Introduction CD7-targeted chimeric antigen receptor (CAR) T-cell therapy has demonstrated efficacy and safety in treating T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). However, evidence remains limited regarding the ability of CD7 CAR-T cells to cross the blood-brain barrier and effectively eradicate central nervous system (CNS) involvement in T-ALL/LBL without exacerbating neurotoxicity. Here we evaluated the efficacy and safety of CD7 CAR-T cells in pediatric and adult patients with relapsed/refractory (R/R) T-ALL/LBL involving the CNS through phase I/II clinical trials (NCT04572308 and NCT04916860). Methods Peripheral blood mononuclear cells were collected via leukapheresis from enrolled patients. A novel approach was used to generate “naturally selected” CD7 CAR (NS7CAR)-T cells that overcome CD7-directed fratricide without gene editing. NS7CAR is a 2nd-generation murine-based CAR-T containing 4-1BB and CD3ζ co-stimulatory domains. Patients who relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT) without active graft-versus-host disease (GVHD) were eligible. All patients received intravenous fludarabine (30mg/m2/d) and cyclophosphamide (300mg/m2/d) (FC) conditioning regimens on day -5 to day -3 prior to NS7 CAR-T infusion. To manage rapidly progressive disease, bridging therapy was allowed between leukapheresis and FC conditioning. CD7 CAR-T cell proliferation and blast clearance in cerebrospinal fluid (CSF) were monitored using flow cytometry (FCM). Results Thirty patients with CNS involvement (24 T-ALL and 6 T-LBL) received NS7CAR T cell infusion. The median age was 23 years (range, 4-44). At enrollment, 13 patients were classified as CNS-3 (WBC ≥5/mcL in CSF), and 17 as CNS-2 (WBC <5/mcL in CSF). Seven patients relapsed from prior allo-HSCT. For high-risk features, 7 patients harbored STIL::TAL1 fusion genes, and 1 patient had TP53 gene mutations. Nineteen patients had blasts in bone marrow (BM), with a median blast of 38%(range:0.5-93%), including 4 patients with blasts>80%. Non-CNS extramedullary disease (EMD) was present in 10 patients. Bridging therapy was administered in 25 patients. All patients received a single dose of NS7 CAR-T cells at a low dose (5×105cells/kg, n=2), and a medium dose (1-1.5×106cells/kg, n=28). Following infusion, 28 patients had available CAR-T cell expansion data in CSF. The median maximum level of NS7CAR-T cells in CSF was 40.5% (range, 0.0-94.9%), which occurred on day 19 (range, 13-51) as detected by FCM. About 1 month post CD7 CAR-T infusion (except 1 evaluated on day 51), 30/30 (100%) patients achieved complete remission (CR) in CSF, and 17/19(89.5%) patients showed minimal residual disease (MRD)-negative CR in BM. For non-CNS EMD, the overall response rate was 9/10 (90%, 6 CR and 3 partial remission). The median follow-up time was 276.5 days (range, 58-1676). The 1-year overall survival (OS) and progression-free survival (PFS) was 83.0% and 52.8%, respectively, and the 3-year OS and PFS were 67.1% and 45.3%. Among the 24 patients achieving CR in both EMD and BM, 18 received consolidation allo-HSCT within 3 months post-CAR-T, with the 1-year OS and PFS of 87.%% and 75.1% and the 3-year rates of 70.0% and 60.1%. Of the remaining 6 CR patients, 4 relapsed within 3 months, 1 was lost to follow-up on day 180, and 1 died on day 75. Eight patients relapsed following CAR-T infusion (6 in BM and 2 in CNS) at a median of 65 days (range, 33-265), of whom 5 patients with STIL::TAL1 fusion genes relapsed within 3 months. Three of 8 patients lost CD7 expression. Specifically, both CNS relapsed patients experienced early relapse on day 56 and day 72, respectively. The majority of (27/30, 90%) patients experienced mild cytokine release syndrome (CRS, grade I, n=26 and grade II, n=1), and 3/30(10%) patients had grade III CRS. Neurotoxicity occurred in 2 patients (6.7%, grade IV), and others had no neurotoxicity. All were effectively managed with corticosteroids and/or tocilizumab. Conclusion This study establishes NS7CAR-T cells as a promising therapeutic option for R/R T-ALL/LBL with CNS involvement, demonstrating a manageable safety profile. Notably, disease relapse primarily occurred at non-CNS sites. We identified STIL::TAL1 fusion as a potential predictor of early relapse. Further studies with extended follow-up and larger cohorts are warranted to better define the long-term efficacy and durability of NS7CAR-T therapy in CNS-involved T-ALL/LBL.

Approximately 30% of acute myeloid leukemia (AML) patients express CD7 on their myeloblasts. We have previously demonstrated that scFv-based “naturally selected” CD7 CAR-T (NS7CAR-T) therapy shows significant efficacy with a favorable safety profile in T-cell lymphoid malignancies. Here we derived dual nanobody-based dVHH NS7CAR-T cells that have superior CD7 binding specificity, affinity to their scFv-based counterparts and improved proliferative capability. In this phase I clinical trial, we evaluated the efficacy and safety of dVHH NS7CAR-T cells in patients with CD7-positive refractory/relapsed (r/r) AML. A cohort of ten patients received dVHH NS7CAR-T cells across two dosage levels of 5×105/kg and 1×106/kg. Before enrollment, patients had undergone a median of 8 (range: 3-17) prior lines of therapy. Seven patients had prior transplants. Following NS7CAR-T cell infusion, 7/10 (70%) patients achieved complete remission (CR). The median observation time was 178 days (28-776 days). Among the seven patients who achieved CR, 3 who relapsed from prior transplants underwent a second allogeneic hematopoietic stem cell transplant (allo-HSCT). One patient remained leukemia-free on day 401, and the other two died on day 241 and day 776 from non-relapse-related causes. Three CR patients without consolidative allo-HSCT relapsed within 90 days. All the nonresponders and relapsed patients had CD7 loss. The treatment was well-tolerated, with 80% experiencing mild cytokine release syndrome and none had neurotoxicity. This trial underscores the potential promising treatment of dVHH NS7CAR-T in providing clinical benefits with a manageable safety profile to CD7-positive AML patients, warranting further investigation. NCT04938115

CD7 Chimeric Antigen Receptor-T cell (CAR-T) therapy demonstrates efficacy in relapsed/refractory (R/R) acute T-lymphoblastic leukaemia (ALL)T-ALL/lymphoblastic lymphoma (LBL), but concerns about T-cell depletion and severe immunodeficiency persist. We compared infection rates and immune cell subsets in 60 R/R T-ALL/LBL patients receiving naturally selected CD7 CAR-T (NS7CAR-T) with 60 R/R B-ALL patients undergoing CD19 CAR-T. Infections were monitored from infusion until allogeneic haematopoietic stem cell transplantation (HSCT) or up to 3 months. Overall infection rates did not significantly differ between groups (36.67% vs. 24.56%, p = 0.24), although the incidence of early immune effector cell-associated haematotoxicity (ICAHT) grade III-IV was higher in the CD7 CAR-T group than in the CD19 CAR-T group (33.9% vs. 16.7%, p = 0.03). Post-CD7 CAR-T infusion analysis showed a significant decline in CD7(+) T cells and an increase in non-CAR-T-derived CD7(-) T cells, particularly non-CAR-T cells, which rose to a median proportion of 84.4% (range: 22.1%-99.9%) by day 28; meanwhile, CD7(-) natural killer (NK) cells approached nearly 100% following the depletion of CD7(+) NK cells. This study indicates that while CD7 CAR-T therapy significantly reduces CD7(+) T cells, it does not lead to increased short-term infection rates. The notable expansion of non-CAR-T-derived CD7(-) T and NK cells helps preserve immune function, highlighting distinct therapeutic mechanisms between CD7 CAR-T and CD19 CAR-T due to their different lineage restrictions.

Introduction Peripheral T-cell lymphomas (PTCL) encompass a broad class of heterogeneous clinicopathologic entities unified in their derivation from a mature, post-thymic T-cell. For patients with relapsed or refractory (R/R) PTCL, outcomes are generally poor, even with an allogeneic hematopoietic stem cell transplantation (allo-HSCT), creating a great need of new therapeutics. CD7 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and a subset of PTCL. We have previously demonstrated that “naturally selected” CD7 CAR-T (NS7CAR-T) therapy shows significant efficacy with a favorable safety profile in 60 patients with T-ALL and T-cell lymphoblastic lymphoma (T-LBL). Here, we conducted a phase I clinical trial (https://clinicaltrials.gov/ NCT04928105) to investigate the safety and efficacy of NS7CAR-T therapy in treating R/R PTCL. Methods Peripheral blood mononuclear cells were obtained from the patients. T-cells were then purified using CD3+ magnetic beads. The second-generation CD7CAR with a 4-1BB costimulatory domain was manufactured following the protocol. Patients who were diagnosed with r/r PTCL and had positive CD7 expression were eligible. Before the CAR-T cell infusion, bridging therapies were permitted for patients with rapid disease progression. All patients received intravenous fludarabine (30mg/m2/d) and cyclophosphamide (300mg/m2/d) lymphodepletion chemotherapy for three consecutive days (day -5 to day -3). The median time from leukapheresis to CAR-T cell infusion was 15 days. Results Between April 2021 and November 2023, 5 patients diagnosed with CD7-positive r/r PTCL were enrolled and received NS7CAR-T cell infusions. The subtypes included PTCL not otherwise specified (PTCL-NOS, n=2), monomorphic epitheliotropic intestinal T cell lymphoma (MEITL, n=1), hepatosplenic T-cell lymphoma (HSTCL, n=1), and NK/T cell lymphoma (n=1). The median age of the enrolled patients was 49 years (39-60 years). Before enrollment, patients had undergone a median of 6 (range: 2-12) prior lines of therapy. One patient had a history of autologous transplant. Regarding the extramedullary disease (EMD) status, 4/5 presented with diffuse EMD, and the other one had localized EMD. Additionally, 3 patients showed minimal residual disease (MRD)-positive involvement in bone marrow (BM) by flow cytometry (FCM) at enrollment, ranging from 0.09 to 1.19%. Four patients received a medium dose (1×106/kg) and 1 received a high dose (2×106/kg). Following infusion, 4/5 patients exhibited good proliferations, with peak levels ranging from 1.61×105 copies/μg to 5.83×105 copies/μg genomic DNA CAR-T cells based on q-PCR and from 57.11% to 93.97% CAR-T cells as detected by FCM, and 1/5 patient had a low proliferation with peak levels of 224 copies/μg genomic DNA CAR-T cells by qPCR and 8.99% by FCM. The median peak time occurred around day 14 (day 14 - day 27) per q-PCR, and around day 14 (day 11 - day 19) by FCM. The median transduction efficiency of the products was 93.5% (range: 89.3%-97.4%). At a median of 34 days post NS7CAR-T cell infusion, three (60%) patients achieved complete remission (CR) per position-emission tomography (PET)/ and/or contrast computed tomography (CT) and BM evaluation, one showed partial remission (PR), and one had no remission (NR). The median observation time was 234 days (range:42-474 days). For the three CR patients, two received consolidation allo-HSCT, including one who remained progression-free survival on day 216, and the other who subsequently relapsed on day 248 and died on day 474 from chronic graft-versus-host disease (GVHD). The remaining CR patient without allo-HSCT progressed to acute leukemia around day 234 and subsequently passed away. The PR patient received salvage allo-HSCT and remained disease-free on day 411. The patient with NK/T cell lymphoma, had NR and subsequently withdrew. Post-infusion, all patients experienced mild cytokine release syndrome (CRS), with four displaying grade I and one having grade II CRS. None of the patients had neurotoxicity. Conclusions Our study highlights that CD7-positive heavy pre-treated R/R PTCL patients could achieve a promising CR and safety profile after CD7-targeted CAR-T therapy, who otherwise have very limited therapy options. More data on additional patients and longer observation times are needed to further evaluate the efficacy and safety of CD7 CAR-T products in treating PTCL.

Background: Limited studies have demonstrated that CD7-targeted chimeric antigen receptor (CAR) T-cell therapy is effective and safe for T-cell acute lymphoblastic leukemia(T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). However, it remains unclear whether CD7 CAR-T cells can penetrate the blood-brain barrier and be effective in treating central nervous system (CNS) T-ALL/LBL without increasing neurotoxicity. Aims: Here we explored the efficacy and safety of NS7CAR T-cells for relapsed or refractory (R/R) T-ALL/LBL with CNS involvement in phase I/II clinical trial (NCT04572308 &NCT04916860). Methods: Peripheral blood mononuclear cells were collected from patients by leukapheresis. A novel fratricide-resistant approach to derive naturally selected anti-CD7 CAR (NS7CAR) T cells was developed using lentiviral transduction of peripheral T cells that could overcome CD7-directed fratricide without additional genetic modifications. NS7CAR is a 2nd generation murine-based CAR-T containing 4-1BB and CD3ζ co-stimulatory domains. Intravenous fludarabine (30mg/m2/d) and cyclophosphamide (300mg/m2/d) were given to all patients on day -5 to day -3 prior to NS7 CAR T cells infusion. The proliferation of CAR-T cells in cerebrospinal fluid (CSF) was detected by flow cytometry (FCM). Results: From Dec. 2020 to Jun. 2022, 10 patients with CNS T-ALL (n=8) and T-LBL (n=2) were enrolled and received NS7CAR T cells. The median age was 14.5 (2-37) years old. Patient characteristics and clinical results were shown in Table 1. Three patients who relapsed from prior allogeneic hematopoietic stem cell transplantation (allo-HSCT) were also enrolled. At enrollment, 6 patients were CNS-3 status (blasts≥5/μL in CSF), and 4 were CNS-2 status (blasts<5/μL in CSF). Eight patients had leukemic cells both in bone marrow (BM) and CNS. Combined extramedullary disease (EMD) except for CNS involvements was found in 5 patients. The transduction efficiency was 91.2% (64.4%-97.4%). A single dose of NSCAR T cells was infused to patients at a low dose (5×105cells/kg, n=2), and a medium dose (1-1.5×106cells/kg, n=8). At a median of day 17 (day 14-day 51) post infusion, 10/10 (100%) achieved complete remission (CR) in CSF. The median follow-up time was 220 days (61-621 days). Two patients had early CNS relapse on day 56 (then received salvage transplant and were still alive at the last follow-up of day 412), and day 72, respectively. For the other 8 patients without CNS relapsed regardless of EMD/BM relapse, 5 who received consolidation/salvage allo-HSCT were still progression-free at a median follow-up of 286 days(188-621days), 1 lost follow-up on day 180, 1 relapsed in BM on day116 and withdrew for other clinical trials, and the other 1 remained CR on day 61. Mild cytokine release syndrome (CRS, ≤grade II) occurred in 8/10 (80%) patients, and 2/10(20%) patients had grade III CRS. One patient had grade IV neurotoxicity, and others did not develop neurotoxicity. All were controlled after the administration of corticosteroids and/or tocilizumab. Following infusion, the median maximum proliferation of NS7CAR T-cells in CSF was 22.56% (0-65.35%), which occurred on day 17(13-33) as detected by FCM. Summary/Conclusion: This study demonstrated that NS7 CAR-T is a promising method in treating patients who had R/R CNS T-ALL/LBL without increasing the risk of severe neurotoxicity. Safety was manageable. However, more data on additional patients and longer observation time are needed to fully evaluate the efficacy of NS7 CAR-T products in patients with CNS involvement.Keywords: CNS, CAR-T, T cell acute lymphoblastic leukemia, relapsed/refractory

Background: chimeric antigen receptor(CAR) T cell therapy achieved a great success in the treatment of B cell acute lymphoblastic leukemia(B-ALL). However, CAR-T post first allogeneic hematopoietic stem cell transplantation (allo-HSCT), donor cells would involve to patients’ immune system, even if same donors’ lymphocytes were used to produce CAR-T cells. To analyze the potential influence of first allo-HSCT, the expansion of CAR-T cells, the clearance of CD19+ cell, and expression of 24 cytokines related to T cell life cycle and graft versus host disease (GVHD) were measured in this study. Aims: To figure out the promising biomarkers may influence the process of CAR-T treatment of HSCT relapsed patients. Methods: 60 patients with B-ALL received CD19-CAR-T treatment in Hebei Yanda Lu Daopei Hospital in 2020 were randomly selected. For 30 patients, CAR-T therapy was performed after the first allo-HSCT failure(group1), and another 30 cases who received CAR-T before allo-HSCT(group2). 22 were females, and 38 were males, with median age 17 (from 2 to 61). All the patients were collected EDTA anti-coagulated PB samples to test CAR-T cells expansion and CD19+ cells clearance by flow cytometer(FCM), and non-anti-coagulated serum to detect 24 kinds of cytomkines by FCM microbeads on before CAR-T(d0+), d4, d7, d11, d15, d20, and d30 after CAR-T. Results: As showed in Figure 1A and 1B, CAR-T cells expanded on d4 and reached the peak on d11, and then decreased and went to smooth from d15 in both group. However, a difference was observed between two groups. The peak was higher and the decrease curve was sharp in group2, and the peak was lower and a plateau was formed from d7 to d15 in group1. CD19+ cells clearance formed similar tracking lines in both groups. The CD19+ cells could not be detected on d14 in both group. The clearance rate might be smoother in group2 than that in group1. However, there were no significant differences based on statistic analyze which might result the bigger variable coefficient and less cases.In Figure 1C, within 24 kinds of cytokines, IL-6, sCD25, MCP-1, REG3a, Elafin, ST-2 and TNFRI were observed higher in group1 than group2 with statistic differences.To avoid the effects of allogeneic cells, all the patients in group2 were treated with autologous CD19 CAR-T cells, and patients in group1 with CD19-CAR-T cells from the same donor. Even though CAR-T expansion and target clearance between two groups were lack of statistic differences, the cytokines related to GVHD were higher in grou1 as expected, which might be the results of absent of anti-rejection drugs for CAR-T expansion. Furthermore, general markers, skin related markers, and GI tract related markers were all increased in group1, which might be partially explain why higher rate of unexpected erythra or gastrointestinal hemorrhage in group1 after CAR-T. Image:Summary/Conclusion: Most CAR-T clinical trials were targeting refractory or relapsed patients, and more markers should be observed not only related to CRS or inflammation, but also related to GVHD during CAR-T treatment especially for those who relapsed after first HSCT. Based on our data, clinicians should be alert about GVHD markers while treating HCT relapsed patients with CAR-T therapy.

While the use of chimeric antigen receptor-T (CAR-T) therapy for T-cell malignancies is in the early stage of clinical trials, it exhibits substantial potential to offer long-term remission for patients with refractory/relapsed (R/R) T-cell malignancies. In our phase I/II clinical trials, 65 pediatric and adult patients with R/R T-cell acute lymphoblastic leukemia and lymphoblastic lymphoma (T-ALL/LBL) were enrolled (NCT04572308 and NCT04916860). Of these, 60 participants (T-ALL 35, T-LBL 25) received a single dose of naturally selected anti-CD7 CAR (NS7CAR) T cells at three levels: a low dose (5 × 105 /kg), a medium dose (1 to 1.5 × 106 /kg), and a high dose (2 × 106 /kg). On day 28, 94.4% of patients achieved deep complete remission (CR) in bone marrow. Among the 32 patients with extramedullary disease, 78.1% showed response, with 56.3% in CR and 21.9% in partial remission. The 2-year overall survival and progression-free survival (PFS) were 63.5% (95% CI 47.7-79.4) and 53.7% (95% CI, 38.9-68.6), with no difference between pediatric and adult patients. PFS was significantly higher among the 37 CR patients who proceeded with consolidation transplant than the 10 patients who did not with 1-year PFS 67.2% (95% CI 51.9-82.4) versus 15.0% (95% CI 0-40.2), p < .0001. Of the 10 CR patients without transplants, eight relapsed, while two sustained CR on day 128, and day 180, respectively. Cytokine release syndrome occurred in 91.7% of patients (grade 1/2 in 80.0%, grade 3/4 in 11.7%) and 5% of patients had neurotoxicity. NS7CAR-T therapy is effective in treating R/R T-ALL/LBL patients with promising PFS while maintaining a manageable safety profile.

Objective:To investigate the relationship between the levels of serum cytokines and chemokines and the prognosis of patients with acute B-ALL after receiving chimeric antigen receptor (CAR)-T cell immunotherapy and acute graft-versus-host disease (aGVHD) in patients after bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:According to the case-control principle, Forty-two patients with B-ALL who received CD19-CAR-T cell immunotherapy bridged to allo-HSCT at Heibei Yanda Ludaopei Hospital from September 18, 2019 to May 9, 2022 were enrolled. Mann-Whitney U test was used to compare the changes of aGVHD-related cytokines and chemokine levels between CAR-T cell immunotherapy and bridging transplantation in different patients at the same time. Their plasma levels of cytokines and chemokines related to aGVHD were monitored at the day before CAR-T therapy and after CAR-T treatment at day 4, 7,14,21,28. The receiver operating characteristic curve was drawn to evaluate the predictive value of cytokines and chemokines in predicting the occurrence and the death of aGVHD patients. Kaplan-Meier method and Log-rank tests were used for Overall survival (OS) analysis. Results:Twenty-four of total 42 patients had aGVHD, of which 11 patients died and 31 patients survived. There was no significant difference in cytokines and chemokines between the aGVHD group and the non-aGVHD group on the day before CAR-T cell treatment. According to statistical analysis, the serum Elafin levels of aGVHD group was higher than that of non-aGVHD group at the 21st day [4 482 (2 811, 6 061) ng/L vs 2 466 (1 948, 3 375) ng/L, Z=3.145, P=0.001] and the 28st day [4 391 (2 808, 5594) ng/L vs 2 463 (1 658, 2 830) ng/L, Z=2.038, P=0.048] separately. At the 14th day, serum cytokines and chemokines levels between the two group were as follows,MIP-1 α [21.02 (12.36, 30.35) ng/L vs 5.56 (3.64, 10.79) ng/L], sCD25 [422.47 (257.99, 1 233.78) IU/ml vs 216.11 (133.75,457.39) IU/ml], Elafin [4 101 (2 393, 5 006) ng/L vs 2 155 (1 781, 3 033) ng/L], IL-6 [119.08 (23.97, 183.43) ng/L vs 8.39 (2.91, 17.42) ng/L] and IL-8 [13.56 (12.50, 24.52) ng/L vs 2.83 (1.73,6.87) ng/L] were at higher levels ( Z=2.653, P=0.007; Z=2.176, P=0. 030; Z=2.058, P=0.041; Z=3.329, P<0.001; Z=3.162, P=0.001). The KM survival curve showed that the cumulative survival rates of patients with higher serum levels of MIP-1α, sCD25, Elafin, IL-6 and IL-8 were lower than those with low levels at day 14, and the difference was statistically significant (χ 2=12.353, 4.890, 6.551, 10.563, 20.755, P<0.05). Conclusion:The outcomes of patients treated with CAR-T cell therapy bridged to allo-HSCT was correlated with serum MIP-1α, sCD25, Elafin, IL-6 and IL-8 levels after receiving CAR-T therapy. High concentrations of MIP-1α, sCD25, Elafin, IL-6 and IL-8 suggest poor prognosis and can be used as biomarkers to suggest appropriate clinical selection of therapy.

Introduction Refractory or relapsed (r/r) acute myeloid leukemia (AML) is associated with a relatively poor prognosis, even in patients who undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT), emphasizing the critical need for novel therapies. Approximately 30% of AML patients express CD7 on their leukemic blasts and malignant progenitor cells. Naturally selected CD7 CAR-T (NS7CAR-T) therapy has shown significant efficacy with a favorable safety profile in T-cell lymphoid malignancies. In a phase I clinical study (https://clinicaltrials.gov NCT04938115), we investigated the safety and efficacy of CAR-T therapy for treating r/r AML patients with CD7-positive disease. Methods Peripheral blood mononuclear cells were obtained from either the patients themselves (n=9) or the transplant donor (n=1) in cases of relapse post-transplant. T-cells were then purified using CD3+ magnetic beads. The second-generation CD7CAR with a 4-1BB costimulatory domain was manufactured following the manufacturer's protocol. Before the CAR-T cell infusion, bridging therapies were permitted for patients with rapid disease progression. All patients received intravenous fludarabine (30mg/m 2/d) and cyclophosphamide (300mg/m 2/d) lymphodepletion chemotherapy for three consecutive days (Day -5 to Day -3). The median time from leukapheresis to CAR-T cell infusion was 15 days. Results Between June 2021 and January 2023, we enrolled 10 patients with CD7-positive r/r AML (CD7 expression >50% with good intensity) and administered NS7CAR-T cell infusions, with 4 receiving a low dose (5×10 5/kg) and 6 receiving a medium dose (1×10 6/kg). Table 1 displays the characteristics of the enrolled patients, revealing a median age of 34 years (7-63 years) and median bone marrow (BM) blasts percentage by flow cytometry (FCM) of 17.0% (2.0-72.7%) at enrollment. One patient presented with diffuse extramedullary disease (EMD). Before enrollment, patients had undergone a median of 9 (range: 3-17) prior lines of therapy. Seven patients had a history of allo-HSCT and the median interval period from the prior transplant to relapse was 12.5 months (3.5-19.5 months). Following infusion, the median peak of circulating NS7CAR-T cells was 2.72×10 5 copies/μg (0.671~5.41×10 5 copies/μg) genomic DNA, which occurred around Day 21 (Day 14 - Day 21) based on q-PCR, with 64.68% (40.08%~92.02%) occurring on Day 17 (Day 11 - Day 21) according to FCM. The median transduction efficiency of the products was 95.6% (70.4%-98.5%). At four weeks post NS7CAR-T cell infusion, 7/10 (70%) patients achieved complete remission (CR) in BM, and 6 of them attained minimal residual disease (MRD)-negative CR. Three patients showed no remission (NR), including 1 with EMD who had partial remission (PR) based on PET-CT evaluation on Day 35. All NR patients were found lost CD7. The median observation time was 178 days (28-752 days). Among the 7 patients who achieved CR, 3 who relapsed from prior transplants underwent consolidative 2 nd allo-HSCT about 2 months after CD7 CAR T-cell infusion. Two patients remained in leukemia-free survival on day 752 and day 315, respectively, while 1 patient died on day 241 due to transplant-related mortality. Among the other 4 patients without consolidative allo-HSCT, 3 relapsed on day 47, day 83, and day 115, respectively (all 3 patients were found to have CD7 loss), and 1 patient died from lung infection. Post-infusion, the majority of patients (80%) experienced mild cytokine release syndrome (CRS), with 7 displaying grade I and 1 having grade II CRS, while 2 patients (20%) experienced grade III CRS. None of the patients had neurotoxicity. Among the 7 patients with prior allo-HSCT, 1 who had a relapse approximately 100 days after prior allo-HSCT developed mild skin graft-versus-host disease following CAR-T therapy. Conclusion Our study highlights the NS7CAR-T therapy as a promising approach for achieving a favorable initial CR in CD7-positive AML patients, even in those who have undergone extensive prior treatments and experienced relapse post allo-HSCT. It potentially could serve as a bridging therapy before transplant. CD7 loss is a major issue either in NR patients or relapsed patients. The safety profile of NS7CAR-T therapy was manageable. However, to comprehensively assess the efficacy of NS7CAR-T in treating CD7-positive AML, further data from a larger cohort of patients and longer follow-up time are essential.

Topic: 25. Gene therapy, cellular immunotherapy and vaccination - Clinical Background: Mixed phenotype acute leukemia (MPAL) is a rare subtype of acute leukemia with features of both acute lymphoblastic leukemia and acute myeloid leukemia. The blast cells of MPAL may have a shared B/T/myeloid phenotype. Once relapsed, the prognosis is extremely poor for MPAL. Aims: We report the safety and efficacy of CD7 CAR-T therapy in treating refractory/relapsed (r/r) MPAL patients with CD7-positive in phase I clinical study (https://clinicaltrials.gov NCT04938115). Methods: Peripheral blood (PB) mononuclear cells were obtained from 10 patients or 1 patient transplant donor who relapsed post-transplant. T-cells were purified using CD3+ magnetic beads or CD4+ and CD8+ magnetic beads. The second-generation CD7 CAR with a 4-1BB costimulatory domain was manufactured according to the manufacturer’s protocol. Prior to the CAR-T cell infusion, patients received bridging chemotherapy to control rapid disease progression, and then all patients received conditioning regimens with intravenous fludarabine (30 mg/m2/d) and cyclophosphamide (300 mg/m2/d) (FC) lymphodepleting chemotherapy for three consecutive days (Day -5 to Day -3). The manufacturing time was 14 days. Results: From November 2020 to September 2022, 11 adult MPAL patients with CD7-positive expression ≥ 80% were enrolled. The median age was 28 years old (range: 18-45). A single dose of CD7 CAR-T cells was infused, 4 with a low dose (1-5 × 105/kg) and 7 with a medium dose (1.0 × 106/kg). The median transduction efficiency of the products was 84.4% (range: 32.0%-98.1%). Characteristics of enrolled patients are shown in Table 1. At enrollment, the median bone marrow (BM) blasts by morphology for the 8 patients with BM blasts were 15.0% (range: 0.5-79.5%), and 7 patients had extramedullary disease (EMD). Four patients had a prior allogeneic hematopoietic stem cell transplantation (allo-HSCT), including 1 who relapsed following a 2nd transplant and had also received CD19 CAR-T cell therapy before enrolling in this trial. On day 28, in BM, 10/11 (90.9%) patients achieved complete remission (CR) or CR with incomplete blood recovery, and all were minimal residual disease (MRD)-negative. Regarding the 7 patients with EMD, the overall response rate (ORR) was 5/7 (71.4%), including 4 CR and 1 partial response (PR), and 2 had no response (NR). The median follow-up time was 222 days (range: 60-791 days). Within 3 months post-CD7 CAR-T cell infusion, 7 patients both in MRD-negative CR and EMD CR received consolidative allo-HSCT, and all remained progression-free after a median follow-up of 561 days (range: 126-791 days), except for 2 who died from relapse and transplant-related mortality (TRM), respectively. For the other 4 patients less than CR, 1 died from progression disease, and 3 received salvage transplantation, of whom 2 died from TRM, and one was still alive at the last follow-up on day 278. The majority of patients developed mild cytokine release syndrome (CRS) (91% ≤ grade II), and only 1 had grade III CRS. None of the patients had neurotoxicity. Following infusion, the median peak of circulating CD7 CAR-T cells in PB was as high as 73.5% (25.8%~90.0%), occurring on day 14 (11-20) by flow cytometry. By qPCR, the median peak of CAR-T copy number was 2.58 ×105 (0.28-7.04 ×105) copies/µg DNA, which occurred on day 18(10-28). Summary/Conclusion: This study demonstrated that CD7-targeted CAR-T therapy was effective with a good safety profile in treating CD7-positive MPAL patients, even for those who relapsed post-transplant. However, more data on additional patients and longer observation time are needed.Keywords: CAR-T, T-ALL, T cell leukemia, Adult

Objective:To investigate the prognostic value of colony forming unit-granulocyte and macrophage (CFU-GM) in allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:Seventy-three patients who received allo-HSCT in Hebei Yanda Lu Daopei Hospital from February 2015 to January 2017 were selected. According to the level of CFU-GM from bone marrow (BM) culture at the time of allo-HSCT, the patients were fit into high CFU-GM group and low CFU-GM group. The overall survival rate (OS) and relapse-free mortality rate (NRM) of patients after transplantation were tested by χ2 test after a follow-up of 37.0 (12.5, 50.5) months. Kaplan-Meier method was used to compare OS and event-free survival (EFS) of patients with different CFU-GM levels. Logistic regression model was used to analyze the prognostic factors. Cox regression model was used to further analyze the prognostic risk of patients.Results:Compared with the low CFU-GM group, the high CFU-GM group had a higher OS (81.40% vs 60.00%, χ2=4.067, P=0.044) and a lower NRM (11.63% vs 36.67%, χ2=6.474, P=0.011). Compared with the low CFU-GM group, the mean OS time (57.6 and 37.1 months, respectively, P=0.039) and the mean EFS time (61.7 and 38.5 months, respectively, P=0.011) were significantly higher in the high CFU-GM group. Logistic regression analysis showed that both the level of CFU-GM and BM MNC were significant influencing factors of OS ( OR=2.917, 95% CI 1.011-8.418, P=0.048 and OR=1.510, 95% CI 1.058-2.154, P=0.023, respectively) and EFS ( OR=4.400, 95% CI 1.336-14.492, P=0.015 and OR=1.447, 95% CI 1.002-2.090, P=0.049, respectively)after transplantation. The level of CFU-GM was an independent risk factor for evaluating EFS ( HR=0.279, 95% CI 0.097-0.805, P=0.018). BM MNC was an independent risk factor for OS ( HR=1.345, 95% CI 1.052-1.720, P=0.018). Conclusion:The level of CFU-GM and BM MNC were related to the prognosis of allo-HSCT. The patients in the high CFU-GM group had higher EFS.

Background Universal CAR-T (UCAR-T) using gene-editing techniques has attracted significant attention due to its prompt availability for patients. To date, there are only a few clinical case reports of universal CAR-T therapy for T-cell malignancies. Here, we explored the efficacy and safety of CD7-targeted UCAR-T (RD13-01) cells for relapsed or refractory (R/R) T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) in a phase I clinical trial (NCT04620655). Methods RD13-01 is an allogeneic, healthy donor-derived, and CRISPR/Cas9-edited anti-CD7 CAR-T cell product. RD13-01 consists of a CAR with a CD7-binding single-chain variable fragment (scFv), a 4-1BB costimulatory domain, CD3ζ signaling domain, a γc intracellular domain, in addition to an NK inhibitory (NKi) ligand containing an EC1-EC2 extracellular domain of E-cadherin fused to a CD28 co-stimulatory domain. The CAR and the NKi ligand are linked by an F2A self-cleaving peptide. Intravenous fludarabine (25-30 mg/m2/d), cyclophosphamide (300mg/m2/d) and etoposide (100 mg/m2/d) were given to all patients on Day -6 to Day -3 prior to CD7 UCAR-T cell infusion. Results Between December 2020 and May 2022, 10 patients (7 males, 3 females), 7 with T-ALL and 3 with T-LBL were enrolled. Median patient age was 16.5 years (range: 2-27 years). Patients had a median of 4.5 (2-6) prior lines of therapies. Two patients relapsed from a previous allogeneic hematopoietic stem cell transplant (allo-HSCT) within 6 months. In addition, 3 patients who showed no response (NR) to prior autologous CD7 CAR-T therapy were also included. Patient characteristics are shown in Table 1. Four patients harbored high-risk genotypes including STIL-TAL1, TP53, BCR-ABL, and others. By morphology, the median bone marrow (BM) blasts at enrollment were 60% (4%-96.5%). Of the eight patients with peripheral blood (PB) blasts, the median blasts were 27% (9%-85%). At enrollment, 8 patients had extramedullary disease (EMD) including 3 with central nervous system (CNS) involvement (1 CNS-1, 2 CNS-3), 3 with diffuse involvement, and 3 had localized EMD. The median transfection efficiency of the RD13-01 product was 64.6% (range: 59.0%-91.2%). A single dose of CD7 UCAR-T cells were infused to the first 3 enrolled patients at 0.5-1×107 cells/kg. Subsequently, 6 patients received a medium dose of 2×107cells/kg, and 1 received a high dose of 4×107cells/kg. By the cut-off date of July 11, 2021, the median observation time was 153 days (range: 28-315 days). On day 28 post infusion, 8/10 (80%) patients achieved complete remission (CR) in BM/PB, and 7 of the 8 patients were minimal residual disease (MRD) negative. Notably, the 3 patients who had failed prior autologous CD7 CAR-T therapy achieved CR. The first enrolled 2 patients who received the low dose showed NR and withdrew on day 28. Among the 7 patients with EMD, 4 achieved EMD CR at the median day 30, 1 had progression disease (PD) and 2, with NR in BM, withdrew prior to EMD evaluation. With a median time of 47 days (40-67 days) following infusion, 6 patients in CR (including 1 MRD +CR) underwent consolidation allo-HSCT, and 4/6 remained progression-free longest up to 315 days. However, 2/6 relapsed without CD7 lost on day 186 and day 200, respectively, and subsequently died. In addition, 1 CR patient died from bacterial infection on day 35. One PD patient received salvage transplantation and died from relapse on day 150. Post infusion, 9/10 patients experienced grade I cytokine release syndrome (CRS) and 1/10 had grade 3 CRS. Only 1 patient with CNS-3 experienced grade 3 neurotoxicity. By qPCR, CD7 UCAR-T cells reached a median peak level of 1.43×106 copies /μg genomic DNA (1.11×105~4.33×106)copies /μg genomic DNA, which occurred on day 12 (7-24 days). The median peak of circulating CD7 UCAR-T was 47.6% (0.01%~83.9%) occurring on day 7 (6-19 days), as measured by flow cytometry (Figure.1). Conclusions Our phase I trial showed that the allogeneic "off-the-shelf” RD13-01 product was safe and dose-dependently effective in treating patients with heavily pretreated T-ALL/LBL, including those with EMD and prior allo-HSCT, those who already failed autologous CD7 CAR-T therapy, and who could not manufacture autologous CAR-T from the patient's own PB due to high blasts in PB and those with rapid disease progression. Long-term observation and more patients are needed to further evaluate the safety and efficacy of CD7 UCAR-T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Objective:To investigate the significance of multicolor flow cytometry (MFC) monitoring of minimal residual disease (MRD) in the course of allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CD19-chimeric antigen receptor(CAR)-T cell immunotherapy for patients with refractory, relapsed B-cell acute lymphoblastic leukemia (r/r B-ALL).Methods:37 patients with r/r B-ALL admitted to Hebei Yanda Lu Daopei Hospital from January to July 2019, aged 15 (6, 19) years old, including 24 males and 13 females, were treated with CD19-CAR-T cell immunotherapy bridging allo-HSCT. MFC with cytoplasmic CD79a antibody to set up B-cell gates was used to monitor patients′ bone marrow (BM), cerebrospinal fluid (CSF), and tissue samples on day 0 (prior to the CAR-T cell immunotherapy), day 15, day 28 post CAR-T cell immunotherapy, and post transplantation.The MRD values of these samples were analyzed to evaluate the residual tumor cells and metastasis. The killing effect of the CAR-T cells was evaluated by the recovery of CD19+B cells before transplantation and the period between the timepoint when CD19+B cells was recovered and the timepoint when CAR-T cells were infused. Peripheral blood CAR-T cells were counted at different time points. Statistic analysis was performed by Kaplan-Meie assay and Log-rank test to analyze the difference of univariate cumulative survival.Results:(1)Among the 37 patients, 8 died and 29 survived. 5 patients relapsed after transplantation, of which 4 relapsed patients died and 1 survived. (2)MFC MRD negative remission rate of the death group was lower than that of the survival group at the following time points: post-CAR-T therapy and prior to transplantation (5/8 vs. 28/29, χ 2=7.540, P=0.006); day 15 of the CAR-T cell reinfusion (3/8 vs. 24/29, χ 2=6.512, P=0.011); day 28 of the reinfusion (3/8 vs. 276/29, χ 2=10.065, P=0.002). The probability of extramedullary MFC MRD positive tumor infiltration in the death group was higher than that in the survival group(7/8 vs. 14/29, χ 2=3.931, P=0.047). After CAR-T cell immunotherapy, the recovery period of CD19-positive cells in the death group, or the time for CAR-T cells to kill CD19-positive cells, was shorter than that in the survival group [42.00 days(30.00,49.00) vs. 55.00 days(41.50,73.50), Z=0.022, P=0.020]. Conclusion:The positive results of MRD by MFC at the following timepoints may predict unfavorable outcomes, such as post-CAR-T therapy and prior to transplantation, day 15 and 28 of the CAR-T cell immunotherapy, which may provide some guidance for clinical management.

Abstract Background In October 2020, we began the clinical trials of CD7 CAR-T treatment for CD7-positive hematological malignancies at our center. We found that the proliferation profile and evolution of CD7 CAR-T cells within 1-month following infusion into patients were quite different from those of CD19 CAR-T cells. From these data, we reasoned that the time to occurrence of CAR-T-cell-related side effects might also differ between the two cellular therapies. Here, we systematically compared the proliferation and CAR-T-cell-related side effects of CD7 CAR-T cells to these of CD19 CAR-T cells. Patients and Methods From October 2020 to June 2021, a total of 30 patients (24 male, 6 female) including 22 with T-cell acute lymphoblastic leukemia (T-ALL), 3 with T-cell lymphoblastic lymphoma (T-LBL), and 5 with mixed phenotype acute leukemia (MPAL) received autologous CD7 CAR-T cells manufactured by the SenlangBio company (https://clinicaltrials.gov NCT04572308, NCT04796441 and NCT04938115). The median follow-up time was 116 days (range: 15-221days). On Day 30, 25/30 patients (83.3%) achieved complete remission (CR)/CR with incomplete blood recovery (CRi). From December 2017 to June 2021, 45 B-ALL patients (19 male, 26 female) received CD19 CAR-T cells, also manufactured by SenlangBio (NCT04792593 and NCT04546893). The median follow-up time was 351 days (range: 15-1110days). On Day 30, 43/45 patients (95.6%) achieved CR/CRi. The median infused CD7 CAR-T cell dose was 1×10 6/kg (range: 0.5-2×10 6/kg), and the median infused CD19 CAR-T cell dose was 3×10 5/kg (range: 0.2-10×10 5/kg). The CD7 or CD19 CAR-T cell ratio in peripheral blood lymphocytes (PBLC) and the CD7 or CD19 B-lymphocyte percentage in PBLC samples from patients were analyzed on days 0, 4, 7, 10, 14, 21, and 30 following CAR-T cell infusion using flow cytometry. Results The presence of CD7 CAR-T cells in the PBLC samples were gradually detected following CD7 CAR-T cell infusion. The CD7 CAR-T cell ratio in PBLC increased significantly on Day 10. CD7 CAR-T cell peak appeared on Day 21 with a peak of 39.14% (range: 0.04%-74.58%), and was still detectable on Day 30 with a high CD7 CAR-T ratio of 7.5% (1.15%-70.41%). The ratio of CD19 CAR-T cells in patient PBLC samples showed a significant increase on Day 7 following infusion, and the CAR-T cell peak appeared on Day 10 with a peak of 14.71% (range: 0.11%-89.33%), and then quickly decreased to 0.23% (range: 0%-82.88%) on Day 21 (Figure 1). As the CAR-T cells increased, the proportion of target cells decreases significantly (Figure 2). However, the rate of decrease of CD19 cells differed from that of CD7 cells. CAR-T cell proliferation is also associated with CAR-T-cell-related adverse effects including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Observing the adverse effects after CD7 CAR-T infusion, we found that fever (incidence rate of 83.8%) occurred on the first 1-3 days following infusion, with a body temperature among patients of about 38°C. After patients' body temperature dropped to approximately normal levels, fever occurred again on Day 10-21 (incidence rate of 77.4%), and a higher temperature of 38-40°C was observed. The adverse event profile coincided with the proliferation of CD7 CAR-T cells we observed. Among the 30 cases, 5 had Grade 2 CRS, 2 had CRS of Grade ≥3, and 1 patient had Grade 3 ICANS. Fever following CD19 CAR-T infusion consisted mainly on Day 7-14 after the infusion (incidence rate of 86.6%), followed by a gradual drop of body temperature to normal after Day 14. Among the 45 patients, 5 had Grade 2 CRS, 5 had CRS of Grade ≥3 and 7 had Grade ≥3 ICANS. Conclusions In this clinical study, we found that the proliferation and evolution of CD7 CAR-T cells are distinct from that of C19 CAR-T cells. CD7 CAR-T cells began to proliferate significantly later following patient infusion and persisted longer compared to CD19 CAR-T cells. We found that patients experienced two rounds of fever, appearing on Day 1-3 and Day 10-21 following CD7 CAR-T infusion, which required more attention and prevention compared to the fever experienced by patients infused with CD19 CAR-T cells. However, the incidence of CRS and ICANS did not increase following CD7 CAR-T infusion. More patients and long-term observation are needed to confirm these results and to improve clinical management of patients treated with CAR-T cellular therapies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objective: To study the clinical efficacy of chimeric antigen receptor T-cell (CART) treatment followed by a second allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with B-cell acute lymphoblastic leukemia (ALL) who relapsed following the first HSCT. Methods: Retrospective analysis of the clinical characteristics and prognosis of 41 patients with B-cell ALL who received a second allo-HSCT from October 2015 to June 2020 in Hebei Yanda Lu Daopei Hospital. After the first HSCT, all patients received CD19-CART, or CD22-CART treatment following a relapse of bone marrow morphology or extramedullary leukemia. Results: A total of 41 patients (male, 21; female, 20) were included in this study. The median age at the second HSCT was 16 (3-46) years. There were 31 cases of bone marrow recurrence (75.6%) , 5 cases of extramedullary recurrence (12.2%) , and 5 cases of bone marrow and extramedullary recurrences (12.2%) . After relapse, 35 patients (85.4%) received CD19-CART treatment, 2 patients received CD22-CART treatment (4.9%) , and 4 patients received CD19-CART and CD22-CART treatments (9.8%) . The expected 3-year overall survival (OS) , leukemia-free survival, cumulative relapse incidence, and non-relapse mortality (NRM) of patients after the second HSCT were 48.9% (95%CI 23.0%-70.6%) , 41.8% (95%CI 17.3%-64.9%) , 8.8% (95%CI 2.9%-26.4%) , and 51.1% (95%CI 31.2%-83.6%) , respectively. The 1-year OS of patients who relapsed ≤6 months and >6 months after the first HSCT were 45.0% (95%CI 12.7%-73.5%) and 75.0% (95%CI 51.4% -88.8%) (P=0.017) , respectively. Conclusion: CART bridging in the second HSCT enables some B-cell ALL patients who relapsed after the first HSCT to achieve long-term survival. However, because of the high NRM, further modifications could help improve the outcome.

Backgrounds As CAR T-cell therapy is a highly personalized therapy, process of generating autologous CAR-T cells for each patient is complex and can still be problematic, particularly for heavily pre-treated patients and patients with significant leukemia burden. Here, we analyzed the feasibility and efficacy in 37 patients with refractory/relapsed (R/R) B-ALL who received CAR T-cells derived from related donors. Patients and Methods From April 2017 to May 2020, 37 R/R B-ALL patients with a median age of 19 years (3-61 years), were treated with second-generation CD19 CAR-T cells derived from donors. The data was aggregated from three clinical trials (www.clinicaltrials.gov NCT03173417; NCT02546739; and www.chictr.org.cn ChiCTR-ONC-17012829). Of the 37 patients, 28 were relapsed following allogenic hematopoietic stem cell transplant (allo-HSCT) and whose lymphocytes were collected from their transplant donors (3 HLA matched sibling and 25 haploidentical). For the remaining 9 patients without prior transplant, the lymphocytes were collected from HLA identical sibling donors (n=5) or haploidentical donors (n=4) because CAR-T cells manufacture from patient samples either failed (n=5) or blasts in peripheral blood were too high (>40%) to collect quality T-cells. The median CAR-T cell dose infused was 3×105/kg (1-30×105/kg). Results For the 28 patients who relapsed after prior allo-HSCT, 27 (96.4%) achieved CR within 30 days post CAR T-cell infusion, of which 25 (89.3%) were minimal residual disease (MRD) negative. Within one month following CAR T-cell therapy, graft-versus-host disease (GVHD) occurred in 3 patients including 1 with rash and 2 with diarrhea. A total of 19 of the 28 (67.9%) patients had cytokine release syndrome (CRS), including two patients (7.1%) with Grade 3-4 CRS. Four patients had CAR T-cell related neurotoxicity including 3 with Grade 3-4 events. With a medium follow up of 103 days (1-669days), the median overall survival (OS) was 169 days (1-668 days), and the median leukemia-free survival (LFS) was 158 days (1-438 days). After CAR T-cell therapy, 15 patients bridged into a second allo-HSCT and one of 15 patients (6.7%) relapsed following transplant, and two died from infection. There were 11 patients that did not receive a second transplantation, of which three patients (27.3%) relapsed, and four parents died (one due to relapse, one from arrhythmia and two from GVHD/infection). Two patients were lost to follow-up. The remaining nine patients had no prior transplantation. At the time of T-cell collection, the median bone marrow blasts were 90% (range: 18.5%-98.5%), and the median peripheral blood blasts were 10% (range: 0-70%). CR rate within 30 days post CAR-T was 44.4% (4/9 cases). Six patients developed CRS, including four with Grade 3 CRS. Only one patient had Grade 3 neurotoxicity. No GVHD occurred following CAR T-cell therapy. Among the nine patients, five were treated with CAR T-cells derived from HLA-identical sibling donors and three of those five patients achieved CR. One patient who achieved a CR died from disseminated intravascular coagulation (DIC) on day 16. Two patients who achieved a CR bridged into allo-HSCT, including one patient who relapsed and died. One of two patients who did not response to CAR T-cell therapy died from leukemia. Four of the nine patients were treated with CAR T-cells derived from haploidentical related donors. One of the four cases achieved a CR but died from infection on day 90. The other three patients who had no response to CAR T-cell therapy died from disease progression within 3 months (7-90 days). Altogether, seven of the nine patients died with a median time of 19 days (7-505 days). Conclusions We find that manufacturing CD19+ CAR-T cells derived from donors is feasible. For patients who relapse following allo-HSCT, the transplant donor derived CAR-T cells are safe and effective with a CR rate as high as 96.4%. If a patient did not have GVHD prior to CAR T-cell therapy, the incidence of GVHD following CAR T-cell was low. Among patients without a history of transplantation, an inability to collect autologous lymphocytes signaled that the patient's condition had already reached a very advanced stage. However, CAR T-cells derived from HLA identical siblings can still be considered in our experience, no GVHD occurred in these patients. But the efficacy of CAR T-cells from haploidentical donors was very poor. Disclosures No relevant conflicts of interest to declare.

Introduction: Cytokine release syndrome (CRS) is a serious side effect of chimeric antigen receptor-T cell (CAR-T) therapy. Effective cytokine monitoring can provide support for early prevention and treatment. As more and more promising markers have been identified to be associated with CRS responses, traditional ELISA method is inapplicable because it's sample-consuming, low flexibility, and difficult to detect multiple cytokines simultaneously. Aimplex kit, a new high-throughput technique based on flow cytometry(FCM) and micro beads has been testified a effective way to monitor multiple cytokines in peripheral blood(PB) samples. If a new suitable cytokines panel is established to monitor the variation curve of multiple cytokines in patients' PBs post CAR-T therapy, study the relationship between clinical symptoms and the lab results, even build a cytokines database of CAR-T therapy, it would offer a promising support to prevent and handle CRS. On the other hand, by studying the relationship between cytokines and activated cells, we might investigate mechanisms of CRS and explore more related markers. Methods: A two tubes panel was designed to detect 24 cytokines, including IFN-γ/IL-1β/IL-2/IL-4/IL-5/IL-6/IL-8/IL-10/IL-12p70/IL-17A/IL-17F/IL-22/TNF-α/TNF-β, and sCD25/GM-CSF/IL-15/MCP-1/GranzymeB/Reg3A/ST2/TNFRSF1A/Elafin/MIP-1 alpha. 50 PB samples from complete response(CR) patients without CAR-T therapy were detested as normal controls to establish the normal values of 24 cytokines. 81 patients who infused CAR-T cell in Hebei Yanda Ludaopei Hospital from January to June 2020 were selected. Serum were collected on 0d, 4d, 7d, 11d, 15d, 20d and 30d after infusion, and 24 cytokines were detected by FCM Aimplex. Of which 31 patients were selected for methodological comparison between FCM and ELISA by detecting four routine cytokines, IL-6, IFN-γ, TNF-α and sCD25. 66 Patients were divided into 2 groups according to clinical manifestations, 60 patients without or mild CRS were classified as low grade CRS group, 6 patients with grade 2 or 3 CRS were classified as high grade CRS group. Results: The comparative test showed that a good relationship between the results of ELISA and Aimplex kit, showing similar time-cytokines variation curve of PBs after CAR-T treatment. The concentrations of IL-2, IL-10, IL-12p70, IL-17A, IL-17F, MCP-1, ST-2s, IL-5, IL-6 and IFN-γ were significantly higher in high CRS grade group. By comparing the proportion of CAR-T+, CD8+ and CD4+ T cells in CD3+ cells and the concentration of cytokines in PB specimens, there were obvious positive correlations between percentages of CAR-T+, CD8+ T cells and most cytokines, and negative correlation between proportion of CD4+ T cells and most cytokines. Conclusions: Detecting 24 kinds of cytokines by Aimplex kit is a promising method, which is time and specimens saving, and can cost-effectively reflect the cytokine situation in PBs from patients post-CAR-T treatment. Increasing the sample size and establishing a cytokines database will be helpful for monitoring, early prevention and control of side effects after CAR-T treatment. Disclosures No relevant conflicts of interest to declare.