Tigit: The Potential Mechanisms of Relapse in Multiple Myeloma Following Anti-BCMA CAR-T Therapy and a Promising Target for Improving CAR-T Efficacy

Background:

Multiple myeloma (MM) remains an incurable disease to date. Chimeric antigen receptor T-cell (CAR-T) therapy targeting B-cell maturation antigen (BCMA) has shown remarkable efficacy in relapsed/refractory MM (RRMM). However, our previous clinical trials and retrospective analyses indicate that despite high response rates, the median progression-free survival (PFS) is only around 7.1 to 15 months following BCMA-CAR-T therapy in RRMM, highlighting inevitable relapse and resistance post-CAR-T therapy. Enhancing and sustaining the antitumor effect of CAR-T cells in vivo is a critical challenge in treating MM.

Methods:

We conducted single-cell RNA sequencing of bone marrow cells from RRMM patients treated with BCMA-CAR-T, analyzing the characteristics and gene expression profiles of tumor-infiltrating T cells. Clinical samples from healthy controls, newly diagnosed MM (NDMM) patients, and MM patients in remission were collected to assess immune checkpoint molecule expression via flow cytometry. CAR-T cells were co-cultured with MM cells, and various assays, including Luciferase assay, CD107a labeling, CFSE assay, and CD45RO/CD62L labeling, were used to evaluate CAR-T cell cytotoxicity, degranulation, proliferative potential, and differentiation state. The effects of TIGIT knockout or anti-TIGIT monoclonal antibody (mAb) on CAR-T cell function and status were investigated. A humanized immune cell-reconstituted mouse model undergoing CAR-T infusion were used to analyze the impact of TIGIT knockout or anti-TIGIT mAb on tumor growth and T-cell exhaustion.

Results:

1. Single-cell RNA sequencing revealed that T cells, especially CD8+ Teff and Pro Teff cells, from early relapse (ER) patients (those who relapsed within one year) exhibited lower cytotoxicity compared to those from durable response (DR) patients (those who did not relapse within one year). T cells from the ER patients, particularly early-differentiated Tn/Tcm and Pro Teff cells, showed signs of exhaustion before CAR-T infusion. Differentially expressed genes (DEGs) between ER and DR groups were significantly enriched in pathways related to T-cell activation, neutrophil regulation, and leukocyte proliferation. Notably, TIGIT expression was significantly higher in T cells from the ER group, suggesting that T-cell exhaustion driven by high TIGIT expression may underlie post-CAR-T therapy relapse in MM patients.
2. Analysis of TIGIT, CD96, CD226, PD-1, LAG-3, and TIM-3 expression in bone marrow tumor-infiltrating T cells from healthy controls, NDMM, and remission patients revealed that only TIGIT expression was higher in the NDMM group compared to the remission and healthy control groups, with significantly higher expression on CD8+ T cells than on CD4+ T cells. This indicates that TIGIT is a significant immune checkpoint in MM.
3. Both TIGIT knockout and anti-TIGIT mAb enhanced CAR-T cell proliferation, degranulation, and cytotoxicity in vitro, improved CAR-T cell differentiation upon tumor antigen exposure, and reversed CAR-T cell exhaustion, with TIGIT mAb showing superior effects.
4. In vivo experiments demonstrated that TIGIT knockout CAR-T cells or anti-TIGIT mAb administration reduced tumor burden in mice and ameliorated CAR-T cell and tumor-infiltrating T-cell exhaustion.

Conclusion:

High TIGIT expression on CAR-T cells and T cells in MM patients may be a key factor in disease progression post-CAR-T therapy. Targeting TIGIT, through anti-TIGIT mAb or gene editing, could be an effective strategy to mitigate T-cell exhaustion and improve the efficacy of CAR-T therapy.