scholarly journals CRISPR/Cas9-mediated TGFβRII Disruption Enhances Anti-tumor Efficacy of Human Chimeric Antigen Receptor T Cells in Vitro

2021 ◽  
Author(s):  
Khadijeh Alishah ◽  
Matthias Birtel ◽  
Elham Masoumi ◽  
Leila Jafarzadeh ◽  
Hamid Reza Mirzaee ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Solid Tumors ◽  
Target Cells ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. Methods: In this study, we hypothesized that knocking out the TGFβ receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFβRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFβRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells.Results: Our experiments demonstrated that TGFβRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFβ in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFβRII KO CAR T-cells has been recorded. TGFβRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFβRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. Conclusion: The TGFβRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

scholarly journals CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Khadijeh Alishah ◽  
Matthias Birtel ◽  
Elham Masoumi ◽  
Leila Jafarzadeh ◽  
Hamid Reza Mirzaee ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Solid Tumors ◽  
Target Cells ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFβ are of great importance: TGFβ is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. Methods In this study, we hypothesized that knocking out the TGFβ receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFβRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFβRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. Results Our experiments demonstrated that TGFβRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFβ in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFβRII KO CAR T-cells has been recorded. TGFβRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFβRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. Conclusion The TGFβRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

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 Lenalidomide Enhances CAR-T Cell Activity Against Solid Tumor Cells

2020 ◽  
Vol 29 ◽  
pp. 096368972092082 ◽  
Author(s):  
Zhixiong Wang ◽  
Guomin Zhou ◽  
Na Risu ◽  
Jiayu Fu ◽  
Yan Zou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cytokine Secretion ◽  
Target Cells ◽  
Functional Assay ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T-cell immunotherapy still faces many challenges in the treatment of solid tumors, one of which is T-cell dysfunction or exhaustion. Immunomodulator lenalidomide may improve CAR T-cell function. In this study, the effects of lenalidomide on CAR T-cell functions (cytotoxicity, cytokine secretion, and cell proliferation) were investigated. Two different CAR T cells (CD133-specific CAR and HER2-specific CAR) were prepared, and the corresponding target cells including human glioma cell line U251 CD133-OE that overexpress CD133 and human breast cancer cell line MDA-MB-453 were used for functional assay. We found that lenalidomide promoted the killing of U251 CD133-OE by CD133-CAR T cells, the cytokine secretion, and the proliferation of CD133-CAR T cells. Lenalidomide also enhanced the cytotoxicity against MDA-MB-453 and the cytokine secretion of HER2-CAR T cells but did not affect their proliferation significantly. Furthermore, lenalidomide may regulate the function of CAR T cells by inducing the degradation of transcription factors Ikaros and Aiolos.

scholarly journals 137 Development of anti-Mucin1 CAR-T against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A146-A146
Author(s):  
Jihyun Lee ◽  
Areum Park ◽  
Jungwon Choi ◽  
Dae Gwan Yi ◽  
Hee Jung Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity

BackgroundChimeric antigen receptor (CAR) -T cell therapies have proven to be effective against various liquid tumors. However, the development of CAR-T against solid tumors has been challenging due to insufficient efficacy and potential on-target off-tumor toxicities caused by low expression of tumor antigens on normal tissues. Testing various affinities of CARs has demonstrated that lower affinity CARs maintain its anti-tumor effect while minimizing safety concerns (1). In order to develop a CAR-T against solid tumors expressing Mucin1, we have screened for Mucin1 binding antibodies and tested their anti-tumor effect in vitro and in vivo. The potential of on-target off-tumor toxicity was also measured in vitro.MethodsAnti-Mucin1 human single chain variable fragments (scFv) were obtained via screening against a scFv display library. Anti-Mucin1 scFvs were incorporated into CARs and in vitro, in vivo functions against various tumor cells expressing Mucin1 were tested. For in vivo studies, tumor bearing NOG mice (HCC1954 cells) received anti-Mucin1 CAR-T cells. Therapeutic efficacy was evaluated by measuring tumor volumes. Potential on-target off-tumor toxicity against Mucin1 on normal cells was tested by investigating the killing effect of anti-Mucin1 CAR-T against cancer cell line (HCC70) and non-tumorigenic breast epithelial cell line (MCF-10A) in co-culture systemsResultsIn vitro activity of anti-Mucin1 CAR-T cells that displayed a range of affinities for Mucin1 (27nM to 320nM) showed similar cytokine secretion levels and cytotoxicity against Mucin-1 expressing tumor cell lines (HCC70 and T47D). Robust anti-tumor activity was also demonstrated in vivo against large tumors (400~500 mm3) with relatively small numbers of CAR-T cells (0.5 x 106 CAR-T cells per mouse). In vivo expansion of CAR-T cells were observed in all scFv-CAR-T cases and accompanied by close to complete regression of tumors within 25 days post CAR-T cell injection. Of the 4 scFv CAR-Ts, 2H08 (with a Kd of 94nM) was tested for activity against normal breast epithelial cells. When 2H08-CAR-T was cocultured with a mixture of HCC70 and MCF-10A cells, they preferentially killed only the Mucin1 overexpressing HCC70 cells leaving MCF-10 cells intact.ConclusionsOur study demonstrates anti-tumor activity of a novel scFv-derived CAR-T recognizing Mucin1 and its effectiveness in large pre-established tumors in vivo. We also demonstrate that 2H08-CAR-T can distinguish between target overexpressing cancer cells and normal epithelial cells, which suggests that by toning down the affinity of CAR against antigen one can improve the safety profile of solid tumor antigen targeting CAR-T cell therapies.ReferenceCastellarin M, Sands C, Da T, Scholler J, Graham K, Buza E, Fraietta J, Zhao Y, June C. A rational mouse model to detect on-target, off-tumor CAR T cell toxicity. JCI Insight 2020; 5:e136012Ethics ApprovalAll experiments were done under protocols approved by the Institutional Animal Care and Use Committee (IACUC) (Study#LGME21-011).ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

scholarly journals Human Hyaluronidase PH20 Potentiates the Antitumor Activities of Mesothelin-Specific CAR-T Cells Against Gastric Cancer

2021 ◽  
Vol 12 ◽  
Author(s):  
Ruocong Zhao ◽  
Yuanbin Cui ◽  
Yongfang Zheng ◽  
Shanglin Li ◽  
Jiang Lv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cell Infiltration ◽  
Car T Cells ◽  
Car T Cell ◽  
T Cell Infiltration ◽  
Car T

T cell infiltration into tumors is essential for successful immunotherapy against solid tumors. Herein, we found that the expression of hyaluronic acid synthases (HAS) was negatively correlated with patient survival in multiple types of solid tumors including gastric cancer. HA impeded in vitro anti-tumor activities of anti-mesothelin (MSLN) chimeric antigen receptor T cells (CAR-T cells) against gastric cancer cells by restricting CAR-T cell mobility in vitro. We then constructed a secreted form of the human hyaluronidase PH20 (termed sPH20-IgG2) by replacing the PH20 signal peptide with a tPA signal peptide and attached with IgG2 Fc fragments. We found that overexpression of sPH20-IgG2 promoted CAR-T cell transmigration through an HA-containing matrix but did not affect the cytotoxicity or cytokine secretion of the CAR-T cells. In BGC823 and MKN28 gastric cancer cell xenografts, sPH20-IgG2 promoted anti-mesothelin CAR-T cell infiltration into tumors. Furthermore, mice infused with sPH20-IgG2 overexpressing anti-MSLN CAR-T cells had smaller tumors than mice injected with anti-MSLN CAR-T cells. Thus, we demonstrated that sPH20-IgG2 can enhance the antitumor activity of CAR-T cells against solid tumors by promoting CAR-T cell infiltration.

scholarly journals 143 Mesothelin (MSLN) targeting allogeneic CAR T cells engineered to overcome tumor immunosuppressive microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A151-A151
Author(s):  
Cecile Schiffer-Mannioui ◽  
Sophie Leduc ◽  
Isabelle Chion-Sotinel ◽  
Diane le Clerre ◽  
Valérie Guyot ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tgfβ Signaling ◽  
Knock Out ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Immunosuppressive Microenvironment

BackgroundChimeric Antigen Receptor (CAR) T cell therapy is emerging as a potential treatment for solid tumors, even if only limited activity has been observed for CAR T therapies to date. Cellular therapies face indeed many hurdles in solid tumors, such as the immunosuppressive microenvironment. TGFβ is an important growth factor of the tumor microenvironment and has been shown to suppress anti-tumor immunity. Gene editing represents a powerful way to enhance properties of CAR T cells and can be used to circumvent the effect of TGFβ signaling. The tumor associated antigen mesothelin (MSLN) is an attractive target for cellular therapy; being expressed at high levels in several tumor types (e.g., pleural mesothelioma and pancreatic cancer) while only modestly expressed in healthy tissues.MethodsUCARTMeso, an allogeneic CAR T cell product targeting MSLN expressing cells is being developed by Cellectis. UCARTMeso bears an anti-MSLN CAR and a triple gene knock-out (KO) for TRAC, CD52 and TGFBR2 genes, all generated using TALEN® gene-editing technology. TRAC KO limits the risk of GvHD, while CD52 KO allows the use of alemtuzumab in the preconditioning regimen. The additional KO of TGFBR2 confers resistance to the immunomodulatory effects of TGFβ within the solid tumor microenvironment.ResultsPreclinical studies showed high specificity of the anti-MSLN CAR, as well as potent anti-tumor activity in vitro against different cell lines expressing MSLN. In addition, this activity was confirmed in mouse studies against pancreatic and pleural mesothelioma tumor models, with comparable activities observed in the latest model upon i.v. or intra-pleural administration of UCARTMeso. Also, we observed that TGFBR2 edited anti-MSLN CAR T cells displayed a blockade in the TGFβ signaling pathway, being able to respond to antigen stimulation in the presence of TGFβ (figure 1).Abstract 143 Figure 1Left panel: TGFβ-induced SMAD2/3 phosphorylation in anti-MSLN CAR T cells. UCARTMeso cells were stained with mesothelin recombinant protein for CAR detection and anti-pSMAD2/3 one hour post exposure to TGFβ. The lack of SMAD2/3 phosphorylation in TGFBR2 KO cells indicates that they are unable to trigger TGFβ signaling. Right panel: Antigen-induced anti-MSLN CAR T cell activation in the presence (blue histogram) or absence (red histogram) of TGFβ. CAR T cells were stained with anti-CD25 antibody and analyzed by flow cytometry 5 days post exposure to antigen ± TGFβ. The data shows that cells not edited at the TGFBR2 locus are unable to be activated upon target exposure in the presence of TGFβ, while edited cells were activated in the presence of TGFβ, triggering CD25 expression at similar levels as those of cells activated in the absence of TGFβ.ConclusionsAltogether, we have demonstrated potent antitumor activity in vitro and in vivo, and that addition of the third knock-out of TGFBR2 gene provide valuable additional properties to UCARTMeso cells, representing a very attractive strategy for their use in the treatment of solid tumors.

scholarly journals 131 Genetic disruption of negative immune regulators to enhance CAR-T efficacy against solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A140-A141
Author(s):  
David Mai ◽  
Omar Johnson ◽  
Carl June
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Transduction Efficiency ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundCAR-T cell therapy has demonstrated remarkable success in hematological malignancies but displays limited efficacy in solid tumors, which comprise most cancer cases. Recent studies suggest that CAR-T cell failure via T cell exhaustion is characterized by decreased surface CAR expression, cytotoxicity, and Th1 cytokine production, leading to reduced antitumor functionality.1 2 3 To address these issues, studies have turned to genetically knocking out or overexpressing targets associated with an exhaustion or effector phenotype, such as PD-1 knockout (KO) and c-Jun overexpression, among other candidates that are typically receptors or transcription factors.4 5 6 However, there are other underexplored factors that mediate various aspects of immune regulation. While genome-wide CRISPR screens may discover such factors, they are unlikely to reveal phenotypes for genes whose function is partially redundant, therefore promising candidates may be missed. Such candidates include post-transcriptional regulators (PTRs) that coordinate immune responses, which are less well-studied in the context of CAR-T cell function. We hypothesized that KO of these PTRs may increase CAR-T cell cytokine activity, phenotype, and persistence, potentially under long-term or exhaustion-inducing conditions, leading to increased tumor control. Ultimately, disruption of negative immune regulators could produce CAR-T cells with enhanced activity and persistence, narrowing the gap between efficacy in hematological and solid tumors.MethodsTo explore whether the disruption of two target PTRs improves solid tumor efficacy, we used CRISPR-Cas9 to genetically delete one or both PTRs in mesothelin-targeting human CAR-T cells and assayed their function in vitro and in vivo in NSG mice.ResultsWe show successful genetic deletion of these genes in post-thymic human T cells and that their disruption does not affect primary expansion (figure 1) or transduction efficiency (figure 2). These KO CAR-T cells display increased expression of co-stimulatory receptors and various cytokines (figure 3). While KO CAR-T cells are functionally similar to WT CAR-T cells in in vitro assays (figure 4), KO CAR-T cells demonstrate superior activity in vivo and can clear large, established tumors compared to WT CAR-T cells at low dose (figure 5).Abstract 131 Figure 1Expansion kinetics of KO CAR-T cellsAbstract 131 Figure 2Transduction efficiency and baseline phenotype of KO CAR-T cellsAbstract 131 Figure 3Costimulatory receptor and cytokine expression of KO CAR-T cellsAbstract 131 Figure 4In vitro cytotoxicity of KO CAR-T cellsAbstract 131 Figure 5In vivo activity of KO CAR-T cellsConclusionsThese results indicate that KO of our target PTRs may improve the potency of CAR-T cells in solid tumors and may have important implications on the development of effective solid-tumor cell therapies.ReferencesJE Wherry and M Kurachi, Molecular and cellular insights into T cell exhaustion, Nature Reviews Immunology 2015;15:486–499.EW Weber, et al. Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science 2021;372:6537.S Kuramitsu et al. Induction of T cell dysfunction and NK-like T cell differentiation in vitro and in patients after CAR T cell treatment. Cell, in revision.BD Choi et al, CRISPR-Cas9 disruption of PD-1 enhances activity of university EGFRvIII CAR T cells in a preclinical model of human glioblastoma. Journal for ImmunoTherapy of Cancer 2019;7:304.RC Lynn et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019;576:293–300.LJ Rupp et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Scientific Reports 2017;7:737.

scholarly journals MDSC Suppression of CAR T Cells Can be Reduced By Targeted Signaling Disruption

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4438-4438 ◽  
Author(s):  
Estelle V Cervantes ◽  
Justin C. Boucher ◽  
Sae Bom Lee ◽  
Kristen Spitler ◽  
Kayla Reid ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Research Funding ◽  
Target Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Counts ◽  
Car T

CAR T cells are genetically modified with an extracellular scFv, transmembrane domain, and intracellular costimulatory and CD3ζ domains. Two treatments received the approval from the FDA for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma. However, CAR T cell persistence remains a problem. A reason for this may be that myeloid cells such as myeloid derived suppressor cells (MDSCs) may be contributing to the reduced persistence of CAR T cells. MDSCs originate from myeloid cells and have been implicated in the suppression of the immune system in the tumor microenvironment. To determine what effect MDSCs might have during CAR T cell production we co-cultured MDSCs during CAR transduction. We found gene transfer was lower for m19z (38.7% vs 46.8%), m1928z (24.1% vs 39.1%), and m19hBBz (35.8% vs 46.2%) CAR T cells co-cultured with MDSCs compared to those that were not (Fig 1A). There was also a reduction in total T cell counts for m19z (58%), m1928z (88%), and m19hBBz (65%) after MDSC co-culture. This data suggests MDSCs present during CAR T cell production can alter gene transfer and total T cell counts. We also investigated the effect MDSCs can potentially have on CAR T cells when present during CAR T cell antigen stimulation. CAR T cells co-cultured with MDSCs in vitro had significant reductions in m19z, m1928z, and m19hBBz CAR T cell mediated killing against target cells (Fig 1B). We also found significantly lower production of IFNγ in m19z, m1928z, and m19hBBz CAR T cells cultured with MDSCs compared to cells cultured with total BM (Fig 1C). After 24hr stimulation with MDSCs and target cells, there was lower expression of activation markers PD1 and LAG3 by CAR T cells compared to culture without MDSCs. This suggests that MDSCs reduce CAR T cell activation, killing, and cytokine production. We also looked at how CAR proliferation after antigen stimulation is affected by MDSC co-culture and found MDSCs significantly reduced CAR proliferation in vitro. To evaluate if we could create a more resistant CAR T cell to MDSC suppression null mutations were incorporated into a CD28 CAR. We mutated the YMNM and PRRP subdomains of CD28 which signal through PI3K and ITK respectively leaving only PYAP active (mut06). When MDSCs were co-cultured with mut06 T cells during production mut06 had a smaller reduction in gene transfer (21% vs 38%) and T cell counts (80% vs 88%) compared to m1928z. Mut06 also had a significantly higher expression of PD1 and TIM3 compared to m1928z after production with MDSCs. In vitro when mut06 was co-cultured with MDSCs it had the same killing ability as m1928z without MDSCs and was significantly better at killing compared to m1928z co-cultured with MDSCs (Fig 1B). To examine the effect of MDSCs on CAR T cells in vivo we injected C57BL6 mice with CAR T cells followed by MDSCs a week later. In these ongoing experiments we found mut06 had significantly higher numbers of CAR T cells in the blood compared to m1928z (Fig1D). Overall our data shows that MDSCs can suppress CAR T cell function when present during production as well as CAR stimulation. It also suggests that by optimizing CD28 CAR signaling using mut06 that we were able to generate a CAR T cell that is more resistant to MDSCs. Furthermore, we may be able to recapitulate the effect of mut06 with targeted inhibitors. Figure 1 Disclosures Davila: GlaxoSmithKline: Consultancy; Novartis: Research Funding; Adaptive: Consultancy; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; Anixa: Consultancy; Atara: Research Funding.

scholarly journals Galunisertib enhances chimeric antigen receptor-modified T cell function

2020 ◽  
Author(s):  
Zhixiong Wang ◽  
Qian Liu ◽  
Na Risu ◽  
Jiayu Fu ◽  
Yan Zou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Cell Function ◽  
Antigen Receptor ◽  
Target Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy still faces the challenge of immunosuppression when treating solid tumors. TGF-β is one of the critical factors in the tumor microenvironment to help tumors escape surveillance by the immune system. Here we tried using the combination of a small molecule inhibitor of TGF-β receptor I, Galunisertib, and CAR T cells to explore whether Galunisertib could enhance CAR T cell function against solid tumor cells. In vitro experiments showed Galunisertib could significantly enhance the specific cytotoxicity of both CD133- and HER2-specific CAR T cells. However, Galunisertib had no direct killing effect on target cells. Galunisertib significantly increased the cytokine secretion of CAR T cells and T cells that do not express CAR (Nontransfected T cells). Galunisertib did not affect the proliferation of T cells, the antigen expression on target cells and CD69 on CAR T cells. We found that TGF-β was secreted by T cells themselves upon activation, and Galunisertib could reduce TGF-β signaling in CAR T cells. Our findings can provide the basis for further preclinical and clinical studies of the combination of Galunisertib and CAR T cells in the treatment of solid tumors.

scholarly journals TMOD-18. EXPLORING THE FACTORS LEAD TO SUCCESS OF CAR T-CELL THERAPY IN GLIOBLASTOMA WITH COMPUTATIONAL MODELING AND IN VITRO DATA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi266-vi266
Author(s):  
Prativa Sahoo ◽  
Xin Yang ◽  
Daniel Abler ◽  
Davide Maestrini ◽  
Vikram Adhikarla ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T-cell therapy is an emerging targeted immunotherapy which has shown success in liquid cancers such as leukemias. CAR T-cells are also being used for the treatment of solid tumors such as glioblastoma, which is a primary brain tumor. Ongoing phase I trials have been designed to evaluate CAR T-cell dosing, scheduling, and route of administration in order to understand and improve the efficacy of CAR T-cell therapy. A better understanding of factors leading to the success of CAR T-cell immunotherapy for solid tumors will be necessary to improve outcomes for patients with solid tumors and to advance the field of CAR T-cell immuno-oncology. Here we use mathematical model to explore factors in determining a successful response to CAR T-cell therapy: proliferation, persistence, and killing capacity of CAR T-cells. Using a novel in vitro experimental apparatus, we are able to measure the density of cancer cells over several days in 15 minute interval time resolution. This highly temporally resolved data provides a unique opportunity to confidently estimate parameters of the model and to provide insights into the dynamics of CAR T-cell proliferation, persistence, and killing capacity. Furthermore we explore the relationship between these factor with CAR T-cell dose level. We will show results from experiments using patient-derived cancer cell lines as well as cancer cells engineered to express specific levels of the target antigen (IL13Rα2) to quantitatively evaluate the roles of proliferation, persistence, and killing in cells with different levels of antigen expression. We will discuss the interpretation of the model parameters and demonstrate the clinical value of this analysis through an application of CAR T-cell treatment tailored to the dynamics of an individual patient’s cancer growth rate.

Sign in / Sign up

Export Citation Format

Share Document