T Cell Engagement without Cytokine Storm: A Novel Bcma x CD3 Antibody Killing Myeloma Cells with Minimal Cytokine Secretion

Introduction

Multiple myeloma (MM) is a plasma cell malignancy with an annual incidence of ~25,000. Improvements in standard of care have extended median survival to greater than 5 years; however, disease refractory to bortezomib and lenalidomide is still associated with a median life expectancy of less than 9 months. Novel therapies are critical to the treatment of these patients. Chimeric antigen receptor T cells (CAR-Ts) targeting B-cell maturation antigen (BCMA) -a protein found exclusively on the surface of plasma cells- have shown efficacy against relapsed/refractory MM in early phase clinical trials. However, toxicity from over-activation of T-cells and large-scale production of CAR-Ts still hinder this approach. Bispecific T-cell engaging antibodies redirecting T cells to BCMA circumvent the latter problem but to date have shown similar T-cell over-activation. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline using in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αBCMA HCA and a library of αCD3 Flic antibodies that show a >2 log range of EC50s for T cell activation in vitro . Of note, the library contained a low-activating αCD3 that induced T-cell dependent lysis of MM cells (when paired with an αBCMA HCA) with minimal cytokine secretion. To characterize the relative efficacy and potential therapeutic window of this unique molecule, we combined both a high- (TNB-384B) and the low-activating (TNB-383B) αCD3 moiety with a high-affinity bivalent αBCMA arm (αBCMA x αBCMA x CD3) and compared these constructs in vitro, in vivo, and ex vivo for T-cell activation, efficacy in killing MM cells, and toxicity.

Methods

T-cell activation was measured via flow cytometry (CD69 expression) and cytokine ELISA (IL-2, INF-ɣ, TNFα, among others) in vitro. Lysis of MM cell lines (RPMI-8226, U266, and H929) was measured via flow cytometry in vitro . Ex vivo, survival of patient MM cells was measured via flow cytometry after incubation with TNB-383B, TNB-384B, or negative control antibody (NCA) with or without supplemental exogenous activated T cells. In vivo, NSG mice were engrafted with RPMI-8226 MM cells and human peripheral blood mononuclear cells (huPBMC), and the mice treated with TNB-383B, TNB-384B, or NCA. Tumor burden was evaluated via Bioluminescent Imaging (BLI). Pharmacokinetic (PK) studies were performed in mice and Cynomolgus monkeys using ELISA.

Results

Both TNB-383B and TNB-384B bound to cell surface BCMA with nanomolar affinity. EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-383B and single-digit picomolar for TNB-384B against MM cell lines in vitro . TNB-383B showed markedly reduced (ie IFN-ɣ) or absent (ie IL-2) cytokine release compared to TNB-384B even at saturating concentrations (Figure 1); these findings were consistent across 10 healthy huPBMC donors. Ex vivo, both TNB-383B and TNB-384B efficiently lysed primary MM cells in the presence and absence of supplementary T-cells. In vivo, both TNB-383B and TNB-384B mediated clearance of established MM tumor from NSG mice at doses as low as 10ng q1w x3 in the presence of huPBMC. PK profiles of both molecules were consistent with those of an IgG in mice (T_1/2 ~5.5 days) and Cyno (T_1/2 ~8.5 days). No activation of T-cells was observed in vitro (Figure 1) or in vivo in the absence of BCMA expressing target cells.

Conclusions

Both low-activating TNB-383B and high-activating TNB-384B αBCMA x αBCMA x αCD3 bispecific antibodies kill multiple myeloma cells in vitro and in vivo in a BCMA-dependent manner, and kill primary patient myeloma cells ex vivo . While TNB-384B showed T-cell activation comparable to other CD3-engaging bispecifics, TNB-383B induced significantly reduced cytokine secretion by T-cells without appreciable reduction of efficacy in vivo or ex vivo. In summary, both TNB-383B and TNB-384B show promise as MM therapeutics differentiated from current T-cell targeted therapies in clinical trials.

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