Abstract No. 129 - Development of a novel bispecific antibody for targeted immunotherapy of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is an ideal target for novel immunotherapies due to its otherwise immunosuppressive intratumoral environment. While the efficacy of bispecific antibodies for immunotherapy is well established for hematological malignancies, their limited circulatory half-lives make them challenging to use for solid tumors. Intratumoral delivery can bypass this issue, potentially making transcatheter-administered bispecific antibody therapy an exciting new paradigm for HCC treatment. We have developed a bispecific antibody that can simultaneously bind to both HCC cells and cytotoxic T cells in order to enhance T-cell mediated tumor lysis. The bispecific antibody targeting the HCC surface antigen glypican-3 (GPC-3) and the T-cell antigen CD-3 was designed as a fusion protein (IDT DNA) and expressed recombinantly in E. coli expression system (Origami Cells) according to previous reports. [1] Specific binding of the antibody against both HCC cells (HepG2) and T cells (Jurkat) was confirmed using cell-surface ELISA assays. [2] Bispecific-triggered activation of T cells (donor CD4 cells) was assessed in vitro using IFN-gamma release ELISA. The bispecific antibody is designed as a 33kD fusion protein consisting of a GPC-3 binding peptide (RLNVGGTYFLTTRQ), previously shown to bind specifically to HCC cells, connected to a CD3-binding single variable fragment domain (ScFV), derived from an OKT3 antibody. [3] The antibody was successfully expressed and purified. It binds to both HCC cells (31.6nM, SD 0.008nM) and T cells (13.2nM, SD 0.006nM) with affinities that are comparable to values reported for other bispecific constructs. [4] The binding to both cell lines were specific and can be inhibited by using excess of either pure GPC-3 binding peptide or full-length OKT3 antibody. The activity of the bispecific antibody is demonstrated by its ability to activate T cells in vitro, resulting in robust IFN-gamma release as measured using ELISA (0.196 Bispecific vs 0.008 Cell Only Control, p = 0.0003). A novel bispecific antibody targeting GPC-3 and CD-3 was successfully synthesized and validated in vitro demonstrating the potential for targeted immunotherapy of HCC.