Developing SEMA4A-directed CAR t cells to overcome low BCMA antigen density in multiple myeloma

Abstract

Abstract Chimeric antigen receptor (CAR) T cell therapies targeting B cell maturation antigen (BCMA) have shown unprecedented activity in patients with relapsed/refractory multiple myeloma (RRMM). Despite initial response, most patients have short disease remissions and commonly relapse. Reduced target antigen expression on tumor cells following CAR therapy is emerging as a critical mechanism contributing to relapse. We quantitatively profile target antigen density in a unique cohort of 41 primary bone marrow (BM) samples from patients who relapsed after BCMA CAR T cell therapy and demonstrate that relapsed multiple myeloma (MM) is preferentially BCMA low. We and others have identified SEMA4A as a promising target in MM (Di Meo et al., Cell Rep Med 2023). Here, we show that SEMA4A is highly expressed in MM patients (n=829 mol/cell and n=832 mol/cell), including those with p53 mutant disease. In patients who relapse after BCMA CAR T cell therapy, the SEMA4A median number of molecules per cell averages 14,000, ensuring optimal CAR T cell activation, whereas BCMA density (avg= 849 mol/cell) is below the level required to trigger CAR T cell activation. No significant correlation in RNA expression is found between SEMA4A and BCMA. Survival analyses in 767 newly diagnosed MM patients suggest that high levels of SEMA4A are associated with worse outcomes. We genetically ablate SEMA4A expression with an inducible CRISPR/Cas9 system in KMS11, U266, OPM2, and AMO.1 MM cell lines.SEMA4AKO MM cells display decreased proliferation and impaired migration compared to controls. In vivo, SEMA4A loss significantly extends the survival of immunocompromised NSG mice bearing human MM cells, reduces MM growth, tissue infiltration and osteoclast formation, suggesting further therapeutic potential of targeting SEMA4A in MM. We generate novel monoclonal antibodies through the immunization of ATX-GXTM mice. 8 antibodies with the best binding profiles are used for CAR construction. SEMA4A-directed CAR T cells specifically eliminate SEMA4A+ cells – one single chain Fragment variant (scFv) leads to the highest (80%) killing activity in vitro and longest (>120 days) mice survival in NSG mice inoculated with MM KMS11 cells. In normal tissues, SEMA4A presents a more favorable profile compared to other immunotherapeutic targets in clinical use. Experimentally, we perform multiplex immunofluorescence analyses followed by confocal microscopy on 17 normal tissues including brain, heart, lung, skin, esophagus, stomach, ovary, kidney and liver. The SEMA4A antibody used for CAR construction does not bind to these normal tissues. Further, we perform flow-cytometric analyses on normal tissues such as CD45- EpCAM+ lung cells demonstrating lack of binding. Antigen density is much higher in MM cells (median 15,000 mol/cell) compared to all normal tissues analyzed (e.g, lung: median 350 mol/cell; skin median 200mol/cell; normal monocytes: 7,251 median mol/cells). Single-cell RNA-seq analyses from >30,000 bone marrow cells and 5 healthy donors show SEMA4A expression at low levels in <25% of myeloid cells. To assess potential toxicity, we develop humanized MM-engrafted mice receiving BCMA or SEMA4A CAR T cells. No statistically significant decrease in CD14+ monocytes compared to controls is observed, indicating that the high anti-myeloma efficacy of SEMA4A CAR T cells occurs without any additional hematopoietic progenitor toxicity than a clinically validated CAR. Finally, we evaluate the efficacy of SEMA4A CAR T cells in BCMAlow MM by creating a panel of H929 MM cells bearing different levels of BCMA, resembling the ones observed in patients. SEMA4A CAR T cells show superior efficacy to BCMA CAR T cells when BCMA levels are low. They effectively kill BM CD138+ cells from patients with BCMAlow MM. Lastly, we infuse sub-curative doses of BCMA CAR T cells into NSG mice injected with MM cells. In this setting of relapse-prone mice, the BCMA number of molecules in progressing H929 MM cells is reduced compared to untreated mice, and treatment with SEMA4A CAR T cells controls the disease up to day 71 post-injection and prolongs mice survival. Our study suggests that low BCMA antigen density mediates resistance to BCMA CAR therapy and, it sets the rationale for developing a SEMA4A-directed CAR T cell therapy in MM patients.