Multiplexed Engineered T Cells Overcoming Antibody-Mediated Transplant Rejection Multi-Specifically Deplete Alloreactive B Cells While Preserving Antiviral Immunity

A major risk for solid organ transplants is antibody-mediated rejection (AMR) by anti-donor HLA antibodies. Engineered T cells harboring a CAR-like receptor (T cells overcoming rejection by antibodies, CORA-Ts) are a novel approach for selectively depleting alloreactive anti-donor HLA B cells while preserving general B-cell immunity. Multiple HLA mismatches require multi-specific CORA-Ts for simultaneous elimination of alloreactive B cells with different donor-HLA specificities.

CORA-Ts are generated by engineering of T cells with chimeric receptors comprising a mismatched donor HLA molecule fused to intracellular 4-1BB/CD3ξ signaling domains to redirect them against alloreactive B cells carrying respective anti-donor HLA B-cell receptors. CORA-Ts based on HLA-B*07:02 were developed and their ability to selectively eliminate anti-HLA-B*07 B cells alongside reduction of antibody release was tested in vitro. Next, the HLA-B*07-based CORA receptor was combined with an established HLA-A*02-based CORA receptor to generate bispecific CORA-Ts. Simultaneous lysis of anti-HLA-A*02 and anti-HLA-B*07 B cells, as well as protection of anti-SARS-CoV-2 B cells to prove maintenance of humoral antiviral immunity was evaluated.

Upon co-cultivation with anti-HLA-B*07 B-cell lines, B*07-CORA-Ts were specifically activated (expression of CD25, CD69, CD137), released pro-inflammatory molecules (e.g. IFN-γ, granzyme B), and exhibited strong cytotoxic activity resulting in effective reduction of anti-HLA-B*07 antibody release. Bispecific A*02/B*07-CORA-Ts exhibited similar effector profiles and comparably strong cytotoxic activity selectively towards both anti-HLA-A*02 and anti-HLA-B*07 B cells, whereby A*02/B*07-CORA-Ts generated by co-transduction were equally effective as compared to mixtures of A*02- and B*07-CORA-T products. Importantly, antiviral B cells secreting anti-SARS-CoV-2 antibodies were not eliminated by CORA-Ts.

Our results show that multi-specific CORA-Ts specifically and effectively eliminate alloreactive B cells with different donor-HLA specificities. They selectively prevent formation of anti-HLA antibodies while preserving antiviral immunity. Offering enormous production and cost benefits, multi-specific CORATs are a potent approach for preventing AMR even in multiple-HLA-mismatched transplant settings, thereby improving long-term graft survival while maintaining overall humoral B-cell immunity.

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