scholarly journals shRNA-Interleukin-6 Modified CD19-Specific Chimeric Antigen Receptor T Cell Significantly Improves the Safety in Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2621-2621
Author(s):  
Liqing Kang ◽  
Xiaowen Tang ◽  
Nan Xu ◽  
Minghao Li ◽  
Jingwen Tan ◽  
...  
Keyword(s):  
T Cells ◽  
Interleukin 6 ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Mouse Serum ◽  
Single Chain ◽  
Car T Cells ◽  
Significant Difference ◽  
Car T ◽  
Short Hairpin

[Background] An urgent need exists to enhance the safety in treating hematologic malignancies with CAR-T therapy by reducing the CAR-T-related cytokine release syndrome (CRS) . Interleukin-6 (IL-6) is a central driver of CRS and neurotoxicity; hence, inhibition of the IL-6 of T cells via gene engineering may improve the safety of CAR-T therapy. [Objective] Investigation of the efficacy and safety of IL-6-targeting short hairpin (sh) RNA in the CART-19 (referred to ssCART-19) to determine whether the IL-6 shRNA in T cells can reduce the severe CRS incidence of ssCART-19 treatment. [Methods]We designed a short hairpin RNA sequence which targets the 3'UTR region of the human IL-6 transcript, and the sequence was added to a CAR construct containing the CD19 target single chain variable fragment (scFv), the EF1a promoter, the co-stimulated domain of 4-1BB and the CD3zeta domain. In vitro study, While there is no significant difference in the transduction efficiency, proliferation ability and cytotoxicity efficacy of ssCART-19 comparing to regular CART-19, there was clear inhibition of the IL-6 expression. IL-6 shRNA mediated gene silence of ssCART-19 significantly inhibited IL-6 gene expression at both the mRNA level (P<0.001) and the soluble cytokines level (P≤0.0001). IL-6 expression profile from ssCART-19 showed consistently maintained the lower level over the entire 150 hours of experiment period compared to regular CART-19 cells (P<0.001 ). And add the supernatants from regular CART-19/Raji co-culture and ssCART-19/Raji co-culture system to the primary induced monocytes, respectively, ssCART-19 could significantly reduce the monocytes derived IL-6 expression levels compared to regular CART-19. In vivo study, the preclinical study showed the consistent results that ssCART-19 significantly reduced the mouse serum IL-6 levels compared to regular CART-19, but with similar anti-tumor efficacy. In the clinical trail, 13 patients with the similar tumor burden baseline administrated with ssCART-19 (n=7) or regular CART-19 (n=6) cells with a dose of 5-10x106 CAR-T cells per kilogram over three consecutive days (10%, 30%, 60% split dose). While all patients from both groups achieved complete response and the CAR-T cells exhibit similar expansion ability, patients treated with ssCART-19 had lower CRS grade and significantly lower IL-6 level in the human serum compared to patients treated with regular CART-19 (the peak value of IL-6, P=0.0285, the IL-6 AUC(0-Tmax), P=0.0217). CRS emerged in 6/6 patients in regular CART-19 cohort and 6/7 patients in ssCART-19 cohort, severe CRS with grade 3 or higher was observed in 83.3% of the patients (5/6) treated with regular CART-19 cohort versus only 42.8% of the patients (3/7) treated with ssCART-19 cohorts. Tocilizumab was given to 66.7 % (4/6) of the patients in the regular CART-19 cohort and two patients needed more than one treatment with tocilizumab. In the regular CART-19 group one patient occurred CRES. There was no CAR T-related death. [Conclusion]Our study demonstrated that inhibition of CAR-T derived IL-6 expression by shRNA interfering technology could significantly reduce the severe CRS incidence without affecting their immune-oncotherapy efficacy in treating r/r B-ALL patients, which may provide a potential technology to improve the safety profile and promote the extended use of the CAR-T therapy without sacrificing efficacy. Disclosures No relevant conflicts of interest to declare.

scholarly journals An Off-the-Shelf™ Fratricide-Resistant CAR-T for the Treatment of T Cell Hematologic Malignancies

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 844-844
Author(s):  
Matt L Cooper ◽  
Jaebok Choi ◽  
Karl W. Staser ◽  
Julie Ritchey ◽  
Jessica Niswonger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Targeted Therapies ◽  
Conflicts Of Interest ◽  
Cell Transplant ◽  
Single Chain ◽  
Car T Cells ◽  
Human T Cell ◽  
Car T ◽  
T Cell Malignancies

Abstract T cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-40% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy. T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B cell leukemia and lymphoma7-9. Thus, a targeted therapy against T cell malignancies represents a significant unmet medical need. However, several challenges have limited the clinical development of CAR-T cells against T cell malignancies. First, the shared expression of target antigens between T effector cells and T cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells, without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients. We hypothesized that deletion of CD7 and the T cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T cell fratricide or induction of GvHD. To generate the CD7 CAR, the anti-CD7 single chain variable fragment (scFv) was created using commercial gene synthesis and cloned into the backbone of a 3rd generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC were confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous bi-allelic deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by both non-homologous end joining (NHEJ) and FACS analyses. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. Of particular importance is that by using two distinct methods for assessing "off-target" nuclease activity across the entire human T cell genome (Guide-seq and probe capture), we could only detect one gene, an intronic modification of RMB33, that was inappropriately targeted using this approach. No obvious genomic rearrangements were detected by these analyses. UCART7 effectively expanded and killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7, however, had no GvHD and, unlike UCART19 controls, effectively cleared primary human T-ALL in NSG mice. Fratricide-resistant and allo-tolerant 'off-the-shelf' UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphomas without a requirement for autologous T cells and represents the first clinically feasible adoptive T cell therapy for T cell malignancies. Disclosures No relevant conflicts of interest to declare.

Construction and Molecular Characterization Of a T-Cell Receptor-Like Antibody and CAR-T Cells Specific For Minor Histocompatibility Antigen HA-1H

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4490-4490
Author(s):  
Yoko Inaguma ◽  
Yasushi Akahori ◽  
Yoshiki Akatsuka ◽  
Yuko Murayama ◽  
Keiko Shiraishi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Immunotherapy ◽  
Conflicts Of Interest ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Minimal Risk ◽  
Single Chain ◽  
Car T Cells ◽  
Car T

Selective graft-versus-tumor (GVT) reactivity with minimal risk of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation is thought to be induced by targeting minor histocompatibility (H) antigens (Ags) expressed only on patients’ hematopoietic cells. Among HLA-A* 02:01 positive patients, minor H Ags such as HA-1 and HA-2 have been shown to be associated with anti-tumor responses with minimal GVHD and explored for application to adoptive immunotherapy. Because preparation of Ag-specific cytotoxic T cell clones (CTLs) or lines for adoptive immunotherapy is labor-intensive and time consuming, the genetic transfer of T-cell receptors (TCRs) directed toward target Ags into T lymphocytes has been used to efficiently generate anti-tumor T cells without the need for in vitro induction and expansion. Alternatively, T cells could be gene-modified with a chimeric antigen receptor (CAR) harnessing a single chain antibody moiety (scFv). The conventional CAR strategy has the limitation of only targeting cell surface Ags on target cells. One possible way to attain intracellular Ag targeting with a CAR is to generate a TCR-like monoclonal antibody (mAb) as a source of scFv. In this study, we sought to generate highly specific mAbs specific for HA-1H minor H Ag by immunizing mice with tetramerized recombinant HLA-A2 incorporating HA-1H minor H Ag peptides and β2-microglobulin (HA-1H/HLA-A2). We hypothesized that the use of HLA-A2 transgenic mice, which should be tolerant to human HLA-A2, would facilitate efficient induction of mAbs specific for peptides presented on HLA-A2. Phage libraries were generated from splenic B cells and screened by panning for clones reactive to plate-bound HA-1H/HLA-A2 in the presence of free MAGEA4/HLA-A2 for competition. Candidate scFv encoded by obtained phage clones were transformed to scFv tetrameric Ab form or introduced into T cells as CAR coupled to CD28 transmembrane and CD3ζ domains (CD28-ζ). A total of 144 clones were randomly selected from 8.1×108 clones that had been recovered after the third panning. Among 144 clones, 18 (12.5%) showed preferential binding to HA-1/HLA-A2, 137 showed similar binding to both pMHC complexes, and 7 showed reactivity to neither of them. One of 18 scFv Abs, clone #131, demonstrated high affinity (KD = 8.34nM) for the HA-1H/HLA-A2 complex. Primary human CD8 T cells transduced with #131 scFv-CD28-ζ were stained with HA-1H/HLA-A2 tetramers as strongly as a CTL clone, EH6, specific for endogenously HLA-A2- and HA-1H-positive cells. Unexpectedly, however, #131 scFv-CD28-ζ CAR-T cells required ∼100-fold higher Ag density when pulsed exogenously to exert cytotoxicity than did the cognate EH6-CTL. In addition, mAb blocking experiments demonstrated that #131 scFv-CD28-ζCAR-T cells were less sensitive to CD8 blockade when they were completely blocked with HA-1H/HLA-A2 tetramer. These data suggest that T cells with higher affinity antigen receptors than TCRs (average KD ranging between 1μM∼100μM) are less able to recognize low density peptide/MHC antigens as reported in the case of affinity-matured TCR or CAR, and that CD8+ CAR-T cells may not be necessarily CD8-dependent possibly due to failure to form complexes with CD3. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel full-human CD22-CAR therapy overcomes resistance to previous CD19/22-CAR regimens in ALL

2021 ◽  
Author(s):  
Yue Tan ◽  
Haodong Cai ◽  
Chuo Li ◽  
Biping Deng ◽  
Weiliang Song ◽  
...  
Keyword(s):  
T Cells ◽  
Lymphoblastic Leukemia ◽  
Considerable Proportion ◽  
Target Antigen ◽  
Single Chain ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Abstract BackgroundCD19- and/or CD22-targeted chimeric antigen receptor (CAR) T cells efficiently induced remission in patients with B acute lymphoblastic leukemia (B-ALL), but a considerable proportion of patients relapsed after both CD19- and CD22-CAR therapies associated with the loss or downregulation of target antigen. Re-infusions of the prior used CAR T cells were usually ineffective. In contrast to the frequent loss of CD19, low level of CD22 is usually present on leukemia cells post CAR therapy, suggesting that newly designed CD22-CAR therapies may be effective in these patients.MethodsA yeast full-human single-chain variable fragment (scFv) library and a high-throughput NFAT reporter assay were utilized to screen several full-human CD22-CAR candidates; CD107 assay and in vitro cytotoxicity assay was used to evaluate the effector function of CAR T cells; membrane proteome assay was conducted to determine the specificity of the CAR toward the target antigen; a leukemia animal models was used to test the in vivo efficacy of CAR T cells. A phase I trial (ChiCTR2000028793) was conducted to assess the safety and effectiveness of CD22-CARFH80 therapy in 8 children with B-ALL resistant to or relapsed after prior CD19- and CD22-CAR treatment.ResultsWe identified a full-human CD22-CAR construct termed CD22CARFH80 which could mediate superior anti-leukemia activity in vitro and in a leukemia animal model and had good specificity to the target antigen. Data from the trial showed that with CD22-CARFH80 T-cell therapy, 6/8 (75%) patients including 2 who had CD22low blasts achieved complete remission; 1 patient had a partial response. CAR T cells efficiently expanded in vivo, while the toxic effect is low in most patients. At a median follow-up of 5 months, 4/6 (57%) patients remained in remission.ConclusionsTherapy with a newly invented CD22-CARFH80 overcomes the resistance to prior versions of CD19- and CD22-CAR formats and elicits potent anti-leukemia responses with an acceptable safety profile, representing a promising salvage regimen for B-ALL that fails in prior CD19- and CD22-CAR treatments.Trial registrationClinicalTrials.gov: ChiCTR2000028793; registered 4 January, 2020. http://www.chictr.org.cn/showproj.aspx?proj=47857

scholarly journals A Single-Arm, Open-Label, Pilot Trial of Autologous CD7-CAR-T Cells for CD7 Positive Relapsed and Refractory T-Lymphoblastic Leukemia/Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3829-3829
Author(s):  
Mingzhi Zhang ◽  
Xiaorui Fu ◽  
Huimin Meng ◽  
Min Wang ◽  
Yu Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Complete Remission ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Pilot Trial ◽  
Open Label ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Introduction: T lymphoblastic leukemia/lymphoma (T-ALL/LBL) is highly aggressive. Although intensive chemotherapies such as ALL-type regimens are commonly used, about half patients eventually relapse and die of T-ALL/LBL. Chimeric antigen receptor (CAR) T cells have given rise to breakthroughs in treating B cell hematological malignancies. However, there are few clinical studies of CAR-T on T cell malignant tumors. CD7, overexpressed in up to 100% of relapsed/refractory T-ALL/LBL, is an attractive therapeutic target for T-lymphoblastic leukemia/lymphoma (T-ALL/LBL). Although several healthy donor derived CD7-CAR-T trials have been reported, autologous CD7-CAR-T cell therapy represents another promising strategy. In current study, a nanobody derived autologous CD7-CAR-T with a highly optimized structure and a clinically feasible strategy to overcome CAR-T cell fratricide and exclude abnormal T cell contamination have been developed. Method This is a single-arm, open label, safety and efficacy pilot study (NCT04004637). Study participants will receive Flu/Cy pre-treatment before CAR-T infusion. CD7 CAR-T was then administered at a single dose of 1.0x10 6 (N=5) or 1.5x10 6 (N=1; Pt#1) or 2x10 6 (N=3; Pt#7, 8,) CAR-T cells/kg. Adverse events were categorized by NCI-CTCAE V5.0. Tumor responses were assessed based on the 2014 Lugano Evaluation Criteria for Lymphoma and 2016 Chinese Guideline for Diagnosis and Treatment of Acute Lymphoblastic Leukemia. Results: Total of 8 patients including 5 r/r T-ALL/LBL and 3 r/r ETP-ALL/LBL (case 4, 5, 6) were assigned to receive CD7 CAR-T cell and completed the efficacy assessment. Overall response rate at 1 month was 100%. In patients followed up for ≥ 3 months, complete remission (CR) rate at 3 month was 75%. Case 1-6 was administered at 1.0×10 6 CAR-T/Kg except for case 1 (1.0×10 6 CAR-T/Kg). Bone marrow (BM) tumor burden of case 1 decreased from 70.03% to 19.51% and case 1 has an OS for nearly two years. Case 2 died due to abdominal infection at 3 month although he was still in MRD- status then. Case 3 had sustained MRD- for 7 months but appeared MRD+ in BM at 8 month, so second CD7 CAR-T infusion had been performed and achieved CR at 14th day again. Case 4 has been achieving CR 12 months. Case 5 and 6 had a DOR (Duration of response) of about 3 and 2 months respectively. In case 1-6, despite case1-2 appeared grade 2 CRS, all other cases only had grade 1. Therefore, in order to further explore the best effective dose, case 7-8 had received dose of 2.0×10 6 CAR-T/Kg. Case 8 are still in complete remission. Unfortunately, case 7 relapsed at 6 month due to CD7 negative blasts. The absence of CD7 on the cell surface enables the tumor to evade CAR T cell-mediated recognition and clearance, despite continued persistence of CAR-T cells. Conclusions: Autologous CD7 CAR-T represents a promising immunotherapy for r/r T-ALL/LBL and exhibited encouraging clinical efficacy and safety in treating r/r T-ALL/LBL and ETP-ALL/LBL. Figure 1Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tandem CD19/CD22 Dual Targets CAR T-Cells Bridging Hematopoietic Stem Cells Transplantation Acquires Robust Remission for Relapsed and Refractory B Acute Lymphoblastic Leukemia Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1753-1753
Author(s):  
Wei Cui ◽  
Xinyue Zhang ◽  
Haiping Dai ◽  
Qingya Cui ◽  
Jia Yin ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Cox Regression ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T

Abstract Background: CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated impressive early response rates in relapse and refractory B acute lymphoblastic leukemia (r/r B-ALL). However, a high rate of patients suffered a relapse, which occurred in a large subset of other trials and confers dismal outcomes. Dual targets approaches are proved to optimize the response rate and prevent CD19 - relapse. Alternatively, limited patients accepted the tandem CD19/CD22 CAR-T therapy, but the clinical trials in a large scale to investigate the tandem CAR-T were rare. Methods: We conducted an open label, single center clinical trial at the First Affiliated Hospital of Soochow University to investigate the efficacy and safety of tandem CD19/CD22 dual targets CAR T-cells for r/r B-ALL. All patients received FC (fludarabine, 30 mg/m2, days 1-3 and cyclophosphamide, 300 mg/m2, days 1-3) based chemotherapy as the lymphodepleting regimen. Median infusion dose of CAR-T cells was 1(0.5-2.5) *10 7 cells/kg. Results: From October 2017 to June 2021, a total of 47 patients were treated with CD19/CD22 CAR T-cells and included in our analysis. Among the 47 patients, primary refractory B-ALL patients account for 44.68%. 27 patients (57.4%) had a high disease burden, with 20% or more blasts in BM. Consequently, at day 28 assessment, 47 patients (100%) got hematological CR and the 40 out of 47 patients (85.1%) achieved minimal residual disease complete remission (MRD -CR). The toxicities of CD19/CD22 CAR T-cells therapy were reversible and clinically manageable. Cytokine reverse syndrome of any grades occurred in 41 of 47 patients (87.23%) and was severe (grade&gt;2) in 8 (17.02%). Immune effector cell-associated neurotoxicity happened in one patient. The most common severe hematological abnormalities were grade 3/4 leukopenia (74.47%). Serious thrombocytopenia and anemia occurred in 48.93% and 57.44% patients. The non-hematological toxicity were reversible with tight monitoring and support care. Within a median follow-up of 21.83 months (range, 2.57 to 42.53), the median overall survival (OS) and leukemia free survival (LFS) for the entire cohort have not reached. The OS rate of all the patients was 93.569% (95%CI, 80.97% to 97.832%) at 6 months, 78.721% (95%CI, 60.719% to 87.625%) at 1 year and 74.578% (95%CI, 55.263% to 84.969%) at 2 years in all patients. The LFS rate was 87.031% (95%CI, 73.375% to 93.958%) and 68.297% (95%CI, 51.419% to 80.365%) at 6 month and 1 year, respectively. The 6-months cumulative incidence of relapse (CIR) was 8.96%, while 1-year CIR was 23.254%. Thirty-four of 47 patients (72.34%) proceeded to a bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT). The OS of HSCT group was 94.118% at 6 months and 80.420% at 1 year. The 6-months OS of no-HSCT group was 83.916%, while 1-year OS was 74.592%. The HSCT group had significantly better LFS and lower CIR than the no-HSCT group (LFS, p=0.0459; CIR, p=0.0267). We initially performed multivariable Cox regression analyses, which shows that better long-term survival in patients with MRD -CR status, as well as bridging allo-HSCT. Conclusions: Tandem CD19/CD22 CAR-T cells are safety and highly effective in inducing CR for r/r B-ALL patients. The consolidative allo-HSCT can provide long-term durable disease control in these patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Impact of the Immunophenotyping Characteristics of Patients' Peripheral Blood on the Manufacturing and Clinical Outcome of CD7-Targeted Chimeric Antigen Receptor T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3830-3830
Author(s):  
Mingzhi Zhang ◽  
Xiaorui Fu ◽  
Huimin Meng ◽  
Min Wang ◽  
Yu Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Significant Difference ◽  
Car T ◽  
The Impact

Abstract Background T cell acute lymphoblastic leukemia and T cell acute lymphoblastic lymphoma (T-ALL/LBL) is a highly aggressive hematological tumor characterized by immature lymphoblasts invading the bone marrow. Treatment options for patients with T-ALL/ LBL are currently limited. Chimeric antigen receptor (CAR)- T cell therapy has opened an era in the treatment of B-cell malignancies. However, the development of CAR-T therapy for T-ALL/LBL faces many challenges. One of them is that therapeutic targets are usually expressed on both tumor and normal T cells, which causes the potential risk of "cell fratricide". Therefore, the difficulty of manufacturing CAR T cells for T-ALL/LBL is dramatically increased. CD7, is 40kD membrane-bound glycoprotein majorly expressed on peripheral T-cells and NK cells and their precursors. CD7 is highly expressed in almost all T-ALL/LBL and considered to be one of the most promising targets for T-ALL/LBL treatment. Patients and Methods This study is based on a phase I clinical trial (NCT04004637) for patients with relapse/refractory CD7 + NK/T cell lymphoma and T-ALL/LBL. To manufacture CAR-T cells, the peripheral blood mononuclear cells (PBMC) were collected from the patients who met the enrollment criteria. The proportion of viability and the ratio of the T cell markers were analyzed. Subsequently, the isolated T cells were co-transduced with CD7 protein expression blocker (PEBL) and CD7-CAR lentiviruses to obtain CD7-CAR-T cells, which can avoid the fratricide of CD7-CAR-T cells. Before the infusion, the phenotypic characteristics and cytotoxicity of CD7-CAR-T products were analyzed. Then peripheral blood (PB) of patients was collected regularly after receiving treatment to analyze the immunophenotyping of T cells. Results From August 2019 to June 2021, 24 leukopaks from patients with CD7-positive T-ALL/LBL were collected, and a total of 32 batches of CD7-CAR T cells were manufactured, with a 78.13% (25/32) successful rate. Among the 7 batches of failure cases, one patient had undergone blood collection twice and CAR-T preparation for three times, but all of three attempts failed (brown icon). Another four patients failed to prepare once. Eight patents were recruited for CD7-CAR-T treatment and 87.5% of complete remission (CR) rate was achieved (7/8), of which a patient (P4, blue icon) has been maintaining CR for more than 15 months. Two other patients, P7 (red icon) and P8 (light red icon), had CD7 - relapse at the time of 6th month and 3rd month after CR, respectively. We divided all samples into successful preparation group (GS), infusion group (GI) and preparation failure group (GF). As shown in Fig. 1A, all three groups exhibited good viability of PBMC. There was no significant difference between GS and GF, but GI was higher than that of GF. The proportion of CD3 + cells in PBMC of GS was significantly lower than that of GF, and GI also showed this feature. Meanwhile, GS and GI both have a higher CD4 +/CD8 + ratio compared with GF. The immunophenotyping results showed CD7-CAR-T products had a majority of the central memory subsets (T CM; 69.41 ± 10.71%) and effect memory subsets (T EM; 28.56 ± 10.19%), with limited number of effector T cell (T E) and naive T cells (T N) (Fig. 1B). The percentage of CAR +CD8 +CD27 + and CD4 +CD25 +CD127 - subsets associated with T cells activation and proliferation, as well as CD223 + and CD279 + subsets related to T cells suppression and exhaustion were lower, except for CD366 + subgroup that also indicated depletion signal (Fig. 1B). In addition, CD7-CAR-T cells showed strong cytotoxicity against CEM (CD7 +) tumor cells accompanied by the release of cytokines, in which the level of IL-2 is extremely low (Fig. 1C). Subsequently, we performed statistics on the proportion of CD3 + and CD4 +/CD8 + cells in the PB of patients after infusion. The proportion of CD3 + cells in the PB of the P4 has been maintained at a high level, and the ratio of CD4 +/CD8 + keeps low (Fig. 1D). P7 showed a significant decrease in the amount of T cells on the 60th day after CAR T infusion, while the ratio of CD4 +/CD8 + showed an upward trend. Conclusion The results indicate that the success rate of CD7-CAR-T manufacturing is positively correlated with higher viability, lower CD3 + and higher CD4 + of PBMC. There was no significant difference among P4 (CR more than 15 months), P7 (CD7 - relapse at 6 th month after CR) and P8 (CD7 - relapse at 3rd month after CR). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals IMMU-48. RATIONAL DEVELOPMENT AND CHARACTERIZATION OF MULTIVALENT TARGETING TANDEM AND PARALLEL CAR T CELLS FOR GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi129-vi129
Author(s):  
Yibo Yin ◽  
Radhika Thokala ◽  
Logan Zhang ◽  
Devneet Kainth ◽  
Leila Haddad ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Interleukin 2 ◽  
Growth Factor Receptor ◽  
High Grade Glioma ◽  
Primary Brain Tumor ◽  
Single Chain ◽  
Car T Cells ◽  
Significant Difference ◽  
Car T

Abstract High grade glioma is the most common malignant primary brain tumor. Despite the best available therapy, the median survival is limited. Chimeric antigen receptor (CAR)-T cells effectively attack target positive tumor cells, demonstrating a promising possible treatment. Based on our previous clinical and pre-clinical studies, we utilized epidermal growth factor receptor variant III (EGFRvIII) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) targeting single-chain variable fragments (scFvs) to generate bi-specific tandem CAR T cells and bi-specific parallel CAR T cells to decrease the potential of antigen escape and tumor recurrence. 5, 10, 15 or 20 flexible amino acids were utilized to link scFvs in tandem CARs. Ribosomal “skipping” 2A peptides were utilized to generate parallel CAR T cells. Flow-based IL13Rα2 staining on glioma stem cells (GSCs) showed 7 out 11 cases positive (64%) heterogeneous intratumoral expression. Tandem CAR T cells and parallel CAR T cells upregulated CD69 expression (P< 0.0001), generated IFNγ, interleukin 2 and TNFα (P< 0.0001), as well as exhibiting cytotoxic activity (P< 0.0001) when compared against un-transduced T cells, in co-culture with either single- or double-target positive GSCs. There was no significant difference between different lengths of linker in tandem CAR T cells. Parallel CAR T cells generated a larger cytokine response (P< 0.0001) and more effectively killed target positive cells (P< 0.05) than tandem CAR T cells. In summary, glioma associated target expression is heterogeneous in GSCs. Multivalent tandem CAR T cells and parallel CAR T cells effectively respond to target expressing tumor cells. The function of tandem CAR T cells is not relative with the length of linker. Parallel CAR T cells are a more promising strategy in generating gene modified multivalent targeting T cells.

scholarly journals Tandem CD19/CD22 Dual Targets CAR-T Cells Therapy Obtains Superior CR Rate Than Single CD19 CAR-T Cells Infusion As Well As Sequential CD19 and CD22 CAR-T Cells Infusion for Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1755-1755
Author(s):  
Sining Liu ◽  
Xinyue Zhang ◽  
Haiping Dai ◽  
Qingya Cui ◽  
Wei Cui ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Significant Difference ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Background: CD19 chimeric antigen receptor T (CAR-T) cells therapy has shown great success in B-cell acute lymphoblastic leukemia (B-ALL). To reduce the possibility of relapse due to CD19 antigen loss, sequential CD19/CD22 and tandem CD19/CD22 dual targets CAR-T cells have been developed. However, the optimal combination strategy of target antigens for CAR-T cells is still uncertain. This study was designed to compare the efficacy and safety of single CD19, tandem CD19/CD22 and sequential CD19/CD22 CAR-T cells therapies in relapsed/refractory(R/R) B-ALL patients. Methods: Between March 2016 and August 2020, a total of 200 patients with R/R B-ALL successfully received 230 CAR-T treatments (30 patients received the second CAR-T therapy and 8 patients received the third CAR-T therapy) were screened in this study. Among them, 168 patients received single CD19 CAR-T therapy, 49 patients received tandem CD19/CD22 CAR-T therapy, and 13 patients received sequential CD19/CD22 CAR-T therapy. ALL patients enrolled in the CD19 CAR-T clinical trials (NCT03919240) or CD19/CD22 CAR-T clinical trials (NCT03614858). Results: The baseline characteristics of patients were similar among the three groups. The complete remission (CR) rates were 82.7% (139/168) in patients who received CD19 CAR-T therapy, 95.9% (47/49) in patients who received tandem CD19/CD22 CAR-T therapy, and 69.2% (9/13) in patients who received sequential CD19/CD22 CAR-T therapy (P=0.012). Tandem CD19/CD22 CAR-T therapy remained one of the significant favorable factors in multivariate logistic regression analysis of CR rate in all patients (hazard ratio, 0.081; 95% CI, 0.010-0.671). Furthermore, minimal residual disease (MRD)-negative CR rates were 66.7%, 81.6% and 61.5%, respectively (P=0.092). There was no significant difference in the incidence of adverse events among the three groups. Severe cytokine release syndrome (CRS, Grade ≥ 3) occurred in 25.0% of patients in CD19 group, 18.4% of patients in tandem CD19/CD22 group, and 23.1% in sequential CD19/CD22 group (P=0.641). There was no significant difference in overall survival (OS) and leukemia-free survival (LFS) among three groups (6-month OS: 83.1%, 90.0% and 88.9%, respectively, P=0.1620; 6-month LFS: 76.2%, 76.2% and 88.9%, respectively, P=0.8179). Univariate and multivariate Cox regression analyses showed that a better LFS related to less frequencies of relapse, lower tumor burden, MRD-negative CR and bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT). Conclusions: Tandem CD19/CD22 dual targets CAR-T cells therapy obtains superior CR rate than single CD19 and sequential CD19/CD22 CAR-T cells therapy. This provides an effective treatment option for R/R B-ALL patients with chemotherapy resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD19 CAR-T Cells Treatment Conferred a Sustained Remission in Patients with Chemotherapy-Refractory MRD in B-ALL

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 48-48
Author(s):  
Wenyi Lu ◽  
Yunxiong Wei ◽  
Yaqing Cao ◽  
Xia Xiao ◽  
Qing Li ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Sustained Remission ◽  
Car T Cells ◽  
Car T ◽  
Duration Of Response ◽  
Cell Acute Lymphoblastic Leukemia

Background: The persistence or recurrence of minimal residual disease (MRD) after chemotherapy predicts relapse of B-cell acute lymphoblastic leukemia (B-ALL). CD19-directed chimeric antigen receptor-modified T (CD19 CAR-T) cells have shown excitable response in B-ALL. Recently, some studies have shown that ALL patients with lower burden had higher CR rate and lower risk of CRS after CAR-T therapy (Park, et al, NEJM. 2018; 378(5):439-448. Lee, et al, Blood.128 (22):Abstract 218.). However, its role in chemotherapy-refractory MRD-positive B-ALL remains unclear. Here we aimed to assess the effectiveness and safety of CD19 CAR-T in MRD-positive B-ALL patients. Methods: Since January 2018, a total of 14 B-ALL patients with persistent (n=8) or recurrent (n=6) MRD were enrolled in the CAR-T clinical trials (ChiCTR-ONN- 16009862 and ChiCTR1800015164). The patients were from two different clinical trials about CAR-T. If patients were treated in an MRD positive state, they would be included in this analysis. All the patients received one or more infusions of autogenous CD19 CAR-T. Results: Median age was 37.5 (13-62) years and 7 patients were female. The median dose of infused CAR-T cells was 6.78´106cells/kg, and 5 patients received more than one infusion. After one cycle of CAR-T infusion, 12 patients achieved MRD-negative remission, leading a response rate of 85.7%. Of the subgroup of 5 Ph-positive patients who subsequently underwent transplantation, 2 patients died due to transplant-related toxic effects, whereas the other 3 patients all currently alive without leukemia. Of the subgroup of 9 Ph-negative patients, 8 patients did not undergo subsequent transplantation (Figure 1). Three patients finally suffered CD19-positive relapse and 1 patient suffered CD19-negative relapse. Importantly, 4 patients (50%) are in ongoing molecular remission without transplantation, with a duration of response averaging 22.9 months (range: 12.1-28.6 months). The most frequent adverse events were fever and hematopoietic toxicities. Ten patients (71.4%) had grade 1 or 2 cytokine release syndrome (CRS) and no patients died of CRS. At a median follow-up time of 599.5 days (range: 172-915 days), the probability of 2-year relapse-free survival and 2-year overall survival was 61.2%±14.0% and 78.6%±11.0%, respectively. Conclusion: In conclusion, pre-emptive CD19 CAR-T treatment is an effective and safe approach and may confer a sustained remission in B-ALL patients with chemotherapy-refractory MRD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Treatment Outcome of Pediatric B-ALL with Bone Marrow and Extramedullary Relapse by Anti-CD19 CAR-T

2021 ◽  
Author(s):  
Xinyu Wan ◽  
Fan Yang ◽  
Xiaomin Yang ◽  
tianyi Wang ◽  
Lixia Ding ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Lentiviral Vector ◽  
Lymphoblastic Leukemia ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T ◽  
Extramedullary Relapse

Background: Anti-CD19 Chimeric Antigen Receptor T-Cell Immunotherapy (19CAR-T) has achieved impressive clinical achievements in both adult and pediatric relapsed/refractory (r/r) B-lineage acute lymphoblastic leukemia (B-ALL). However, the application and effect of CAR-T therapy in B-ALL patients with extramedullary relapse are rarely issued even disqualified in some clinical trials. Here, we examined the efficacy of 19CAR-T in patients with both bone marrow and extramedullary involvement. Methods: CAR-T cells were generated by a lentiviral vector transfection into primary human T lymphocytes to express anti-CD19 and anti-CD22 single chain antibody fragments (scFvs) with the cytoplasmic domains of 4-1BB and CD3ζ. Patients diagnosed as r/r B-ALL with extramedullary origination were infused with anti-CD19 CAR-T cells. The clinical responses were evaluated by bone marrow aspiration, imaging, and flow cytometry examination. Results: A total of 8 patients received 19CAR-T infusion and all of them acquired complete remission (CR), in which only 1 patient was bridged to hematopoietic stem cell transplantation (HSCT). Even though there were 3 patients relapsed after infusion, they received 19/22CAR-T infusion sequentially and acquired the second remission. To date, 5 patients are continuous CR, and all patients are still alive. The mean follow-up time was 21.9 months while the 24-month estimated event-free survival (EFS) is 51.4%. Conclusions: Anti-CD19 CAR-T therapy can lead to clinical remission for extramedullary relapsed pediatric B-ALL patients. However, the problem of CD19+ relapses after CAR-T remained to be solved. For patients relapsing after CAR-T, the second CAR-T therapy suggests creating another opportunity of remission for subsequent HSCT.

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