This year we have continued to identify novel mAbs in several formats as Fabs, scFvs and eAds against cancer-related proteins. These mAbs were tested for their activity against cancer cells in vitro and some of them were also used as tools for conjugation with nanoliposomes and for development of novel approaches for multiple site-specific conjugations of bioactive molecules to antibody fragments. We have also developed several large libraries of eAds which can be used as a source of binders to various targets. The major accomplishments are summarized below. 1) We have previously proposed to use isolated CH2 domains as scaffolds for construction of libraries containing diverse binders, and identified and characterized a stabilized scaffold containing an additional disulfide bond. This scaffold was further characterized and found that by shortening the N-terminus it could be additionally stabilized. This new scaffold is being explored together with the others for generation of libraries of eAds. A specific binder to the membrane proximal external region of the HIV-1 envelope glycoprotein gp41 was selected and is being characterized. 2) Yeast-displayed libraries of engineered CH2 domains were constructed and are being tested for selection of binders. We are also expressing human neonatal Fc receptor (FcRn) to test binding of some of the engineered CH2 domains with the specific aim to generate nAbs that can bind specifically to a cancer-related protein and simultaneously to the FcRn. 3) Previously, we designed a novel type of eAds library containing CDRs in non-cognate positions where H1 was replaced by a library of human light chain CDR3s (L3s) thus combining three most diversified fragments (L3, H3 and H2) in one VH scaffold. The new library was used for selection of eAds against cancer-related proteins, and the selected binders are being characterized. 5) We have continued to characterize our previously identified human mAbs against DR4 and DR5. We also identified a new mAb against the folate receptor beta. This mAb is highly specific and binds with high avidity to cell-surface associated receptor. Currently it is being tested in a mouse model. 6) We have continued to perform high-throughput sequencing of large portions of the antibody repertoires of humans (the human antibodyome) and analyze large number of sequences. We believe that knowledge of the complete antibodyome will have implications for research, diagnosis, prevention and treatment of cancer. It can help for deeper understanding of the B cell (system) biology and diseases, help develop new diagnostic methods based on individual antibodyomes, help predict individual immune responses to immunization and therapeutics as well as provide information for the design of novel therapeutics. Most implications can not be predicted due to the very nature of an omic science.
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