Biomedical subjects
Publications and source records attributed to Stefan Dübel.
Recombinant antibody technology has revolutionized the development of antibody therapeutics. This minireview offers an overview of enabling technologies and future prospects of this rapidly progressing field.
BACKGROUND: Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. RESULTS: The lysozyme specific single chain Fv (scFv) fragment D1.3 was successfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41 degrees C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 microg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli. CONCLUSION: High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.
ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.
Monoclonal antibodies (Mab) are the fastest growing group of biopharmaceuticals in development. For production in mammalian cells, the four polypeptide chains of the immunoglobulin diheterotetramer must be assembled prior to exit from the endoplasmic reticulum. Various recombinant Mab expression vectors have been developed utilizing mono-and bicistronic expression cassettes encoded on one or two plasmids. However, there are only few studies providing information on the type of vector design optimal for stable or transient production of recombinant IgG. Consequently, in this study, we have constructed a series of mammalian expression vectors for the production of recombinant human or chimeric IgG antibodies with different expression cassette designs. Versions for monocistronic and bicistronic expression with different promoters and cistron arrangements were generated. Antibody production levels were evaluated in transiently transfected 293T and CHO-K1 cells. Furthermore, stable CHO cell lines were generated and analyzed for antibody production levels and stability. Our results indicate that compared to monocistronic expression, EMCV IRES-mediated bicistronic expression constructs yield similar antibody expression levels and show long-term stability in CHO cell lines. Addition of a third cistron encoding YFP was shown to facilitate screening and isolation of clones using a FACS sorter.
Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection.
The enrichment of open reading frames (ORFs) from large gene libraries and the presentation of the corresponding polypeptides on filamentous phage M13 (phage display) is frequently used to identify binding partners of unknown ORFs. In particular phage display is a valuable tool for the identification of pathogen-related antigens and a first step for the development of new diagnostics and therapeutics. Here, we introduce a significant improvement of phage-based ORF enrichment by using Hyperphage, a helperphage with a truncated gIII. The methods allow both the enrichment of ORFs from cDNA libraries and the display of the corresponding polypeptides on phage, thus combining ORF enrichment with a screening for binding in one step without any further subcloning steps. We demonstrated the benefits of the method by isolating the sequences encoding two predicted immunogenic epitopes of the outer membrane protein D encoding gene (ompD) of Salmonella typhimurium. Here, we showed that when using a mixture of three constructs with only one containing an ORF solely this correct construct could be reisolated in phage particles. Further; both epitopes were detected by enzyme-linked immunosorbent assay (ELISA), demonstrating correct translation of fusion proteins. Furthermore, the enrichment system was evaluated by the enrichment of ORFs from total cDNA of lymphocytes. Here, we could show that 60% of the phage contained ORFs, which is an increase of an order of magnitude compared with conventional phage expression system. Together these data show that the Hyperphage-based enrichment system significantly improves the enrichment of ORFs and directly allows the display of the corresponding polypeptide on bacteriophage M13.
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After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains approximately 25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell. Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging. Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods.
A major source of human antibodies are phage display libraries, which are constructed from various genetic sources. Antibodies are expressed as scFV and Fab antibody fragments using various vector systems. This review offers a comprehensive overview of M13 phage display antibody vectors and discusses their applications.
A multitude of systems for the presentation of foreign peptides or proteins on the surface of microorganisms has been developed within the past two decades. However, the majority of the bacterial surface display systems are devoted to the presentation of heterologous antigens to the immune system (vaccine generation). Bacteria are the preferable hosts for the generation of vast genetic repertoires, and their genetic manipulation and cultivation is easy. As a consequence, they provide promising systems for large-scale functional screenings, e.g. for enzyme activity or protein-protein interactions. This review will focus on examples of microbial surface display used for the screening of combinatorial repertoires. Further, we discuss future opportunities and promising candidate proteins not yet employed for that task.
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Despite the availability of antibody libraries for the selection of receptor molecules, the large number of established and well-characterized hybridoma lines still represent a useful source for recombinant antibody genes. This protocol describes the PCR amplification, cloning, and a small-scale expression test for the generation of scFv fragments from hybridoma cell lines. Particular emphasis was placed on frequently observed problems and pitfalls of this method.
The aim of this study was to establish a method to obtain antibodies against cell-surface molecules of hematopoietic stem and progenitor cells in situ. A phage-displayed scFv antibody library with a size of 2 x 10(6) clones was constructed from spleens of mice immunized with living KG1a human leukemia cells. Living cell panning was used to screen anti-KG1a cell antibody fragments. After four rounds of panning, 27 out of 126 scFv fragments showed detectable binding to KG1a cells without cross-reaction to non-hematopoietic cell lines. Individual clones were analyzed by cell-based ELISA and flow cytometry for their binding specificity. Their sequence analysis showed highest homology to mouse gamma heavy chain subgroup II and mouse Kappa subgroup III genes, with four amino acids difference in VH and identical VL. Further, a fusion protein of scFv 5C1 to core-streptavidin was cloned and produced. It was used to search for cell-surface antigen on immunoblots and to test its effect on KG1a cell growth in cell culture. The scFv5C1::core-streptavidin fusion protein recognized a molecule with MW 85/125 KDa in immunoblots of KG1a cell membrane proteins and inhibited KG1a cell homoaggregation in cell cultures. The results validate an efficient approach of making antibodies against living cells and searching for functional inhibitors of cell-surface molecules.
cTAGE-1 is a cutaneous-T-cell-lymphoma-specific tumor antigen recently identified by serologic identification of antigens by recombinant expression cloning. This study was aimed at identifying and characterizing related genes. Rapid amplification of cDNA ends and DNA screening led to five new members of the cTAGE gene family belonging to four different genes, two of which were differentially spliced (cTAGE-1/2 and cTAGE-5). Expression analysis using reverse transcription polymerase chain reaction revealed that cTAGE-1, cTAGE-1B, and cTAGE-5A expression was restricted to testis and tumor tissues, whereas the other cTAGE members were found in two to eight other normal tissues (of 27 tissues tested). Tumor-specific protein expression of cTAGE-5 was confirmed by Western blotting. Sero-reactivity against cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B was found only in tumor patients (cutaneous T cell lymphoma and melanoma). The immunogenic epitope of cTAGE-1 was determined by using epitope mapping and sera of two cutaneous T cell lymphoma patients. Moreover, cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B could be detected in most types of tumor tissues and cell lines at variable frequencies, including those of cutaneous T cell lymphoma, melanoma, head and neck squamous cell carcinoma, breast carcinoma, and colon carcinoma. We conclude that cTAGE-1 and cTAGE-5 are new cancer germline antigens and that tumor-specific splicing of cTAGE genes may lead to further candidate proteins for specific immunotherapy of cutaneous T cell lymphoma and other malignancies.
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Filamentous bacteriophages are nonlytic, male-specific bacteriophages which infect Escherichia coli carrying an F-episome. The molecular mechanism of infection remains elusive, including the role of the major coat protein pVIII. In order to evaluate the contributions of major coat protein pVIII in the process of infection, two phage display libraries were generated. One library consisted of random amino acids at positions 2, 4, 5, 8, 11 and 12 of the N-terminus of major coat protein pVIII. The second library was generated by randomizing these positions as well as position 1. All these residues were previously shown to be exposed at the surface of the virions by being accessible to ligands. The infectivity of randomly selected mutant phages was analyzed. The present results demonstrate that phages modified at these positions can be correctly assembled and secreted into the exoplasm, although the efficiency was slightly lower than that of wild-type phage. Their infectivity varied greatly, and a general structural pattern underlying infectivity did not emerge. However, residual differences were observed between infectious and defective phage; in general, uncharged polar amino acids present at positions 5 and 11 of the N-terminus of pVIII reduced phage infectivity, whereas polar residues at position 8 facilitated infection. The first position of pVIII is remarkably critical for infection; when this alanine was substituted with other residues, most of the phages lost their infectivity. These results shed new light on the true complexity of random peptide pVIII phage display libraries.
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