First-in-human trial of allogeneic CD6-CAR tregs in patients with chronic graft-versus-host disease after allogeneic hematopoietic cell transplantation

Abstract Chronic graft-versus-host disease (cGVHD) remains the leading cause of late nonrelapse mortality and morbidities affecting quality of life (QoL) in patients who received allogeneic hematopoietic cell transplantation (HCT). While currently available drugs for cGVHD treatment achieve high overall response rates (ORR) and can improve QoL, failure-free survival (FFS) remains suboptimal, with high risk of infections. To address this unmet need, we developed a novel cellular therapy based on engineering regulatory T cells, isolated from HCT donors, expressing a CD6-targeting chimeric antigen receptor (CAR) with a novel CTLA-4 intracellular domain (CD6-CAR Tregs). In vitro, CD6-CAR Tregs immunomodulate T-cell effector function, and in vivo, prevented GVHD onset in a NSG mice xenograft model adoptively transferred with human PBMCs. A GMP-compliant manufacturing platform was developed and initiated a phase I clinical trial to evaluate the safety and feasibility of donor-derived CD6-CAR Tregs in patients with steroid-refractory or dependent cGVHD (NCT05993611). This trial employed a standard 3+3 dose-escalation design including 0.1, 0.3, 1.0, and 3.0 x106 CAR-Tregs/kg. To our knowledge, this represents the first clinical investigation of a genetically engineered human CAR-Treg product. Sixpatients have been treated in the study, assessable for the primary endpoint of safety based on dose limiting toxicity (DLT) and adverse event (AE) evaluation within the first 28 days. Three patients are evaluable for ORR at 9 months, and 4 patients at 6 months. The median duration of follow-up for patients is 6 months (range: 1-11). The median age of patients is 59 years (range 18-64). All received peripheral blood stem cell graft (PBSC) graft from a matched sibling donor, 4 female, 2 male, median age 56.2 years (range 25-67). HCT followed the treatment for ALL (n=3), MPN (n=2), or AML (n=1) using myeloablative (n=4) or reduced intensity regimen (n=2), and all patients received calcineurin inhibitor-based prophylaxis. The median time to cGVHD onset was 11.5 months (range 6-36) and time from cGVHD and CD6-CAR Tregs was 7.1 years (range 1.4-10.8). Median prior lines of therapy was 3.5 (range 2-7), including belumosudil (n=5) and ruxolitinib (n=4). Scleroderma was the most common organ involvement (n=5), followed by oral (n=4), ocular (n=3), joint fascia (n=2), and lung (n=1), with most patients (n=4) having multiorgan (≥ 3 organ) involvement. CD6-CAR Tregs were successfully manufactured, cryopreserved, and infused without prior lymphodepleting chemotherapy. Three patients at DL1 and DL2 received 6.4 (+/-0.2) and 24.1 (+/-7.1) x106 CAR+ cells, respectively. One patient in DL1 received a second infusion at DL2, 5 months after DL1, based on the initial safety/efficacy, seeking to consolidate a clinical response. No CRS, ICANS or infections appeared during the DLT period. The most common AEs during the DLT period were: cytopenias (n=9; neutropenia=4, lymphopenia=1, low platelets=3, anemia=1), sinus tachycardia (n=5), dyselectrolytemia (n=3), hypertension (HTN, n=1), and fatigue (n=2). Grade 3 AEs were noted in two patients (lymphopenia, HTN) without DLT or Grade 4/5 AEs. No growth factor or RBC transfusion was required, and one patient received a platelet transfusion for a count of 43,000/mm3. All patients with a 3-month or longer follow-up (n=5) experienced partial response (PR) based on NIH consensus criteria with continued FFS, including 3 patients with a 9-month follow-up associated with an improvement in patient-reported outcomes. CD6-CAR Tregs were at the peak levels within one month after infusion (DL1, 1856 (+/-1511) vector copy#/ml blood). Changes in serum cytokines were variable between patients (DL1), and IL-4 elevation ≥10-fold above baseline was the only finding commonly detected in two participants. One patient showed a ≥10-fold decrease from baseline in IL-1RA, MIG, and IL-8. T-cell depletion in PBMC was not detected at DL1. Evidence suggests that CD6-CAR Tregs persist up to two months after infusion, as observed in a skin biopsy from one patient. In summary, CD6-CAR Tregs can be successfully manufactured and administered to patients without significant toxicity, including CRS or ICANS. No hematologic malignancy was observed, and salvage therapy for GVHD has not been required. These early clinical findings support CD6-CAR Tregs as a promising cellular therapy with the potential to re-educate an imbalanced immune system in cGVHD.