Multiple Myeloma Persister Cells Surviving Bispecific Antibodies and CAR-T Cells Exhibit New CD45+ Expression

Introduction: Multiple myeloma (MM) is a largely incurable disease characterized by periods of therapeutic response, remission, and relapse. To extend MM patient survival, several T cell redirection therapies (TCRTs), including chimeric antigen receptor T cells (CAR-Ts) and bispecific antibodies (bsAbs) have been FDA-approved. However, most patients ultimately relapse after TCRTs. Therefore, characterization of post-TCRT myeloma is critical to develop therapies for the growing post-TCRT population. The typical clinical MM cell phenotype exhibits loss of CD45, but here we show the novel finding that MM persisting and relapsing after TCRTs upregulates CD45. This is particularly interesting in light of recent research showing CD45 expression allows circulating tumor cells to resist T cell attack (Yang et al, Signal Transduct Target Ther. 2024).

Methods: Bone marrow aspirates were obtained from MM patients with informed consent and approval of the Institutional Review Board. Myeloma Drug Sensitivity Testing (My-DST) was performed as previously described using 1nM anti-BCMA bsAbs elranatamab and teclistamab, anti-GPRC5D talquetamab, and anti-CD38 SAR442257 (Walker et al, Blood Adv. 2020). Persister MM cell phenotype surviving bsAb treatment was analyzed via flow cytometry. Phenotypes observed in bsAb ex vivo culture were validated using MM primary specimens before and after TCRTs. To determine if CD45 upregulation was mediated by a secreted factor, transwell plates with 0.4 µm pores were seeded with 1:1 PBMCs and H929 MM cells with SAR442257 and H929s alone in the insert. Transwells were incubated for 48 h and both the insert and base cells were separated and analyzed via flow cytometry. Imaging flow cytometry was used to visualize CD45 expression after T cell stimulation by Dynabeads.

Results: While investigating therapeutic response to TCRT in bone marrow using My-DST, we discovered that CD38+CD138+ MM cells persisting after 48 hours of treatment with anti-BCMA or anti-CD38 bsAb-therapy exhibited new surface expression of CD45 that was not present on untreated controls. Identical results were observed in MM cells persisting in culture with anti-BCMA CAR-T cells. Primary MM persisting after treatment with SAR442257 expressed significantly more CD45 in 11/17 (64.7%) sensitive samples and only 1/9 (11%) resistant samples. Similarly, primary MM persisting after treatment with elranatamab expressed significantly more CD45 in 4/7 (57%) sensitive samples and 0/5 (0%) resistant samples. Also, MM persisting after teclistamab and talquetamab expressed significantly more CD45 in 2/3 (67%) and 1/2 (50%) sensitive samples and 0/3 (0%) and 0/3 (0%) resistant samples respectively. To determine if this phenomenon was conserved in patients treated with a variety of TCRTs, we investigated CD45 in primary aspirates before initiation of BCMA CAR-T and following relapse. An average of 28.9% of pre-CAR-T MM cells were CD45+ compared to 72.1% of post-CAR-T MM cells (p-value <0.0001). All patient samples with paired pre- and post- timepoints (n=3) had a significant increase in percent of MM cells CD45+ post-CAR-T. This phenomenon was further characterized using H929s cultured with PBMCs. Indirect contact in transwell coculture with PBMCs, H929s, and bsAbs significantly increased CD45 on H929 cells. Size-based fractionation revealed the CD45-inducing factor to be <50 kDa. Stimulated T cells alone were found sufficient to induce CD45 on H929s after 72 h culture. Imaging flow cytometry revealed singular focal CD45 patches of expression on the MM cell surface, and confirmed this observation is not due to T cell-MM doublets. Similar to the above observations in patients, MM cells cultured with unstimulated T cells averaged 18% CD45+ vs. 79% with stimulated T cells. Bulk-RNA-Seq confirmed the gene for CD45 was upregulated alongside an increase in LAG-3 expression and IL-6/JAK/STAT signaling. Increased LAG-3 expression was also confirmed at the protein level on H929 cells surviving culture with SAR442257.

Conclusion: These data support that (1) CD45 expression increases at the RNA and protein level on MM persisting after TCRT, (2) this is caused by a soluble factor secreted by stimulated T cells, and (3) LAG-3 may be a potential target for post-TCRT MM. The CD45+LAG-3+ phenotype aligns with previous reports of an immunosuppressive response cancer cells may use to provide protection from T cell attack.