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Chimeric antigen receptor T (CAR-T) cell therapies have demonstrated high response rate and durable disease control for the treatment of B cell malignancies. However, in the case of solid tumors, CAR-T cells have shown limited efficacy, which is partially attributed to intrinsic defects in CAR signaling. Here, we construct a double-chain chimeric receptor, termed as synthetic T cell receptor (TCR) and antigen receptor (STAR), which incorporates antigen-recognition domain of antibody and constant regions of TCR that engage endogenous CD3 signaling machinery. Under antigen-free conditions, STAR does not trigger tonic signaling, which has been reported to cause exhaustion of traditional CAR-T cells. Upon antigen stimulation, STAR mediates strong and sensitive TCR-like signaling, and STAR-T cells exhibit less susceptibility to dysfunction and better proliferation than traditional 28zCAR-T cells. In addition, STAR-T cells show higher antigen sensitivity than CAR-T cells, which holds potential to reduce the risk of antigen loss-induced tumor relapse in clinical use. In multiple solid tumor models, STAR-T cells prominently outperformed BBzCAR-T cells and generated better or equipotent antitumor effects to 28zCAR-T cells without causing notable toxicity. With these favorable features endowed by native TCR-like signaling, STAR-T cells may provide clinical benefit in treating refractory solid tumors.

Background Cytomegalovirus (CMV) infection is an important infectious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to the immune-suppressive state of the transplant recipient. Patients are likely to be resistant to traditional antiviral drugs, such as ganciclovir and foscarnet, or have intolerable adverse reactions to these drugs, including bone marrow suppression and nephrotoxicity. CMV-specific CD8 + and CD4 + T-cell functional defects are the main reasons for the emergence of refractory CMV infection. Thus, it is possible to cure refractory CMV infection using CMV-specific TCR-T cell therapy via a direct antiviral effect and indirect T cell mediated immune reconstruction. Objectives and Methods We conducted a single-arm, open-label, phase I clinical trial (https://clinicaltrials.gov, NCT04153279) at the Hebei Yanda Lu Daopei Hospital. This study was designed to assess the safety and feasibility of CMV-specific TCR-T cell therapy in allo-HSCT patients with refractory CMV viremia or viral disease. We screened multiple TCRs with specificity for CMV-PP65 or IE1 polypeptides restricted by HLA-A*11:01, 24:02 and 02:01, and constructed lentiviral vectors containing CMV-specific TCRs. Peripheral blood (PB) stem cells were obtained from healthy HSCT donors for the TCR-T cell preparation. After stimulation for 24 hours, T-cells were infected with lentivirus carrying HLA-matched CMV TCR, then cultured with medium containing IL-7/IL-15 for a total of 7-9 days before harvest. After infusion, CMV TCR-T cell proliferation and persistence in PB was detected by Q-PCR. Results From December 24, 2019 to January 1, 2021, 9 patients were enrolled and as a result 7 patients including 5 with refractory CMV viremia and 2 with CMV disease successfully received the TCR-T therapy. Among the 7 patients who received TCR-T, the median age was 11 years (5-45 years of age) and 5 were male (71.4%). The diagnosis included 5 cases of acute lymphoblastic leukemia (ALL), 1 case of acute myeloid leukemia (AML) and 1 case of mixed phenotype acute leukemia (MPAL) . Patients underwent either haplo-identical transplantation (n=6) or unrelated donor transplantation (n=1). The median time for patients to CMV viremia diagnosis was 28 days (21-231 days). Patients received a median dose of 3 × 10 5 TCR-T cells/kg body weight (1-10 × 10 5 TCR-T cells/kg body weight). The mean proportion of CMV-specific TCR-T in the cultured cell products was 35.5% (18.0-68.9%). Among the 7 patients, successful follow up and data evaluation was available for 6 patients. One patient (P3) withdrew early due to uncontrolled lung infection. The median follow-up time for the entire cohort after HSCT was 199 days (144-479 days). Only 1 patient experienced Grade 1/2 cytokine release syndrome (CRS) with mild hypotension and fever. No immune effector cell-associated neurotoxicity syndrome (ICANS) or TCR-T cell-related graft-versus-host disease (GVHD) occurred in any patient. A total of 6 patients achieved complete response (CMV DNA-negative plasma) after TCR-T cell infusion. The median time from CMV-specific TCR-T infusion to the first CMV clearance was 41 days (19-91 days). The median time for CMV TCR-T cells to reach their first peak was 21 days (10-28 days) with a median copy number of 3.85×10 4 copies/μg genomic DNA (range: 1.93×10 4-7.75×10 4 copies/μg DNA) (Figure 1). CMV TCR T-cells persisted up to 3 months with detectable copy number. The levels of cytokines IL-2, TNF-α, IL-6, IL-8 and IFN-γ in PB were relatively low. Conclusion This study demonstrates the safety and feasibility of CMV-specific TCR-T cells for refractory CMV infection after HSCT. We show that adoptive transfer of CMV-specific TCR-T cells can lead to complete CMV clearance in patients who have undergone hematopoietic stem cell transplantation (HSCT) and with good tolerability and safety. Longer-term observation of these patients and larger patient studies are warranted to demonstrate the efficacy and safety of CMV TCR-T cells. Given that immune defect-related CMV infections are common and life threatening for HSCT patients, we see a potential future for CMV-specific TCR-T cell therapy for the treatment and prevention of CMV infection following HSCT or other organ transplantation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Chimeric antigen receptor (CAR) T -cell therapy has demonstrated high response rates among patients with B cell malignancies yet remission durability and safety could be improved. We have developed a novel double-chain chimeric receptor Synthetic T Cell Receptor and Antigen Receptor (STAR) consisting of 2 protein modules each containing an antibody light or heavy chain variable region, the T Cell Receptor (TCR) a or b chain constant region fused to the OX-40 co-stimulatory domain, with the 2 modules linked by a self-cleaving Furin-p2A sequence that allows the modules to be proteolytically separated and reconstituted (Fig. 1A). Here, we report pre-clinical and first-in-human phase I trial results of CD19 STAR-T cell therapy for CD19+ R/R B-ALL. Methods Peripheral blood (PB) mononuclear cells were obtained from healthy donors and patients for the pre-clinical and clinical studies, respectively. T-cells were transduced with the STAR lentiviral vector. A leukemia xenograft mouse model was used to assess the STAR T-cell antitumor function. For the clinical trial, from Dec. 2019 to Jun. 2020, 18 CD19+ R/R B-ALL patients (M/F 10:8) with a median age of 22.5 years (range: 6-68) were enrolled (NCT03953599). Patients received a conditioning regimen of IV fludarabine (25mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days followed by a single STAR T-cell infusion. Once patients achieved complete remission (CR), they were given the option to proceed to consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT) or not. Results In preclinical studies, we found CD19 STAR T-cells to be superior to conventional CAR (BBz CAR) measured by the following parameters: 1) faster/stronger T-cell activation within 3 hours (76.67±2.621% vs 46.4±9.318%; p=0.0253); 2) higher cytokine production (4100.92±174.4 pg/ml vs 2556.78±563.39 pg/ml; p1:1, p In the phase I trial, the median observation time was 69 (20-180) days. The median pre-treatment bone marrow (BM) blast level was 7.0% (0.1%-86.6%). All 18 patients received a single infusion of STAR T-cells at a median dose of 1×106/kg (5×105/kg-2.5×106/kg): low dose (5×105/kg) (n=3), medium dose (1×106/kg) (n=8) and high-dose (2-2.5×106/kg) (n=7). Three early enrollees subsequently received a second consolidation infusion of STAR T-cells at 1×106/kg (n=2) and 2×106/kg (n=1). The median STAR T-cell production time was 9 (7-13) days with a transduction efficacy of 57.4% (41.0%-78.2%). Two weeks post STAR T-cell infusion, 18/18 (100%) patients achieved CR with a negative minimal residual disease (MRD) status. After a median of 57 (43-66) days following STAR T-cell therapy, 8/18 patients made a choice to pursue consolidation allo-HSCT and all have remained in CR after a median follow-up of 110 (75-180) days. Of the 10 patients who did not undergo allo-HSCT, 1 relapsed on day 58 and died from relapse on day 63. This patient had a pre-CAR T-cell BM blast level of 86.6% with central nervous system leukemia. Another patient became MRD-positive with 0.09% blasts on day 30 per flow cytometry (FCM). The other 8 patients have remained in CR. Despite the achievement of a high CR rate, cytokine release syndrome (CRS) occurred only in 10/18 (55.6%) patients with 8 Grade I, and 2 Grade II CRS. Two patients developed Grade III neurotoxicity. After STAR T-cell infusion, CD19 STAR T-cells in PB were followed by qPCR and FCM. We saw high in vivo proliferation and persistence regardless of the infusion dose. The median peak level was reached on day 8.5 (day 4-10) with 4.9×104 (0.104-175×104) copy number/ug PB genomic DNA detectable at 6 months. Conclusion This study demonstrates the superiority of STAR T-cells compared to conventional CAR T-cells in terms of signaling capacity, cytokine production capability and anti-tumor potency in an animal model. The Phase I first-in-human study demonstrated technical feasibility, clinical safety and efficacy of STAR-T in treating CD19+ R/R B-ALL. A high CR could be achieved on day 14 with low toxicity. Longer-term observation of these patients and studies of larger patient cohorts are warranted. Disclosures No relevant conflicts of interest to declare.