In Vivo clonal dynamics of alloreactive CAR T cells reveal mechanisms limiting gvhd risk following allogeneic CAR T cell therapy Free

Introduction: Allogeneic CAR T cell therapy holds curative potential for patients with relapsed or refractory B-cell malignancies that have progressed after allogeneic hematopoietic stem cell transplantation (alloHSCT). However, its broader clinical application remains constrained by the risk of graft-versus-host disease (GvHD). In a fully human leukocyte antigen (HLA)-mismatched lymphoma model, allogeneic CAR T cells expressing a CD28-costimulated CD19 CAR (28z CD19 CAR) demonstrated potent anti-tumor activity without causing a significant increase in GvHD (Ghosh et al. Nat. Med. 2017). The limited alloresponses were attributed to aberrant hyperactivation of alloreactive CAR T cells, driven by cumulative CAR and TCR signaling, resulting in progressive loss of effector function, reduced proliferative potential, and eventual clonal deletion. While this study offers preclinical evidence for the low GvHD rate observed in early-phase trials, the in vivo dynamics of alloreactive CAR T cells and the mechanisms underlying GvHD in real-world settings remain poorly understood. Here, we investigate the potential of alloreactive CAR T cells to induce GvHD in selected HLA-mismatched patients following allogeneic CAR T cell therapy.

Methods: We identified 20 patients with relapsed or refractory B-cell malignancies post-alloHSCT treated with allogeneic CD19 CAR T cells (NCT01087294). To study alloreactive T cell responses, we developed a novel cell-based platform capable of efficiently displaying recipient-mismatched HLA molecules and selectively expanding rare alloreactive T cells from original transplant donor PBMCs. These donor-derived alloreactive T cells were engineered with a 28z CD19 CAR construct and co-cultured with CAR targets alone, TCR targets alone, or both simultaneously. In parallel, we used the same cell-based platform to identify and analyze alloreactive CAR T cells from post-infusion patient PBMCs. By integrating the high-resolution Beacon Discovery™ technology with single-cell TCR and RNA sequencing, we performed real-time functional and phenotypic profiling of dominant alloreactive CAR T cell clones, enabling the reconstruction of their in vivo trajectories and transcriptional states over time.

Results: Despite 70% (14/20) of patients having a prior history of GvHD following alloHSCT, no new cases of acute GvHD were observed after CAR T cell infusion, including in two responders who received CAR T cells manufactured from a 9/10 HLA-matched unrelated donor, as previously reported (Brudno et al. J Clin Oncol. 2016). We found that CAR expression on alloreactive T cells, derived from donor apheresis PBMCs, preserved both CAR- and TCR-mediated cytotoxicity when each target was presented individually. However, TCR target recognition was markedly impaired when alloreactive CAR T cells were exposed to both CAR and TCR antigens simultaneously. Notably, CAR engagement alone was sufficient to induce significant contraction and functional exhaustion of alloreactive CAR T cells. Interestingly, inhibition of the PI3K/AKT/mTOR axis—the primary downstream pathway activated by CD28 costimulation—improved alloreactive CAR T cell persistence and effector function, suggesting that CAR engagement can induce a dysregulated downstream signaling. Additionally, using our cell-based platform, we longitudinally tracked dominant alloreactive CAR T cell clones from early expansion through later time points following allogeneic CAR T cell administration. To isolate the specific effects of CAR integration, non-transduced alloreactive T cells derived from unmanipulated donor apheresis PBMCs served as baseline controls. Compared to their non-CAR counterparts, post-infusion alloreactive CAR T cells followed a distinct trajectory marked by sustained hyperactivation, pronounced exhaustion, and altered intracellular signaling, showing a substantial decline over time.

Conclusions: These findings provide mechanistic evidence that CAR integration into alloreactive T cells mitigates GvHD risk by impairing their persistence and thereby limiting alloimmune responses. Our study represents the first comprehensive in-human characterization of alloreactive CAR T cells, offering critical insights to guide the development of safer, more effective next-generation allogeneic CAR T cell therapies and to support their broader clinical application.