Construction Of Two Bispecific Antibodies For Targeting Both CD20and HLA-DR And The Study Of Their Biological Functions

Rituximab, an anti-CD20antibody, was approved by the US FDA as the first therapeutic monoclonal antibody against B-cell lymphoma. Despite the unprecedented success of Rituximab in treating NHL, a subgroup of patients does not respond, and in patients with initial response early relapses occur, indicating Rituximab resistance. These areas of unmet clinical need highlight the requirement to explore improved treatments for these patients.HLA-DR is highly expressed on a variety of hematological malignancies and has been actively pursued for antibody-based lymphoma therapy. hL243γ1is a humanized IgG1anti-HLA-DR mAb, recognizing a conformational epitope in the a chain of HLA-DR. It has been reported that, in contrast to type I Rituximab-like anti-CD20mAbs, hL243γ1can directly induce more potent cell death.CD20and HLA-DR antigens are highly expressed on a variety of B-cell lymphomas. It has also been shown that CD20and HLA-DR are physically and functionally coupled on B cells. Importantly, the combination of anti-HLA-DR mAb and Rituximab was more effective than Rituximab alone, implying that combination therapy may be more effective in patients.Bispecific antibodies simultaneously bind two different antigens and may have the potential to provide sufficient therapeutic benefit for cancer immunotherapy. BsAbs usually do not occur in nature, but are generated by artificial method. Genetic engineering has led to the development and optimization of various BsAbs.Objective:We aim to develop two genetically engineered BsAbs, CD20-HLA-DR DVD-Ig and CD20-HLA-DR CrossMabCH1-CL for targeting both CD20and HLA-DR, to further improve the efficacy of antibody therapy for B-cell lymphoma. Methods:1. Molecular cloning of CD20-HLA-DR DVD-Ig using Rituximab and hL243yl and construction of light-chain and heavy-chain cDNA of DVD-Ig.2. A pair of constructed heavy chain expression vector and light chain expression vector was co-transfected in FreeStyle293-F cells. DVD-Ig protein from culture medium was purified by Protein A and verified by SDS-PAGE analysis.3. Molecular cloning of CD20-HLA-DR CrossMabCH1-CL using Rituximab and hL243yl and construction or modification of two light chains and two heavy chains of the CrossMab.4. Constructed two heavy chain expression vectors and two light chain expression vectors were co-transfected in FreeStyle293-F cells. CrossMab protein from culture medium was purified by Protein A and verified by SDS-PAGE analysis.5. Cell binding of CD20-HLA-DR DVD-Ig and CD20-HLA-DR CrossMabCH1-CL was assessed in vitro cellular assays by flow cytometry.6. Homotypic adhesion followed by cell death was verified in vitro cellular assays.Results:1. We designed CD20-HLA-DR DVD-Ig for argeting CD20and HLA-DR, and constructed each light chain (CMV/R-IgKappa/CD20-HLA-DR DVD-Ig LC) and heavy chain (CMV/R-IgG1/CD20-HLA-DR DVD-Ig HC) of the DVD-Ig containing two variable domains in tandem through a short peptide linkage.2. CD20-HLA-DR DVD-Ig was produced by transient expression in FreeStyle293-F cells cells and was obtained in high purity via protein A purification.3. Based on the "knobs-into-holes" and "crossover" technology, we designed CD20-HLA-DR CrossMabCH1-CL against CD20and HLA-DR, and constructed or modified four chains as follows:CMV/R-IgG1/HC(1)、CMV/R-IgKappa/LC(1) CMV/R-IgG,/HC(2).CMV/R-IgKappa/LC(2).4. CD20-HLA-DR CrossMabCH1-CL was produced by transient expression in FreeStyle293-F cells cells and was obtained in high purity via protein A purification.5. CD20-HLA-DR DVD-Ig and CD20-HLA-DR CrossMabCH1-CL showed an apparent higher binding avidity than Rituximab and hL243γ1.6. CD20-HLA-DR DVD-Ig and CD20-HLA-DR CrossMabCH1-CL directed to CD20and HLA-DR can elicit homotypic adhesion followed by cell death in B-cell lymphoma.Conclusion:We successfully constructed and characterized two formats of genetically engineered BsAbs, CD20-HLA-DR DVD-Ig and CD20-HLA-DR CrossMabCH1-CL for targeting CD20and HLA-DR. Both of the tow BsAbs exhibited stabilities comparable to natural antibodies. CD20-HLA-DR DVD-Ig and CD20-HLA-DR CrossMabCH1-CL can simultaneously bind CD20and HLA-DR of B-cell lymphoma and elicit homotypic adhesion followed by induction of cell death in B-cell lymphoma. Furthermore, treatment of CD20-HLA-DR DVD-Ig or CD20-HLA-DR CrossMabCH1-CL alone can directly elicit more potent cell death than either Rituximab or hL243γ1alone.