Tumor Reactive T Cells Drive Clinical Responses to Multiple Myeloma

T cell receptors (TCRs) play a pivotal role in mediating anti-tumor responses through the recognition of cancer antigens. Although well-characterized in solid tumors, their function in hematological malignancies is less understood due to the high abundance of virus-specific bone marrow T cells (BMTCs) and technical challenges to deorphanize human TCRs at scale. Additionally, the clinical relevance of T cells with anti-tumor reactivity remains unclear for multiple myeloma (MM). Here, we aimed to chart BMTC specificities and transcriptional profiles in MM patients and assessed their significance in mediating clinical anti-tumor responses.

We collected BM biopsies and blood samples from 62 MM patients. We utilized longitudinal single-cell RNA and V(D)J sequencing to characterize the phenotype and clonality of over 500,000 BMTCs. Additionally, tumor-reactive T cells were identified by screening 187,015 T cells representing 132,501 unique TCR clonotypes for their reactivity against single autologous MM cells using a newly developed microfluidics-based screening method. We deorphanized putative tumor-reactive TCRs using patient-specific peptide libraries of tumor MHC class I and II immunopeptidomes. By synthesizing, cloning, and re-expressing these TCRs, we examined recognition of MM versus healthy patient cells. We further differentiated TCRs that cross-reacted with viral antigens (CMV, EBV, Influenza, SARS-CoV-2) from those that demonstrated bona fide MM-specificity.

We found distinct heterogeneity in patient BMTCs, with specific transcriptional signatures linked to anti-tumor functions as opposed to virus recognition or bystander activity. Deeper profiling of T cells with MM-specificity revealed a transcriptionally unique, ITGB1 (CD29)-expressing T cell subset recognizing cancer-associated antigens (CAAs) or peptides from non-canonical translation products (nuORFs). Additionally, we recurrently identified personalized neoantigens within the immunoglobulin (Ig) hypervariable region of MM clones. T cells targeting such Ig-derived antigens were found in 70% of analyzed patients. Further, a minimal epitope derived from cancer/testis antigen 2 (CTAG2) was recognized by TCRs from multiple MM patients spanning four different HLA supergroups. This data suggests the frequent occurrence of both private neoantigens derived from the hypermutated MM-Ig and public, MHC-promiscuous antigens as targets of endogenous anti-myeloma T cell responses. Surprisingly, the clonal expansion of these tumor-specific TCRs, present in only about 1.2% of assayed BMTCs but detectable in all newly diagnosed MM patients, correlated with enhanced clinical responses.

To further investigate the clinical relevance of our findings, we developed and benchmarked a transcriptional signature of anti-tumor reactivity tailored to the unique transcriptional phenotype of lymphocytes within the bone marrow environment. Applying this signature to two independent patient cohorts, we found that early detection of tumor-reactive TCRs correlates with improved responses to current SoC induction/consolidation treatment with Daratumumab-VTd (HR = 0.32, p < 0.0001) as well as BCMAxCD3 bispecific T cell engager administration (HR = 0.36, p = 0.0005) in relapsed/refractory MM.

To determine if tumor-reactive TCRs are transplanted and persist long-term, we analyzed stem cell grafts from MM patients undergoing autologous stem cell transplantation (ASCT; NCT03617731) and traced individual TCRs up to two years post-ASCT. We found that tumor-reactive TCRs were enriched in these grafts, preferentially transplanted, and maintained long-term persistence, potentially contributing to the therapeutic value of ASCT. Furthermore, the presence of these TCRs in the bone marrow correlated with long-term clinical responses, indicating their role in sustaining anti-myeloma immunity.

Here, we systematically profiled and consistently detected T cells with endogenous anti-tumor reactivity in MM patients. Our data suggest that tumor-reactive TCRs play a critical role in mediating anti-myeloma responses and are influenced by both intrinsic properties and microenvironmental factors. Ongoing studies aim to further elucidate how these TCRs contribute to long-term immune surveillance and therapeutic responses in MM, with potential implications for the development of more effective immunotherapies.