Alexander J. Howard Biol 509 Dr. Mourad IPFW Biology November 5, 2010 Monoclonal antibodies isolated without screening by analyzing the variable gene repertoire of plasma cells. Sai T. Reddy, Xin Ge, Aleksandr E. Miklos, Randall A. Hughes, Seung Hyun Kang , Kam Hon Hoi, Constantine Chrysostomou, Scott P. Hunicke-Smith, Brent L. Iverson, Philip W. Tucker, Andrew D. Ellington & George Georgiou. Nat. Biotechnol. 28:9, 965-969 (2010) Introduction & Objective: Monoclonal antibodies (mAb) are protein molecules that are microscopically identical with respect to their polypeptide sequence and consequently their ability to bind with high affinity to a specific antigen. Preparations of high purity mAb have proven invaluable in a diverse number of applications, including medicine, biochemistry, chemical engineering, and as diagnostic reagents in clinical & molecular biology research. Currently there are a plethora of mAb drugs available for treatment of autoimmune disorders like Crohn’s disease & rheumatoid arthritis (infliximab, adalimumab), acute organ rejection (basiliximab, daclizumab), cancers like B cell leukemia, nonHodgkin’s lymphoma & squamous cell carcinomas (alemtuzumab, rituximab, nimotuzumab & cetuximab), and as anticoagulation and antiviral agents (abciximab, palivizumab). The use of mAb as catalytic enzymes has numerous potential applications in both medicine (antibody directed enzyme prodrug therapy) and materials technology. Lastly, the importance of mAbs in general molecular biology research is overwhelming, as they allow for detection, characterization, and quantification of specific substances of interest (ELISA, western immunoblotting). The objective of the current study was to demonstrate a novel method for mAb production without the need for affinity screening at the antibody-antigen level. The authors eliminate the physical screening process entirely by taking advantage of the highly efficacious B cell selection processes of affinity maturation and somatic hypermutation that occur naturally in mammalian immune responses. Early approaches to antibody production involved the immunization of animals with antigen followed by recovery and purification of immunoglobulins from the serum. With the advent of hybridoma technology in 1975 by Köhler & Milstein it was possible to create immortalized B cell lines for proliferation in vitro. Once high affinity Ab secreting hybridomas were identified and selected, the subsequent clonal populations of cells derived from them all express the same identical Ab. In this sense, the antibody produced is monoclonal because all cells are clones of one original B cell hybridoma. Isolation of high affinity antibody producing B cells is laborious because a large number of potentially useful B cells must be isolated and individually exposed to antigen to determine binding affinity. Although recombinant antibody discovery methods such as phage display libraries solve the problem of immortalizing B cell clones in vitro, they still rely on direct interactions between receptor molecules and antigen to isolate and enrich high affinity genetic material. Techniques like these consume time, labor, and materials, mostly due to the large number of variables involved in the screening process. Experiments & Results: The general experimental strategy is best summarized in six steps. In the first step, female Balb/C mice are immunized with antigens (human complement serine protease, C1s; human Bcell regulator of IgH transcription, Bright; chicken egg ovalbumin, OVA), and after six days plasma cells (CD45R- CD138+) are extracted and isolated. Second, mRNA is isolated and V gene cDNA is synthesized from highly expressed transcripts using reverse transcription. In the third step the VL and VH genes are amplified from the cDNA generated in step two. In the fourth step, high throughput DNA sequencing is used to generate frequency distributions for the most abundant complementarity determining region 3 (CDR3) sequences. This bioinformatic analysis of previously obtained V gene sequence data is reveals the most common CDR3 sequences using a simple frequency rank system (Supplementary Fig. 3). In the fifth step, the highest ranking CDR3 sequences are used to construct synthetic mAb genes, which are then cloned into suitable vectors for expression in mammalian and bacterial cells. Finally, in step six, bacterial and mammalian cells are transformed with the recombinant mAb genes (using vectors generated in the previous step) and expressed as either single-chain variable fragments (scFvs) in E. coli or full size recombinant IgG in HEK293F mammalian cell lines. Finally, antigen binding assays are performed to evaluate the binding affinity of recombinant mAbs. The sequence data obtained from VL and VH cDNA library preparations relied on homologies to conserved framework regions to identify the most highly represented CDR3s for each immunized mouse BMPC repertoire. Heavy chain CDRs (CDRH3) were found to be more diverse than light chain CDR3s (CDRL3), most likely as a result of an additional heavy chain gene recombination event (V-D-J vs V-J). In all immunized mice, the top four most highly represented CDRH3 sequences on average represented 10-20% of the total repertoire. Further, to avoid the problem of heavy and light chain pairing, and the process of screening all together, the authors hypothesized that VL and VH sequences from the same mouse that are represented at similar frequencies arise from the same plasma cell. To test this hypothesis, VL and VH sequences were paired in this manner and used as templates for automated gene synthesis. They were then expressed as scFvs in E. coli, and the bacterial cell lysates were analyzed by ELISA. The results indicated that ~78% of scFvs created in this manner were highly antigen specific (21 of 27 antibodies generated from the six immunized mice). High specificity binding was indicated in samples demonstrating more than threefold higher ELISA signal on antigen coated wells compared to wells coated with an unrelated protein antigen. Mouse C1s-2 showed the highest serum antibody titers; antibodies from this animal were selected for biophysical characterization by surface plasmon resonance. Certain pairings of VL and VH genes from this animal generated an IgG (expressed in HEK293F cells) with subnanomolar binding affinities. Conclusion: This series of experiments presents a novel method for mAb production, with results attesting to the full functionality of mAbs created in this manner. The most distinguishing feature of the research strategy is best illustrated in the context of the central dogma of molecular biology. The antibody coding genetic material undergoes several conversions; the most outstanding step in this series is when the physically “real” cDNA is converted into pure information in the form of sequence data. Using computers, this data is analyzed and manipulated with ease, and then used as a schematic for the creation of new biological material in the form of synthetic genes. Put simply, this method converts biological materials into intangible information, and then back again to tangible biological materials. There are several advantages to this approach for mAb production. Firstly, although sophisticated automation, software, and robotics are use in several steps to facilitate sequencing and gene reconstruction, the overall reagent and labor costs are small compared to other methods. Thus, for a dedicated mAb producing facility, the improved efficiency will lower the price of services while helping pay for start-up costs. Secondly, the method could potentially be used in primates, including humans. This would allow for the production of fully humanized, highly specific mAb instead of chimeric recombinant antibodies. Third, this technology may prove valuable for antibody discovery using complex antigens like bacterial and viral pathogens. Lastly, the potential future applications of this technology are virtually endless; the bioinformatic analysis step can be expanded and modified to create custom engineered multivalent antibodies that may be hybrid protein molecules with respect to their function. For example it may be possible to create mAbs with multiple enzymatic properties, and the ability to act as allosteric or direct activators and/or inhibitors of other proteins, DNA, and RNA.