BACKGROUND While Chimeric Antigen Receptor T cell (CAR-T) therapies and bispecific antibodies (bAbs) can lead to remarkable responses in patients with Relapsed/Refractory Multiple Myeloma (RRMM), the disease remains incurable. We hypothesized that patients with Smoldering Multiple Myeloma (SMM) possess a more preserved immune fitness compared to those with RRMM, and might therefore derive greater benefit from immunotherapeutic interventions administered at this earlier disease stage. Indeed, preliminary data from clinical trials of patients with high-risk SMM (HRSMM) have demonstrated unprecedented responses to immunotherapy with Teclistamab (TEC) and Ciltacabtagene autoleucel (cilta) supporting this hypothesis (Nadeem et al, ASH 2023 and 2024). However, no prior studies have systematically characterized immune cell fitness before and after T cell-redirecting therapies across patients with distinct disease stages (SMM vs. RRMM). Here, we present results from a single-cell sequencing study of immune responses to cilta and TEC in asymptomatic patients with HRSMM compared to those with RRMM.

METHODS We performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on a total of 123 CD138-neg samples (peripheral blood, PB, n=105; bone marrow, BM, n=18) from patients with HRSMM (n=30, cilta n=6; TEC, n=24) and relapsed/refractory MM (RRMM, n=27, cilta n =19; TEC, n=8). Patients with HRSMM were enrolled in the Phase II CAR-PRISM (NCT05767359) and Immuno-PRISM (NCT05469893) studies, whereas patients with RRMM received cilta or TEC as standard of care. Publicly available scRNA/TCR-seq datasets of healthy donor PB (n=10), and PB (n=19) or BM (n=24) from patients with RRMM who received BCMA-targeted T cell-redirecting therapies were also integrated into the final dataset. A total of 122,783 and 126,830 T cells were analyzed for the CAR-T and bAbs cohorts, and CAR-T cells were isolated in silico based on the smoothed-expression of construct transcripts. Day28 (D28) post-CAR-T data were used to compare patients with HRSMM and RRMM, as this was the earliest post-treatment available timepoint for the latter.

RESULTS At D28, patients with HRSMM (n=6) treated with cilta showed higher frequency of CAR-T cells with a CD4+ phenotype expressing high levels of stemness genes (TCF7, LEF1) (q=0.026), and lower frequency of exhausted (q=0.036) and cytotoxic CD8+ phenotypes (q=0.007), compared to patients with RRMM (n=14). Patients with HRSMM, who showed increased TCR repertoire diversity at baseline compared to RRMM (p=0.02), also showed more polyclonal CAR-T cells at D28. Furthermore, CAR-T cells from patients with HRSMM showed increased expression of T cell activation genes (CXCR4, CD69) at D28 compared to RRMM. In contrast, CAR-T cells from patients with RRMM expressed higher levels of genes related to cytotoxic differentiation (EOMES, TBX21, GZMB) and exhaustion (LAG3, TIGIT, HAVCR2), suggesting a more differentiated, and potentially less functional, profile. Notably, most of the transcriptional differences observed at D28 between CAR-T cells from patients with HRSMM and RRMM mirrored those observed in pre-treatment endogenous T cells, indicating that baseline T cell states are a key determinant of CAR-T cell fate. Nevertheless, only patients with HRSMM exhibited significant upregulation of a T cell activation signature in CAR-T cells at D28 compared to baseline (q=0.07). Additionally, patients with HRSMM showed greater expansion of CD14+ monocytes and increased activation of endogenous T cells post-treatment with CAR-T cells compared to RRMM. Similarly, patients with HRSMM treated with TEC exhibited a significant post-treatment increase in activated CD8⁺ T cells frequency (q=0.028), T cell clonality (p=0.03), and T cell activation signatures expression (q<0.05) in PB at the time of Measurable Residual Disease negativity, while patients with RRMM displayed a more heterogeneous immunological profile.

CONCLUSION Our study reveals that patients with HRSMM exhibit enhanced immune responses to both CAR-T cells and bAbs compared to patients with RRMM. In patients with HRSMM, CAR-T cells exhibit higher stemness, polyclonality, and CD4+ predominance, while TEC-treated T cells show stronger induction of activation and clonal expansion, compared to those with RRMM. In conclusion, these results suggest that the greater pre-treatment immune capacity in patients with HRSMM favorably shapes immune responses to T cell-redirecting therapies.

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2025
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