Our research focuses on the generation of recombinant antibodies, mostly fully human, that can be used in the diagnosis and treatment of cancers and other human diseases, or to capture or neutralize toxins. We create antibody display libraries in single-chain variable fragment domain format (scFv) or fragment antigen-binding domain (Fab) format and express them in phage or yeast display platforms. Lead antibody candidates are isolated from these libraries by successive rounds of selection against natural or recombinant proteins or other soluble target antigens, or on specific cell lines. Selected antibody leads are expressed as fragments, full-length immunoglobulin, or in a designed molecular format via mammalian cell line or other protein expression systems for further in-vivo and in-vitro characterization. Our Research We create antibody display libraries by obtaining immunoglobulin (Ig) heavy and light chain variable region genes from mRNA that was prepared from human volunteers or immunized animals, or using chemically synthesized genes. These genes are cloned into phage display or yeast display vectors for antibody fragment display, either on the surface of phage or on yeast. Yeast surface display is also used for the construction and display of antigen cDNA libraries constructed from target cell lines, and for display of recombinant antigens to be targeted by antibodies. The cDNA libraries are used for identification of antigens bound by antibodies that have been selected on tumor cell lines. Recombinant target antigens displayed on yeast include the extracellular domains of receptor tyrosine kinases, Fc receptors, engineered integrin domains, and nontoxic domains of protein toxins, which are used for antibody selection and mapping the epitopes of the selected antibodies. Recombinant scFv and Fab antibody fragments are expressed in E. coli K12 strains containing bacterial expression plasmids, and then purified from culture. To make full-length antibody, Ig genes are cloned into a mammalian expression vector where expression is driven by the CMV promoter. Vector DNA is transfected into CHO cells for stable expression, or COS7 or HEK293 cells for transient expression. Cloning and mammalian expression is also done for some cell surface proteins used as antigens for antibody selection, including integrin avb8 and TGF beta, extracellular domains of the oncogenic receptor tyrosine kinases (RTKs) such as EGFR, ErbB2, ErbB3, ErbB4, and EpHA2. In vitro characterization of selected antibody clones is performed by assays against their targets, including FACS, ELISA, surface plasmon resonance, quartz crystal microbalance, kinetic exclusion assay (KinExA), meso-scale electrochemiluminescence (MSD), and EM (electron microscopy). Antibody fragments are also crystallized in complex with their targets for structural analysis.
