scholarly journals A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ehsan Razeghian ◽  
Mahyuddin K. M. Nasution ◽  
Heshu Sulaiman Rahman ◽  
Zhanna R. Gardanova ◽  
Walid Kamal Abdelbasset ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Genome Editing ◽  
The United States ◽  
Special Focus ◽  
Tumor Escape ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractTo date, two chimeric antigen receptors (CAR)-T cell products from autologous T cells have been approved by The United States Food and Drug Administration (FDA). The case-by-case autologous T cell generation setting is largely considered as a pivotal restraining cause for its large-scale clinical use because of the costly and prolonged manufacturing procedure. Further, activated CAR-T cells mainly express immune checkpoint molecules, including CTLA4, PD1, LAG3, abrogating CAR-T anti-tumor activity. In addition, CAR-T cell therapy potently results in some toxicity, such as cytokine releases syndrome (CRS). Therefore, the development of the universal allogeneic T cells with higher anti-tumor effects is of paramount importance. Thus, genome-editing technologies, in particular, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 are currently being used to establish “off-the-shelf” CAR-T cells with robust resistance to immune cell-suppressive molecules. In fact, that simultaneous ablation of PD-1, T cell receptor alpha constant (TRAC or TCR), and also β-2 microglobulin (B2M) by CRISPR-Cas9 technique can support the manufacture of universal CAR-T cells with robust resistance to PD-L1. . Indeed, the ablation of β2M or TARC can severely hinder swift elimination of allogeneic T cells those express foreign HLA-I molecules, and thereby enables the generation of CAR-T cells from allogeneic healthy donors T cells with higher persistence in vivo. Herein, we will deliver a brief overview of the CAR-T cell application in the context of tumor immunotherapy. More importantly, we will discuss recent finding concerning the application of genome editing technologies for preparing universal CAR-T cells or cells that can effectively counter tumor escape, with a special focus on CRISPR-Cas9 technology.

scholarly journals Chimeric Antigen Receptor T Cell Therapy Pipeline at a Glance: A Retrospective and Systematic Analysis from Clinicaltrials.Gov

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5629-5629 ◽  
Author(s):  
Eider F Moreno Cortes ◽  
Caleb K Stein ◽  
Paula A Lengerke Diaz ◽  
Cesar A Ramirez-Segura ◽  
Januario E. Castro
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
The United States ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Background: Chimeric Antigen Receptor (CAR) T cell therapy is a promising cancer immunotherapy that is growing exponentially. The doubling time of medical knowledge in 2010 was 3.5 years, and the projection for 2020 is just 73 days. In the last five years, the number of PubMed publications on cancer applications of CAR T cells has tripled. Therefore, to remain updated in the field represents a challenge for patients, care providers and researchers. In this review we provide a focused summary of the currently ongoing clinical trials, with a comprehensive overview of advances in CAR T cell therapy, beyond CD19, emphasizing on antigenic targets, development phases, and leading sponsor pharmaceutical companies. Methods: We retrieved the available data from the national registry of clinical trials (clinicaltrials.gov) using the following keywords: "CAR T cell", "CAR T cell and cancer", "chimeric antigen receptor", "CAR T AND tumor antigen", 'CAR T cell antigens", "Tumor antigens targeted by CAR T cells", "engineered T cells", "modified T cell", "CAR T cells in Cancer", "CAR T cell therapy", "CAR T cell therapy AND Cancer" until December 31, 2018 and manually excluded the trials unrelated to CAR T-cell therapies on cancer, by reviewing the detailed information provided on the website as well as preliminary data published. Results: The analysis included 271 clinical trials posted on the clinicaltrials.gov website from the United States by the cut-off date. For efficacy analysis, we retrieved information from 52 trials, by NCT number on a PubMed search. The majority of CAR T clinical research is focused on hematological cancer (57%), followed by CNS 8%, GI 6%, Skin 5%, Genitourinary 4%, Breast 4%, Gynecologic 4%, Respiratory 3%, Sarcoma 2%, Mesothelioma 2% and others 5%. The most used target in CAR T cell therapy and the leaders in phase 3 trials are CD19 (42%) and BCMA (12%), followed by CD20, NY-ESO-1, Mesothelin, HER2, GD2, MAGE-A3 and CD30. An essential step in CAR T cell therapy development is the selection of the right antigen/target. Here, we provide an overview of the clinically relevant targets that are actively being using by clinical trials in the United States. For example, CD19 appears to be a leading target regarding CAR T cell therapy on cancer with 116 trials (42% of total CAR T cells trials) on going just in the United States with a significant increment in the previous years. Similarly, with BCMA is one of the targets with more phase 3 trials (Figure 1) with promising results on patients with Multiple Myeloma with and the objective response of 85%, CR 45%, and PFS of 11.8 months. Second-generation CARs with either CD28 or 4-1BB as costimulatory signaling domain are preferred, with 4-1BB being the most commonly chosen. Conclusions: Our findings show growing trends in the development of CAR T cell-based therapies, combination and possible retargeting therapies in the future for solid tumor and hematologic malignances; taking into account the amount of important information and the complexity of the database, we have developed this analysis to understand how to generate in the future a friendly platform for researchers and patients to have an detailed overview of the clinical trials in cellular therapies Disclosures No relevant conflicts of interest to declare.

scholarly journals Allogeneic CAR T Cells: An Alternative to Overcome Challenges of CAR T Cell Therapy in Glioblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Darel Martínez Bedoya ◽  
Valérie Dutoit ◽  
Denis Migliorini
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Solid Tumors ◽  
Tumor Escape ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy has emerged as one of the major breakthroughs in cancer immunotherapy in the last decade. Outstanding results in hematological malignancies and encouraging pre-clinical anti-tumor activity against a wide range of solid tumors have made CAR T cells one of the most promising fields for cancer therapies. CAR T cell therapy is currently being investigated in solid tumors including glioblastoma (GBM), a tumor for which survival has only modestly improved over the past decades. CAR T cells targeting EGFRvIII, Her2, or IL-13Rα2 have been tested in GBM, but the first clinical trials have shown modest results, potentially due to GBM heterogeneity and to the presence of an immunosuppressive microenvironment. Until now, the use of autologous T cells to manufacture CAR products has been the norm, but this approach has several disadvantages regarding production time, cost, manufacturing delay and dependence on functional fitness of patient T cells, often reduced by the disease or previous therapies. Universal “off-the-shelf,” or allogeneic, CAR T cells is an alternative that can potentially overcome these issues, and allow for multiple modifications and CAR combinations to target multiple tumor antigens and avoid tumor escape. Advances in genome editing tools, especially via CRISPR/Cas9, might allow overcoming the two main limitations of allogeneic CAR T cells product, i.e., graft-vs.-host disease and host allorejection. Here, we will discuss how allogeneic CAR T cells could allow for multivalent approaches and alteration of the tumor microenvironment, potentially allowing the development of next generation therapies for the treatment of patients with GBM.

scholarly journals CS-1 Re-Directed Central Memory T Cell Therapy for Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1114-1114 ◽  
Author(s):  
Xiuli Wang ◽  
ChingLam W Wong ◽  
Ryan Urak ◽  
Wen-Chung Chang ◽  
Elizabeth E. Budde ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
The United States ◽  
Central Memory ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Multiple myeloma (MM), a plasma cell malignancy, accounts for approximately 1 percent of all cancers and slightly more than 10 percent of hematologic malignancies in the United States. Approximately 20,000 new cases will be diagnosed this year and over 11,000 people will die from this disease. Current therapies for MM often induce remission, but nearly all patients eventually relapse and die. T-cell mediated anti-tumor therapies using genetically modify T cells with specific chimeric antigen receptors (CARs) have non-overlapping activity, toxicity and tumor resistance profiles compared to conventional chemotherapeutic agents. The main challenge in designing a CAR T cell immunotherapeutic approach is identifying the best antigen for tumor targeting. CS-1 is a cell surface glycoprotein of the signaling lymphocyte activation molecule (SLAM) receptor family that is highly and selectively expressed on normal plasma cells and MM cells, with lower expression on NK cells and little or no expression on normal tissues. This unique expression pattern and proven clinical benefit of CS-1 monoclonal antibody for the treatment of relapsed MM makes CS-1 a good target for CAR T cell therapy. Central memory T cells (TCM) from PBMC were isolated using a two-step process on the AutoMACS device to first deplete CD14+, CD45RA+ and CD25+ cells, then to positively select CD62L+ cells. These TCM undergo anti-CD3/CD28 bead stimulation and transduction with a lentiviral vector encoding CS-1 CAR containing a CD28 co-stimulatory domain and two mutations on IgG4 linker CH-2 portion to ensure enhanced potency and persistence after adoptive transfer. Gene modified CS-1 CAR T cells specifically lysed MM.1S, a MM cell line, in 4-hour 51Cr release assays and all the CAR+ cells expressed 107a upon co-cultured with the MM.1S cells. To investigate the potency of the CS-1CAR T cells, 2x106 MM.1S cells that were engineered to express GFP firefly luciferase were inoculated into NSG mice by tibia injection. 7 days post tumor engraftment, 1x106 CS-1 CAR T cells were intravenously injected into the tumor bearing mice. In contrast to untreated and mock cell treated mice in which tumor progressed rapidly systemically, single intravenous infusion of CS-1 CAR T cells induced dramatic tumor regression and significantly prolonged survival. In addition to CS-1, CD44v6 and BCMA are antigens that have also been shown to be over-expressed on MM tumor cells. We therefore compared the two CARs with CS-1 CAR for their anti-MM activity. Based on our studies, targeting CS-1 resulted in the best efficacy (Figure 1) and would be an attractive strategy for development of a clinical trial. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Laura Castelletti ◽  
Dannel Yeo ◽  
Nico van Zandwijk ◽  
John E. J. Rasko
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Malignant Mesothelioma ◽  
Oncolytic Viruses ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

AbstractMalignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.

scholarly journals Metabolic and Mitochondrial Functioning in Chimeric Antigen Receptor (CAR)—T Cells

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1229
Author(s):  
Ali Hosseini Rad S. M. ◽  
Joshua Colin Halpin ◽  
Mojtaba Mollaei ◽  
Samuel W. J. Smith Bell ◽  
Nattiya Hirankarn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Metabolic State ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized adoptive cell therapy with impressive therapeutic outcomes of >80% complete remission (CR) rates in some haematological malignancies. Despite this, CAR T cell therapy for the treatment of solid tumours has invariably been unsuccessful in the clinic. Immunosuppressive factors and metabolic stresses in the tumour microenvironment (TME) result in the dysfunction and exhaustion of CAR T cells. A growing body of evidence demonstrates the importance of the mitochondrial and metabolic state of CAR T cells prior to infusion into patients. The different T cell subtypes utilise distinct metabolic pathways to fulfil their energy demands associated with their function. The reprogramming of CAR T cell metabolism is a viable approach to manufacture CAR T cells with superior antitumour functions and increased longevity, whilst also facilitating their adaptation to the nutrient restricted TME. This review discusses the mitochondrial and metabolic state of T cells, and describes the potential of the latest metabolic interventions to maximise CAR T cell efficacy for solid tumours.

scholarly journals Engineering Next-Generation CAR-T Cells for Better Toxicity Management

2020 ◽  
Vol 21 (22) ◽  
pp. 8620
Author(s):  
Alain E. Andrea ◽  
Andrada Chiron ◽  
Stéphanie Bessoles ◽  
Salima Hacein-Bey-Abina
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Receptors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Safety Strategies ◽  
Life Threatening ◽  
Preclinical And Clinical Studies

Immunoadoptive therapy with genetically modified T lymphocytes expressing chimeric antigen receptors (CARs) has revolutionized the treatment of patients with hematologic cancers. Although clinical outcomes in B-cell malignancies are impressive, researchers are seeking to enhance the activity, persistence, and also safety of CAR-T cell therapy—notably with a view to mitigating potentially serious or even life-threatening adverse events like on-target/off-tumor toxicity and (in particular) cytokine release syndrome. A variety of safety strategies have been developed by replacing or adding various components (such as OFF- and ON-switch CARs) or by combining multi-antigen-targeting OR-, AND- and NOT-gate CAR-T cells. This research has laid the foundations for a whole new generation of therapeutic CAR-T cells. Here, we review the most promising CAR-T cell safety strategies and the corresponding preclinical and clinical studies.

IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi102-vi103
Author(s):  
Tomás A Martins ◽  
Marie-Françoise Ritz ◽  
Tala Shekarian ◽  
Philip Schmassmann ◽  
Deniz Kaymak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Differential Expression Analysis ◽  
Dependent Manner ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.

scholarly journals Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3921-3921 ◽  
Author(s):  
Cesar Sommer ◽  
Hsin-Yuan Cheng ◽  
Yik Andy Yeung ◽  
Duy Nguyen ◽  
Janette Sutton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cells ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

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