Background/Purpose: The autoreactive B cell compartment in systemic lupus erythematosus (SLE) is characterized by expansion of B cells expressing immunoglobulin heavy variable gene 4-34 (IGHV4-34) B cell receptors (BCRs). This innately autoreactive B-cell population carries the 9G4 idiotope (9G4) and is a major source of disease-relevant autoantibodies in lupus, cold agglutinin disease (CAD), and other autoimmune diseases. While CD19-CAR-T cells have transformed the treatment of refractory SLE, pan-B cell depleting therapies carry an increased risk of infection. By contrast, precision immunotherapies that deplete autoreactive B cells, while sparing other B cells, may treat SLE without causing immunosuppression. To achieve this, we developed synthetic immune receptor T-cell therapies to selectively eliminate 9G4 B cells in patients with SLE (A).

Methods: Ramos B cells were CRISPR-Cas9-edited to replace their endogenous BCR with monoclonal BCRs from patients with SLE (“SLE 9G4 Ramos C1-C3”), CAD, or APS. Homology-directed repair template, encoding an anti-9G4 CAR or anti-9G4 cTCR, and Cas12a-gRNA ribonucleoprotein targeting TCR genes were transfected into activated T cells purified from PBMCs of healthy donors and patients with SLE. CAR/cTCR expression was quantified by flow cytometry, and edited T cells were isolated by positive selection. The potency and specificity of engineered T cells against Ramos B cells were quantified by live-cell imaging, flow cytometry, CellTrace Violet dye dilution to measure T-cell proliferation, and interferon (IFN)-γ ELISA. SLE PBMCs were treated with engineered T cells, and depletion of IgG 9G4 B cells was visualized by anti-9G4 FluoroSpot. Autoantibody depletion was quantified using custom 9G4 and dsDNA ELISAs.

Results: Anti-9G4 CAR-T cells and anti-9G4 cTCR-T cells, integrating anti-9G4 antibody fragments into a re-engineered TCR (A), eliminated autoreactive SLE-9G4 Ramos B cells (B-D) and their secreted anti-dsDNA autoantibodies (E-F) with equal potency (p >0.99), while sparing non-9G4 Ramos B cells. Despite ~17-fold lower IFN-γ secretion compared to CAR-T cells (315 [SD 41] vs 5237 [SD 570] pg/mL for cTCR2 vs CAR, p< 0.0001), anti-9G4 cTCR-T cells achieved equal cytotoxicity in one-time and repeated B cell stimulation assays (G). Increased cytokine release by CAR-T cells was paralleled by greater T-cell proliferation following 9G4 B cell exposure (weighted mean of generations 3.24 vs 1.67 for CAR vs cTCR2, p< 0.0001). Notably, 9G4 B cell-independent proliferation was also observed in CAR-T cells, but not with cTCR-T cells (37.7% vs 5.1% T cells divided; weighted mean of generations 1.28 vs 0.14, p< 0.0001) (H-I). In co-culture with SLE PBMCs, donor-matched cTCR-T cells and CAR-T cells depleted primary human 9G4 B cells in a dose-dependent manner.

Conclusion: We describe precision cellular immunotherapies for the depletion of autoreactive, 9G4 B cells in SLE, CAD, and other autoimmune diseases. Both anti-9G4 CAR-T cells and anti-9G4 cTCR-T cells completely and efficiently eliminated primary or cancerous 9G4 B cells. cTCR-T cells, as compared to CAR-T cells, have preferable cytokine release/safety characteristics for the treatment of patients with autoimmune diseases (J).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/precision-targeting-of-autoreactive-9g4-b-cells-in-systemic-lupus-erythematosus-using-engineered-chimeric-antigen-receptor-car-and-chimeric-t-cell-receptor-ctcr-t-cells/