First-in-human Phase I study of autologous CD5-knockout anti-CD5 CAR-T cells in CD5⁺ hematologic malignancies

Abstract Introduction CD5 is broadly expressed across hematologic malignancies, notably in ~85% of T-cell tumors and is associated with poorer prognosis in certain B-cell tumors, making it a promising target for CAR-T cell therapy. Two primary clinical studies have reported on CD5-targeted CAR-T therapy: one using autologous CAR-T cells without CD5 editing (NCT03081910) and one using allogeneic CD5-knockout (CD5-KO) CAR-T cells (NCT05032599). To further explore its clinical utility, we conducted a Phase I trial (NCT04767308) of autologous CD5-KO anti-CD5 CAR-T cells (CAR55ko-T) in patients with relapsed and refractory (r/r) CD5⁺ hematologic malignancies. Methods Patients received a single dose of CAR55ko-T cell infusion after a three-day FC lymphodepletion regimen, followed by a 1-day rest. Dose levels evaluated were 1.0 × 10⁶, 2.0 × 10⁶, and 1.0 × 10⁵ cells/kg. Adverse events (AEs) were graded using CTCAE v5.0, and treatment responses were assessed per Lugano 2014 criteria. CAR-T kinetics were measured by ddPCR and flow cytometry. Lymphocyte subset dynamics were analyzed via multicolor flow cytometry. The study was conducted in accordance with the Declaration of Helsinki, and all participants provided informed consent. Results Seven patients (4 males, 3 females) were treated from February 2021 to October 2021, including 1 ALCL, 2 AITL, 1 SPTCL, 2 MCL, and 1 DLBCL. Median age was 47 years (range: 17–67), with a median of 3 prior therapy lines (range: 2–6). Patient 2 had previously received anti-CD19/22 CAR-T, and Patient 3 had undergone autologous HSCT. The overall response rate was 85.7%, with 4 CR and 2 PR. Median PFS and OS were 170 and 205 days, respectively. Patient 1 remained in remission 902 days post-infusion. Notably, CD5+T cells were absent at the disease relapse or resistance. CAR55ko-T cells expanded robustly and persisted long-term. Median peak copy number was 87,178 copies/μg DNA (range: 31,618–118,000), with a median time to peak of 14 days. During a median follow-up period of 124 days, CAR-T cells remained detectable in all patients, with persistence 809 days in patient 1. CRS and ICANS were generally mild; only one case of grade 3 CRS occurred. The most common ≥ grade 3 AEs were cytopenia: neutropenia (100%), lymphopenia (100%), thrombocytopenia (78%), and anemia (89%). Notably, grade 4 lymphopenia and neutropenia recured after one-month post-infusion. Consistently, EBV (6/7) and CMV (4/7) reactivations were frequent. Four patients had bacterial infections, resulting in to two deaths. Uncommon AEs were observed, including rash (71.4%), skin tingling (71.4%) and autoimmune-associated AEs: xerostomia (28.6%), hypothyroidism (14.3%), and adrenal insufficiency (14.3%). Immune reconstitution process was monitored. Following the transient nadir, CD3+ T cells recovered to pre-lymphodepletion levels within 1 month and maintained stable. Notably, the reconstituted T cells was CD5 negative. The CD4/CD8 ratio decreased rapidly and remained 1 throughout the entire follow-up. B cells were rapidly depleted and failed to recover during long-term follow-up. NK and NKT cells showed transient decline but rebounded to baseline. Conclusion CD5-KO anti-CD5 CAR-T therapy demonstrates promising efficacy in treating CD5+ hematological malignancies, including both T-cell and B-cell tumors. The knockout of CD5 enhances CAR-T persistence and therapeutic durability. However, sustained CD5+ T cells depletion and expansion of CD5- T cells may impair immune regulation and infection control in patients. These findings highlight the need to modulate the CAR-T persistence duration clinical application and support the consideration of consolidative HSCT therapy in patients achieving remission.