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CD3-engaging bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells are potent therapeutic approaches for redirecting patient T cells to recognize and kill tumors. Here we describe a fully human bsAb (REGN5458) that binds to B-cell maturation antigen (BCMA) and CD3, and compare its antitumor activities vs those of anti-BCMA CAR T cells to identify differences in efficacy and mechanism of action. In vitro, BCMAxCD3 bsAb efficiently induced polyclonal T-cell killing of primary human plasma cells and multiple myeloma (MM) cell lines expressing a range of BCMA cell surface densities. In vivo, BCMAxCD3 bsAb suppressed the growth of human MM tumors in murine xenogeneic models and showed potent combinatorial efficacy with programmed cell death protein 1 blockade. BCMAxCD3 bsAb administration to cynomolgus monkeys was well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines. The antitumor efficacy of BCMAxCD3 bsAb was compared with BCMA-specific CAR T cells containing a BCMA-binding single-chain variable fragment derived from REGN5458. Both BCMAxCD3 bsAb and anti-BCMA CAR T cells showed similar targeted cytotoxicity of MM cell lines and primary MM cells in vitro. In head-to-head in vivo studies, BCMAxCD3 bsAb rapidly cleared established systemic MM tumors, whereas CAR T cells cleared tumors with slower kinetics. Thus, using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects.

The Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality.A rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum.BM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation.Quantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates.

Improving therapies for multiple myeloma (MM) remains a high medical need because of the significant morbidity and mortality of the disease. Targeted immunotherapies represent a promising opportunity to fill this clinical need. B cell maturation antigen (BCMA) is an attractive cell-surface target for MM due to its consistent expression on MM patient malignant plasma cells and expression limited in normal tissue primarily to plasma cells. Redirection of a patient's T cells to recognize tumors by CD3-binding bispecific molecules or through the generation of chimeric antigen receptor (CAR) T cells, has shown preliminary evidence of clinical activity. Bispecific antibodies concurrently engage a tumor antigen on cancer cells and the CD3 signaling machinery on T cells, bringing the tumor cell and T cell into proximity and facilitating T cell activation and tumor cell killing. By contrast, CAR T cell therapy involves re-infusion of the patient's own T cells after ex vivo engineering to express CARs targeting tumor antigens and triggering T cell signaling. Here we describe the generation of REGN5458, a human bispecific antibody that binds to BCMA and CD3. In vitro, REGN5458 efficiently activates T cells and induces polyclonal T cell killing of myeloma cell lines with a range of BCMA cell-surface densities, and also induces cytotoxicity of primary human plasma cells. Similar to gamma-sectretase inhibitors, incubation of myeloma cell lines with REGN5458 increased surface levels of BCMA. In xenogenic studies, after BCMAhigh NCI-H929 and BCMAlow MOLP-8 MM cells were co-implanted with PBMC and grown subcutaneously in immunodeficient NOD/SCID/L2Rgamma-deficient (NSG) mice, REGN5458 doses as low as 0.4 mg/kg significantly suppressed the growth of both tumors. Using aggressive, systemic xenogenic tumor models, in which NSG mice were engrafted with PBMC and intravenously injected with BCMAhigh OPM-2 cells or BCMAlow MOLP-8 cells expressing luciferase, REGN5458 reduced tumor burden and suppressed tumor growth at doses as low as 0.4 mg/kg. In immunocompetent mice genetically engineered to express human CD3, REGN5458 inhibited the growth of syngeneic murine tumors expressing human BCMA at doses as low as 0.04 mg/kg. Finally, as REGN5458 binds to cynomolgus CD3 and BCMA and mediates cytotoxicity of primary cynomolgus plasma cells, the pharmacology of REGN5458 was evaluated in cynomolgus monkeys. REGN5458 administration was well-tolerated, resulting in a mild inflammatory response characterized by transiently increased CRP and serum cytokines. Importantly, REGN5458 treatment led to the depletion of BCMA+ plasma cells in the bone marrow, demonstrating cytotoxic activity in non-human primates. The anti-tumor efficacy of REGN5458 was compared to BCMA-specific CAR T cells using 2nd generation CAR lentiviral constructs containing a single-chain variable fragment binding domain from REGN5458's BCMA binding arm and 4-1BB and CD3z signaling domains. Human PBMC-derived T cells were transduced to express this CAR and expanded. Both REGN5458 and the BCMA CAR T cells demonstrated similar targeted cytotoxicity of myeloma cell lines and primary patient blasts in vitro, and were capable of clearing established systemic OPM-2-luciferase myeloma tumors in NSG mice, but with different kinetics: treatment with REGN5458 resulted in rapid clearance of tumors within 4 days, whereas treatment with BCMA CAR T cells allowed tumors to continue to grow for 10-14 days following injection before rapidly inducing tumor clearance. Thus, REGN5458 exerts its therapeutic effect rapidly after injection, using effector T cells that are already in place. In contrast, BCMA CAR T cells require time to traffic to the tumor site and expand, before exerting anti-tumor effects. Collectively, these data demonstrate the potent pre-clinical anti-tumor activity of REGN5458 that is comparable to that of CAR T cells, and provide a strong rationale for clinical testing of REGN5458 in patients with MM. Disclosures Dilillo: Regeneron Pharmaceuticals: Employment. Olson:Regeneron Pharmaceuticals: Employment. Mohrs:Regeneron Pharmaceuticals: Employment. Meagher:Regeneron Pharmaceuticals: Employment. Bray:Regeneron Pharmaceuticals: Employment. Sineshchekova:Regeneron Pharmaceuticals: Employment. Startz:Regeneron Pharmaceuticals: Employment. Retter:Regeneron Pharmaceuticals: Employment. Godin:Regeneron Pharmaceuticals: Employment. Delfino:Regeneron Pharmaceuticals: Employment. Lin:Regeneron Pharmaceuticals: Employment. Smith:Regeneron Pharmaceuticals: Employment. Thurston:Regeneron Pharmaceuticals: Employment. Kirshner:Regeneron Pharmaceuticals: Employment.