BCMA Mutational Screening Reveals Differential Cytotoxic Activity between T-Cell Engagers in Preclinical Co-Culture Models

Background: Multiple Myeloma (MM) is an incurable hematological malignancy with treatments comprising combination regimens, typically featuring immunomodulatory agents (IMiDs) due to their potent dual mechanism of direct anti-tumor activity and immunomodulatory effects. The introduction of immune-based therapies such as targeted bispecific antibodies and CAR T-cell therapy, has led to significant improvement in long-term patient survival with R/R disease. B-cell maturation antigen (BCMA) is one of the key immunotherapeutic targets in MM. MM patients often relapse after BCMA-directed therapies and tumor-intrinsic factors such as, truncations and point mutations on BCMA have been identified as resistance mechanisms. This study expands the list of potential mutations leading to T cell engager (TCE) resistance and a comparison of the preclinical efficacy of three TCEs in a co-culture model of cells expressing these mutant BCMA variants.

Methods: An alanine mutation screen was utilized to generate point mutations in each extracellular BCMA amino acid, and 53 stable K562 cell lines were generated, each expressing a BCMA mutant or the wild-type sequence. Co-culture assays were performed to test the efficacy of three T-cell engagers, teclistamab, elranatamab, and alnuctumab. T-cells were isolated from a healthy donor for the co-culture assay. Incucyte cytotox red dye was utilized to measure cell death in the co-culture assay over a period of 96 hours.

Results: Across the panel of 53 cell lines, we observed altered surface expression (greater than +/- two-fold change) of BCMA by flow cytometry in 26 cell lines, where some mutants were more highly expressed (as high as ~3 fold), while others had nearly undetectable BCMA surface levels. Co-culture assays were performed in duplicates at a target:effector ratio of 1:1, with increasing concentrations of the TCEs and the cellular cytotoxicity was measured. With the WT BCMA, alnuctamab and elranatamab induced cytotoxicity at a lower concentration (elranatamab: 10ng/ml, alnuctamab: 0.1ng/ml) in comparison to teclistamab (1000ng/ml). Some of the cell lines where surface BCMA expression was drastically reduced were minimally responsive to TCE-mediated cell death (n=5). Confirming prior literature, positive control R27P did not respond to teclistamab and elranatamab whereas alnuctumab retained some cytotoxic potential. Apart from R27P, we identified 15 novel mutants that had unique specificity towards one or more of the TCEs. For instance, L17A retained sensitivity to elranatamab only. Overall, elranatamab and alnuctamab sustained sensitivity to 90% of the mutants and teclistamab to 85% of the mutants.

Conclusion: Taken together, these data identified that point mutations in BCMA can alter the surface expression of BCMA and these mechanisms are under active investigation. In the context of a WT expressing system, elranatamab and alnuctamab were more sensitive in inducing T-cell mediated killing in comparison to teclistamab. Finally, we identified novel mutations which can abrogate TCE activity, however most of the mutants tested in our system demonstrated sustained sensitivity to TCEs albeit the extent varied between each TCE with elranatamab and alnuctamab appearing to have broader activity compared to teclistamab.