scholarly journals TRAIL-R1-Targeted CAR-T Cells Exhibit Dual Antitumor Efficacy

2021 ◽  
Vol 8 ◽  
Author(s):  
Yaru Nai ◽  
Li Du ◽  
Meiying Shen ◽  
Tingting Li ◽  
Jingjing Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Death Receptor ◽  
Antitumor Efficacy ◽  
Single Chain ◽  
Tumor Cell Death ◽  
Positive Tumor Cell ◽  
Normal Tissues ◽  
Car T Cells ◽  
Car T ◽  
Dual Mechanism

Tumor necrosis factor–related apoptosis-inducing ligand receptor 1 (TRAIL-R1) has limited expression in normal tissues but was highly expressed in various types of tumors, making it an attractive target for cancer immunotherapy. Here, we utilized the single-chain variable fragment (scFv) from our previously identified TRAIL-R1–targeting monoclonal antibody (TR1419) with antitumor efficacy and produced the TR1419 chimeric antigen receptor (CAR) T cells. We characterized the phenotypes and functions of these CAR-T cells and found that the third-generation TR1419-28BBζ CAR-T cells exhibited greater target sensitivity and proliferative capability, with slightly higher PD-1 expression after antigen stimulation. Importantly, we found that the TR1419 CAR-T cells could induce TRAIL-R1–positive tumor cell death via a dual mechanism of the death receptor–dependent apoptosis as well as the T-cell–mediated cytotoxicity. Altogether, the TR1419 CAR-T cells could serve as a promising strategy for targeting the TRAIL-R1–positive tumors.

scholarly journals TRAIL-R1-targeted chimeric antigen receptor T cells exhibit dual antitumor effects

2021 ◽  
Author(s):  
AiShun Jin ◽  
Yaru Nai ◽  
Li Du ◽  
Meiying Shen ◽  
Tingting Li ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Human Monoclonal Antibody ◽  
Death Receptor ◽  
Target Antigen ◽  
Single Chain ◽  
Antitumor Effects ◽  
Apoptotic Pathway ◽  
Car T Cells ◽  
Car T

Tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) has limited expression in normal tissues but highly expression in a broad range of tumors, making it an attractive target for cancer immunotherapy. We have previously prepared a fully human monoclonal antibody targeting TRAIL-R1 (TR1419), which can specifically induce apoptosis in antigen-positive tumor cells. Here, we prepared the TR1419CAR-T cells using the single chain variable fragment (scFv) from TR1419, which were evaluated for the phenotypes and function. The TR1419CAR-T cells induced cytolysis of TRAIL-R1-positive tumor cells not only via activation of the death receptor-dependent apoptotic pathway, but also via T-cell mediated cytotoxicity. Furthermore, compared to the second-generation TR1419-28ζ and TR1419-BBζ CAR-T cells, the third-generation TR1419-28BBζ CAR-T cells had greater sensitivity to target antigen, exhibited a better proliferative ability, but showed slightly higher PD-1 expression after antigen stimulation. Altogether, TR1419CAR-T cells, especially TR1419-28BBζCAR-T cells could be a promising treatment strategy for TRAIL-R1 positive tumors.

Combinatorial IL13Rα2 chimeric antigen receptor-T cells plus checkpoint blockade to treat solid tumors in murine and canine models.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 152-152
Author(s):  
Yibo Yin ◽  
Alina Boesteanu ◽  
Chong Xu ◽  
Avery Posey ◽  
Danielle Cook ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Combined Treatment ◽  
Checkpoint Blockade ◽  
Single Chain ◽  
Normal Tissues ◽  
Car T Cells ◽  
Car T ◽  
Canine Tumors

152 Background: Glioblastoma (GBM) is a deadly cancer, with a 5-year survival rate of less than 10%. T cells can be redirected to kill cancer cells using chimeric antigen receptors (CAR), a promising method to treat solid tumors. IL13Rα2 is expressed in many solid tumors but not normal tissues, and provides a target for CAR T cells as a powerful immunotherapy treatment. Methods: Human and canine tumors were screened for IL13Rα2 expression by flow cytometry and RT-PCR. Different IL13Rα2 single-chain variable fragments (scFv) were tested in CARs in vitro against human and canine recombinant IL13Rα2 protein to identify a cross-reactive clone. IL13Rα2 CARs expressed in human and canine T cells and showed antigen-specific stimulation by cytokine secretion and target cell lysis. Solid tumors were used to establish in vivo subcutaneous and orthotopic xenograft models in NSG mice. Mice received a single treatment of 2-5 million CAR T cells IV with or without anti PD-1, CTLA-4 or TIM3 mAb checkpoint blockade. Results: IL13Rα2 was detected on three human GBM (D270, U251 and U87), four canine osteosarcomas (BW-, CS-, MC- and SK-KOSA) and two canine lung cancer cell lines (Cacal3, Cacal5). Two different IL13Rα2 CAR were generated that recognized human IL13Rα2 and not IL13Rα1, one also recognized canine IL13Rα2. Five million IL13Rα2 CAR T-cells delivered IV were able to eliminate established GBM tumors. Use of fewer CAR T cells initially controlled tumor growth, followed by tumor outgrowth correlated with expression of T cell exhaustion markers. Combined treatment with CARs plus checkpoint blockade mAb delivered IP had synergistic effects, restoring anergic T-cell function and improving tumor treatment. Conclusions: IL13Rα2 is highly expressed on human and canine tumors, but not normal tissues. IL13Rα2 specific CAR T cells successfully respond to human and canine tumors and treat GBM in a xenograft mouse model, although this effect was transient at lower treatment doses. Addition of checkpoint blockade mAbs to CAR therapy was beneficial for the treatment of solid tumors. We plan to utilize this treatment in a canine spontaneous cancer preclinical model and translate into clinical trials for patients with GBM.

scholarly journals IMMU-17. USE OF MRNA FOR SAFE AND EFFECTIVE GD2-DIRECTED CAR T CELLS TO TREAT DIFFUSE MIDLINE GLIOMAS

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i30-i31
Author(s):  
Jessica Foster ◽  
Crystal Griffin ◽  
Allison Stern ◽  
Cameron Brimley ◽  
Phillip Storm ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Regression ◽  
Single Chain ◽  
Tumor Cell Death ◽  
T Cell Therapy ◽  
Cellular Models ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T cells targeting the disialoganglioside GD2 have garnered interest as an effective therapeutic for treating diffuse midline glioma (DMG). However, prior studies raised significant concerns of neurotoxicity and fatality when using virally transduced CAR T cells against these midline tumors. Building upon our prior work optimizing mRNA for use in CAR T cells (Hum Gen Ther, 2019), we hypothesized transient GD2-directed mRNA CAR T cells could be successfully employed for safe and effective treatment of DMG. GD2-directed CAR T cells were created using mRNA encoding the 14G2a single chain variable fragment paired with 41BB and CD3-zeta co-stimulatory domains and transfected into human T cells. GD2-directed CAR T cells were tested against a panel of DMG cell lines and two murine xenograft models of DMG: 7316-6349 and SU-DIPG13P*. In all DMG cellular models, GD2-directed mRNA CAR T cells induced significant tumor cell death compared to CD19-directed mRNA CAR T cell controls. In vivo, mRNA CAR T cells were delivered locoregionally using an indwelling infusion catheter to allow for repeated dosing. Four intratumoral doses of 5 x 106 GD2-directed mRNA CAR T cells induced significant tumor regression measured by bioluminescence in DMG model 7316-6349 (p<0.0001). In addition, GD2-directed mRNA CAR T cells prolonged survival of mice harboring the aggressive DMG model SU-DIPG13P* by 61% (mean survival 29 days versus 18 days, p<0.01) following four intratumoral doses of 4 x 106 CAR T cells. No GD2-directed CAR T cell treatment-related deaths or toxicities were observed. These data highlight the utility of using mRNA to titrate CAR T cell therapy in the brain, and establish GD2-directed mRNA CAR T cells as a safe and effective method for treating DMG.

112 Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A124-A124
Author(s):  
Letizia Giardino ◽  
Ryan Gilbreth ◽  
Cui Chen ◽  
Erin Sult ◽  
Noel Monks ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Single Chain ◽  
High Affinity ◽  
Car T Cells ◽  
Car T ◽  
Animal Care ◽  
Cytotoxic Function

BackgroundChimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity, including on-target off-tumor antigen binding, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CAR bearing a low and high affinity single-chain variable fragment (scFv,) binding to the same epitope and cross-reactive with murine GPC3. We characterized low and high affinity CAR-T cells immunophenotype and effector function in vitro, followed by in vivo efficacy and safety studies in hepatocellular carcinoma (HCC) xenograft models.ResultsCompared to the high-affinity construct, the low-affinity CAR maintained cytotoxic function but did not show in vivo toxicity. High-affinity CAR-induced toxicity was caused by on-target off-tumor binding, based on the evidence that high-affinity but not low-affinity CAR, were toxic in non-tumor bearing mice and accumulated in organs with low expression of GPC3. To add another layer of safety, we developed a mean to target and eliminate CAR-T cells using anti-TNFα antibody therapy post-CAR-T infusion. This antibody functioned by eliminating early antigen-activated CAR-T cells, but not all CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from anti-tumor efficacy.ConclusionsSelecting a domain with higher off-rate improved the quality of the CAR-T cells by maintaining cytotoxic function while reducing cytokine production and activation upon antigen engagement. By exploring additional traits of the CAR-T cells post-activation, we further identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that would eliminate early activated CAR-T following antigen engagement in vivo. By combining the reduced affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.Ethics ApprovalAll animal experiments were conducted in a facility accredited by the Association for Assessment of Laboratory Animal Care (AALAC) under Institutional Animal Care and Use Committee (IACUC) guidelines and appropriate animal research approval.

scholarly journals Bispecific c-Met/PD-L1 CAR-T Cells Have Enhanced Therapeutic Effects on Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Wei Jiang ◽  
Tao Li ◽  
Jiaojiao Guo ◽  
Jingjing Wang ◽  
Lizhou Jia ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Carcinoma Cells ◽  
Therapeutic Effects ◽  
Single Chain ◽  
Antitumor Activities ◽  
Hepatocellular Carcinoma Cells ◽  
Car T Cells ◽  
Car T

T cells expressing chimeric antigen receptors, especially CD19 CAR-T cells have exhibited effective antitumor activities in B cell malignancies, but due to several factors such as antigen escape effects and tumor microenvironment, their curative potential in hepatocellular carcinoma has not been encouraging. To reduce the antigen escape risk of hepatocellular carcinoma, this study was to design and construct a bispecific CAR targeting c-Met and PD-L1. c-Met/PD-L1 CAR-T cells were obtained by lentiviral transfection, and the transfection efficiency was monitored by flow cytometry analysis. LDH release assays were used to elucidate the efficacy of c-Met/PD-L1 CAR-T cells on hepatocellular carcinoma cells. In addition, xenograft models bearing human hepatocellular carcinoma were constructed to detect the antitumor effect of c-Met/PD-L1 CAR-T cells in vivo. The results shown that this bispecific CAR was manufactured successfully, T cells modified with this bispecific CAR demonstrated improved antitumor activities against c-Met and PD-L1 positive hepatocellular carcinoma cells when compared with those of monovalent c-Met CAR-T cells or PD-L1 CAR-T cells but shown no distinct cytotoxicity on hepatocytes in vitro. In vivo experiments shown that c-Met/PD-L1 CAR-T cells significantly inhibited tumor growth and improve survival persistence compared with other groups. These results suggested that the design of single-chain, bi-specific c-Met/PD-L1 CAR-T is more effective than that of monovalent c-Met CAR-T for the treatment of hepatocellular carcinoma., and this bi-specific c-Met/PD-L1 CAR is rational and implementable with current T-cell engineering technology.

A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3094-3094 ◽  
Author(s):  
Alena A. Chekmasova ◽  
Holly M. Horton ◽  
Tracy E. Garrett ◽  
John W. Evans ◽  
Johanna Griecci ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Drug Product ◽  
Single Chain ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

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.

scholarly journals Allogeneic gene-edited HIV-specific CAR-T cells secreting PD-1 blocking scFv enhance anti-HIV activity in vitro

2022 ◽  
Author(s):  
Hanyu Pan ◽  
Jing Wang ◽  
Huitong Liang ◽  
Zhengtao Jiang ◽  
Lin Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
Envelope Glycoprotein ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Patient Specific ◽  
Single Chain ◽  
Car T Cells ◽  
Car T ◽  
Hiv Envelope ◽  
Anti Hiv

HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. However, the T-cell exhaustion and the patient-specific autologous paradigm of CAR-T hurdled the clinical application. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells and a 3BNC117-E27 CAR (3BE CAR) construct that enables the expression of PD-1 blocking scFv E27 and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV envelope glycoprotein (Env). In comparison with T cells expressing 3BNC117-CAR alone, 3BE CAR-T cells showed greater anti-HIV potency with stronger proliferation capability, higher killing efficiency (up to ~75%) and enhanced cytokine secretion in the presence of HIV envelope glycoprotein-expressing cells. Furthermore, our approach achieved high levels (over 97%) of the TCR-deficient 3BE CAR-T cells with the functional inactivation of endogenous TCR to avoid graft-versus-host disease without compromising their antiviral activity relative to standard anti-HIV CAR-T cells. These data suggest that we have provided a feasible approach to large-scale generation of "off-the-shelf" anti-HIV CAR-T cells in combination with antibody therapy of PD-1 blockade, which can be a powerful therapeutic candidate for the functional cure of HIV.

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.

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