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1.Libraries of synthetic antibodies:
A major advance in oncology over the last decade has been the emergence of monoclonal antibodies as effective therapeutics. Our lab has been involved in developing the latest frontier in antibody therapeutics: synthetic antibody libraries with man-made antigen-binding sites.
|[Fig1 – hybridoma vs. synthetic abs]: Construction of antibody libraries from natural or synthetic diversity. (a) Antibody libraries from natural repertoires are derived by harvesting VH and VL genes from naive B cells. B-cell maturation (1) involves the rearrangement of germline antibody genes in pro-B cells to produce naive B cells that contain diverse, functional antibody genes61. The gene encoding the heavy chain is formed first by the joining of three diversity elements (VH, D and JH), which together encode the variable domain, and a constant element (Cμ), which encodes the constant region of immunoglobulin M (IgM). Subsequently, the gene encoding a κ (shown) or λ light chain is formed by the joining of two diversity elements (Vκ and Jκ, or Vλ and Jλ for λ light chains) that encode the variable domain and a constant segment (Cκ or Cλ) that encodes the constant domain. Gene segments that encode leader sequences (L) direct secretion of both chains. For library construction, mRNA from naive B cells is reverse transcribed to produce cDNA (2). VH and VL repertoires are amplified from the cDNA using PCR (3), and these are combined in a phage-display vector (4) to produce phage-displayed antibody repertoires (5). (b) For the construction of synthetic antibody repertoires, insights from structural and functional analyses of functional antibodies (1) are used to design synthetic oligonucleotides (2) that introduce chemically and spatially defined diversity into the CDR loops (3). The synthetic CDR repertoires are incorporated into defined VH and VL framework genes in phage-display vectors (4) to produce phage-displayed antibody repertoires (5).|
|2. Alternative antibody frameworks:|
Synthetic antibody technology gives the freedom to explore other frameworks for their ability to bind antigens. The full-length antibody framework consists of a constant region (Fc) and a variable region (Fab).
|[Fig2 – structure of IgG and domains]: Crystal structure of a full-length antibody (IgG), a heterotetramer of two heavy chains (yellow) and two light chains (blue). The antigen-binding site (red) is formed by six hypervariable loops (three each from the light and heavy chain) or CDRs. The antigen-binding unit (Fab) can be reduced further to an Fv, consisting of a VL and VH monomer where the two variable domains are linked, producing a stable scFv. The simplest antigen-binding unit is the VH domain, found in natural camelid antibodies. For synthetic antibody library construction, diversity is introduced into the CDR loops, and the remaining regions of the variable domains serve as a framework to maintain the structure of the antigen-binding site.|
|3. Generation of synthetic antibodies by high throughput phage display selections:|
To produce synthetic antibodies, our antibody libraries are displayed on phage screened against a desired target antigen in an in vitro setting. Thus, this technology allows for rapid affinity maturation and specificity optimization of the selected antibodies.
|[Fig3-High throughput flowchart]: Flow chart summary of the high-throughput Fab generation process|
|4. Modulation of cell signaling with synthetic antibodies, and antibodies as potential therapeutics and reagents for biological research:|
Some of the best targets for antibodies are cell surface receptors involved in signal transduction pathways. Several of these pathways are deregulated in cancer and other diseases, and have been the target of chemical drugs for a number of years.
|FIG4-Immunofluorescence staining with Fab-YSv1 (red) performed on A673 cells expressing murine VEGFGFP (green). VEGF-GFP and Fab-YSv1 staining co-localize in the extracellular space formed between cell-to-cell contacts (merge, yellow). The Fab-YSv1 staining was completely abolished bypre-incubation of the antibody with excess recombinant hVEGF (C VEGF panels). The scale bar represents 20 mm. (d) Immunoprecipitations performed on media collected from metabolically labeled A673 cells. Fab-YSv1 and monoclonal antibody A4.6.1 show comparable specificity, as evidenced by identical patterns of bands for precipitated hVEGF isoforms. Anti-GFP polyclonal antibody was used as a negative control|