Target Antigen-Expressing T Cells Facilitate CAR-T Cell Eradication of Residual CD19+ B Malignant Cells

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated remarkable efficacy in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL). However, the infusion of CAR-T cells alone may not thoroughly eradicate tumor cells, particularly in instances of low tumor burden, due to the insufficient stimulation required for the activation and proliferation of CAR-T cells , leading to the potential failure of CAR-T therapy. We postulated that the activation of CAR-T cells is contingent upon the concentration of the target antigen and proposed a novel strategy to rescue CAR-T cell activation through the use of target antigen-expressing T cells (TAETs). Underpinned by this hypothesis, we devised an in vitro model to assess target cell dosage and an in vivo mouse model to simulate the clinical situation of minimal residual disease (MRD), which collectively demonstrated that the immune response of anti-CD19 CAR-T cells is reliant on the dosage of the target antigen, with activation occurring only upon reaching a threshold level of target cells. The supplementary infusion of engineered CD19+ TAET cells alongside anti-CD19 CAR-T cells rescued the antitumor capabilities of the latter in scenarios with a limited number of residual tumor cells, facilitating deep clearance of these residual cells. In vitro studies revealed that CD19+ TAET cells exert a comparable influence on the activation of anti-CD19 CAR-T cells to that of B-cell tumor cell lines such as Raji and Nalm6, as well as normal B cells. The expression levels of CD107a on CAR-T cells stimulated by CD19+ TAET cells mirrored those stimulated by Raji, Nalm6, and normal B cells. We also found that CD19+ TAET cells specifically enhance the cytotoxic functions of anti-CD19 CAR-T cells. Our target cell dosage-dependent study revealed that both the activation and cytotoxic effects of anti-CD19 CAR-T cells are significantly influenced by the number of target-expressing cells, with CD19+ TAET cells effectively stimulating CAR-T cells to perform cytotoxic functions even when the quantity of Raji cells was inadequate. Additionally, we observed that the expansion ability of anti-CD19 CAR-T cells is dependent on the dosage of target-expressing cells, with CD19+ TAET cells being instrumental in stimulating this expansion. We innovatively established an in vivo mouse model with a low tumor burden to mimic the clinical scenario of minimal residual disease (MRD). Initially, we monitored the tumor burden in mice injected with different concentrations of Raji cells, selecting those with the minimal concentration （2.5E+04 cells/mice）sufficient for tumor formation as our residual tumor cell model. Subsequently, using this model, mice were treated with either anti-CD19 CAR-T cells alone or in conjunction with CD19+ TAET cells. The in vivo study demonstrated that in the presence of CD19+ TAET cells, anti-CD19 CAR-T cells maintained robust antitumor activity in the residual tumor cell model, effectively inhibiting tumor growth and achieving deep elimination of tumor cells. Our study, for the first time, clearly elucidated three pivotal findings: (a) The activation of anti-CD19 CAR-T cells is dependent on the target antigen dosage, and when the number of target cells falls below the activation threshold, CAR-T cells are not effectively activated, leading to the neglect of target cells. (b) Even when the number of tumor cells is below the activation threshold for CAR-T cells, the inclusion of CD19+ TAET cells can effectively salvage the antitumor efficacy of CAR-T cells, enabling the eradication of tumor cells. (c) Given that CD19+ TAET cells cannot proliferate, CAR-T refusion supplemented with CD19+ TAET cells in scenarios of low tumor burden can significantly enhance the safety of clinical applications, deeply clear tumor cells, and confer long-term benefits to patients. In fact, our recently published phase I/II study has substantiated the therapeutic impact of employing TAET cells in conjunction with CAR-T cells to achieve complete molecular response (CMR)(bloodadvances.2022009072). Furthermore, this combined treatment strategy is highly flexible and scalable, applicable to various targets, target cell types, and effector cell types, to tackle a range of clinical issues.