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Cytomegalovirus (CMV) infection remains a major cause of mortality after hematopoietic stem cell transplantation (HSCT). Current treatments, including antiviral drugs and adoptive cell therapy with CMV-specific cytotoxic T lymphocytes (CTLs), only show limited benefits in patients. T-cell receptor (TCR)-T cell therapy offers a promising option to treat CMV infections. Here, using tetramer-based screening and single-cell TCR cloning technologies, we identified various CMV antigen-specific TCRs from healthy donors, and generated TCR-T cells targeting multiple pp65 epitopes corresponding to three major HLA-A alleles. The TCR-T cells showed efficient cytotoxicity toward epitope-expressing target cells in vitro. After transfer into immune-deficient mice bearing pp65

We developed a T-cell-receptor (TCR) complex-based chimeric antigen receptor (CAR) named Synthetic TCR and Antigen Receptor (STAR). Here, we report pre-clinical and phase I clinical trial data (NCT03953599) of this T-cell therapy for refractory and relapsed (R/R) B-cell acute lymphoblastic leukemia (B-ALL) patients. STAR consists of two protein modules each containing an antibody light or heavy chain variable region and TCR α or β chain constant region fused to the co-stimulatory domain of OX40. T-cells were transduced with a STAR-OX40 lentiviral vector. A leukemia xenograft mouse model was used to assess the STAR/STAR-OX40 T cell antitumor activity. Eighteen patients with R/R B-ALL were enrolled into the clinical trial. In a xenograft mouse model, STAR-T-cells exhibited superior tumor-specific cytotoxicity compared with conventional CAR-T cells. Incorporating OX40 into STAR further improved the proliferation and persistence of tumor-targeting T-cells. In our clinical trial, 100% of patients achieved complete remission 4 weeks post-STAR-OX40 T-cell infusion and 16/18 (88.9%) patients pursued consolidative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Twelve of 16 patients (75%) remained leukemia-free after a median follow-up of 545 (433-665) days. The two patients without consolidative allo-HSCT relapsed on Day 58 and Day 186. Mild cytokine release syndrome occurred in 10/18 (55.6%) patients, and 2 patients experienced grade III neurotoxicity. Our preclinical studies demonstrate super anti-tumor potency of STAR-OX40 T-cells compared with conventional CAR-T cells. The first-in-human clinical trial shows that STAR-OX40 T-cells are tolerable and an effective therapeutic platform for treating R/R B-ALL.

Stem cells maintain adult tissue homeostasis under physiological conditions. Uncontrolled stem cell proliferation will lead to tumorigenesis. How stem cell proliferation is precisely controlled is still not fully understood. Phosphorylation of Yun is essential for ISC proliferation. Yun is essential for the proliferation of normal and transformed intestinal stem cells. Our mass spectrometry and biochemical data suggest that Yun can be phosphorylated at multiple residues in vivo. Interestingly, we show that the phosphorylation among these residues is likely interdependent. Furthermore, phosphorylation of each residue in Yun is important for its function in ISC proliferation regulation. Thus, our study unveils the important role of post-translational modification of Yun in stem cell proliferation.

Significance Stem cells maintain tissue homeostasis. We identified a factor, Yun, required for proliferation of normal and transformed intestinal stem cells in adult Drosophila . Yun acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. The Yun/PHB complex acts downstream of EGFR/MAPK signaling and affects the levels of E2F1 to regulate intestinal stem cell proliferation. The role of the PHB complex in cell proliferation is evolutionarily conserved. Our results provide insight into the underlying mechanisms of how stem cell proliferation is properly controlled during tissue homeostasis and tumorigenesis.

Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. Here, from an RNA interference (RNAi) screen in adult

Age-related macular degeneration (AMD) is currently the leading cause of blindness worldwide. Previously, we identified ubiquitin-protein ligase E3D (UBE3D) as an AMD-associated protein for East Asian populations, and here we further demonstrate that UBE3D could be associated with DNA damage response.The established I-SceI-inducible GFP reporter system was used to explore the effect of UBE3D on homologous recombination. Immunoprecipitation-mass spectrometry (MS) was used to explore potential UBE3D-interacting proteins and validated with coimmunoprecipitation assays and the pulldown assays. Micrococcal nuclease (MNase) assays were used to investigate the function of UBE3D on heterochromatin de-condensation upon DNA damage. An aged mouse model of blue light-induced eye damage was constructed, and electroretinography (ERG) and optical coherence tomography (OCT) were performed to compare the differences between wild-type and UBE3D+/- mice.First, we show that GFP-UBE3D is recruited to damage sites by PCNA, through a PCNA-interacting protein (PIP) box. Furthermore, UBE3D interacts with KAP1 via R377R378 and oxidation of the AMD-associated V379M mutation abolishes KAP1-UBE3D binding. By MNase assays, UBE3D depletion reduces the chromatin relaxation levels upon DNA damage. In addition, UBE3D depletion renders less KAP1 recruitment. Compared with wild type, blue light induces less damage in UBE3D+/- mice as measured by ERG and OCT, consistent with our biochemical results.Hence, we propose that one potential mechanism that UBE3D-V379M contributes to AMD pathogenesis might be via defective DNA damage repair linked with oxidative stress and our results offered a potential direction for the treatment of AMD.

The tumor microenvironment (TME) in gastric cancer (GC) has been shown to be important for tumor control but the specific characteristics for GC are not fully appreciated. We generated an atlas of 166,533 cells from 10 GC patients with matched paratumor tissues and blood. Our results show tumor-associated stromal cells (TASCs) have upregulated activity of Wnt signaling and angiogenesis, and are negatively correlated with survival. Tumor-associated macrophages andLAMP3+DCs are involved in mediating T cell activity and form intercellular interaction hubs with TASCs. Clonotype and trajectory analysis demonstrates that Tc17 (IL-17+CD8+T cells) originate from tissue-resident memory T cells and can subsequently differentiate into exhausted T cells, suggesting an alternative pathway for T cell exhaustion. Our results indicate thatIL17+cells may promote tumor progression throughIL17,IL22, andIL26signaling, highlighting the possibility of targetingIL17+cells and associated signaling pathways as a therapeutic strategy to treat GC.

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.