Chemically Programmed Bispecific Antibodies in Diabody Format

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.

Design and Production of Bispecific Antibodies

“…Adding another cysteine residue to the end of each heavy-chain improves stability by forming a C-terminal disulfide bridge (see Figure 2a) [41]. Compared to a BiTE, DART molecules are able to retain potency for both in vitro and in vivo administration as well but can be produced at scale with lower aggregation rates [21,42]. A recent comparison by Moore et al of the in vitro ability of CD19xCD3 DART and BiTE molecules to kill B-cell lymphoma found that DART molecules outperformed BiTE molecules consistently.…”

Harnessing a catalytic lysine residue for the one-step preparation of homogeneous antibody-drug conjugates

“…The antibody has been used extensively to target antigens on cancer cells after being equipped with a targeting peptide, peptidomimetic, or other small molecule using 1,3-diketone or β-lactam functionalized linkers. A variation of these h38C2-based chemically programmed antibodies 39 are chemically programmed bispecific antibodies that recruit and activate T cells 40 . The highly efficient conjugation reaction only requires an equimolar ratio of compound for complete covalent conjugation and does not modify other Lys residues due to the hydrophobic pocket being selective for hapten-derived compounds.…”

Synthetic Cancer‐Targeting Innate Immune Stimulators Give Insights into Avidity Effects

“…A number of modified antibodies of varying sizes that retain antigen‐binding activity and, in some cases, immune stimulatory activity have been developed. These include nanobodies, minibodies, diabodies, and scFv (single‐chain fragment‐variable) modified antibodies, among others . Bispecific constructs have also been developed; these include CovX‐bodies, in which two peptides targeting the vascular endothelial growth factor (VEGF) and angiopoietin‐2 were fused to an antibody scaffold designed to provide long half‐life and biodistribution, and BiTE (bispecific T‐cell engaging) antibodies, single‐chain bispecific antibody (Bis‐scFv) constructs that target tumor cells and activate T‐cells .…”