Introduction It has been made great commercial success in hematological malignancies by chimeric antigen receptor (CAR) T cell therapy. However, high rate of adverse events (AEs) still remains an issue. Non-viral Genome Targeting CAR (gtCAR) delivery technology, which uses CRISPR-Cas9 to insert CAR DNA to TRAC locus specifically in human T cells, produces moderate CAR-T cells compared with conventional ones. Earlier results of SC-gtCAR19, an anti-CD19 gtCAR-T candidate from an investigator initiated Phase I clinical trial (ChiCTR2000031942) demonstrated a favorable benefit-risk profile in relapsed/refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL) patients. Reported here are updated long-term follow-up results. Objective The primary objective is to assess safety. Secondary objective is to evaluate efficacy. Level of CAR-T cell in blood was explored. Method This is a single center, open-label and single-arm trial. Patients or allogeneic donors underwent leukapheresis and their CD3+ T cells were selected and modified by non-viral vector to produce SC-gtCAR19. A standard lymphodepletion chemotherapy was performed. The median dose was 3.89×106CAR+ cells/kg (0.58-8.31×106/kg). AEs were graded according to CTCAE v5.0. Cytokine Release Syndrome (CRS) and neurological events (NEs) were graded by ASTCT consensus grading. Relapse defined as bone marrow (BM) blast or B-ALL related mutation gene positive from nonexistence regardless of specific value, and extramedullary disease (EMD) occurrence. Result As of May 1st 2022, twenty-two r/r B-ALL patients received SC-gtCAR19 (Table 1). The median age was 29 (13-61), and the median number of previous regimen was 5 (2-16). The median baseline BM blast after preconditioning was 44.1% (0.3-99%), particularly 73% of patents with high disease burden (>25% leukemic blast). Five patients (23%) once had stem cell transplantation (SCT), and 14 (64%) have B-ALL related chromosomal and molecular abnormalities. One patient has EMD. No patient has been treated by any other CD19 related therapy before enrollment. The most common AEs of any grade were anaemia (100%), pyrexia (91%), neutrophil count decreased (91%), and white blood cell count decreased (86%) (Table 2). CRS occurred in 20 patients (91%), and 4 of them (18%) with grade 3 CRS. The median time to CRS onset was 7 days (IQR 7-14), and the median duration of any grade CRS was 3 days (IQR 2-5). Three patents developed NEs, one was grade 2 and 2 were grade 3 (temporary seizure, related to infection and patient's cerebral infarction before enrollment). No fatal or life-threatening events happened. No Tocilizumab administered, four patients used vasopressors and 2 steroids for treatment of CRS. All patients were eligible for efficacy analysis. At Day 28, the overall response rate was 100%, with 9 (41%) patients achieving complete remission (CR), ten (45%) achieving complete remission with incomplete haematological recovery (CRi), and 3 (14%) with blast-free hypoplastic. Among them, twenty patients (91%) achieved minimum residual disease (MRD) negative CR, and those 2 patients with MRD positive CR were assessed undetectable BM MRD in Day 90. Patient with EMD, the nodal lesions in her breast disappeared at Day 60. At a median follow-up of 19.9 months (4.5-26.6), nineteen patients relapsed, within which 7 (36.8%) antigen escape relapse. The median duration of remission for 19 patients achieving CR/CRi was 8 months (2.5-NE). All patients received a consolidation treatment by Lenalidomide 3 months after infusion on condition of CR status until disease progression/death, and all of them did not receive SCT afterwards before relapse. As of the data cutoff, two patients (9%) had ongoing CR with median follow-up of 22.4 months. The median relapse-free survival for 19 patients was 8.9 months (2.9-NE) (Figure 1). The median OS was 24.2 months (3.7-NE) (Figure 2). CAR T cell level measured by cell copies percentage per peripheral blood mononuclear cells peaked at median 12 days (IQR 10-13) (Figure 3). The median peak percentage of copies was 53% (IQR 26.5-72.1%). Conclusion This study is the first clinical trial applied a gtCAR-T candidate for r/r B-ALL treatment. SC-gtCAR19 has a better risk-tolerance. Despite TCR gene knocked-out, gtCAR-T cell proliferation and its function both short and long term may not be influenced. We illustrate that this novel approach is feasible in terms of manufacture and clinical application. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction: It has been made great clinical progresses in hematological malignancies by chimeric antigen receptor (CAR) T cell therapy which utilizes virus vector for manufacture. However, there're still issues unresolved, for instance, sophisticated virus production process, deadly Cytokine Release Syndrome (CRS) side-effect, and high recurrence rate, which probably limit the availability of CAR-T therapy. Non-viral Genome Targeting CAR-T (nvGT CAR-T) may provide a feasible solution to those unmet needs mentioned above. We used CRISPR-Cas9 and non-viral vector to insert anti-CD19 CAR DNA to a specific genome locus in human T cells, which in theory, produces more moderate CAR-T cells compared with conventional CAR-T cells. The efficacy of anti-CD19 nvGT CAR-T cells had been demonstrated in our previous pre-clinical studies, and in this Phase I clinical trial (ChiCTR2000031942), its safety and efficacy in relapsed/refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL) patients were explored. Objective: The primary objective of this Phase I trial is to assess safety, including evaluation of adverse events (AEs) and AEs of special interest, such as CRS and neurotoxicity. Secondary objective is to evaluate efficacy as measured by the ratio of complete remission (CR). Method: Peripheral blood mononuclear cells were collected from patients or allogeneic donors, then CD3+ T cells were selected and modified by nvGT vector to produce anti-CD19 CAR-T, then administrated to patients with r/r B-ALL. Up to July 2020, twelve patients with r/r B-ALL had been enrolled in this study and 8 patients completed their treatments and entered follow-up period. For 8 patients with follow-up data, the median age was 33 years (range, 13 to 61), and the median number of previous regimens was 5 (range, 2 to 11). The median baseline percentage of bone marrow (BM) blast is 72% (range, 24.5% to 99%). Among those subjects, 2 patients once have been conducted autologous or allogeneic hematopoietic stem cell transplantation (Auto-HSCT or Allo-HSCT), and 2 patients experienced serious infection before CAR-T infusion. No patient has been treated by any other CAR-T therapy before enrollment. Baseline characteristics refer to Table 1. Administering a lymphodepleting chemotherapy regimen of cyclophosphamide 450-750 mg/m2 intravenously and fludarabine 25-45 mg/m2 intravenously on the fifth, fourth, and third day before infusion of anti-CD19 nvGT CAR-T, all patients received an infusion at dose of 0.55-8.21×106/kg (Table 1). Result: Until day 30 post CAR-T cell infusion, 8/8 (100%) cases achieved CR and 7/8 (87.5%) had minimal residual disease (MRD)-negative CR (Table 1). Anti-bacterial and anti-fungal were performed in patients SC-3, SC-4 and SC-5 after CAR-T cell infusion, which seems no influence on efficacy. Patient SC-7 was diagnosed as T-cell Acute Lymphoblastic Leukemia before Allo-HSCT but with recent recurrence of B-ALL, which was MRD-negative CR on day 21 post nvGT CAR-T therapy. Up to July 2020, all cases remain CR status. CRS occurred in all patients (100%) receiving anti-CD19 nvGT CAR-T cell, including 1 patient (12.5%) with grade 3 (Lee grading system1) CRS, two (25%) with grade 2 CRS, and 5 (62.5%) with grade 1 CRS. There were no cases of grade 4 or higher CRS (Table 1). The median time to onset CRS was 9 days (range, 1 to 12 days) and the median duration of CRS was 6 days (range, 2 to 9 days). None developed neurotoxicity. No fatal or life-threatening reactions happened and no Tocilizumab and Corticosteroids administered following CAR-T treatment. Data including body temperature (Figure 1), CAR-positive T cell percentage (Figure 2), Interleukin-6 (IL-6) and Interleukin-8 (IL-8) (Figure 3 and 4), C-reactive Protein (CRP) (Figure 5), Lactate Dehydrogenase (LDH) (Figure 6), and Procalcitonin (PCT) (Figure 7), are in accordance with the trend of CRS. Conclusion: This Phase I clinical trial primarily validates the efficacy of this novel CAR-T therapy, however, it still needs time to prove its durability. Surprisingly, we find that nvGT CAR-T therapy is seemingly superior than viral CAR-T therapy in terms of safety. All subjects which are high-risk patients with high tumor burden had low grade CRS, even a few patients sent home for observation post infusion with limited time of in-patient care. Furthermore, patients could tolerate a higher dose without severe adverse events, which probably bring a better dose-related efficacy. Disclosures No relevant conflicts of interest to declare.