Abstract 5658: MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies

Abstract

Abstract The generation of bispecific molecules, in which two target-specific moieties are engineered into a single entity, offers an attractive approach to optimize biological activity which may improve clinical outcomes. Among various formats of bispecific molecules, the heterodimeric Fc currently is a favorable scaffold for the design of bispecific antibodies because of its structural similarity to the natural antibody. However, development of molecules based on this concept is challenged by the presence of potential homodimer contamination, reduced yield, and stability loss relative to the natural Fc. Here, we report the development of the MG-Ig platform by grafting a single chain fragment variable (scFv) onto the hinge region of a full-size immunoglobulin with a different specificity to produce a trivalent IgG-like bispecific antibody that retains natural homodimer Fc. Whereas the MG-Ig platform is evolved from the Dock-and-Lock (DNL) technology which exploits the interaction between the anchoring domain (AD) of A kinase anchor proteins and the dimerization and docking domain (DDD) of regulatory subunit of cAMP-dependent protein kinase, the main distinctions include 1) the AD-fused scFv monomer is judiciously designed to graft onto the DDD dimer inserted into the hinge region of IgG, forming a bispecific antibody with three (1+2) binding arms and 2) all modular subunits are expressed in a single host cell and self-assemble to generate the bioactive conjugate in situ. To validate the method, bispecific T-cell engagers, designated 3scFv × hL0125C (CD3 × Trop2) and 3scFv × hACD20C (CD3 × CD20), which redirect T cells to Trop2 and CD20, respectively, have been produced with high yield and purity. Flow cytometry analysis demonstrated the bispecific binding of 3scFv × hL0125C to both CD3 on Jurkat and Trop2 on ME-180 with unaltered affinities. These T-cell redirecting bispecific antibodies mediated dose-dependent killing of target cancer cells when co-incubated with human PBMCs. Based on the 60-h viability assays using MTS, the IC50 of 3scFv × hL0125C was about 6 pM for MDA-MB-468, 13 pM for MDA-MB-231, 20 pM for BT-474, and 18 pM for HCT116. In 3D culture of HCT116 cells, the formation of multicellular tumor spheroids was impeded by 3scFv × hL0125C at 19.5 pM or higher, which is consistent with the cytotoxicity assays for tumor cells grown in monolayer. In addition, 3scFv × hL0125C was stable in human plasma for 7 days at 37°C, retaining bispecific integrity and binding activity. These data are promising and merit further development of MG-Ig bispecific antibodies for preclinical and clinical studies. Citation Format: Donglin Liu, Li Zeng, Berenice E. Liu, Chien-Hsing Chang. MG-Ig (molecularly grafted immunoglobulin): An alternative platform for production of bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5658.