Late re-expansion and high-level persistence of CAR T-cells can be driven by single CAR+ T-cell clones without clinically evident CAR+ T-cell lymphoma Free

Chimeric antigen receptor (CAR) T-cells have proven highly efficacious in the treatment of relapsed/refractory B-cell malignancies. Despite the great success in cancer treatment, CAR T-cell therapy is also associated with a unique spectrum of toxicities that drive morbidity and mortality. Since the engineered CAR is delivered through lentiviral or gamma-retroviral vector integration, insertional mutagenesis could modify the cellular genome and possibly cause malignant transformation of the transduced T cells. Overall, malignant transformation of CAR T-cells seemed to be extremely rare, but the recently reported cases of CAR-expressing T cell lymphomas (Kobbe et al., 2024; Harrison et al., 2025) refueled the discussion to which extent insertional mutagenesis, clonal hematopoiesis, or the CAR with its strong proliferative signal contribute to lymphomagenesis. We therefore reanalyzed our large and longitudinal CAR T-cell monitoring data towards identification of the rate of clonal evolution and possible oncogenesis events in patients post CAR T-cell therapy.

Here, we closely followed CAR T-cell expansion and persistence in > 100 cancer patients with longitudinal measurement after CAR T-cell therapy and identified two patients with large B-cell lymphoma (LBCL) who exhibited uncommon, late re-expansion of anti-CD19 CAR T-cells with sustained persistence, defined as >20 CAR-T cells/µL over a period of at least four weeks. Furthermore, CAR T-cell re-expansion was also observed in a patient with solid cancer (endometrial carcinoma, Pt3) who was treated with anti-Claudin.6 CAR T-cells. All three patients experienced initial CAR T-cell expansion peaking within two weeks post-infusion, followed by a typical contraction phase and a re-expansion at later time (Pt1: month 8; Pt2 and Pt3: week 7 each). In both LBCL patients, CAR T-cells at re-expansion showed a different immune phenotype with dominant CD4 expression (Pt1) or loss of CD4 and CD8 coexpression (Pt2). scRNA/TCR sequencing revealed high clonal diversity at the initial CAR-T cell peak, but a shift to clonal dominance with the second CAR T-cell expansion. Notably, CAR T-cells in Pt1 persisted at atypical high numbers of > 200/µl for the last 14 months, and the patient remained in complete remission for over 36 months with no clinical symptoms except mild thrombocytopenia. Pt2 initially achieved a partial response, but despite CAR T-cell re-expansion, showed progressive CD19-positive LBCL two months post treatment. Interestingly, the dominant CAR T-cell clone in Pt2 exhibited reduced expression of effector molecules (granzyme B, perforin, granulysin) and loss of cytotoxic functionality. Targeted DNA sequencing of sorted CAR T-cell clones detected mutations that were predicted to be pathogenic in DNMT3A (Pt1: c.2165G>A; Pt2: c.1903C>T) and in TET2 genes (Pt2: c.3921_3922del). CAR T-cell re-expansion in the patient with solid cancer occurred directly after cytomegalovirus (CMV) reactivation and was primarily driven by oligoclonal expansion of CD8+ CAR T-cells. Notably, a significant proportion of the expanded CD8+ CAR T-cells exhibited CMV reactivity, suggesting TCR-driven activation and expansion of virus-reactive T cells exclusively in the CAR⁺ T cell population, suggesting a growth advantage of CAR-transduced T cells even when engaged through their TCR.

Taken together, we observed late clonal CAR T-cell re-expansion in two LBCL patients without any evidence for development of CAR+ T cell lymphoma. Furthermore, we identified CAR T-cells that underwent expansion in response to the TCR antigen rather than the CAR target. Further research is needed to determine how frequently clonal CAR-T cell expansion occurs without resulting in malignant transformation and lymphomagenesis, and to identify the underlying drivers of such expansions which may have therapeutic consequences.