Programmed cell death protein-1 (PD-1)-mediated immunosuppression has been proposed to contribute to the limited clinical efficacy of chimeric antigen receptor T (CAR-T) cells in solid tumors. We generated PD-1 and T cell receptor (TCR) deficient mesothelin-specific CAR-T (MPTK-CAR-T) cells using CRISPR-Cas9 technology and evaluated them in a dose-escalation study. A total of 15 patients received one or more infusions of MPTK-CAR-T cells without prior lymphodepletion. No dose-limiting toxicity or unexpected adverse events were observed in any of the 15 patients. The best overall response was stable disease (2/15 patients). Circulating MPTK-CAR-T cells peaked at days 7–14 and became undetectable beyond 1 month. TCR-positive CAR-T cells rather than TCR-negative CAR-T cells were predominantly detected in effusion or peripheral blood from three patients after infusion. We further confirmed the reduced persistence of TCR-deficient CAR-T cells in animal models. Our results establish the preliminary feasibility and safety of CRISPR-engineered CAR-T cells with PD-1 disruption and suggest that the natural TCR plays an important role in the persistence of CAR-T cells when treating solid tumors.

Purpose: Although chimeric antigen receptor T-cell (CAR-T) therapy development for B-cell malignancies has made significant progress in the last decade, broadening the success to treating T-cell acute lymphoblastic leukemia (T-ALL) has been limited. We conducted two clinical trials to verify the safety and efficacy of GC027, an “off-the-shelf” allogeneic CAR-T product targeting T-cell antigen, CD7. Here, we report 2 patients as case reports with relapsed/refractory T-ALL who were treated with GC027. Patients and Methods: Both the trials reported here were open-label and single-arm. A single infusion of GC027 was given to each patient after preconditioning therapy. Result: Robust expansion of CAR-T cells along with rapid eradication of CD7+ T lymphoblasts were observed in the peripheral blood, bone marrow, and cerebrospinal fluid. Both patients achieved complete remission with no detectable minimal residual disease. At data cutoff, 30 September 2020, 1 of the 2 patients remains in ongoing remission for over 1 year after CAR T-cell infusion. Grade 3 cytokine release syndrome (CRS) occurred in both patients and was managed by a novel approach with a ruxolitinib-based CRS management. Ruxolitinib showed promising activity in a preclinical study conducted at our center. No graft-versus-host disease was observed. Conclusions: The two case reports demonstrate that a standalone therapy with this novel CD7-targeted “off-the-shelf” allogeneic CAR-T therapy may provide deep and durable responses in select patients with relapsed/refractory T-ALL. GC027 might have a potential to be a promising new approach for treating refractory/relapsed T-ALL. Further studies are warranted.

IntroductionThere is no curative treatment available for patients with chemotherapy relapsed or refractory CD19+ B cells-derived acute lymphoblastic leukaemia (r/r B-ALL). Although CD19-targeting second-generation (2nd-G) chimeric antigen receptor (CAR)-modified T cells carrying CD28 or 4-1BB domains have demonstrated potency in patients with advanced B-ALL, these 2 signalling domains endow CAR-T cells with different and complementary functional properties. Preclinical results have shown that third-generation (3rd-G) CAR-T cells combining 4-1BB and CD28 signalling domains have superior activation and proliferation capacity compared with 2nd-G CAR-T cells carrying CD28 domain. The aim of the current study is therefore to investigate the safety and efficacy of 3rd-G CAR-T cells in adults with r/r B-ALL.Methods and analysisThis study is a phase I clinical trial for patients with r/r B-ALL to test the safety and preliminary efficacy of 3rd-G CAR-T cells. Before receiving lymphodepleting conditioning regimen, the peripheral blood mononuclear cells from eligible patients will be leukapheresed, and the T cells will be purified, activated, transduced and expanded ex vivo. On day 6 in the protocol, a single dose of 1 million CAR-T cells per kg will be administrated intravenously. The phenotypes of infused CAR-T cells, copy number of CAR transgene and plasma cytokines will be assayed for 2 years after CAR-T infusion using flow cytometry, real-time quantitative PCR and cytometric bead array, respectively. Moreover, several predictive plasma cytokines including interferon-γ, interleukin (IL)-6, IL-8, Soluble Interleukin (sIL)-2R-α, solubleglycoprotein (sgp)130, sIL-6R, Monocyte chemoattractant protein (MCP1), Macrophage inflammatory protein (MIP1)-α, MIP1-β and Granulocyte-macrophage colony-stimulating factor (GM-CSF), which are highly associated with severe cytokine release syndrome (CRS), will be used to forecast CRS to allow doing earlier intervention, and CRS will be managed based on a revised CRS grading system. In addition, patients with grade 3 or 4 neurotoxicities or persistent B-cell aplasia will be treated with dexamethasone (10 mg intravenously every 6 hours) or IgG, respectively. Descriptive and analytical analyses will be performed.Ethics and disseminationEthical approval for the study was granted on 10 July 2014 (YLJS-2014-7-10). Written informed consent will be taken from all participants. The results of the study will be reported, through peer-reviewed journals, conference presentations and an internal organisational report.Trial registration numberNCT02186860.

Introduction Chimeric Antigen Receptor T cells (CAR-T) therapy, e.g. B Cell Maturation Antigen (BCMA)-directed CAR-T has provided an encouraging modality for relapsed and refractory management of multiple myeloma (MM). However, a significant portion of patients still relapse with progressive disease after monospecific anti-BCMA CAR-T treatment. It has been demonstrated that CD19-directed CAR-T was effective in certain MM patients, likely due to CD19 expression on subsets of MM cells, and/or undetectable level of CD19 on MM cells. In addition, it has been reported that CD19 could express on the myeloma progenitor cells. To further improve the efficacy and to reduce relapse, we have designed a bispecific CAR-T targeting both BCMA and CD19. In addition to the conventionally-manufactured BCMA-CD19 CAR-T, the bispecific CAR-T was also successfully manufactured in our newly developed FasT CAR-T platform, which shortened the production time to one day. Here we report the results from pre-clinical studies and early results from the first-in-human clinical study. Methods The BCMA-CD19 bispecific CAR was constructed by linking BCMA and CD19 scFv, joined by a CD8 hinge, transmembrane domain, co-stimulatory domain and CD3. CAR-T cells were produced using either the conventional process (GC012) or the FasT CAR-T platform (GC012F). Peripheral blood (PB) mononuclear cells were obtained by leukapheresis either from healthy donors for the pre-clinical study or from patients for the clinical trial. T cells were isolated and used for CAR-T manufacturing. A xenograft mouse model was used to determine the efficacy in vivo. From March 2019 to July 2019, 5 adult relapsed/refractory MM patients (Age 50-59), who had previously received multiple lines of therapies, were enrolled (Table). Among them, 2 had extramedullary diseases. One patient did not receive lymphodepletion, and all other 4 patients received i.v. fludarabine and cyclophosphamide for 3 days. All patients received a single infusion of CAR-T cells, either at dose 1x106/Kg (DL1) (2 patients) or at dose 2x106/Kg (DL2) (3 patients), and the dose escalation is still ongoing. The endpoints of the exploratory trial were to evaluate the safety, feasibility, PK, and clinical efficacy of BCMA-CD19 bispecific CAR-T. Results In pre-clinical study, BCMA-CD19 bispecific CAR-T were very effective in killing CD19+ and/or BCMA + target cells including MM cell lines RPMI8226 and MM.1s (Fig 1). Increased IFN production and CD107a up-regulation were also observed. We demonstrated that BCMA-CD19 CAR-T completely eliminated BCMA+ MM cell line RPMI8226, MM.1s, and CD19+ ALL cell line Nalm6 in in vivo xenograft models. Additionally BCMA-CD19 CAR-T cells were shown to be more cytotoxic than single CAR-T both in vitro and in vivo. BCMA-CD19 CAR-T manufactured in the FasT CAR-T platform was more effective in eliminating MM in a xenograft model (Fig. 1). In the clinical study, the median observation time was 68 days (27-144 days up to 2019/7/30). Five patients were evaluated between 15-59 days post CAR-T infusion. Despite the relatively short disease evaluation time, all 5 patients responded to the treatment: 1 patient achieved sCR, 3 achieved VGPR and 1 achieved PR. Notably, although patient KM001 did not receive any pre-conditioning, however, the patient achieved sCR status on Day 15 and has maintained sCR up to now (129 days). CAR-T PK in the PB was monitored by qPCR and flow cytometry. The CAR-T proliferation peak was reached on Day 10 (D7-D14), and the median peak copy number was 34,039 (12,897-128,775) copies /ug DNA (Fig. 2). Remarkably, despite the encouraging clinical response to the CAR-T treatment, no severe adverse events were encountered during the observation period. Three patients experienced only grade 1 cytokine release syndrome (CRS) and no subject suffered from neurotoxicity of any level (Table). Conclusion Pre-clinical data demonstrated BCMA-CD19 CAR-T cells are effective in eliminating MM tumor cells both in vitro and in vivo. The first-in-human clinical trial also showed extraordinary safety profile and efficacy of BCMA-CD19 bispecific CAR-T in treating R/R MM. The long-term benefit and effect on relapse are being further studied. Bispecific CAR-T manufacturing on the FasT CAR-T platform is successful and has been shown to be more potent. A clinical study to evaluate safety and efficacy of FasT BCMA-CD19 CAR-T is ongoing. Disclosures No relevant conflicts of interest to declare.

To improve clinical outcomes and shorten the vein-to-vein time of chimeric antigen receptor T (CAR-T) cells, we developed the FasT CAR-T (F-CAR-T) next-day manufacturing platform. We report the preclinical and first-in-human clinical studies evaluating the safety, feasibility, and preliminary efficacy of CD19 F-CAR-T in B-cell acute lymphoblastic leukemia (B-ALL). CD19 F-CAR-T cells demonstrated excellent proliferation with a younger cellular phenotype, less exhaustion, and more effective tumor elimination compared to conventional CAR-T cells in the preclinical study. In our phase I study (NCT03825718), F-CAR-T cells were successfully manufactured and infused in all of the 25 enrolled pediatric and adult patients with B-ALL. CD19 F-CAR-T safety profile was manageable with 24% grade 3 cytokine release syndrome (CRS) and 28% grade 3/4 neurotoxicity occurring predominantly in pediatric patients. On day 14, 23/25 patients achieved minimal residual disease (MRD)-negative complete remission (CR), and 20 subsequently underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) within 3 months post F-CAR-T therapy. Fifteen of 20 patients were disease-free with a median remission duration of 734 days. One patient relapsed and 4/20 died from transplant-related mortality. Of the three patients who did not undergo allo-HSCT, two remained in CR until 10 months post-F-CAR-T. Our data indicate that anti-CD19 FasT CAR-T shows promising early efficacy for B-ALL. Further evaluations in larger clinical studies are needed.