Enhanced antitumor efficacy of next-generation B7-H3 chimeric antigen receptor T cells containing CD28, CD137 and CD27 costimulatory domains in multiple myeloma

Abstract

Background: Multiple myeloma (MM) remains an incurable disease despite significant advancements in treatment strategies. Chimeric antigen receptor (CAR) T cell therapies targeting B-cell maturation antigen have demonstrated clinical promise; however, their effectiveness is often limited by disease relapse, partly due to CAR T cell exhaustion. B7-homolog 3 (B7-H3), an immune checkpoint molecule that is overexpressed in MM and may suppress T cell function, represents a potential alternative target to improve CAR T cell efficacy and enhance disease control. Methods: We engineered and characterized three generations of B7-H3-specific CAR T cells, second (B7H3.CAR2), third (B7H3.CAR3) and next-generation (B7H3.CAR-NG), each incorporating distinct costimulatory domains. B7H3.CAR2 contains the CD28 costimulatory domain, B7H3.CAR3 combines CD28 and CD137, and B7H3.CAR-NG incorporates CD28, CD137, and CD27 costimulatory modules. Their expression and function were evaluated in vitro using MM cell lines with differential B7-H3 expression. CAR T cell phenotype, cytotoxic activity, persistence and cytokine secretion were assessed through both short- and long-term coculture assays. Results: B7-H3 expression levels varied across MM cell lines, with MM.1S exhibiting the highest and NCI-H929 the lowest expression. Second-, third- and next-generation B7-H3-specific CAR T cells (B7H3.CAR2, B7H3.CAR3, and B7H3.CAR-NG) were successfully generated, with CAR expression rates of 31.27 ± 8.63%, 29.90 ± 8.86% and 37.27 ± 8.69%, respectively. All three CAR T cell types selectively lysed B7-H3-positive MM.1S cells in an antigen density-dependent manner while sparing B7-H3-negative SupT1 cells. Among them, B7H3.CAR-NG T cells showed the highest cytotoxicity, lysing 53.22 ± 9.28% of MM.1S cells at a 1:1 effector-to-target ratio, compared to 11.66 ± 1.62% of SupT1 cells. In long-term cocultures, CAR-NG T cells demonstrated superior tumor control and persistence, likely due to a higher frequency of central memory T cells. Cytokine analysis revealed elevated secretion of effector molecules by CAR-NG T cells, indicating enhanced antitumor functionality. Conclusions: B7-H3-specific CAR T cells exhibit potent antitumor activity against MM, with the next-generation construct (B7H3.CAR-NG) demonstrating superior cytotoxicity, persistence and cytokine production. These findings support the potential of B7H3.CAR-NG T cells as a promising therapeutic strategy for MM.