Dual targeting of BCMA and B7-H3 with CAR T cells and bispecific protein engagers enhances anti-myeloma activity

Abstract

Background B cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T cells have shown remarkable activity in relapsed/refractory multiple myeloma (R/R MM). However, therapeutic resistance often arises due to antigen heterogeneity and immune checkpoint upregulation. B7-H3, an immune checkpoint protein overexpressed in MM and linked to T cell dysfunction and poor prognosis, represents a promising complementary target. This study evaluates a dual targeting approach that combines αBCMA CAR T cells with a recombinant αB7-H3/CD3 bispecific protein engager (BiPE) to enhance anti-myeloma activity. Methods A cell-based system was engineered to continuously produce αB7-H3/CD3 BiPE. Fourth-generation αBCMA CAR T cells (CAR4; incorporating CD28, 4–1BB, CD27, and CD3ζ signaling domains) were combined with recombinant BiPE, and their anti-myeloma activities were assessed in vitro using cytotoxicity, proliferation, immunophenotyping, cytokine profiling, and tumor re-challenge assays. Results In BCMA⁺ /B7-H3^high MM cells (MM1.S), CAR T + BiPE co-treatment yielded stronger cytotoxicity than CAR T cells alone. In BCMA⁺/B7-H3^low cells (H929), the combination markedly outperformed either monotherapy, particularly at low effector-to-target ratios. Enhanced tumor killing was accompanied by increased T cell activation (CD69), proliferation, effector memory differentiation, and secretion of IFN-γ, TNF-α, IL-2, FasL, granzyme B, and perforin. BiPE exposure initially upregulated exhaustion and inhibitory checkpoint markers (PD-1, PD-L1, B7-H3, TIGIT, and LAG-3), but these effects were transient, which declined upon tumor re-challenge with preserved central memory phenotype. Conclusions Dual targeting of BCMA and B7-H3 with CAR T cells and BiPE synergistically enhances T cell activation, effector function, and cytotoxicity against MM. This combinatorial strategy has the potential to overcome resistance mechanisms and represents a promising therapeutic approach for improving outcomes in MM and other BCMA⁺/B7-H3⁺ malignancies.