BCMA CART Therapy Failure in Myeloma Is Associated with Immunosuppressive S100A8/9 Overexpression and Can be Ameliorated with Novel Monoclonal Antibodies

Background Anti-BCMA CAR-T therapy is an effective therapy with high response rates in refractory patients and got recent approvals for earlier lines of therapy in myeloma. However, disease relapses can occur after CAR-T and loss of target antigen (BCMA) only occurs in minority of CAR-T therapy failures; implicating the immunosuppressive environment in disease relapse. Our multimodal CITE-seq data from a cohort of (N=20) CAR-T treated patients (ASH 2023 Abstract #652) as well as others (Dhodapkar Blood Cancer Discovery 2022) show an increase in Myeloid Derived Suppressor Cells (MDSCs) one month after CAR-T infusion in patients that have unfavorable outcomes. Novel targets against MDSCs would be critical for management of patients with CAR-T failure. Results: We analyzed the transcriptome of CAR-T relapse associated MDSCs in primary myeloma patient bone marrows (N=20) and identified S100A8/A9 (alarmin, calprotectin) as highly expressed in MDSCs. Aberrant production of S100A8/A9 by myeloid cells has been shown to promote an immunosuppressive environment in cancer and we observed high levels of S100A8/A9 levels in plasma from bone marrow aspirates in CAR-T recipients using a custom ELISA assay. Next, we evaluated the impact of S100A8/A9 on the activation and function of anti-BCMA CAR-T cells and endogenous T cells in vitro. Using custom spectral flow cytometry panels, we captured the phenotype, cytokine production and antigen specific cytotoxicity of the CAR-T cells in the presence of S100A8/A9 and rescue with anti-S100A8/A9 monoclonal antibodies. Addition of exogenous S100A8/A9 directly reduced CAR-T specific killing of MM1S cells in a dose dependent manner in-vitro (P=0.016). This was associated with reduced CD69+ (activation marker) and cytokine expression (interferon-gamma, IL-2, TNF-alpha, GM-CSF) and increased expression of checkpoint markers, specifically TIGIT and TIM3 in antigen specific CAR-T cells. Additionally, exposure of endogenous T cells to physiological levels of S100A8/A9 lead to a significant increase in their differentiation into IL-10 secreting Treg cells, which further inhibited CAR-T dependent killing of myeloma cells in co-culture assays. S100A8 and S100A9 proteins can form heterodimers (alarmin or calprotectin) that can be potent stimulators of MDSC development. Since there are no potent and specific inhibitors of S100A8/A9 heterodimers, we generated specific monoclonal neutralizing antibodies against the heterodimers. Rabbits were inoculated with human S100A8/9 heterodimer and single B cell cloning yielded multiple monoclonal antibody clones with nanomolar blocking potency that inhibit binding of S100A8/9 to RAGE, TLR4, CD69 and other cognate receptors in vitro. Further, we developed chimeric human monoclonal antibodies that demonstrated potent inhibition of MDSC development from primary human PBMCs in vitro. The monoclonal antibodies also inhibited development of T regulatory cells from primary human PBMCs in chronic stimulation assays. These antibodies were then tested for efficacy in CAR-T cell killing assays using exhausted BCMA directed CAR-T cells. Strikingly, pre-incubation with anti-S100A8/A9 monoclonal antibodies was able to rescue the myeloma cells directed CAR-T cytotoxicity to baseline levels. Conclusions: Taken together, our results indicate that S100A8/A9 is a potential inhibitor of CAR-T cell function. We show that S100A8/A9 decreases CAR-T cytotoxicity directly by inhibiting their activation & cytokine production, and indirectly by inducing differentiation of endogenous T cells into immunosuppressive Tregs. Monoclonal antibodies targeting S100A8/A9 rescue CAR-T cell cytotoxicity and are a novel approach to overcome myeloid derived CAR-T cell resistance with the potential of extending efficacy of cellular therapy for improving long term patient outcomes.