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Recombinant Antibodies (recombinant + antibody)
Terms modified by Recombinant Antibodies Selected AbstractsInduction of central T cell tolerance: Recombinant antibodies deliver peptides for deletion of antigen-specific CD4+8+ thymocytesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2005Karoline, Western Schjetne Abstract In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self-antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen-presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II-specific Troybodies with the ,2315 T cell epitope were injected into ,2315 -specific TCR transgenic mice, a profound deletion of CD4+8+ thymocytes was observed. MHC class II-specific Troybodies were 10,100-fold more efficient than non-targeting peptide Ab, and 500-fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II-specific Troybodies with the OVA323,339 T cell epitope were injected into OVA-specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II-specific Troybodies for 4,weeks, had reduced antigen-specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases. [source] Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodiesDRUG DEVELOPMENT RESEARCH, Issue 3 2004Reed J. Harris Abstract Stable therapeutic antibody dosage forms present production technology challenges, particularly when high-concentration formulations are needed to meet the elevated dose requirements that are generally required for successful antibody therapy. Solid dosage forms, such as lyophilized powders, are generally more stable than liquid formulations. High-concentration drug products can be achieved by reconstitution of the lyophilisate in a smaller volume than its initial (pre-lyophilization) volume, but requires a significant vial overfill. High-concentration liquid formulations are becoming feasible as new techniques and technologies become available. Analytical methods to detect subtle molecular variations have been developed to demonstrate manufacturing consistency. Some molecular heterogeneity is contributed by conserved sites, such as Asn297 glycosylation and the loss of heavy chain C-terminal Lys residues. Characteristics that affect potency, stability, or immunogenicity must be elucidated for each therapeutic antibody. Drug Dev. Res. 61:137,154, 2004. © 2004 Wiley-Liss, Inc. [source] Induction of central T cell tolerance: Recombinant antibodies deliver peptides for deletion of antigen-specific CD4+8+ thymocytesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2005Karoline, Western Schjetne Abstract In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self-antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen-presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II-specific Troybodies with the ,2315 T cell epitope were injected into ,2315 -specific TCR transgenic mice, a profound deletion of CD4+8+ thymocytes was observed. MHC class II-specific Troybodies were 10,100-fold more efficient than non-targeting peptide Ab, and 500-fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II-specific Troybodies with the OVA323,339 T cell epitope were injected into OVA-specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II-specific Troybodies for 4,weeks, had reduced antigen-specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases. [source] What is the current evidence for antigen involvement in the development of chronic lymphocytic leukemia?HEMATOLOGICAL ONCOLOGY, Issue 1 2006Gerard Tobin Abstract For many years it has been evident that B-cell chronic lymphocytic leukemia (CLL) displays preferential usage of individual immunoglobulin (Ig) variable heavy chain (VH) genes. The VH1-69 gene was the first to be reported overrepresented in a large number of CLL patients, where the VH1-69+ CLL rearrangements showed characteristic molecular features, such as unmutated VH genes, usage of specific diversity/joining gene segments, and a longer than average complementarity determining region (CDR) 3 with certain common amino acid motifs. Also, biased usage of the VH3-07 and VH4-34 genes with specific rearrangement characteristics was reported in CLL. These findings led to the speculation that antigens could be involved during CLL development by triggering proliferation of B-cells with specific B-cell receptors (BCRs) leading to an increased risk of transforming events. Recently, we characterized a subset of CLL utilizing the VH3-21 gene that also displayed peculiar Ig features, e.g. very short and homologous CDR3s, predominant , expression and preferential V,2-14 gene usage. This VH3-21+ subgroup also had poor prognosis despite the fact that two-thirds of cases carried mutated VH genes. Moreover, we and others have thereafter described further CLL subsets with very similar heavy and light chain gene rearrangement features. These latter findings of subsets expressing restricted BCRs have emphasized the hypothesis that antigens could play a role during the pathogenesis of CLL. Interestingly, recombinant antibodies produced from these restricted subsets showed similar cytoplasmatic reactivity within each group, thus suggesting recognition of a limited number of autoantigens. Further characterization of antigens is now necessary in order to understand their nature and exact role in CLL development. Copyright © 2005 John Wiley & Sons, Ltd. [source] Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2005Alexandru C. Lazar Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species. Copyright © 2005 John Wiley & Sons, Ltd. [source] A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancerTHE PROSTATE, Issue 13 2006Ursula Elsässer-Beile Abstract BACKGROUND Prostate-specific membrane antigen (PSMA) is an excellent candidate for targeting prostate cancer by virtue of its restricted expression on prostatic epithelial cells and its upregulation on prostatic carcinoma cells. PSMA is expressed on the cell surface displaying a specific three-dimensional structure. Therefore, only antibodies with a high cell binding activity will have an important impact on antibody-based imaging and therapy. METHODS Monoclonal antibodies (mAbs) and single chain antibody fragments (scFvs) were prepared from spleen cells of mice that had been immunized either with purified PSMA or a cell lysate of prostate cancer LNCaP cells containing native PSMA. mAbs and scFvs were screened for reactivity with purified PSMA and binding to PSMA-expressing LNCaP cells. RESULTS From mice immunized with purified PSMA, we obtained three mAbs (K7, K12, D20) and four scFvs (G0, G1, G2, G4), which were highly reactive with the isolated antigen, but showed weak or no reaction with viable LNCaP cells. From mice immunized with unpurified LNCaP lysate, we obtained three mAbs (3/E7, 3/F11, 3/A12), and one scFv (A5), which were reactive with purified PSMA, also showing a strong and specific binding to viable LNCaP cells and PSMA-transfected cells. CONCLUSIONS Our results suggest that only the mAbs and scFvs, that were elicited with unpurified LNCaP lysate and not with purified PSMA will be useful agents for diagnostic imaging and therapeutic applications of prostate cancer. Prostate 66: 1359,1370, 2006. © 2006 Wiley-Liss, Inc. [source] Intrathecal pathogenic anti,aquaporin-4 antibodies in early neuromyelitis optica,ANNALS OF NEUROLOGY, Issue 5 2009Jeffrey L. Bennett MD Objective The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis. Methods Fluorescence-activated cell sorting and single-cell reverse transcriptase polymerase chain reaction were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE). Results Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis, and complement and Ig deposition. Interpretation Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specfic Ig is synthesized intrathecally at disease onset and directly contributes to CNS pathology. AQP4 is now the first confirmed antigenic target in human demyelinating disease. Ann Neurol 2009;66:617,629 [source] Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid,ANNALS OF NEUROLOGY, Issue 6 2009Gregory P. Owens PhD Objective Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. Methods We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. Results Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. Interpretation The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein. Ann Neurol 2009;65:639,649 [source] Varicella zoster virus is not a disease-relevant antigen in multiple sclerosis,ANNALS OF NEUROLOGY, Issue 4 2009Mark P. Burgoon PhD Herpesvirions and varicella zoster virus (VZV) DNA were recently reported in all 15 cerebrospinal fluid (CSF) samples from patients with relapsing-remitting multiple sclerosis (MS) obtained within 1 week of exacerbation. Using identical electron microscopic and polymerase chain reaction techniques, including additional primer sets representing different regions of the VZV genome, we found no herpesvirions or VZV DNA in MS CSF or acute MS plaques. Although enzyme-linked immunosorbent assay analysis demonstrated a higher titer of VZV antibody in MS CSF than in inflammatory control samples, recombinant antibodies prepared from clonally expanded MS CSF plasma cells did not bind to VZV. VZV is not a disease-relevant antigen in MS. Ann Neurol 2009;65:474,479 [source] Expression of Human Recombinant Antibody Fragments Capable of Partially Inhibiting the Phospholypase Activity of Crotalus durissus terrificus VenomBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 2 2009Juliana G. Oliveira It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A2 with high enzymatic activity. The phospholipases A2 are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema-inducing, hypotensive, platelet-aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A2. In this study, we used a non-immune human single-chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema-inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single-chain fragment variable libraries in the production of antivenoms. [source] Triple light chain antibodies: Factors that influence its formation in cell cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Natalia Gomez Abstract THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species,Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression,evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC),correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production,evaluated as the ratio of the cell-specific production rate of GSH (qGSH) to the cell-specific production rate of THIOMAB (qp),corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and qGSH/qp ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high qGSH/qp ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748,760. © 2009 Wiley Periodicals, Inc. [source] Comparative transcriptome analysis to unveil genes affecting recombinant protein productivity in mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Joon Chong Yee Abstract Low temperature culture (33°C) has been shown to enhance the specific productivity of recombinant antibodies in Chinese hamster ovary (CHO) cells but did not affect antibody productivity in hybridoma (MAK) cells. We probed the transcriptional response of both cells undergoing temperature shift using cDNA microarrays. Among the orthologous gene probes, common trends in the expression changes between CHO and MAK are not prominent. Instead, many transcriptional changes were specific to only one cell line. Notably, oxidative phosphorylation and ribosomal genes were downregulated in MAK but not in CHO. Conversely, several protein trafficking genes and cytoskeleton elements were upregulated in CHO but remained unchanged in MAK. Interestingly, at 33°C, immunoglobulin heavy and light chain showed no significant changes in CHO, but the immunoglobulin light chain was downregulated in MAK. Overall, a clear distinction in the transcriptional response to low temperature was seen in the two cell lines. To further elucidate the set of genes responsible for increased antibody productivity, the expression data of low temperature cultures was compared to that of butyrate treatment which increased specific antibody productivity in both cell lines. Genes which are commonly differentially expressed under conditions that increased productivity are likely to reflect functional classes that are important in the productivity changes. This comparative transcriptome analysis suggests that vesicle trafficking, endocytosis and cytoskeletal elements are involved in increased specific antibody productivity. Biotechnol. Bioeng. 2009;102: 246,263. © 2008 Wiley Periodicals, Inc. [source] Design and engineering human forms of monoclonal antibodiesDRUG DEVELOPMENT RESEARCH, Issue 3 2004Manuel L. Penichet Abstract The antibody molecule has multiple properties that make it a key component of the immune response. These include its ability to recognize a vast array of different foreign substrates and to interact with and activate the host effector systems. Antibodies with defined specificities may serve as "magic bullets" for the diagnosis and therapy of multiple diseases. With the development of the hybridoma technology, it was possible to produce rodent (mouse or rat) monoclonal antibodies that are the product of a single clone of antibody producing cells and have only one antigen binding specificity. However, the therapeutic use of rodent monoclonals antibodies in humans is limited by their immunogenicity, short circulating half-life, and inability to efficiently trigger human effector mechanisms. However, it proved difficult to produce human monoclonal antibodies using the same methods. To address these problems genetic engineering and expression systems have instead been used to produce chimeric, humanized, and totally human antibodies as well as antibodies with novel structures and functional properties. In addition, the use of yeast and human artificial chromosome vectors for animal transgenesis has allowed the development of animal models that produce antigen specific antibodies that are totally human. As a consequence, recombinant antibody-based therapies are now used to treat a variety of clinical conditions including infectious diseases, inflammatory disorders, and cancer. This article summarizes and compares different strategies for designing and engineering human antibodies and their derivatives. Drug Dev. Res. 61:121,136, 2004. © 2004 Wiley-Liss, Inc. [source] Attovial-based antibody nanoarraysPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2009Peter Ellmark Abstract Antibody array-based technology is a powerful emerging tool in proteomics, but to enable global proteome analysis, antibody array layouts with even higher density has to be developed. To this end, we have further developed the first generation of a nanoarray platform, based on attoliter-sized vials, attovials, which we have characterized and used for the detection of complement factor C1q in human serum samples. Finally, we demonstrated proof-of-concept for individual functionalization of the attovials with a recombinant antibody. [source] Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfectionBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2008Sebastien Chenuet Abstract Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc. [source] Promoting ,-secretase cleavage of beta-amyloid with engineered proteolytic antibody fragmentsBIOTECHNOLOGY PROGRESS, Issue 4 2009Srinath Kasturirangan Abstract Deposition of beta-amyloid (A,) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of A, levels by various therapeutic approaches is actively being pursued. A potentially non-inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze A, at its ,-secretase site. We have previously identified a light chain fragment, mk18, with ,-secretase-like catalytic activity, producing the 1,16 and 17,40 amino acid fragments of A,40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking ,-secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for A,. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the ,-secretase site of A,. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3- and 6-fold increase in catalytic activity (kcat/KM) toward the synthetic A, substrate compared to the original scFv primarily due to an expected decrease in KM rather than an increase in kcat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for A,. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble A, levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009 [source] | ||||||||||||||||