Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers.

    PubMed

    Lace, Michael J; Anson, James R; Klussmann, Jens P; Wang, Dong Hong; Smith, Elaine M; Haugen, Thomas H; Turek, Lubomir P

    2011-02-01

    Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.

  16. Human Papillomavirus Type 16 (HPV-16) Genomes Integrated in Head and Neck Cancers and in HPV-16-Immortalized Human Keratinocyte Clones Express Chimeric Virus-Cell mRNAs Similar to Those Found in Cervical Cancers ▿

    PubMed Central

    Lace, Michael J.; Anson, James R.; Klussmann, Jens P.; Wang, Dong Hong; Smith, Elaine M.; Haugen, Thomas H.; Turek, Lubomir P.

    2011-01-01

    Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture. PMID:21123375

  17. Design and Construction of Chimeric VP8-S2 Antigen for Bovine Rotavirus and Bovine Coronavirus

    PubMed Central

    Nasiri, Khadijeh; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza; Zibaee, Saeed

    2016-01-01

    Purpose: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Methods: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. Results: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni–NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. Conclusion: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure. PMID:27123423

  18. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies.

    PubMed

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Zhang, Kai; Batt, Micheal; Fitchett, Jonathan R; He, Dongmei; Rick, Heather L; Conner, Elaine M; Demarest, Stephen J

    2015-01-01

    Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing CH1 with Cα and CL with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.

  19. Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes

    PubMed Central

    van der Geld, Ymke M; Hellmark, Thomas; Selga, Daina; Heeringa, Peter; Huitema, Minke G; Limburg, Pieter C; Kallenberg, Cees G M

    2007-01-01

    Aim In this study, we employed chimeric human/mouse Proteinase 3 (PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice. Method Rats and mice were immunised with recombinant human PR3 (HPR3), recombinant murine PR3 (mPR3), single chimeric human/mouse PR3 (HHm, HmH, mHH, mmH, mHm, Hmm) or pools of chimeric proteins. Antibodies to mPR3 and HPR3 were measured by ELISA. Antibodies to rat PR3 were determined by indirect immunofluorescence (IIF) on rat white blood cells. Urinalysis was performed by dipstick analysis. Kidney and lung tissue was obtained for pathological examination. Results In mice, immunisation with the chimeric human/mouse PR3 Hmm led to an autoantibody response to mPR3. Rats immunised with the chimeric human/mouse PR3 Hmm, HmH and mmH, or a pool of the chimeric human/mouse PR3 proteins, produced antibodies selectively binding to rat granulocytes as detected by IIF. No gross pathological abnormalities could be detected in kidney or lungs of mice or rats immunised with chimeric human/mouse PR3. Conclusion Immunisation with chimeric human/mouse proteins induces autoantibodies to PR3 in rats and mice. Chimeric proteins can be instrumental in developing experimental models for autoimmune diseases. PMID:17644551

  20. Marker antibody expression stratifies Crohn's disease into immunologically homogeneous subgroups with distinct clinical characteristics

    PubMed Central

    Vasiliauskas, E; Kam, L; Karp, L; Gaiennie, J; Yang, H; Targan, S

    2000-01-01

    BACKGROUND—Perinuclear antineutrophil cytoplasmic antibodies (pANCA) have been detected in a clinically distinct Crohn's disease subpopulation. Antibodies to Saccharomyces cerevisiae (ASCA) have been demonstrated in the majority of patients with Crohn's disease.
AIMS—To examine the relationship between selective marker antibody expression in Crohn's disease and disease onset, location, and clinical behaviour patterns.
METHODS—Sera from 156 consecutive patients with established Crohn's disease were evaluated in a blinded fashion for the presence of ASCA and ANCA. Clinical profiles were generated by investigators blinded to immune marker status.
RESULTS—Using multiple regression analyses, higher ASCA levels were shown to be independently associated with early age of disease onset as well as both fibrostenosing and internal penetrating disease behaviours. Higher ANCA levels were associated with later age of onset and ulcerative colitis-like behaviour. Substratification of the Crohn's disease population using selective ANCA and ASCA expression (high levels of a single marker antibody): (1) distinguished homogeneous subgroups that manifested similar disease location and behaviours; and (2) identified patients with more aggressive small bowel disease.
CONCLUSIONS—The findings suggest that by taking into account the magnitude of the host immune response, Crohn's disease can now be stratified on an immunological basis into more homogeneous clinically distinct subgroups, characterised by greater uniformity among anatomical distribution of disease and disease behaviour.


Keywords: antineutrophil cytoplasmic antibody; anti-Saccharomyces cerevisiae antibody; Crohn's disease; inflammatory bowel disease; ulcerative colitis PMID:10986208

  1. Iron as the Key Modulator of Hepcidin Expression in Erythroid Antibody-Mediated Hypoplasia

    PubMed Central

    Fernandes, J. C.; Garrido, P.; Ribeiro, S.; Rocha-Pereira, P.; Bronze-da-Rocha, E.; Belo, L.; Costa, E.; Reis, F.; Santos-Silva, A.

    2014-01-01

    Erythroid hypoplasia (EH) is a rare complication associated with recombinant human erythropoietin (rHuEPO) therapies, due to development of anti-rHuEPO antibodies; however, the underlying mechanisms remain poorly clarified. Our aim was to manage a rat model of antibody-mediated EH induced by rHuEPO and study the impact on iron metabolism and erythropoiesis. Wistar rats treated during 9 weeks with a high rHuEPO dose (200 IU) developed EH, as shown by anemia, reduced erythroblasts, reticulocytopenia, and plasmatic anti-rHuEPO antibodies. Serum iron was increased and associated with mRNA overexpression of hepatic hepcidin and other iron regulatory mediators and downregulation of matriptase-2; overexpression of divalent metal transporter 1 and ferroportin was observed in duodenum and liver. Decreased EPO expression was observed in kidney and liver, while EPO receptor was overexpressed in liver. Endogenous EPO levels were normal, suggesting that anti-rHuEPO antibodies blunted EPO function. Our results suggest that anti-rHuEPO antibodies inhibit erythropoiesis causing anemia. This leads to a serum iron increase, which seems to stimulate hepcidin expression despite no evidence of inflammation, thus suggesting iron as the key modulator of hepcidin synthesis. These findings might contribute to improving new therapeutic strategies against rHuEPO resistance and/or development of antibody-mediated EH in patients under rHuEPO therapy. PMID:25580431

  2. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis

    PubMed Central

    Dolzhikova, Inna V.; Shcherbinin, Dmitry N.; Zubkova, Olga V.; Ivanova, Tatiana I.; Tukhvatulin, Amir I.; Shmarov, Maxim M.; Logunov, Denis Y.; Naroditsky, Boris S.; Gintsburg, Aleksandr L.

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. PMID:26962869

  3. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

    PubMed

    Burmistrova, Daria A; Tillib, Sergey V; Shcheblyakov, Dmitry V; Dolzhikova, Inna V; Shcherbinin, Dmitry N; Zubkova, Olga V; Ivanova, Tatiana I; Tukhvatulin, Amir I; Shmarov, Maxim M; Logunov, Denis Y; Naroditsky, Boris S; Gintsburg, Aleksandr L

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection.

  4. Lack of Interference with Immunogenicity of a Chimeric Alphavirus Replicon Particle-Based Influenza Vaccine by Preexisting Antivector Immunity

    PubMed Central

    Vajdy, Michael; Lian, Ying; Perri, Silvia; Greer, Catherine E.; Legg, Harold S.; Galli, Grazia; Saletti, Giulietta; Otten, Gillis R.; Rappuoli, Rino; Barnett, Susan W.; Polo, John M.

    2012-01-01

    Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens. PMID:22623651

  5. Preparation of Polyclonal Antibody and Expression Analysis of GR in Tomato

    NASA Astrophysics Data System (ADS)

    Xie, Yuanhong; Zhu, Benzhong; Luo, Yunbo; Chen, Xiangning; Zhang, Hongxing

    The fruit ripening of Green-ripe (Gr) mutant tomato was inhibited dramatically. To determine the expression patterns of Gr in tomato, we first produced the polyclonal antibody of Gr protein. RT-PCR was used to amplify the Gr gene from green ripe tomato fruit. And the PCR product was subcloned into prokaryotic protein expression vectors pET-30a to generate recombinant plasmid. The Gr protein was induced by IPTG in BL21 (DE3) and purified by Ni-NTA agarose column. The anti-Gr serum was produced by immunizing rabbits, and the titer of the anti-Gr serum was above 5000 by ELISA analysis. Purified by the DEAE-52 ion-column, the high purification level of anti-Gr polyclonal antibody was obtained. Furthermore, RT-CPR was used in the RNA level to demonstrate that the expression of Gr gene was specialized in some cultures of tomato. For example, the expressions of Gr were higher in seed, flower and green ripe fruit than others, and the expression level were reduced by exogenous ethylene treatment in the flower and green ripe fruit. Moreover, Polyclonal antibody of Gr was used to investigate the expression pattern of Gr in protein level by the Western blotting. Our results show that the expression level of Gr in protein level was complied with the expressions in RNA. So, we suggested that the regulation of Gr was transcriptional.

  6. Oligomer-targeting with a conformational antibody fragment promotes toxicity in Aβ-expressing flies

    PubMed Central

    2014-01-01

    Introduction The self-assembly of Aβ peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer’s disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments. Results We show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aβ-expressing Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aβ(1–40) and not with those expressing Aβ(1–42) or the arctic variant of Aβ(1–42) This finding is consistent with the binding preference of KW1 for Aβ(1–40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation. Conclusions While our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents. PMID:24725347

  7. Spontaneous reversal of acquired autoimmune dysfibrinogenemia probably due to an antiidiotypic antibody directed to an interspecies cross-reactive idiotype expressed on antifibrinogen antibodies.

    PubMed Central

    Ruiz-Arguelles, A

    1988-01-01

    A young man with a long history of abnormal bleeding was seen in January 1985. Coagulation tests showed dysfibrinogenemia and an antifibrinogen autoantibody was demonstrable in his serum. This antibody, when purified, was capable of inhibiting the polymerization of normal fibrin monomers, apparently through binding to the alpha fibrinogen chain. 6 mo later the patient was asymptomatic, coagulation tests were normal, and the antifibrinogen autoantibody was barely detectable. At this time, affinity-purified autologous and rabbit antifibrinogen antibodies were capable of absorbing an IgG kappa antibody from the patient's serum, which reacted indistinctly with both autologous and xenogeneic antifibrinogen antibodies in enzyme immunoassays. It has been concluded that the patient's dysfibrinogenemia was the result of an antifibrinogen autoantibody, and that later on an anti-idiotype antibody, which binds an interspecies cross-reactive idiotype expressed on anti-human fibrinogen antibodies, inhibited the production of the antifibrinogen autoantibody which led to the remission of the disorder. Images PMID:3262127

  8. [Cloning and expression of VLRB of Lampetra japonica and generation of the corresponding monoclonal antibodies].

    PubMed

    Wu, Fen-Fang; Ma, Ning; Chen, Li-Yong; Su, Peng; Li, Qing-Wei

    2012-04-01

    The agnathans (lampreys and hagfishes) are representatives of the jawless vertebrates. The receptor molecules of adaptive immune system in lampreys are different from the antigen receptors in mammal vertebrates. The unique receptor molecules of lampreys are known as variable lymphocyte receptors (VLR). There are three types of VLRs in lampreys, VLRA, VLRB, and VLRC. Multimeric antigen-specific VLRB antibodies are secreted by VLRB+ lymphocytes and constitute the major components of the humoral arm of the lamprey adaptive immune system. Oligomeric VLRB antibodies are composed of four or five disulfide-linked dimeric subunits, which are similar to IgM antibodies in structure and function. In this study, the conservative c-terminal of Lampetra japonica VLRB was cloned and expressed in BL21 E. coli. The recombinant VLRB protein was purified by Ni2+ affinity chromatography column. After Balb/c mice immunity, cell fusion, the positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA). Finally, the hybridoma cells that produced specific anti-VLRB monoclonal antibodies were obtained. In order to get a large number of antibodies against VLRB, the hybridoma cells were injected into the abdominal cavity of Balb/c mice and the antibodies were purified by protein G sepharose. The results of ELISA indicated that the valence of anti-VLRB antibodies was 1:40000. Western blotting assay showed that the antibodies were able to detect both recombinant VLRB and secreted VLRB in lamprey sera. Flow cytometry analysis also revealed the existence of VLRB on the surface of lymphocytes. In summary, the anti-VLRB monoclonal antibodies provided a major tool for studying lamprey adaptive immune system.

  9. Evaluation of a chimeric multi-epitope-based DNA vaccine against subgroup J avian leukosis virus in chickens.

    PubMed

    Xu, Qingqing; Cui, Ning; Ma, Xingjiang; Wang, Fangkun; Li, Hongmei; Shen, Zhiqiang; Zhao, Xiaomin

    2016-07-19

    The prokaryotic expressed recombinant chimeric multi-epitope protein X (rCMEPX) had been evaluated with good immunogenicity and protective efficacy against subgroup J avian leukosis virus (ALV-J) in our previous study. In the present research, we cloned the chimeric multi-epitope gene X into the eukaryotic expression vector pVAX1 to evaluate its potency as a DNA vaccine. The purified recombinant gp85 protein and rCMEPX were used as positive controls and a DNA prime-protein boost strategy was also studied. Six experimental groups of 7-day-old chickens (20 per group) were immunized intramuscularly three times at 2weeks interval with PBS, gp85, rCMEPX, pVAX1, pVAX-X and pVAX-X+rCMEPX respectively. The antibody titers and cellular immune responses were assayed after immunization. The efficacy of immunoprotection against the challenge of ALV-J NX0101 strain was also examined. The results showed that the DNA vaccine could elicit both neutralizing antibodies and cellular responses. Immune-challenge experiments showed good protection efficacy against ALV-J infection. Particularly, the regimen involving one priming pVAX-X and twice recombinant rCMEPX boosting, induced the highest antibody titers in all immunized groups. Our results suggest that the constructed chimeric multi-epitope DNA has potential for a candidate vaccine against ALV-J when used in proper prime-boost combinations. The data presented here may provide an alternative strategy for vaccine design in chicken ALV-J prevention.

  10. Chimeric mitochondrial peptides from contiguous regular and swinger RNA.

    PubMed

    Seligmann, Hervé

    2016-01-01

    Previous mass spectrometry analyses described human mitochondrial peptides entirely translated from swinger RNAs, RNAs where polymerization systematically exchanged nucleotides. Exchanges follow one among 23 bijective transformation rules, nine symmetric exchanges (X ↔ Y, e.g. A ↔ C) and fourteen asymmetric exchanges (X → Y → Z → X, e.g. A → C → G → A), multiplying by 24 DNA's protein coding potential. Abrupt switches from regular to swinger polymerization produce chimeric RNAs. Here, human mitochondrial proteomic analyses assuming abrupt switches between regular and swinger transcriptions, detect chimeric peptides, encoded by part regular, part swinger RNA. Contiguous regular- and swinger-encoded residues within single peptides are stronger evidence for translation of swinger RNA than previously detected, entirely swinger-encoded peptides: regular parts are positive controls matched with contiguous swinger parts, increasing confidence in results. Chimeric peptides are 200 × rarer than swinger peptides (3/100,000 versus 6/1000). Among 186 peptides with > 8 residues for each regular and swinger parts, regular parts of eleven chimeric peptides correspond to six among the thirteen recognized, mitochondrial protein-coding genes. Chimeric peptides matching partly regular proteins are rarer and less expressed than chimeric peptides matching non-coding sequences, suggesting targeted degradation of misfolded proteins. Present results strengthen hypotheses that the short mitogenome encodes far more proteins than hitherto assumed. Entirely swinger-encoded proteins could exist.

  11. Integrative Expression System for Delivery of Antibody Fragments by Lactobacilli▿ †

    PubMed Central

    Martín, M. Cruz; Pant, Neha; Ladero, Victor; Günaydın, Gökçe; Andersen, Kasper Krogh; Álvarez, Beatriz; Martínez, Noelia; Alvarez, Miguel A.; Hammarström, Lennart; Marcotte, Harold

    2011-01-01

    A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection. PMID:21257814

  12. The level of HER2 expression is a predictor of antibody-HER2 trafficking behavior in cancer cells.

    PubMed

    Ram, Sripad; Kim, Dongyoung; Ober, Raimund J; Ward, E Sally

    2014-01-01

    The receptor tyrosine kinase HER2 is known to play a central role in mitogenic signaling, motivating the development of targeted, HER2-specific therapies. However, despite the longstanding use of antibodies to target HER2, controversies remain concerning antibody/HER2 trafficking behavior in cancer cells. Understanding this behavior has direct relevance to the mechanism of action and effective design of such antibodies. In the current study, we analyzed the intracellular dynamics of trastuzumab, a marketed HER2-targeting antibody, in a panel of breast and prostate cancer cell lines that have a wide range of HER2 expression levels. Our results reveal distinct post-endocytic trafficking behavior of antibody-HER2 complexes in cells with different HER2 expression levels. In particular, HER2-overexpressing cells exhibit efficient HER2 recycling and limited reductions in HER2 levels upon antibody treatment, and consequently display a high level of antibody persistence on their plasma membrane. By contrast, in cells with low HER2 expression, trastuzumab treatment results in rapid antibody clearance from the plasma membrane combined with substantial decreases in HER2 levels and undetectable levels of recycling. A cell line with intermediate levels of HER2 expression exhibits both antibody recycling and clearance from the cell surface. Significantly, these analyses demonstrate that HER2 expression levels, rather than cell origin (breast or prostate), is a determinant of subcellular trafficking properties. Such studies have relevance to optimizing the design of antibodies to target HER2.

  13. Development of a recombinant, chimeric tetravalent dengue vaccine candidate.

    PubMed

    Osorio, Jorge E; Partidos, Charalambos D; Wallace, Derek; Stinchcomb, Dan T

    2015-12-10

    Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24-67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8(+) T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.

  14. Expression of two cross-reactive idiotypes on mouse antibodies against bromelain-treated mouse erythrocytes.

    PubMed Central

    Kawaguchi, S

    1987-01-01

    Two cross-reactive anti-idiotype (Id) antibodies were previously prepared from sera of rabbits immunized with mouse monoclonal antibodies against bromelain-treated mouse erythrocytes (BrMRBC). Most of the anti-BrMRBC plaque-forming cells (PFC) were suppressed by either of the two anti-Id antibodies. The Id profiles of anti-BrMRBC PFC were almost identical among various cell populations in a strain, but different among various mouse strains. Mouse sera contained both of the Id-bearing immunoglobulins Ig, and a significant part of the Id-bearing Ig were eliminated by absorption with BrMRBC. Nude BALB/c mice were almost equal to normal BALB/c mice in the Id patterns of anti-BrMRBC PFC and in the concentrations of the Id-bearing Ig. The injections of anti-Id antibodies into suckling mice suppressed, specifically, the development of the B cells to produce the homologous Id-bearing Ig, but the injection of Id-bearing monoclonal antibodies barely affected Id expression. It is suggested that the two Id are encoded in germ-line genes of mice, and are expressed independently of each other and Id-anti-Id regulations by T cells or B cells. PMID:3327804

  15. Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

    PubMed Central

    Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

    2012-01-01

    Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

  16. Pituitary expression of CTLA-4 mediates hypophysitis secondary to administration of CTLA-4 blocking antibody.

    PubMed

    Iwama, Shintaro; De Remigis, Alessandra; Callahan, Margaret K; Slovin, Susan F; Wolchok, Jedd D; Caturegli, Patrizio

    2014-04-02

    Hypophysitis is a chronic inflammation of the pituitary gland of unknown (primary forms) or recognizable (secondary forms) etiology, such as the use of ipilimumab in cancer immunotherapy. Ipilimumab, which blocks the T cell inhibitory molecule CTLA-4 (cytotoxic T lymphocyte antigen-4), induces hypophysitis in about 4% of patients through unknown mechanisms. We first established a model of secondary hypophysitis by repeated injections of a CTLA-4 blocking antibody into SJL/J or C57BL/6J mice, and showed that they developed lymphocytic infiltration of the pituitary gland and circulating pituitary antibodies. We next assessed the prevalence of pituitary antibodies in a cohort of 20 patients with advanced melanoma or prostate cancer, 7 with a clinical diagnosis of hypophysitis, before and after ipilimumab administration. Pituitary antibodies, negative at baseline, developed in the 7 patients with hypophysitis but not in the 13 without it; these antibodies predominantly recognized thyrotropin-, follicle-stimulating hormone-, and corticotropin-secreting cells. We then hypothesized that the injected CTLA-4 antibody could cause pituitary toxicity if bound to CTLA-4 antigen expressed "ectopically" on pituitary endocrine cells. Pituitary glands indeed expressed CTLA-4 at both RNA and protein levels, particularly in a subset of prolactin- and thyrotropin-secreting cells. Notably, these cells became the site of complement activation, featuring deposition of C3d and C4d components and an inflammatory cascade akin to that seen in type II hypersensitivity. In summary, the study offers a mechanism to explain the pituitary toxicity observed in patients receiving ipilimumab, and highlights the utility of measuring pituitary antibodies in this form of secondary hypophysitis.

  17. Thionin-D4E1 chimeric protein protects plants against bacterial infections

    DOEpatents

    Stover, Eddie W; Gupta, Goutam; Hao, Guixia

    2017-08-08

    The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

  18. Induction of type I interferon secretion through recombinant Newcastle disease virus expressing measles virus hemagglutinin stimulates antibody secretion in the presence of maternal antibodies.

    PubMed

    Kim, Dhohyung; Martinez-Sobrido, Luis; Choi, Changsun; Petroff, Natasha; García-Sastre, Adolfo; Niewiesk, Stefan; Carsillo, Thomas

    2011-01-01

    Measles virus (MV) vaccine effectively protects seronegative individuals against infection. However, inhibition of vaccine-induced seroconversion by maternal antibodies after vaccination remains a problem, as it leaves infants susceptible to MV infection. In cotton rats, passive transfer of MV-specific IgG mimics maternal antibodies and inhibits vaccine-induced seroconversion. Here, we report that immunization in the presence of passively transferred IgG inhibits the secretion of neutralizing antibodies but not the generation of MV-specific B cells. This finding suggested that MV-specific B cells require an additional stimulus to mature into antibody-secreting plasma cells. In order to provide such a stimulus, we generated a recombinant Newcastle disease virus (NDV) expressing the MV hemagglutinin (NDV-H). In contrast to MV, NDV-H induced high levels of type I interferon in plasmacytoid dendritic cells and in lung tissue. In cotton rats immunized with NDV-H, neutralizing antibodies were also generated in the presence of passively transferred antibodies. In the latter case, however, the level and kinetics of antibody generation were reduced. In vitro, alpha interferon stimulated the activation of MV-specific B cells from MV-immune spleen cells. NDV infection (which induces alpha interferon) had the same effect, and stimulation could be abrogated by antibodies neutralizing alpha interferon, but not interleukin 6 (IL-6). In vivo, coapplication of UV-inactivated MV with NDV led to increased MV-specific antibody production in the presence and absence of passively transferred antibodies. These data indicate that MV-specific B cells are being generated after immunization in the presence of maternal antibodies and that the provision of alpha interferon as an additional signal leads to antibody secretion.

  19. Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

    PubMed Central

    Koolivand, Davoud; Bashir, Nemat Sokhandan; Behjatnia, Seyed Aliakbar; Joozani, Raziallah Jafari

    2016-01-01

    The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection. PMID:27721695

  20. Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

    PubMed

    Magaña-Ortíz, Denis; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

    2017-08-31

    Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104 kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

  1. A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice.

    PubMed

    de Oliveira, Natasha Rodrigues; Jorge, Sérgio; Gomes, Charles Klazer; Rizzi, Caroline; Pacce, Violetta Dias; Collares, Thais Farias; Monte, Leonardo Garcia; Dellagostin, Odir Antônio

    2017-03-01

    Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP.

  2. Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

    PubMed

    de Hoog, Hans-Peter M; Lin JieRong, Esther M; Banerjee, Sourabh; Décaillot, Fabien M; Nallani, Madhavan

    2014-01-01

    G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.

  3. VH gene expression is restricted in anti-IgG antibodies from MRL autoimmune mice

    PubMed Central

    1986-01-01

    Antibodies directed against IgG and DNA are found in the sera of autoimmune MRL/Mp lpr/lpr mice. Little is known of the molecular mechanisms underlying expression of such autoantibodies. We have investigated the binding diversity and pattern of VH gene expression in a panel of murine anti-IgG antibodies. We constructed eight hybridoma clones secreting IgM antibodies that bound to mouse IgG by using spleen cells from MRL/Mp lpr/lpr mice varying in age from 4 to 15 wk; one clone was derived from a 32-wk-old MRL +/+ mouse. The monoclonal IgM products exhibited varying binding specificities for intact mouse IgG, fragments of mouse IgG [Fc, Fab, (Fab')2], and heterologous IgG. Two of these antibodies crossreacted with B and/or Z DNA. Probes from seven of eight identified mouse VH gene families (7183, S107, Q52, J558, J606, 36-60, and 3609) were hybridized under high-stringency conditions with cytoplasmic RNA blots from each clone. Six clones hybridized only with the probe from the five-member 36-60 family. The remaining three clones crosshybridized with the 36-60 probe and the probe from the 60 member J558 family, perhaps reflecting somatic mutation from the original germline VH gene resulting in recognition by a probe from another family, in addition to the probe from the original germline family. Our results indicate that spontaneous MRL lpr/lpr anti-IgG antibodies are encoded predominantly by the 36-60 VH gene family and imply a nonrandom selection of this VH gene family in the production of these antibodies. PMID:3093628

  4. In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33 Acute Myeloid Leukemia.

    PubMed

    Dutour, A; Marin, V; Pizzitola, I; Valsesia-Wittmann, S; Lee, D; Yvon, E; Finney, H; Lawson, A; Brenner, M; Biondi, A; Biagi, E; Rousseau, R

    2012-01-01

    Genetic engineering of T cells with chimeric T-cell receptors (CARs) is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV-) specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34(+) hematopoietic progenitors. Moreover, after intravenous administration into CD33(+) human acute myeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone.

  5. ALK gene copy number gain and immunohistochemical expression status using three antibodies in neuroblastoma.

    PubMed

    Kim, Eun Kyung; Kim, Sewha

    2016-03-17

    Anaplastic lymphoma kinase (ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC positive rate in ALK1 and 5A4 antibodies (p= < 0.001 and 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

  6. ALK Gene Copy Number Gain and Immunohistochemical Expression Status Using Three Antibodies in Neuroblastoma.

    PubMed

    Kim, Eun Kyung; Kim, Sewha

    2017-01-01

    Anaplastic lymphoma kinase ( ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC-positive rate in ALK1 and 5A4 antibodies ( P < 0.001 and P = 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

  7. Engineered antibodies take center stage.

    PubMed

    Huston, J S; George, A J

    2001-01-01

    The start of the post-genomic era provides a useful juncture for reflection on the state of antibody engineering, which will be a critical technology for relating function and pathology to genomic sequence in biology and medicine. The phenomenal progress in deciphering the human genome has given significant impetus to the application of engineered antibodies in proteomics. Thus, advances in phage display antibody libraries can now help to define novel gene function and the measurement of abnormal protein expression in pathological states. Furthermore, intrabody and antibody engineering provide vehicles for the development of molecular medicines of the future. In addition to these new directions, antibody engineering has begun to show concrete success in its long-term efforts to develop targeted immunotherapies for cancer and other diseases. The cornerstones of clinical development are the detailed academic clinical trials that continue to push the boundaries of engineered antibodies into the real world. The field displays a healthy impatience for practical results, as research accelerates with concerted efforts to transfer preclinical insights into clinical trials. Growing private and governmental expenditures will lead to the rapid expansion of life-saving immunotherapeutic agents. The present review developed from our effort to report on the 11th Annual International Conference on Antibody Engineering (3-6 December 2000). This annual meeting is a forum for discussions on the latest advances in antibody engineering groups from around the world, and now includes the broader agenda of engineering in molecular immunology. In bringing scientists together to exchange ideas at this open forum, new collaborations and the threads of new discoveries are woven. For example, Professors Gerhard Wagner (Harvard Medical School), Dennis Burton (Scripps Research Institute), and Peter Hudson (CSIRO, Melbourne, Australia) gave exciting insights on structural immunobiology that had

  8. Prism adaptation changes perceptual awareness for chimeric visual objects but not for chimeric faces in spatial neglect after right-hemisphere stroke.

    PubMed

    Sarri, Margarita; Kalra, Lalit; Greenwood, Richard; Driver, Jon

    2006-06-01

    Prism adaptation can ameliorate some symptoms of left spatial neglect after right-hemisphere stroke. The mechanisms behind this remain unclear. Prism therapy may increase exploration towards the contralesional side, yet without improving perceptual awareness, as apparently for the left side of chimeric face stimuli (Ferber et al. 2003). However, other prism studies suggest that perceptual awareness might be improved (e.g., Maravita et al., 2003). We tested the impact of prism therapy on visual awareness for the left side of chimeric objects as well as chimeric faces, in three neglect patients. Prism therapy dramatically improved awareness for the identity of the left side of chimeric non-face objects, but had no effect on judging expressions for chimeric faces. The latter may thus be unique in showing no prism benefit.

  9. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    PubMed Central

    2009-01-01

    Background RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3

  10. Effect of antibodies on the expression of Plasmodium falciparum circumsporozoite protein gene.

    PubMed

    Jesuíno, B S; Casimiro, C; do Rosário, V E; Silveira, H

    2006-01-01

    Antibodies are known to play an important role in the control of malaria infection. However, they can modulate parasite development enhancing infection. The effect of anti-Plasmodium antibodies on the expression of circumsporozoite protein gene (csp) was investigated. Plasmodium falciparum 3D7 in vitro cultures were submitted to: i) anti- circumsporozoite protein monoclonal antibody (anti-CSP-mAb) [1microg/ml, 0.1microg/ml, 0.01microg/ml and 0.001microg/ml] and ii) purified IgG Fab fragment from a pool of malaria patients [1mg/ml and 1microg/ml]; and compared to control cultures. After 24h the number of ring infected erythrocytes was determined in order to calculate invasion efficacy. At 48h culture supernatant was collected, and the amount of circumsporozoite protein determined by ELISA, parasitaemia was calculated and cells were processed for RNA preparation. Expression of csp gene was quantified using Real time RT-PCR. There was an increase in parasite growth when treated with lower anti-CSP-mAb concentration, which was associated with lower csp expression, while 1mug/ml anti-CSP-mAb treatment presented a growth inhibitory effect accompanied by high csp expression.

  11. Effect of antibodies on the expression of Plasmodium falciparum circumsporozoite protein gene

    PubMed Central

    Jesuíno, B S; Casimiro, C; do Rosário, V E; Silveira, H

    2006-01-01

    Antibodies are known to play an important role in the control of malaria infection. However, they can modulate parasite development enhancing infection. The effect of anti-Plasmodium antibodies on the expression of circumsporozoite protein gene (csp) was investigated. Plasmodium falciparum 3D7 in vitro cultures were submitted to: i) anti- circumsporozoite protein monoclonal antibody (anti-CSP-mAb) [1μg/ml, 0.1μg/ml, 0.01μg/ml and 0.001μg/ml] and ii) purified IgG Fab fragment from a pool of malaria patients [1mg/ml and 1μg/ml]; and compared to control cultures. After 24h the number of ring infected erythrocytes was determined in order to calculate invasion efficacy. At 48h culture supernatant was collected, and the amount of circumsporozoite protein determined by ELISA, parasitaemia was calculated and cells were processed for RNA preparation. Expression of csp gene was quantified using Real time RT-PCR. There was an increase in parasite growth when treated with lower anti-CSP-mAb concentration, which was associated with lower csp expression, while 1μg/ml anti-CSP-mAb treatment presented a growth inhibitory effect accompanied by high csp expression. PMID:16421624

  12. Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery.

    PubMed

    Chicoine, L G; Montgomery, C L; Bremer, W G; Shontz, K M; Griffin, D A; Heller, K N; Lewis, S; Malik, V; Grose, W E; Shilling, C J; Campbell, K J; Preston, T J; Coley, B D; Martin, P T; Walker, C M; Clark, K R; Sahenk, Z; Mendell, J R; Rodino-Klapac, L R

    2014-02-01

    Duchenne muscular dystrophy is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus (AAV) will ultimately require a vascular approach to broadly transduce muscle cells. We tested the impact of preexisting AAV antibodies on microdystrophin expression following vascular delivery to nonhuman primates. Rhesus macaques were treated by isolated limb perfusion using a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of the three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus, and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Microdystrophin expression was visualized in AAV, rhesus serotype 74 sero-negative animals (mean: 48.0 ± 20.8%) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high-level transduction (60.8 ± 18.0%), which is comparable with that of AAV sero-negative animals (53.7 ± 7.6%), whereas non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of preexisting immunity (natural infection) to AAV.

  13. Expression characteristics and specific antibody reactivity of diverse cathepsin F members of Paragonimus westermani.

    PubMed

    Ahn, Chun-Seob; Na, Byoung-Kuk; Chung, Dong-Ll; Kim, Jeong-Geun; Kim, Jin-Taek; Kong, Yoon

    2015-02-01

    Paragonimiasis, caused by the lung fluke Paragonimus, is a major food-borne helminthic disease. Differential diagnosis of paragonimiasis from tuberculosis and other infectious granulomas in the lung is a prerequisite to proper management of patients. Cysteine proteases of Paragonimus westermani (PwCPs) invoke specific antibody responses against patient sera, while antibody capturing activity of different PwCPs has not been comparatively analyzed. In this study, we observed the expressional regulation of 11 species of different PwCPs (PwCP1-11). We expressed recombinant PwCPs and assessed diagnostic reliability employing sera from patients with P. westermani (n=138), other trematodiases (n=80), cestodiases (n=60) and pulmonary tuberculosis (n=20), and those of normal controls (n=20). PwCPs formed a monophyletic clade into cathepsin F and showed differential expression patterns along with developmental stages of worm. Bacterially expressed recombinant PwCPs (rPwCPs) exhibited variable sensitivity of 38.4-84.5% and specificity of 87.2-100% in diagnosing homologous infection. rPwCPs recognized specific antibodies of experimental cat sera as early as 3 or 6weeks after infection. Patient sera of fascioliasis, Schistosomiasis japonicum and clonorchiasis demonstrated weak cross-reactions. Our results demonstrate that diverse PwCPs of the cathepsin F family participate in inducing specific antibody responses. Most P. westermani cathepsin F, except for PwCP2 (AAF21461), which showed negligible antibody responses, might be applicable for paragonimiasis serodiagnosis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. Antibodies to probe endogenous G protein-coupled receptor heteromer expression, regulation, and function

    PubMed Central

    Gomes, Ivone; Gupta, Achla; Bushlin, Ittai; Devi, Lakshmi A.

    2014-01-01

    Over the last decade an increasing number of studies have focused on the ability of G protein-coupled receptors to form heteromers and explored how receptor heteromerization modulates the binding, signaling and trafficking properties of individual receptors. Most of these studies were carried out in heterologous cells expressing epitope tagged receptors. Very little information is available about the in vivo physiological role of G protein-coupled receptor heteromers due to a lack of tools to detect their presence in endogenous tissue. Recent advances such as the generation of mouse models expressing fluorescently labeled receptors, of TAT based peptides that can disrupt a given heteromer pair, or of heteromer-selective antibodies that recognize the heteromer in endogenous tissue have begun to elucidate the physiological and pathological roles of receptor heteromers. In this review we have focused on heteromer-selective antibodies and describe how a subtractive immunization strategy can be successfully used to generate antibodies that selectively recognize a desired heteromer pair. We also describe the uses of these antibodies to detect the presence of heteromers, to study their properties in endogenous tissues, and to monitor changes in heteromer levels under pathological conditions. Together, these findings suggest that G protein-coupled receptor heteromers represent unique targets for the development of drugs with reduced side-effects. PMID:25520661

  15. Construction of human naive antibody gene libraries.

    PubMed

    Hust, Michael; Frenzel, André; Meyer, Torsten; Schirrmann, Thomas; Dübel, Stefan

    2012-01-01

    Human antibodies are valuable tools for proteome research and diagnostics. Furthermore, antibodies are a rapidly growing class of therapeutic agents, mainly for inflammation and cancer therapy. The first therapeutic antibodies are of murine origin and were chimerized or humanized. The later-developed antibodies are fully human antibodies. Here, two technologies are competing the hybridoma technology using transgenic mice with human antibody gene loci and antibody phage display. The starting point for the selection of human antibodies against any target is the construction of an antibody phage display gene library.In this review we describe the construction of human naive and immune antibody gene libraries for antibody phage display.

  16. Development of a chimeric DNA-RNA hammerhead ribozyme targeting SARS virus.

    PubMed

    Fukushima, Akiko; Fukuda, Noboru; Lai, Yimu; Ueno, Takahiro; Moriyama, Mitsuhiko; Taguchi, Fumihiro; Iguchi, Akifumi; Shimizu, Kazushi; Kuroda, Kazumichi

    2009-01-01

    Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To confirm its activity, in vitro cleavage reactions were performed with the synthesized ribozyme. Effects of the chimeric ribozyme were evaluated on multiplication of MHV. Effects of the chimeric ribozyme on expression of SARS-CoV were evaluated in cultured 3T3 cells. The synthetic ribozyme cleaved the synthetic target MHV and SARS-CoV RNA into fragments of predicted length. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited the expression of SARS-CoV RNA in 3T3 cells transfected with the recombinant plasmid. The chimeric DNA-RNA ribozyme targeting SARS-CoV significantly inhibited MHV viral activity and expression of recombinant SARS RNA in vitro. These findings indicate that the synthetic chimeric DNA-RNA ribozyme could provide a feasible treatment for SARS. Copyright (c) 2009 S. Karger AG, Basel.

  17. [Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

    PubMed

    Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

    2012-08-01

    To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2

  18. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

    PubMed

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J; Tuckey, Corinna; Riggs, Paul D; Colussi, Paul A; Noren, Christopher J; Taron, Christopher H; DeLisa, Matthew P; Berkmen, Mehmet

    2015-08-27

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named 'cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.

  19. Epitope expression in nine commercial kits for the determination of anti-thyroid peroxidase (TPO) antibodies.

    PubMed

    Whitham, K; Patel, D; Ward, A M

    1999-01-01

    Anti-thyroid peroxidase (TPO) antibodies, from patients with autoimmune disease, bind predominantly to two neighbouring, non-identical, conformational domains referred to as domains A and B. In recent years a number of ELISA assays have been developed for the detection of anti-TPO antibodies, however, considerable variation between the different commercial assay kits has been documented in inter-laboratory surveys (UK NEQAS). This investigation assessed the differences between nine commercial ELISA assays currently available in the UK. The anti-TPO kits varied in terms of their imprecision and accuracy and in the density of coated antigen. Recombinant antigen containing kits demonstrated partial destruction of the B epitope, possibly due to the close proximity of both epitope regions in the recombinant molecule. None of the kits expressed only one epitope although there were differences in the degrees of expression of each epitope. Clinicians should be aware of the variability of the numbers generated, when interpreting test results.

  20. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

    PubMed Central

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J.; Tuckey, Corinna; Riggs, Paul D.; Colussi, Paul A.; Noren, Christopher J.; Taron, Christopher H.; DeLisa, Matthew P.; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

  1. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections.

    PubMed

    Yang, Zhenhua; Wu, Rui; Li, Robert W; Li, Ling; Xiong, Zhongliang; Zhao, Haizhong; Guo, Deyin; Pan, Zishu

    2012-04-01

    A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.

  2. Construction and expression of single-chain Fv antibody against human bladder carcinoma.

    PubMed

    Yu, L Z; Xiao, S; Huang, H L; Gu, Z; Gu, F L; Guo, Y L

    1996-01-01

    We designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable-region genes from genomic DNA by polymerase chain reaction (PCR). The genomic DNA was extracted from hybridoma BDI-1 cells, which secreted a monoclonal antibody (mAb) against human bladder carcinoma. The primers contained special restriction sites that allowed the variable-region genes to be easily cloned for sequencing and expression. The recombinants were sequenced by Sanger's method. It was proved that the full lengths of the VH and VK genes were 366 and 324 bp, respectively. Compared with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from the rearrangement of VH, Dsp2.2 and JH4. The VK gene was VK subgroup IV and from VK and JK4. The VH and VK genes was inserted expression vector pWAI80. By inducement, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected. We suggest that ScFv antibody recognized the antigen specifically.

  3. A recombinant chimeric protein containing B chains of ricin and abrin is an effective vaccine candidate

    PubMed Central

    Wang, Junhong; Gao, Shan; Zhang, Tao; Kang, Lin; Cao, Wuchun; Xu, Na; Liu, Wensen; Wang, Jinglin

    2014-01-01

    Both ricin toxin (RT) and abrin toxin (AT) are 2 important toxin agents as potantial bioweapons. A dual subunit vaccine against RT and AT exposure is a promising option for developing prophylactic vaccination. In this study, we constructed a dual vaccine with RT B chain and AT B chain named RTB-ATB. The RTB-ATB chimeric protein was expressed in Escherichia coli (E. coli), and the purified protein was used to evaluate the immune response by a 2 × 2 × 2 × 2 factorial design. The main effects included dose of RTB-ATB, route of immunization injection, immunization time interval, and dose of native toxins challenge. For 2 × LD50 challenge of RT or AT, 100% of the RTB-ATB immunized mice survived and regained or exceeded their initial weights within 10 days. For 4 × LD50 challenge, different routes of immunization injection caused significant difference (P < 0.05), intraperitoneal (i.p.) administration of immunogen protected mice better than the subcutaneous (s.c.) administration. In conclusion, when administered i.p. to mice with 25 μg per mouse and immunization time interval Π in the absence of adjuvant, the chimeric protein elicited a stronger immune response and protected the animals from a dose of native toxins which was 4 times higher than their LD50 in unvaccinated mice. Besides, the RTB-ATB chimeric protein could induce specific neutralizing antibodies against these 2 toxins. We anticipate that this study will open new possibilities in the preparation of RTB-ATB dual subunit vaccine against the exposure to deadly RT and AT. PMID:24509607

  4. A recombinant chimeric protein containing B chains of ricin and abrin is an effective vaccine candidate.

    PubMed

    Wang, Junhong; Gao, Shan; Zhang, Tao; Kang, Lin; Cao, Wuchun; Xu, Na; Liu, Wensen; Wang, Jinglin

    2014-01-01

    Both ricin toxin (RT) and abrin toxin (AT) are 2 important toxin agents as potantial bioweapons. A dual subunit vaccine against RT and AT exposure is a promising option for developing prophylactic vaccination. In this study, we constructed a dual vaccine with RT B chain and AT B chain named RTB-ATB. The RTB-ATB chimeric protein was expressed in Escherichia coli (E. coli), and the purified protein was used to evaluate the immune response by a 2 × 2 × 2 × 2 factorial design. The main effects included dose of RTB-ATB, route of immunization injection, immunization time interval, and dose of native toxins challenge. For 2 × LD(50) challenge of RT or AT, 100% of the RTB-ATB immunized mice survived and regained or exceeded their initial weights within 10 days. For 4 × LD(50) challenge, different routes of immunization injection caused significant difference (P < 0.05), intraperitoneal (i.p.) administration of immunogen protected mice better than the subcutaneous (s.c.) administration. In conclusion, when administered i.p. to mice with 25 μg per mouse and immunization time interval Π in the absence of adjuvant, the chimeric protein elicited a stronger immune response and protected the animals from a dose of native toxins which was 4 times higher than their LD(50) in unvaccinated mice. Besides, the RTB-ATB chimeric protein could induce specific neutralizing antibodies against these 2 toxins. We anticipate that this study will open new possibilities in the preparation of RTB-ATB dual subunit vaccine against the exposure to deadly RT and AT.

  5. A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors.

    PubMed

    Chatterjee, Samit; Lesniak, Wojciech G; Gabrielson, Matthew; Lisok, Ala; Wharram, Bryan; Sysa-Shah, Polina; Azad, Babak Behnam; Pomper, Martin G; Nimmagadda, Sridhar

    2016-03-01

    Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.

  6. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

    PubMed

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H

    1980-06-01

    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.

  7. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

    PubMed Central

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H

    1980-01-01

    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen. PMID:6968260

  8. Global selection of Plasmodium falciparum virulence antigen expression by host antibodies

    PubMed Central

    Abdi, Abdirahman I.; Warimwe, George M.; Muthui, Michelle K.; Kivisi, Cheryl A.; Kiragu, Esther W.; Fegan, Gregory W.; Bull, Peter C.

    2016-01-01

    Parasite proteins called PfEMP1 that are inserted on the surface of infected erythrocytes, play a key role in the severe pathology associated with infection by the Plasmodium falciparum malaria parasite. These proteins mediate binding of infected cells to the endothelial lining of blood vessels as a strategy to avoid clearance by the spleen and are major targets of naturally acquired immunity. PfEMP1 is encoded by a large multi-gene family called var. Mutually-exclusive transcriptional switching between var genes allows parasites to escape host antibodies. This study examined in detail the patterns of expression of var in a well-characterized sample of parasites from Kenyan Children. Instead of observing clear inverse relationships between the expression of broad sub-classes of PfEMP1, we found that expression of different PfEMP1 groups vary relatively independently. Parasite adaptation to host antibodies also appears to involve a general reduction in detectable var gene expression. We suggest that parasites switch both between different PfEMP1 variants and between high and low expression states. Such a strategy could provide a means of avoiding immunological detection and promoting survival under high levels of host immunity. PMID:26804201

  9. Expression of chimeric receptor CD4ζ by natural killer cells derived from human pluripotent stem cells improves in vitro activity but does not enhance suppression of HIV infection in vivo.

    PubMed

    Ni, Zhenya; Knorr, David A; Bendzick, Laura; Allred, Jeremy; Kaufman, Dan S

    2014-04-01

    Cell-based immunotherapy has been gaining interest as an improved means to treat human immunodeficiency virus (HIV)/AIDS. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) could become a potential resource. Our previous studies have shown hESC and iPSC-derived natural killer (NK) cells can inhibit HIV-infected targets in vitro. Here, we advance those studies by expressing a HIV chimeric receptor combining the extracellular portion of CD4 to the CD3ζ intracellular signaling chain. We hypothesized that expression of this CD4ζ receptor would more efficiently direct hESC- and iPSC-derived NK cells to target HIV-infected cells. In vitro studies showed the CD4ζ expressing hESC- and iPSC-NK cells inhibited HIV replication in CD4+ T-cells more efficiently than their unmodified counterparts. We then evaluated CD4ζ expressing hESC (CD4ζ-hESC)- and iPSC-NK cells in vivo anti-HIV activity using a humanized mouse model. We demonstrated significant suppression of HIV replication in mice treated with both CD4ζ-modified and -unmodified hESC-/iPSC-NK cells compared with control mice. However, we did not observe significantly increased efficacy of CD4ζ expression in suppression of HIV infection. These studies indicate that hESC/iPSC-based immunotherapy can be used as a unique resource to target HIV/AIDS. © 2013 AlphaMed Press.

  10. Chimeric Genes as a Source of Rapid Evolution in Drosophila melanogaster

    PubMed Central

    Rogers, Rebekah L.; Hartl, Daniel L.

    2012-01-01

    Chimeric genes form through the combination of portions of existing coding sequences to create a new open reading frame. These new genes can create novel protein structures that are likely to serve as a strong source of novelty upon which selection can act. We have identified 14 chimeric genes that formed through DNA-level mutations in Drosophila melanogaster, and we investigate expression profiles, domain structures, and population genetics for each of these genes to examine their potential to effect adaptive evolution. We find that chimeric gene formation commonly produces mid-domain breaks and unites portions of wholly unrelated peptides, creating novel protein structures that are entirely distinct from other constructs in the genome. These new genes are often involved in selective sweeps. We further find a disparity between chimeric genes that have recently formed and swept to fixation versus chimeric genes that have been preserved over long periods of time, suggesting that preservation and adaptation are distinct processes. Finally, we demonstrate that chimeric gene formation can produce qualitative expression changes that are difficult to mimic through duplicate gene formation, and that extremely young chimeric genes (dS < 0.03) are more likely to be associated with selective sweeps than duplicate genes of the same age. Hence, chimeric genes can serve as an exceptional source of genetic novelty that can have a profound influence on adaptive evolution in D. melanogaster. PMID:21771717

  11. How should chimerism be decoded?

    PubMed

    Ferrand, Christophe; Perruche, Sylvain; Robinet, Eric; Martens, Anton; Tiberghien, Pierre; Saas, Philippe

    2003-05-15

    To date, the significance of chimerism has not been fully understood. In particular, microchimerism can be associated with allograft acceptance or rejection. Several factors may influence the immunologic consequences of chimerism. In this review, the major factors influencing these consequences are briefly described. Subsequently, the different methods available for detecting and tracking donor-derived cells are listed. These techniques have been mainly developed concomitantly with nonmyeloablative hematopoietic allografts to monitor immunosuppression. Finally, the authors suggest how these methods may help to improve the understanding of microchimerism in solid organ transplantation.

  12. EXPRESSION OF CYP4F2 IN HUMAN LIVER AND KIDNEY: ASSESSMENT USING TARGETED PEPTIDE ANTIBODIES

    PubMed Central

    Hirani, Vandana; Yarovoy, Anton; Kozeska, Anita; Magnusson, Ronald P.; Lasker, Jerome M.

    2008-01-01

    P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61–74 (WGHQGMVNPTEEG) and 65–77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly-variable CYP4F2 expression in liver (16.4 ± 18.6 pmol/mg microsomal protein; n = 29) and kidney cortex (3.9 ± 3.8 pmol/mg; n = 10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r ≥ 0.63; p < 0.05) with leukotriene B4 and arachidonate ω-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate ω-hydroxylase in human liver. PMID:18662666

  13. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    PubMed

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis.

  14. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  15. Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia.

    PubMed

    Nakazawa, Yozo; Matsuda, Kazuyuki; Kurata, Takashi; Sueki, Akane; Tanaka, Miyuki; Sakashita, Kazuo; Imai, Chihaya; Wilson, Matthew H; Koike, Kenichi

    2016-03-16

    Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML. We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34(+) cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34(+) cells. GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34(+) cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34(+) cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34(+) cell proliferation, particularly CD34(+)CD38(-) cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34(+) cell colony growth. Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.

  16. [New antibodies in cancer treatment].

    PubMed

    Pestalozzi, B C; Knuth, A

    2004-09-22

    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  17. A Chimeric Pneumovirus Fusion Protein Carrying Neutralizing Epitopes of Both MPV and RSV

    PubMed Central

    Wen, Xiaolin; Pickens, Jennifer; Mousa, Jarrod J.; Leser, George P.; Lamb, Robert A.; Crowe, James E.; Jardetzky, Theodore S.

    2016-01-01

    Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are paramyxoviruses that are responsible for substantial human health burden, particularly in children and the elderly. The fusion (F) glycoproteins are major targets of the neutralizing antibody response and studies have mapped dominant antigenic sites in F. Here we grafted a major neutralizing site of RSV F, recognized by the prophylactic monoclonal antibody palivizumab, onto HMPV F, generating a chimeric protein displaying epitopes of both viruses. We demonstrate that the resulting chimeric protein (RPM-1) is recognized by both anti-RSV and anti-HMPV F neutralizing antibodies indicating that it can be used to map the epitope specificity of antibodies raised against both viruses. Mice immunized with the RPM-1 chimeric antigen generate robust neutralizing antibody responses to MPV but weak or no cross-reactive recognition of RSV F, suggesting that grafting of the single palivizumab epitope stimulates a comparatively limited antibody response. The RPM-1 protein provides a new tool for characterizing the immune responses resulting from RSV and HMPV infections and provides insights into the requirements for developing a chimeric subunit vaccine that could induce robust and balanced immunity to both virus infections. PMID:27224013

  18. Chimeric enzymes with improved cellulase activities

    DOEpatents

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  19. Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics

    PubMed Central

    Wilmes, Gwendolyn M; Carey, Kimberly L; Hicks, Stuart W; Russell, Hugh H; Stevenson, Jesse A; Kocjan, Paulina; Lutz, Stephen R; Quesenberry, Rachel S; Shulga-Morskoy, Sergey V; Lewis, Megan E; Clark, Ethan; Medik, Violetta; Cooper, Anthony B; Reczek, Elizabeth E

    2014-01-01

    Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures. PMID:24758837

  20. Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer.

    PubMed

    Wang, G; Qiu, J; Wang, R; Krause, A; Boyer, J L; Hackett, N R; Crystal, R G

    2010-08-01

    Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody (mAb) directed against an extracellular region of the human epidermal growth-factor receptor type 2 (HER2) protein. We hypothesized that a single adeno-associated virus (AAV)-mediated genetic delivery of an anti-HER2 antibody should be effective in mediating long-term production of anti-HER2 and in suppressing the growth of human tumors in a xenograft model in nude mice. The adeno-associated virus gene transfer vector AAVrh.10alphaHER2 was constructed based on a non-human primate AAV serotype rh.10 to express the complementary DNAs for the heavy and light chains of mAb 4D5, the murine precursor to trastuzumab. The data show that genetically transferred anti-HER2 selectively bound human HER2 protein and suppressed the proliferation of HER2(+) tumor cell lines. A single administration of AAVrh.10alphaHER2 provided long-term therapeutic levels of anti-HER2 antibody expression without inducing an anti-idiotype response, suppressed the growth of HER2(+) tumors and increased the survival of tumor bearing mice. In the context that trastuzumab therapy requires frequent and repeated administration, this strategy might be developed as an alternate platform for delivery of anti-HER2 therapy.

  1. Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection.

    PubMed

    Kapoor, Reetika; Mandal, Bikash; Paul, Prabir Kumar; Chigurupati, Phaneendra; Jain, Rakesh Kumar

    2014-02-01

    Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (∼40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (∼50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (∼40kDa and ∼50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses.

  2. Restricted light chain subgroup expression on human rheumatoid factor paraproteins determined by monoclonal antibodies.

    PubMed Central

    Mageed, R A; Walker, M R; Jefferis, R

    1986-01-01

    Two hybridoma antibodies specific for determinants on the V kappa light chain subgroup have been produced and characterized. Antibodies C7 and B12 reacted with distinct V kappa epitopes irrespective of association with heavy chain class or subclass. Epitopes recognized by C7 and B12 were expressed on the light chain of IgG, IgA, and IgM and Bence-Jones paraproteins from the V kappa subgroup. However, a preferential association of both epitopes with IgM RF paraproteins was demonstrated. Hybridomas C7 and B12 reacted with 12/12 and 10/12 IgM RF paraproteins, respectively, but only with 3/6 IgM paraproteins, with no RF activity. Both epitopes C7 and B12, were immunodominant and conformation dependent, being detected by HA, HAI and ELISA on intact light chain but not isolated VK. PMID:2432001

  3. Molecular characterization of antibodies bearing Id-460. II. Molecular basis for Id-460 expression

    PubMed Central

    1985-01-01

    Id-460+ immunoglobulins can be induced in vivo by immunization with dinitrophenyl (DNP) or P. pneumotropica and form two nonoverlapping groups of antibodies with respect to antigen binding specificity. In this study, using Id-460+ antibodies of differing antigen binding specificities, we compared on the molecular genetic level the five gene segment combinations (VH, DH, JH, VL, and JL) that encode the variable regions of these idiotype-positive immunoglobulins. The Id-460 determinant appears to be a conformational or combinatorial determinant encoded by VH460 and VK1 crosshybridizing genes. DH, JH, and JK gene segments appear to have no measurable effect upon expression of Id-460. Finally, antigen binding specificity does not appear to simply localize to any particular gene segment but may in part be the result of somatic mutation and/or VDJH junctional sequences, whose length correlates roughly with antigen binding specificity. PMID:3932578

  4. Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies.

    PubMed

    Hu, Zhonglan; Chen, Zhenyu; Huang, Nongyu; Teng, Xiu; Zhang, Jun; Wang, Zhen; Wei, Xiaoqiong; Qin, Ke; Liu, Xiao; Wu, Xueping; Tang, Huan; Zhu, Xiaofeng; Cui, Kaijun; Li, Jiong

    2015-03-01

    Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl β-D-1-thio galactoside (IPTG) at 37°C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined.

  5. [Prokaryotic expression and antibody preparation of human GALNT3-sol protein].

    PubMed

    Kong, Yun; Gao, Hai-tao; Li, Shu-fang; Wang, Peng; Gu, Li

    2011-10-01

    In order to detect the expression of GALNT3 in various tumor tissues, the prokaryotic expression vector of human GALNT3-sol (a truncation of GALNT3 being deleted of the hydrophobic trans-membrane domain) was constructed, and then the recombinant GALNT3-sol protein was expressed and purified from E.coli, followed by the preparation of polyclonal antibody against GALNT3-sol and characterization of its properties. The human cDNA of GALNT3-sol (1 755 bp)was amplified from MKN45 cell line and cloned into expression vector pET5b/GALNT3-sol, then transformed into E.coli BL21(DE3), in which the GALNT3-sol protein was induced by IPTG and then purified by Electrophoresis.Mice were immunized with the purified protein and the anti-serum was collected at different time intervals.Properties of the anti-serum were further detected by ELISA and Western blot. The prokaryotic expression vector of pET15b/GALNT3-sol was constructed successfully.Human GALNT3-sol protein was expressed in E.coli after IPTG induction.The titer of the obtained anti-serum reached 1:25 600, and its specificity was proved by Western blot. Human GALNT3-sol protein can be successfully expressed in E.coli, and the specific anti-human GALNT3-sol antibody can be obtained by immunization of mice, which makes it possible to further investigate the role of GALNT3 in the progression of various tumors.

  6. Adaptive impact of the chimeric gene Quetzalcoatl in Drosophila melanogaster.

    PubMed

    Rogers, Rebekah L; Bedford, Trevor; Lyons, Ana M; Hartl, Daniel L

    2010-06-15

    Chimeric genes, which form through the genomic fusion of two protein-coding genes, are a significant source of evolutionary novelty in Drosophila melanogaster. However, the propensity of chimeric genes to produce adaptive phenotypic changes is not fully understood. Here, we describe the chimeric gene Quetzalcoatl (Qtzl; CG31864), which formed in the recent past and swept to fixation in D. melanogaster. Qtzl arose through a duplication on chromosome 2L that united a portion of the mitochondrially targeted peptide CG12264 with a segment of the polycomb gene escl. The 3' segment of the gene, which is derived from escl, is inherited out of frame, producing a unique peptide sequence. Nucleotide diversity is drastically reduced and site frequency spectra are significantly skewed surrounding the duplicated region, a finding consistent with a selective sweep on the duplicate region containing Qtzl. Qtzl has an expression profile that largely resembles that of escl, with expression in early pupae, adult females, and male testes. However, expression patterns appear to have been decoupled from both parental genes during later embryonic development and in head tissues of adult males, indicating that Qtzl has developed a distinct regulatory profile through the rearrangement of different 5' and 3' regulatory domains. Furthermore, misexpression of Qtzl suppresses defects in the formation of the neuromuscular junction in larvae, demonstrating that Qtzl can produce phenotypic effects in cells. Together, these results show that chimeric genes can produce structural and regulatory changes in a single mutational step and may be a major factor in adaptive evolution.

  7. Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses

    PubMed Central

    McCormick, Kara; Jiang, Zhiyong; Zhu, Longchao; Lawson, Steven R.; Langenhorst, Robert; Ransburgh, Russell; Brunick, Colin; Tracy, Miranda C.; Hurtig, Heather R.; Mabee, Leah M.; Mingo, Mark; Li, Yanhua; Webby, Richard J.

    2015-01-01

    Background and Objectives Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes. Methods and Results Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates. Conclusion This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines. PMID:26061265

  8. Construction and Immunogenicity Evaluation of Recombinant Influenza A Viruses Containing Chimeric Hemagglutinin Genes Derived from Genetically Divergent Influenza A H1N1 Subtype Viruses.

    PubMed

    McCormick, Kara; Jiang, Zhiyong; Zhu, Longchao; Lawson, Steven R; Langenhorst, Robert; Ransburgh, Russell; Brunick, Colin; Tracy, Miranda C; Hurtig, Heather R; Mabee, Leah M; Mingo, Mark; Li, Yanhua; Webby, Richard J; Huber, Victor C; Fang, Ying

    2015-01-01

    Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes. Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates. This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.

  9. Comparison of different blood sample processing methods for sensitive detection of low level chimerism by RHD real-time PCR assay.

    PubMed

    Javadi, Ahmad; Verduin, Esther P; Brand, Anneke; Schonewille, Henk

    2013-01-01

    The rhesus D blood group, which is expressed on the red blood cells (RBC) of 85% of the Caucasian population, is one of the most immunogenic RBC antigens, inducing D antibody formation in up to 20-80% of D-negative transfusion recipients and about 10% of pregnancies at risk. Pregnancy-induced D-antibodies can persist for many years, but the mechanisms underlying this persistence are unclear. The LOTUS study, a long-term follow-up study of mothers from severely affected children with hemolytic disease of the fetus and newborn investigates, among other endpoints, whether persistent feto-maternal chimerism is associated with long-term maternal anti-D persistence. We questioned which blood sample processing method should be used to detect low levels of RHD chimerism with the highest sensitivity and specificity using qPCR. After optimization of primer and probe concentrations for singleplex RHD exon 5 and 7 qPCR, sensitivity, specificity and efficiency of RHD and DYS1 qPCR were investigated in artificial chimeric samples. Sensitivity of DYS1 was one log higher (0.0001%) in enriched mononuclear cell fractions as compared with whole blood. Comparable linear sensitivity (0.007%) and mean efficiency (84-99%) for RHD qPCR were observed in all samples regardless whether whole blood or pre- or post-mixing of cellular fractions had been used. We conclude that RHD chimerism using singleplex exon 5 and 7 qPCR is linearly detectable down to 1.0 GE, without an advantage of fraction enrichment.

  10. Tissue-specific expression and dietary regulation of chimeric mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase/human growth hormone gene in transgenic mice.

    PubMed

    Serra, D; Fillat, C; Matas, R; Bosch, F; Hegardt, F G

    1996-03-29

    We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels of hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium-, and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice.

  11. Chimerism in transfusion medicine

    PubMed Central

    Brunker, Patricia AR

    2013-01-01

    Transfusion therapy is complicated by the production of alloantibodies to antigens present in the donor and lacking in the recipient through the poorly-understood but likely multi-factorial process of alloimmunization. The low prevalence of alloimmunization in transfused patients (6.1%)1 suggests that processes central to immunologic tolerance may be operating in the vast majority of transfused patients who do not produce alloantibodies. Using RhD as a prototype, evidence is reviewed that the ability to make antibodies to red blood cell (RBC) antigens may result in part from immunologic tolerance acquired in utero. These ideas are extended to other examples of maternal microchimerism (MMc) of other non-inherited maternal antigens (NIMA). An evolutionary argument is offered that multi-generational immunity supports the hypothesis that MMc may partly explain the “non-responder” phenotype in RBC alloimmunization. PMID:24196285

  12. Chimeric aptamers in cancer cell-targeted drug delivery

    PubMed Central

    Kanwar, Jagat R; Roy, Kislay; Kanwar, Rupinder K

    2011-01-01

    Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern Pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities. PMID:21955150

  13. Chimeric Antigen Receptor Therapy for Cancer

    PubMed Central

    Barrett, David M.; Singh, Nathan; Porter, David L.; Grupp, Stephan A.; June, Carl H.

    2014-01-01

    Improved outcomes for patients with cancer hinge on the development of new targeted therapies with acceptable short-term and long-term toxicity. Progress in basic, preclinical, and clinical arenas spanning cellular immunology, synthetic biology, and cell-processing technologies has paved the way for clinical applications of chimeric antigen receptor– based therapies. This new form of targeted immunotherapy merges the exquisite targeting specificity of monoclonal antibodies with the potent cytotoxicity and long-term persistence provided by cytotoxic T cells. Although this field is still in its infancy, clinical trials have already shown clinically significant antitumor activity in neuroblastoma, chronic lymphocytic leukemia, and B cell lymphoma, and trials targeting a variety of other adult and pediatric malignancies are under way. Ongoing work is focused on identifying optimal tumor targets and on elucidating and manipulating both cell- and host-associated factors to support expansion and persistence of the genetically engineered cells in vivo. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer. PMID:24274181

  14. Intracellular Reprogramming of Expression, Glycosylation, and Function of a Plant-Derived Antiviral Therapeutic Monoclonal Antibody

    PubMed Central

    Lee, Kyung-Jin; Kim, Young-Kwan; So, Yang-Kang; Ryu, Jae-Sung; Oh, Seung-Han; Han, Yeon-Soo; Ko, Kinarm; Choo, Young-Kug; Park, Sung-Joo; Brodzik, Robert; Lee, Kyoung-Ki; Oh, Doo-Byoung; Hwang, Kyung-A; Koprowski, Hilary; Lee, Yong Seong; Ko, Kisung

    2013-01-01

    Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAbPs), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAbP SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAbP SO57 with KDEL (mAbPK) were significantly higher than those of mAbP SO57 without KDEL (mAbP) regardless of the transcription level. The Fc domains of both purified mAbP and mAbPK and hybridoma-derived mAb (mAbH) had similar levels of binding activity to the FcγRI receptor (CD64). The mAbPK had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAbP had mainly Golgi type glycans (96.8%) similar to those seen with mAbH. Confocal analysis showed that the mAbPK was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAbP with KDEL in the ER. Both mAbP and mAbPK disappeared with similar trends to mAbH in BALB/c mice. In addition, mAbPK was as effective as mAbH at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAbP by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant. PMID:23967055

  15. Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

    PubMed

    Gadue, Paul; Gouon-Evans, Valerie; Cheng, Xin; Wandzioch, Ewa; Zaret, Kenneth S; Grompe, Markus; Streeter, Philip R; Keller, Gordon M

    2009-09-01

    The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

  16. Targeting Pan-Resistant Bacteria With Antibodies to a Broadly Conserved Surface Polysaccharide Expressed During Infection

    PubMed Central

    Skurnik, David; Davis, Michael R.; Benedetti, Dennis; Moravec, Katie L.; Cywes-Bentley, Colette; Roux, Damien; Traficante, David C.; Walsh, Rebecca L.; Maira-Litràn, Tomas; Cassidy, Sara K.; Hermos, Christina R.; Martin, Thomas R.; Thakkallapalli, Erin L.; Vargas, Sara O.; McAdam, Alexander J.; Lieberman, Tami D.; Kishony, Roy; LiPuma, John J.; Pier, Gerald B.; Goldberg, Joanna B.; Priebe, Gregory P.

    2012-01-01

    Background New therapeutic targets for antibiotic-resistant bacterial pathogens are desperately needed. The bacterial surface polysaccharide poly-β-(1-6)-N-acetyl-glucosamine (PNAG) mediates biofilm formation by some bacterial species, and antibodies to PNAG can confer protective immunity. By analyzing sequenced genomes, we found that potentially multidrug-resistant bacterial species such as Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomonas maltophilia, and the Burkholderia cepacia complex (BCC) may be able to produce PNAG. Among patients with cystic fibrosis patients, highly antibiotic-resistant bacteria in the BCC have emerged as problematic pathogens, providing an impetus to study the potential of PNAG to be targeted for immunotherapy against pan-resistant bacterial pathogens. Methods The presence of PNAG on BCC was assessed using a combination of bacterial genetics, microscopy, and immunochemical approaches. Antibodies to PNAG were tested using opsonophagocytic assays and for protective efficacy against lethal peritonitis in mice. Results PNAG is expressed in vitro and in vivo by the BCC, and cystic fibrosis patients infected by the BCC species B. dolosa mounted a PNAG-specific opsonophagocytic antibody response. Antisera to PNAG mediated opsonophagocytic killing of BCC and were protective against lethal BCC peritonitis even during coinfection with methicillin-resistant Staphylococcus aureus. Conclusions Our findings raise potential new therapeutic options against PNAG-producing bacteria, including even pan-resistant pathogens. PMID:22448004

  17. Chimeric Genes in Deletions and Duplications Associated with Intellectual Disability

    PubMed Central

    Monfort, Sandra; Roselló, Mónica; Oltra, Silvestre; Caro-Llopis, Alfonso

    2017-01-01

    We report on three nonrelated patients with intellectual disability and CNVs that give rise to three new chimeric genes. All the genes forming these fusion transcripts may have an important role in central nervous system development and/or in gene expression regulation, and therefore not only their deletion or duplication but also the resulting chimeric gene may contribute to the phenotype of the patients. Deletions and duplications are usually pathogenic when affecting dose-sensitive genes. Alternatively, a chimeric gene may also be pathogenic by different gain-of-function mechanisms that are not restricted to dose-sensitive genes: the emergence of a new polypeptide that combines functional domains from two different genes, the deregulated expression of any coding sequence by the promoter region of a neighboring gene, and/or a putative dominant-negative effect due to the preservation of functional domains of partially truncated proteins. Fusion oncogenes are well known, but in other pathologies, the search for chimeric genes is disregarded. According to our findings, we hypothesize that the frequency of fusion transcripts may be much higher than suspected, and it should be taken into account in the array-CGH analyses of patients with intellectual disability. PMID:28630856

  18. A Comparative Antibody Analysis of Pannexin1 Expression in Four Rat Brain Regions Reveals Varying Subcellular Localizations

    PubMed Central

    Cone, Angela C.; Ambrosi, Cinzia; Scemes, Eliana; Martone, Maryann E.; Sosinsky, Gina E.

    2012-01-01

    Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and

  19. Immune deficiency enhances expression of recombinant human antibody in mice after nonviral in vivo gene transfer.

    PubMed

    Kitaguchi, Kohji; Toda, Mikako; Takekoshi, Masataka; Maeda, Fumiko; Muramatsu, Tatsuo; Murai, Atsushi

    2005-10-01

    A cDNA encoding human antibody against hepatitis B virus was expressed in normal and severe combined immune deficiency (SCID) mice to clarify whether or not host immune status affects circulating levels of the recombinant human antibody (RhAb) after nonviral in vivo gene transfer. For transferring genes, either electroporation (EP) or hydrodynamics-based transfection (HD) was employed. The former was applied to the leg muscle to express the gene, while the latter primarily targeted foreign gene expression in the liver. The expressed RhAb was secreted into the blood circulation, and its existence was assayed by ELISA. Prior to the investigation of host immune status, suitable forms of plasmid expression vectors and types of electrodes were determined in normal mice. Results showed that the vector encoding both the light and heavy chains driven by the CMV promoter had the highest plasma RhAb concentrations, and a pair of pincette-type electrodes conferred the best performance. In both EP and HD, the SCID state showed an increased and prolonged RhAb production in the blood circulation due probably to suppressed recognition of RhAb as a foreign protein to the host animal. The difference in gene transfer methods demonstrated a characteristic pattern: an early and sharp rise followed by a relatively rapid decrease in HD, in contrast to a gradual rise followed by a plateau level maintained in EP. As a result, with the same amount of gene transferred, the plasma RhAb concentrations for the first 7 or 8 weeks were higher in HD than EP, while the reverse was true for the latter period. Multiple gene transfer contributed to maintaining and prolonging high RhAb concentrations in plasma by both methods with similar characteristic patterns accompanying the respective gene transfer method. These results suggest the importance of host immunological potency for maintaining plasma RhAb concentrations if these gene transfer technologies are used for clinical and therapeutic purposes.

  20. Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

    PubMed

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Leitolis, Amanda; Crestani, Sandra; Foti, Leonardo; de Souza, Wayner Vieira; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2017-10-01

    Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania, a pathogen with high similarity to T. cruzi, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD. Copyright © 2017 American Society for Microbiology.

  1. A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection.

    PubMed

    Wang, Haiying; Sun, Xingmin; Zhang, Yongrong; Li, Shan; Chen, Kevin; Shi, Lianfa; Nie, Weijia; Kumar, Raj; Tzipori, Saul; Wang, Jufang; Savidge, Tor; Feng, Hanping

    2012-08-01

    The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.

  2. Analysis of HPV-1 E4 gene expression using epitope-defined antibodies.

    PubMed Central

    Doorbar, J; Evans, H S; Coneron, I; Crawford, L V; Gallimore, P H

    1988-01-01

    Six monoclonal antibodies (mAbs) have been raised against the E4 proteins of HPV-1. Five of these were found to recognize denaturation-resistant epitopes as determined by Western blotting--and their binding sites were identified by determining their reactivity against a panel of bacterial E4--beta-galactosidase fusion proteins which contained progressive deletions at the C-terminal end of the E4 region. The five mAbs were found to bind to four distinct sites. By using these epitope-defined mAbs, along with anti-peptide antibodies raised against putative N- and C-terminal E4 sequences, we have determined the relationships between the eight distinct polypeptides (mol. wt 10/11 kd, 16/17 kd, 21/23 kd and 32/34 kd) previously shown to be expressed from the E4 gene of HPV-1 in productively infected papillomas. The 17 kd E4 polypeptide appears to be the product of a spliced mRNA encoding five amino acids from open reading frame (ORF) E1 joined onto 120 from the E4 ORF. The 16 kd and 10/11 kd proteins, which may be derived from this, lack sequences (approximately 15 and 70 amino acids respectively) encoded by the 5' end of the E4 gene. The 32/34 kd proteins were detected by all antibodies which reacted with the 16/17 kd polypeptides, suggesting that they represent dimers of the latter species. The 21/23 kd polypeptides, however, do not appear to be simple dimers of the 10/11 kd protein as previously predicted, and reacted with antibodies whose epitopes mapped in the N-terminal half of the E4 protein.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2456213

  3. Structure of idiotopes associated with antiphenylarsonate antibodies expressing an intrastrain crossreactive idiotype

    PubMed Central

    1989-01-01

    We have explored the structural basis of idiotopes associated with the major idiotype (CRIA) of A/J anti-p-azobenzenearsonate antibodies, with emphasis on the regions of contact with anti-idiotypic antibody. The analysis was facilitated by a recent description of the three- demensional structure of the Fab portion of a CRIA-related antibody molecule. Direct binding measurements failed to reveal idiotopes associated exclusively with the L chain. However, the L chain participated in the formation of approximately 80% of the idiotopes recognized by polyclonal anti-Id. This indicates that multiple complementarity-determining regions (CDRs) participate in the formation of idiotopes. The affinity of anti-Id for CDRs on L chains must be appreciable but insufficient to permit direct binding (i.e., less than approximately 10(4) M-1). Approximately 20-35% of polyclonal anti-Id reacted with high affinity with H chains recombined with non-CRIA- related L chains. This interaction was found to involve the D region as well as one or both CDRs in the VH segment, again indicating the contribution of multiple CDRs. It is suggested that a typical idiotope may be similar in size to that of protein epitopes whose three- dimensional structures are known; such epitopes comprise a substantial fraction of the surface area occupied by the CDRs of an antibody. The expression of an idiotope recognized by the mAb AD8, which interacts with the VH segment, was found to be unaffected by major changes in the neighboring D and VL regions. This observation is relevant to efforts to predict three-dimensional structure from the amino acid sequence of CRIA+ molecules. PMID:2507724

  4. Obtaining anti-type 1 melatonin receptor antibodies by immunization with melatonin receptor-expressing cells.

    PubMed

    Cordeiro, Nelia; Wijkhuisen, Anne; Savatier, Alexandra; Moulharat, Natacha; Ferry, Gilles; Léonetti, Michel

    2016-01-01

    Antibodies (Abs) specific to cell-surface receptors are attractive tools for studying the physiological role of such receptors or for controlling their activity. We sought to obtain such antibodies against the type 1 receptor for melatonin (MT1). For this, we injected mice with CHO cells transfected with a plasmid encoding human MT1 (CHO-MT1-h), in the presence or absence of an adjuvant mixture containing Alum and CpG1018. As we previously observed that the immune response to a protein antigen is increased when it is coupled to a fusion protein, called ZZTat101, we also investigated if the association of ZZTat101 with CHO-MT1-h cells provides an immunogenic advantage. We measured similar levels of anti-CHO and anti-MT1-h Ab responses in animals injected with either CHO-MT1-h cells or ZZTat101/CHO-MT1-h cells, with or without adjuvant, indicating that neither the adjuvant mixture nor ZZTat101 increased the anti-cell immune response. Then, we investigated whether the antisera also recognized murine MT1 (MT1-m). Using cloned CHO cells transfected with a plasmid encoding MT1-m, we found that antisera raised against CHO-MT1-h cells also bound the mouse receptor. Altogether our studies indicate that immunizing approaches based on MT1-h-expressing CHO cells allow the production of polyclonal antibodies against MT1 receptors of different origins. This paves the way to preparation of MT1-specific monoclonal antibodies.

  5. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    PubMed

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

  6. Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials.

    PubMed

    Ramanayake, Saumya; Bilmon, Ian; Bishop, David; Dubosq, Ming-Celine; Blyth, Emily; Clancy, Leighton; Gottlieb, David; Micklethwaite, Kenneth

    2015-09-01

    Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification. We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period. Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer. We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor Induce Remissions of B-Cell Malignancies That Progress After Allogeneic Hematopoietic Stem-Cell Transplantation Without Causing Graft-Versus-Host Disease

    PubMed Central

    Brudno, Jennifer N.; Somerville, Robert P.T.; Shi, Victoria; Rose, Jeremy J.; Halverson, David C.; Fowler, Daniel H.; Gea-Banacloche, Juan C.; Pavletic, Steven Z.; Hickstein, Dennis D.; Lu, Tangying L.; Feldman, Steven A.; Iwamoto, Alexander T.; Kurlander, Roger; Maric, Irina; Goy, Andre; Hansen, Brenna G.; Wilder, Jennifer S.; Blacklock-Schuver, Bazetta; Hakim, Frances T.; Rosenberg, Steven A.; Gress, Ronald E.

    2016-01-01

    Purpose Progressive malignancy is the leading cause of death after allogeneic hematopoietic stem-cell transplantation (alloHSCT). After alloHSCT, B-cell malignancies often are treated with unmanipulated donor lymphocyte infusions (DLIs) from the transplant donor. DLIs frequently are not effective at eradicating malignancy and often cause graft-versus-host disease, a potentially lethal immune response against normal recipient tissues. Methods We conducted a clinical trial of allogeneic T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Patients with B-cell malignancies that had progressed after alloHSCT received a single infusion of CAR T cells. No chemotherapy or other therapies were administered. The T cells were obtained from each recipient’s alloHSCT donor. Results Eight of 20 treated patients obtained remission, which included six complete remissions (CRs) and two partial remissions. The response rate was highest for acute lymphoblastic leukemia, with four of five patients obtaining minimal residual disease–negative CR. Responses also occurred in chronic lymphocytic leukemia and lymphoma. The longest ongoing CR was more than 30 months in a patient with chronic lymphocytic leukemia. New-onset acute graft-versus-host disease after CAR T-cell infusion developed in none of the patients. Toxicities included fever, tachycardia, and hypotension. Peak blood CAR T-cell levels were higher in patients who obtained remissions than in those who did not. Programmed cell death protein-1 expression was significantly elevated on CAR T cells after infusion. Presence of blood B cells before CAR T-cell infusion was associated with higher postinfusion CAR T-cell levels. Conclusion Allogeneic anti-CD19 CAR T cells can effectively treat B-cell malignancies that progress after alloHSCT. The findings point toward a future when antigen-specific T-cell therapies will play a central role in alloHSCT. PMID:26811520

  8. Petunia AGAMOUS enhancer-derived chimeric promoters specify a carpel-, stamen- and petal-specific expression pattern sufficient for engineering male and female sterility in tobacco

    USDA-ARS?s Scientific Manuscript database

    Previous studies have shown that the AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer specifies a carpel- and stamen-specific pattern of expression in its native host species but not in heterologous species, such as tobacco which restricts its application in the engin...

  9. Effects of platelet glycoprotein IIb/IIIa receptor blockade by a chimeric monoclonal antibody (abciximab) on acute and six-month outcomes after percutaneous transluminal coronary angioplasty for acute myocardial infarction. EPIC investigators.

    PubMed

    Lefkovits, J; Ivanhoe, R J; Califf, R M; Bergelson, B A; Anderson, K M; Stoner, G L; Weisman, H F; Topol, E J

    1996-05-15

    Percutaneous transluminal coronary angioplasty (PTCA) for acute myocardial infarction is an attractive alternative to thrombolysis, but is still limited by recurrent ischemia and restenosis. We determined whether adjunctive platelet glycoprotein IIb/IIIa receptor blockade improved outcomes in patients undergoing direct and rescue PTCA in the Evaluation of c7E3 for Prevention of Ischemic Complications (EPIC) trial. Of the 2,099 patients undergoing percutaneous intervention who randomly received chimeric 7E3 Fab (c7E3) as a bolus, a bolus and 12-hour infusion, or placebo, 42 underwent direct PTCA for acute myocardial infarction and 22 patients had rescue PTCA after failed thrombolysis. The primary composite end point comprised death, reinfarction, repeat intervention, or bypass surgery. Outcomes were assessed at 30 days and 6 months. Baseline characteristics were similar in direct and rescue PTCA patients. Pooling the 2 groups, c7E3 bolus and infusion reduced the primary composite end point by 83% (26.1% placebo vs 4.5% c7E3 bolus and infusion, p = 0.06). No reinfarctions or repeat urgent interventions occurred in c7E3 bolus and infusion patients at 30 days, although there was a trend toward more deaths in c7E3-treated patients. Major bleeding was increased with c7E3 (24% vs 13%, p = 0.28). At 6 months, ischemic events were reduced from 47.8% with placebo to 4.5% with c7E3 bolus and infusion (p = 0.002), particularly reinfarction (p = 0.05) and repeat revascularization (p = 0.002). We conclude that adjunctive c7E3 therapy during direct and rescue PTCA decreased acute ischemic events and clinical restenosis in the EPIC trial. These data provide initial evidence of benefit for glycoprotein IIb/IIIa receptor blockade during PTCA for acute myocardial infarction.

  10. Expression of POTE protein in human testis detected by novel monoclonal antibodies

    SciTech Connect

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R.; Pastan, Ira

    2008-01-25

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2{gamma}C, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2{gamma}C and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

  11. Expression of POTE protein in human testis detected by novel monoclonal antibodies.

    PubMed

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R; Bera, Tapan K; Pastan, Ira

    2008-01-25

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.

  12. Expression of POTE protein in human testis detected by novel monoclonal antibodies

    PubMed Central

    Ise, Tomoko; Das, Sudipto; Nagata, Satoshi; Maeda, Hiroshi; Lee, Yoomi; Onda, Masanori; Anver, Miriam R.; Bera, Tapan K.; Pastan, Ira

    2008-01-01

    The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis and placenta. We produced 10 monoclonal antibodies (MAbs) against 3 representative POTE paralogs: POTE-21, POTE-2γC and POTE-22. One reacted with all 3 paralogs, 6 MAbs reacted with POTE-2γC and POTE-22, and 3 MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis. PMID:17996727

  13. Strategic deployment of CHO expression platforms to deliver Pfizer's Monoclonal Antibody Portfolio.

    PubMed

    Scarcelli, John J; Shang, Tanya Q; Iskra, Tim; Allen, Martin J; Zhang, Lin

    2017-05-08

    Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  14. PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody.

    PubMed

    Gargouri-Bouzid, Radhia; Jaoua, Leïla; Rouis, Souad; Saïdi, Mohamed Najib; Bouaziz, Donia; Ellouz, Radhouane

    2006-06-01

    A synthetic gene encoding a single chain Fv fragment of an antibody directed against the nuclear inclusion a (NIa) protein of potato virus Y (PVY) was used to transform two commercial potato cultivars (Claustar and BF15). The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. Immunoblot analysis showed that most of the resulting transgenic plants accumulate high levels of the transgenic protein. Furthermore, a majority of the selected transgenic lines showed an efficient and complete protection against the challenge virus after mechanical inoculation with PVYO strain. Two transgenic lines showed an incomplete resistance with delayed appearance of symptoms accompanied by low virus titers, whereas one line developed symptoms during the first days after inoculation but recovered rapidly, leading to a low virus accumulation rate. These results confirm that expression of scFv antibody is able to inhibit a crucial step in the virus multiplication, such as polyprotein cleavage is a powerful strategy for engineered virus resistance. It can lead to a complete resistance that was not obtained previously by expression of scFv directed against the viral coat protein.

  15. Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y.

    PubMed

    Rouis, Souad; Lafaye, Pierre; Jaoua-Aydi, Leila; Sghaier, Zidani; Ayadi, Hammadi; Gargouri-Bouzid, Radhia

    2006-10-01

    Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.

  16. Stable transgenic expression of IL-2 and HSV1-tk by single and fusion tumor cell lines bearing EWS/FLI-1 chimeric genes.

    PubMed

    Staege, Martin S; Gorelov, Victor; Bulankin, Andrej; Fischer, Ute; Dumon, Kristoffel; Hohndorf, Lars; Hattenhorst, Uwe; Kramm, Christof; Burdach, Stefan

    2003-03-01

    In augmenting systemic anti-tumor immune response, the authors evaluated the genetic modification of Ewing family tumor (EFT) cell lines for use as allogeneic vaccines. EFT cell lines A673 and RD-ES were transfected with cDNAs for human interleukin (IL)-2 and/or HSV1 thymidine kinase (HSV1-tk), respectively. Clones with high and stable secretion of IL-2 alone or with coexpression of functional HSV1-tk were obtained and their features were analyzed. IL-2 expressing clones derived from the A673 cell line demonstrated decreased expression of HLA class I molecules compared with the parental cell line and corresponding clones derived from RD-ES. However, IFN-gamma could upregulate the expression of HLA class I antigens by IL-2 transfected A673 cells. Ganciclovir induced apoptosis in double-transfected cell clones. IL-2/HSV1-tk cells continued to produce and release IL-2 after initial ganciclovir treatment. After gamma-irradiation, transfected clones released bioactive IL-2 in a quantity sufficient to activate T and natural killer cells in culture. A polyvalent allogeneic vaccine was also obtained using fusion of two different transgenic cell lines. The resulting hybrids inherited antigenic and transgenic characteristics of both parental cell lines. It is presumed that the cell lines generated here could be used as allogeneic vaccines for treatment of patients with EFTs.

  17. Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef

    SciTech Connect

    Bouzar, Baya Amel; Rea, Angela; Hoc-Villet, Stephanie; Garnier, Celine; Guiguen, Francois; Jin Yuhuai; Narayan, Opendra; Chebloune, Yahia . E-mail: ychebloune@kumc.edu

    2007-08-01

    Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

  18. Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

    PubMed

    Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

    2015-10-01

    A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting.

  19. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    SciTech Connect

    Bujak, Emil; Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah; Neri, Dario

    2014-09-10

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  20. Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

    PubMed

    Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

    2017-02-10

    Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

  1. Construction of a mouse Aos1-Uba2 chimeric SUMO-E1 enzyme, mAU, and its expression in baculovirus-insect cells

    PubMed Central

    Nakayama, Tomofumi; Yuasa, Eri; Kanemaru, Ayumi; Saito, Masayuki; Saitoh, Hisato

    2014-01-01

    Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1. PMID:24637489

  2. Mimicry of erythropoietin and interleukin-6 signalling by an antibody/cytokine receptor chimera in murine myeloid 32D cells.

    PubMed

    Kawahara, Masahiro; Ueda, Hiroshi; Tsumoto, Kouhei; Kumagai, Izumi; Nagamune, Teruyuki

    2007-04-01

    We hav