Chemically Programmed Bispecific Antibodies in Diabody Format

Even Walseng,Christopher G Nelson,Junpeng Qi,Alex R Nanna,William R Roush,Rajib K Goswami,Subhash.C Sinha,Terrence R Burke,Christoph Rader

doi:10.1074/jbc.m116.745588

Abstract

Chemically programmed bispecific antibodies (biAbs) endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system. Here we report a platform of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells. Two different antibody technologies were merged together to make a novel chemically programmed biAb. This was achieved by combining the humanized anti-hapten monoclonal antibody (mAb) h38C2 with the humanized anti-human CD3 mAb v9 in a clinically investigated diabody format known as Dual-Affinity Re-Targeting (DART). We show that h38C2 × v9 DARTs can readily be equipped with tumor-targeting hapten-derivatized small molecules without causing a systemic response harming healthy tissues. As a proof of concept, we chemically programmed h38C2 × v9 with hapten-folate and demonstrated its selectivity and potency against folate receptor 1 (FOLR1)-expressing ovarian cancer cells in vitro and in vivo Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy.

Highlights

Programmed bispecific antibodies endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system
BiAbs in Dual-Affinity Re-Targeting (DART) format are comprised of two polypeptides that are linked at their C termini via a disulfide bridge, where each polypeptide contains one of two cognate variable light and heavy chain domains that form the antigen or hapten binding site [25] (Fig. 1A)
Cognate plasmid pairs encoding the h38C2 ϫ v9 DARTs hv-L and hv-H were transiently co-transfected into human embryonic kidney (HEK) 293 cells and the DARTs were purified from concentrated supernatants by immobilized metal ion affinity chromatography (IMAC) followed by size exclusion chromatography (SEC)

Summary

Chemically Programmed Bispecific Antibodies

Favorable features of the BiTE format are attributed to: (i) its small size (ϳ50 kDa), which brings target and effector cells into close proximity to enable cytolytic synapses; and (ii) the monovalent engagement of the T-cell receptor (TCR) complex, which prevents systemic activation of effector cells in the absence of target cells [22]. Folate was used as a representative tumor-cell targeting small molecule as FOLR1 is a clinically investigated target for both mAbs and small molecules in cancer therapy in general and in the treatment of ovarian cancer in particular [27,28,29]. Both chemically programmed and conventional FOLR1 ϫ CD3 DARTs selectively and potently killed FOLR1-expressing human ovarian cancer cell lines in vitro and in vivo. Our findings support the notion of broad therapeutic utility of chemically programmed biAbs at the interface of immunology and chemistry

Results

Fluorescence intensity

Discussion

Blue fluorescence

Experimental Procedures