Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy

Maciej Kujawski,John E Shively,Wen-Hui Lee,Lindsay Williams,Nagarajan Vaidehi,Nicole Bowles,Harry Li,Junie Chea,Patty Wong,Kofi Poku,Lin Li,Paul Yazaki,Supriyo Bhattacharya

doi:10.1186/s12885-019-6056-8

Maciej Kujawski, John E Shively + Show 11 more

Open Access

https://doi.org/10.1186/s12885-019-6056-8

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Abstract

BackgroundBispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance.MethodsAn intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy.ResultsThe interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice.ConclusiondbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Highlights

Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen single chain Fv (scFv) fragments, are important therapeutic agents for the treatment of cancer
Generation of dbBiTES Building on the approach developed by Lum et al [4] who randomly cross-linked two intact antibodies using Traut’s reagent and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate to redirect T-cell therapy to tumor targets, we first generated bispecific antibodies by cross-linking two intact antibodies at their hinge regions using Click chemistry (Scheme 1)
To distinguish the product from conventional BiTEs built from monovalent single chain Fv fragments, we suggest the name Dual binding bivalent bispecific T-cell engager (dbBiTE), for dual specific bivalent BiTE

Summary

Introduction

Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. In spite of the high heterogeneity of the resulting bispecific antibodies, advantages of the approach were that only microgram amounts of antibody were required for coating the T-cells and the therapy was considered a cell-based therapy by the FDA since it involved infusion of T-cells and not antibody infusion. This approach used clinically tested humanized antibodies as a starting point, avoiding

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