Overcoming Tumor Heterogeneity by Ex Vivo Arming of T Cells Using Multiple Bispecific Antibodies

Abstract

PurposeTumoral heterogeneity is a hallmark of tumor evolution and cancer progression, being a longstanding challenge to targeted immunotherapy. Ex vivo armed T cells (EATs) using IgG-[L]- scFv bispecific antibodies (BsAbs) are potent tumor-specific cytotoxic effectors. To improve the anti-tumor efficacy of EATs against heterogeneous solid tumors, we explored multi-antigen targeting approaches. MethodsEx vivo expanded T cells were armed with BsAbs built on the IgG-[L]-scFv platform, where an anti-CD3 (huOKT3) scFv was attached to the carboxyl end of both light chains of a tumor specific IgG. Multispecificity was created by combining monospecific EATs, combining BsAbs on the same T cell, or combining specificities on the same antibody. Three multi-antigens targeting EAT strategies were tested: (1) pooled EATs (simultaneous combination of monospecific EATs or alternate EATs (alternating combination of monospecific EATs), (2) dual-EATs or multi- EATs (T cells simultaneously armed with [≥] 2 BsAbs), and (3) TriAb-EATs [T cells armed with BsAb specific for two tumor targets besides CD3 (TriAb)]. The properties and efficiencies of these 3 strategies were evaluated by flow cytometry, in vitro cytotoxicity, cytokine release assays, and in vivo studies performed in BALB-Rag2-/-IL-2R-{gamma}c-KO (BRG) mice xenografted with cancer cell line (CDX) or patient-derived tumor (PDX). ResultsMulti-EATs retained target antigen specificity and anti-tumor potency. Cytokine release with multi-EATs in the presence of tumor cells was substantially less than when multiple BsAbs were mixed with unarmed T cells. When tested against CDXs or PDXs, dual- or multi-EATs effectively suppressed tumor growth without clinical toxicities. Most importantly, dual- or multi- EATs were highly efficient in preventing clonal escape while mono- or TriAb- EATs were not as efficient. ConclusionArming T cells with multiple BsAbs enabled multi-specific T cell immunotherapy which overcomes tumor heterogeneity without excessive cytokine release.