Specific CAR-T Cells Research Articles

PD1CD28 chimeric molecule enhances EGFRvⅢ specific CAR-T cells in xenograft experiments in mouse models.

Over the years, CAR-T cell therapy has achieved remarkable success in treating hematological malignancies. However, this efficacy has not been replicated in the context of glioblastoma (GBM). In this study, a PD1CD28 chimeric molecule was introduced into EGFRvⅢ-directed CAR-T cells, generating EGFRvⅢ-P2A-PD1CD28 CAR-T cells. Notably, this modification significantly increased IL-2 secretion and enhanced antigen-dependent activation of CAR-T cells, especially when programmed cell death ligand 1 (PD-L1) was present in vitro. In addition, the in vivo xenograft experiments revealed that the PD1CD28 chimeric molecule played a pivotal role in reducing recurrence rates, effectively controlling recurrent tumor volume, and ultimately prolonging the survival of mice. Collectively, these findings suggest that EGFRvⅢ-directed CAR-T cells co-expressing the PD1CD28 chimeric molecule have the potential to significantly enhance the treatment efficacy against GBM.

Binary oncolytic adenovirus in combination with HER2-specific autologous CAR VST for treatment of advanced HER2-positive solid tumors (VISTA).

TPS2679 Background: Urgent therapies are needed for patients with refractory Human Epidermal Growth Factor Receptor 2 (HER2) positive solid malignancies. Our group has previously shown potent antitumor effect of a binary oncolytic/helper-dependent adenovirus (CAdVEC) with dual expression of IL-12 and PD-L1 blocker. Initial preclinical and clinical studies with direct tumor injection of CAdVEC has shown it to be safe and result in increased infiltration of CD8 T cells into the tumor microenvironment, with antitumor effect on locoregional and distant metastatic sites.1 Based on these results, we describe an ongoing study with addition of HER2-specific autologous chimeric antigen receptor (CAR)-T cell therapy to CAdVEC. Methods: This is a single arm, dose escalation phase I clinical trial guided by Bayesian Optimal Interval Design (BOIN) for patients with advanced HER2 positive solid tumors. Patients who are deemed unsuitable for curative treatments and progressed after at least one standard first line therapy are eligible. HER2 positivity is determined by IHC and defined as ≥2+. Patients are required to have at least one tumor site appropriate for intratumor injection and radiographically measurable disease per RECIST v1.1. Patients with autoimmune disease requiring systemic corticosteroids greater than 10mg/day and those with active/untreated CNS metastasis are excluded. Patients will receive increasing doses of CAdVEC intratumor injection alone (first two dose levels) or in combination with HER2 specific autologous CAR-T cells (dose levels 3-7). A total of 45 patients are planned. The primary endpoint of the study is to evaluate safety and maximum tolerated dose as assessed by incidence of dose limiting toxicities (DLT) of CAdVEC intratumor injection in combination with HER2-specific autologous CAR T cells. Secondary endpoints include anti-tumor activity of the combination measured by overall response rate (ORR), disease control rate (DCR), median progression free survival (PFS) and median overall survival (OS). Exploratory endpoints include assessing the immunogenicity of combination therapy by determining the impact of treatment on cellular and humoral immunity, as well as assessing long-term persistence and functional status of HER2 CAR T-cells. Correlative analysis of levels of adenovirus antibodies with clinical outcomes and immunological findings are also planned. This study is currently enrolling. 1. Wang D, Porter CE, Lim B, et al: Science Advances 9, 2023. Clinical trial information: NCT03740256 .

CRISPR/Cas9-mediated glucocorticoid receptor knockout effectively enhances antitumor efficacy of ROR1 specific CAR-T cells in advanced adrenocortical carcinoma

CRISPR/Cas9-mediated glucocorticoid receptor knockout effectively enhances antitumor efficacy of ROR1 specific CAR-T cells in advanced adrenocortical carcinoma

Functional single-cell screening of chimeric antigen receptor (CAR)-T cells based on multiple cytokines using Nanovial Technology

Abstract CAR-T therapies, while potent against some cancers, face challenges including toxicities and tumor relapse, particularly in solid tumors with hostile TMEs and sub-optimal CAR designs. A major obstacle is the lack of efficient functional screening methods for CAR-T cells, crucial for T-cell-based immunotherapy progress. Our study introduces a novel functional screening approach using hydrogel microparticles (Nanovials) to analyze millions of individual CAR-T cells based on antigen specificity and functional cytokine secretion. We modified Nanovials with Mesothelin (MSLN) antigen, enriching and binding specific CAR-T cells. Additionally, Nanovials were adapted with antibodies to detect key cytokines (IFN-gamma, GranzymeB) from activated CAR-T cells, identified through fluorescent markers. This setup allows for analyzing and sorting CAR-T cells with unique functional traits using standard flow cytometers. Using the Nanovial assay we distinguished MSLN CAR-T variants by cytokine profiles, revealing minor CAR engineering differences and enabling early functional assessment. This technique identified distinct CAR-T cell subpopulations within patient samples, surpassing traditional bulk analysis methods like ELISA and cell killing assays. In summary, our Nanovial-based method offers a scalable, efficient solution for CAR-T cell functional screening, enhancing CAR design and improving therapeutic manufacturing quality control.

Abstract LB219: DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays

Abstract Gastrointestinal cancers, including gastric cancer (GC) and colorectal cancer (CRC), continue to be devastating diseases despite advances in screening, early detection, and removal of pre-neoplastic and early cancerous lesions. Even with advancements in targeted chemotherapy and immunotherapies, advanced and metastatic diseases continue to be responsible for the high mortality associated with GC and CRC. Newer, safer, and more effective therapies to prevent, inhibit, or reverse advanced disease progression are urgently needed. Doublecortin-like kinase 1 (DCLK1) is a microtubule associated protein kinase that has been reported to drive tumor initiation, progression, metastasis, and drug resistance in many solid tumor cancers including GC and CRC, where it is upregulated in patient tumors and associated with poor overall survival. An anti-DCLK1 monoclonal antibody has demonstrated potential therapeutic activity against CRC. In this study, we established patient-derived organoids (PDOs) to evaluate the effects of inhibiting DCLK1 kinase activity (DCLK1-IN-1) or targeting DCLK1 with a highly specific CAR-T cells on the treated PDOs. Two GC PDOs were treated with various concentrations of DCLK1 kinase inhibitor DCLK1-IN-1 (0-32 μM) for 72 hrs, viability of the PDOs were determined, histochemistry and immunohistochemistry staining were used to image the live cells and DCLK1 expression. For CRC PDOs, five CRC PDOs were cultured in the presence or absence of PBMC, treated with Mock CAR-T cells or DCLK1 CAR-T cells (three doses) for 4 days. Tumor size, tumor total area, and viability of these PDOs were determined. Here, we demonstrate that treating GC PDOs with DCLK1 kinase inhibitor resulted in a significant dose-dependent inhibition in PDO numbers in both PDOs (two-way ANOVA P<0.0001), a decrease of cell viability, revealing IC50 values of 7.566 and 9.958 μM respectively, and a near total loss of GC PDO viability at 10 μM using CellTiterGlo assay. In CRC PDOs, two of five CRC PDOs showed a significant decrease in total tumor size, tumor areas, and an increase in cell death measured by the DRAQ7 method. Compared to Mock CAR-T cells, treatment with DCLK1 CAR-T cells resulted in a dose dependent decrease of tumor size and total tumor area, with more than a 50% reduction at the 50000 cells dose (p<0.05). Treatment with DCLK1 CAR-T cells also resulted in a dose dependent increase in cell death, with approximately 20% cell death without PBMC, and 50% cell death with PBMC at the 50000 cells dose (p<0.05). These results suggest that targeting DCLK1 represents a potential novel therapeutic strategy for GI cancers, especially in combination with FDA approved drugs: 5-fluorouracil (5-FU) or Gefitinib. Citation Format: Dongfeng Qu, Jinsen Shi, Zhiyun Cao, Eddie BAnnerman-Menson, Nathaniel Weygant, Courtney Houchen. DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB219.

Synthetic biology approaches for enhancing safety and specificity of CAR-T cell therapies for solid cancers

CAR-T cell therapies have been successful in treating numerous hematologic malignancies as the T cell can be engineered to target a specific antigen associated with the disease. However, translating CAR-T cell therapies for solid cancers is proving more challenging due to the lack of truly tumor-associated antigens and the high risk of off-target toxicities. To combat this, numerous synthetic biology mechanisms are being incorporated to create safer and more specific CAR-T cells that can be spatiotemporally controlled with increased precision. Here, we seek to summarize and analyze the advancements for CAR-T cell therapies with respect to clinical implementation, from the perspective of synthetic biology and immunology. This review should serve as a resource for further investigation and growth within the field of personalized cellular therapies.

Abstract 741: Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity

Abstract The expression of ILT (LILRB) family members and their major ligand HLA-G within solid tumors are associated with immune suppression and poor patient survival. Blocking multiple ILT family members might be more potent than targeting individual members by overcoming compensatory resistances within tumor microenvironment. Based on this hypothesis, we developed a monoclonal antibody (mAb) LITCHI 7 which reacts to three major ILT family members, ILT2, ILT4 and ILT5, and blocks ligand binding to these receptors. LITCHI 7 exhibits a broad binding capability to a wide range of human myeloid and T cell subsets that express ILT2, ILT4, and/or ILT5. Binding to monocyte-derived dendritic cells (moDC), LITCHI 7 released the inhibition on activating Fcγ receptor. On LPS-treated macrophages, LITCHI 7 enhanced inflammatory macrophage differentiation. The effect is primarily mediated by blocking ILT4 since ILT4 mono-specific mAb is as potent as cross-reactive LITHI 7 in modulating myeloid differentiation although both ILT2 and ILT5 are highly expressed by different myeloid subsets. The result was further confirmed by CRISPR Cas9 knock down of ILT2 or ILT5. In the absence of either ILT2 or ILT5, the accumulation of CD14+ monocytes and inflammatory macrophages were not changed during differentiation. While different ILT family members are generally enriched on myeloid cells, ILT2 is also expressed on NK cells and effector memory CD45RA+ T cells (Temra). Blocking ILT2 by LITCHI 7 enhanced TCR signaling when co-culturing the MART-1 specific T cell reporter line with MART-1/HLA-G positive melanoma cells. The effect of LITCHI 7 is mediated by ILT2 blockade since knocking out ILT2, but not ILT4 and ILT5, reduced accumulation of Temra in activated PBMC. To assess whether immune modulation of LITCHI 7 can be translated into anti-tumor activity by blocking different ILT family members, tumor killing assays were performed using innate and adaptive immune effector cells. LITCHI7 enhanced NK cell-mediated cytotoxicity on HLA-G+ tumor cells by blocking ILT2. Both ILT2 and ILT4 blockade play a role in macrophage phagocytosis on lymphoma cells since LITCHI 7 as well as individual anti-ILT2 or ILT-4 mAbs increased macrophage phagocytosis to a similar level. Finally, LITCHI 7 could boost in vitro T cell-mediated killing of CD19+ lymphoma by CD19 specific CAR-T cells in the presence of CD33+ myeloid cells. In summary, LITCHI 7 can enhance both innate and adaptive anti-tumor immunity through ILT2 or ILT4 blockade directly on effector cells or indirectly through myeloid cell reprogramming. As clinical trials are ongoing by targeting ILT2 and ILT4, blocking multiple ILT family members through a single cross-reactive mAb may provide broad anti-tumor effects and advantages when combining with additional oncology treatments. Citation Format: Shiming Ye, Dong Zhang, Brenal Singh, Jaishree Bankoti, Diane Cohen, Siusze Tan, Donghee Choi, Jonathan Hickson, Min-Zu Wu, Urvi Kolhatkar, Mikhail Binnewies, Wei Jin, Jason Hall, John Engelhardt, Alexander Shoemaker. Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 741.

Abstract 4009: Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy

Abstract Century Therapeutics is developing NECTIN4 targeted induced Pluripotent Stem Cell (iPSC) derived Chimeric Antigen Receptor (CAR) T cell therapy. NECTIN4 targeted antibody drug conjugate enfortumab vedotin, while generally well tolerated, causes severe skin adverse events in some patients, thought to be driven by on-target off-tumor toxicity against NECTIN4 displaying skin keratinocytes. We include a safety switch in our first clinical-stage iPSC-derived CAR-NK cell therapy, CNTY-101, which provides an antibody target for elimination of the engineered cells should that be necessary. An additional method for reducing the incidence and severity of this type of adverse event is through the incorporation of an inhibitory CAR, also known as a NOT-gate, directed against antigen expressed on normal skin keratinocytes, but the optimal surface protein to target with such an inhibitory CAR for a NECTIN4 targeted therapy is unknown. Here, we identify DSG1 (Desmoglein-1) as a top inhibitory CAR target for NECTIN4 targeted CAR-T cell therapy by conducting a multi-omics analysis of public single-cell RNAseq, bulk RNAseq, and protein microarray immunohistochemistry datasets. We find that DSG1 is constitutively and uniformly displayed on the surface of skin keratinocytes in the layers of epidermis where they display NECTIN4, and DSG1 gene expression in normal skin is uniformly high across individuals. Conversely, DSG1 mRNA and protein are rarely expressed in NECTIN4 expressing cancer indications including bladder, breast, non-small cell lung, ovarian, and pancreatic. Further, autoimmune antibodies specific to DSG1 have been described, suggesting that DSG1 is targetable by a CAR in situ. Finally, DSG1 is not expressed by our iPSC-derived CAR-T cells. While NECTIN4 expression is greatest in skin keratinocytes, epithelial cells in other tissues express NECTIN4 at lower levels. In this study, we compare the expression levels of NECTIN4 to targets associated with tissue and cell-type specific on-target off-tumor toxicity in primary CAR-T cell clinical trials. Our findings support the strategy of DSG1-directed inhibitory CAR incorporation into NECTIN4 specific CAR-T cell therapeutic candidates. Citation Format: Matthew S. Hall, Christopher M. Dower, Michael Miller, John Wheeler, Jill Carton, Ohad Manor, Hyam Levitsky. Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4009.

Gp350-Targeted CAR-T Therapy in EBV-Positive NK/T-Cell Lymphoma: Pre-Clinical Development of gp350CAR-T

Introduction: Epstein-Barr virus (EBV) positive NK/T cell lymphoma is a subtype of non-Hodgkin's lymphoma strongly associated with EBV infection. EBV infection has been reported in 85-100% of cases. It has been suggested to be central in pathogenesis. The incidence of EBV+ NK/T cell lymphoma is increasing worldwide, particularly in Asia and Latin America, and it has a poor prognosis with few effective treatment options. Therefore, novel approaches for treating EBV-associated NK/T cell lymphoma are urgently needed. Developing chimeric antigen receptor (CAR)-T cells therapy targeting EBV specific protein in the context of this EBV-positive aggressive lymphoma will provide a novel therapeutic strategy for patients with limited treatment options. Targeting EBV lytic protein for control EBV in vivo replication and tumorigenesis have been explored lately. The gp350 protein is a major glycoprotein on the surface of EBV during its lytic phase, after fusion between virus and host cell membrane, gp350 is left on the host cell surface. The distinct surface distribution pattern of gp350 in both freshly infected host cells and the lytic phase host cells renders it an attractive target for immunotherapy. Method and Result: We developed EBV specific CAR-T cells targeting gp350. We utilized single-chain variable fragments (scFvs) derived from four novel, gp350-binding, neutralizing monoclonal antibodies (mAbs) (12C10, 7D11, 6A12, 7A8) to generate the CAR constructs. We demonstrated that these scFvs, when fused to a second-generation CAR backbone consisting of 4-1BB and CD3ζ signal domains, are efficiently expressed on the surface of transduced T cells. These gp350 CAR-T cells exhibit specific activation and cytotoxic effects in vitro. Of note, the clone 6A12 scFv of CAR displayed the most potent cytotoxicity effects in gp350 positive cancer coculture toxicity assays. To optimize the CAR structure further, we designed various linker and transmembrane domains based on the clone 6A12 scFv, including CD8 linker, IgG4 hinge, IgG1 Fc linker, and CD28 linker and transmembrane domains. Our cytotoxicity assay screening indicated that the CD8 linker and transmembrane domain resulted in the most advanced cytotoxicity and target specificity (Figure A, B). To assess gp350 CAR-T efficacy in vivo, we established a T-cell leukaemia animal model in NOD immune-deficient mice through intravenous injection of Jurkat-gp350-luc cells. On post xenograft day 7, the Jurkat-gp350-luc xenografted mice were separated into three groups and each group with 5 mice (except PBS group with 2 mice). CAR-T and T cell were infused by tail vein injection with mock T 2x10^6/mouse, 6A12CAR-T 2x10^6/mouse and PBS control. The tumor growth in mice was monitored by in vivo luminescence with IVIS imaging system. Upon engraftment, Jurkat-gp350-luc cells established disseminated leukaemia expanding preferentially in the spine, femur, head, and pelvis, with few cells in peripheral blood. Mice receiving control mock T cells or without T cell PBS control developed rapid disease progression and were euthanized by day 35 after Jurkat-gp350-luc engraftment. In contrast, mice receiving gp350 CART cells were significantly protected from rapid progression (Figure C, D). Conclusion: In pre-clinical development, we designed, constructed, and screened gp350 CAR-T cells to demonstrate its efficacy in vitro and in vivo against and kill gp350 positive leukaemia cells and prevent formation and development leukaemia in xenograft mice model.

EXTH-08. IMMUNOTHERAPEUTIC TARGETING OF FIBROBLAST ACTIVATION PROTEIN (FAP) IN TREATMENT REFRACTORY GLIOBLASTOMA USING NOVEL CAR-T CELL THERAPY

Abstract Glioblastoma (GBM), the most common malignant primary adult brain tumor, is characterized by extensive cellular and genetic heterogeneity. The standard of care therapy and clinical trials have not been successful in improving patients’ survival which underscores an urgent need for developing new effective therapies. In silico and in vitro analysis have revealed high level of Fibroblast Activation Protein (FAP) expression in GBM tissue by multiple cell populations including endothelial cells, fibroblasts, stromal and tumor cells, but not normal brain tissue, making FAP a safe and suitable therapeutic target. As chimeric antigen receptor (CAR) expressing T cells have shown promising results against multiple cancers, we aimed to target FAP in GBM by FAP specific CAR-T cells generated using a novel GMP-ready FAP-CAR construct produced based on a virus-free CAR gene transfer using advanced sleeping beauty transposon technology and pre-clinically test their anti-tumor efficacy. Our data revealed that co-culturing CAR-T cells with FAP+ GBM cells (U87) resulted in increased surface expression of T-cell activation markers. Moreover, FAP CAR-T mediated cytotoxicity against FAP+ GBM cells was also observed in the co-culture of GBM and CAR-T cells. For further validation, the effect of FAP CAR-T cells will be tested on patient-derived GBM organoids. The in vivo efficacy of FAP CAR-T cells is currently under investigation and we anticipate that the treatment of GBM tumor-bearing mice with FAP CAR-Ts results in extended survival and tumor burden reduction. These rigorously obtained data show high level of efficacy for FAP-specific CAR-T cells against GBM suggesting that clinical development of this therapeutic modality can provide a novel therapeutic strategy for GBM patients. Therefore, upon successful completion of the preclinical study, we plan to conduct a phase I dose-escalation trial of autologous FAP-CAR T cells for patients with recurrent Glioblastoma.

Efficacy and Safety of Avatrombopag on Thrombocytopenia after Chemotherapy in Acute Leukaemia

Background: Thrombocytopenia following chemotherapy is a common and serious complication that leads to an increased risk of bleeding and poor prognosis for patients with acute leukaemia. Current therapeutic strategies consist of platelet transfusion, recombinant human interleukin-11(rhIL-11) and recombinant human thrombopoietin (rhTPO) injection, but these strategies are not satisfactory for some patients and the treatment remains challenging. Avatrombopag is a novel thrombopoietin receptor agonist, which had been approved for thrombocytopenia in patients with chronic liver disease who are scheduled to undergo a surgery in the USA, Europe, and China, and in patients with chronic immune thrombocytopenia who were unresponsive to previous treatments in the USA and Europe. This study aims to determine the efficacy and safety of avatrombopag on thrombocytopenia after chemotherapy of acute leukaemia, and to increase the evidence for clinical use of avatrombopag. Methods: The clinical data of 24 acute leukaemia patients with grade 3/4 chemotherapy-induced thrombocytopenia (CIT) for 3-28 days were collected from February 2022 to February 2023. All patients received avatrombopag orally at a dose of 40 mg / day. Platelet (PLT) transfusion was allowed when the platelet level was less than 20×10 9/L or with bleeding events. If platelet count increased to 100 ×10 9/L or 50 ×10 9/L more than baseline, which was defined as complete response (CR), treatment of avatrombopag was stopped. Results: The median age of 24 patients with CIT was 38 (range 17‒69) years, with 14 patients diagnosed with acute myeloid leukemia (AML), 9 patients diagnosed with acute lymphoblastic leukemia (ALL), and 1 patient diagnosed with mixed phenotype acute leukemia (MPAL). Chemotherapy regiments for patients with AML included decitabine combined with venetoclax (n=5), decitabine combined with arabinoside (n=3), arsenic trioxide combined with retinoic acid (n=3), azacytidine combined with venetoclax (n=2), azacytidine combined with medium-dose arabinoside (n=1). Chemotherapy regiments for patients with ALL included CD19 specific CAR-T cells (n=3), decitabine (n=2), blinatumomab (n=2), IVP (idarubicin, vindesine, and prednisone) (n=1), azacytidine combined with venetoclax and chidamide (n=1). Chemotherapy regiment for the patient with MPAL was decitabine combined with venetoclax (n=1). At baseline, 10 patients experienced grade 3 thrombocytopenia after chemotherapy, while 14 patients experienced grade 4 thrombocytopenia after chemotherapy. The median lowest platelet count was 19×10 9/L (range 1-47×10 9/L). During the treatment period, 14 patients received platelet transfusion. The median time to reach the CR standard was 6 (range 3-26) days, and no patients stopped treatment due to side effect or drug intolerance. Compared with using avatrombopag before, the levels of platelet count were significantly increased ( P=0.000 1). The optimal platelet count of 22 patients reached the standard of CR after 7 days of drug withdrawal. Conclusion: Avatrombopag is effective on increasing platelet counts in patients with acute leukaemia after chemotherapy, with a good safety profile. It is a suitable therapeutic option for thrombocytopenia after chemotherapy.

Tyrosine kinase signaling-independent MET-targeting with CAR-T cells

BackgroundRecent progress in cancer immunotherapy encourages the expansion of chimeric antigen receptor (CAR) T cell therapy in solid tumors including hepatocellular carcinoma (HCC). Overexpression of MET receptor tyrosine kinase is common in HCC; however, MET inhibitors are effective only when MET is in an active form, making patient stratification difficult. Specific MET-targeting CAR-T cells hold the promise of targeting HCC with MET overexpression regardless of signaling pathway activity.MethodsMET-specific CARs with CD28ζ or 4-1BBζ as co-stimulation domains were constructed. MET-CAR-T cells derived from healthy subjects (HS) and HCC patients were evaluated for their killing activity and cytokine release against HCC cells with various MET activations in vitro, and for their tumor growth inhibition in orthotopic xenograft models in vivo.ResultsMET-CAR.CD28ζ and MET-CAR.4-1BBζ T cells derived from both HS and HCC patients specifically killed MET-positive HCC cells. When stimulated with MET-positive HCC cells in vitro, MET-CAR.CD28ζ T cells demonstrated a higher level of cytokine release and expression of programmed cell death protein 1 (PD-1) than MET-CAR.4-1BBζ T cells. When analyzed in vivo, MET-CAR.CD28ζ T cells more effectively inhibited HCC orthotopic tumor growth in mice when compared to MET-CAR.4-1BBζ T cells.ConclusionWe generated and characterized MET-specific CAR-T cells for targeting HCC with MET overexpression regardless of MET activation. Compared with MET-CAR.4-1BBζ, MET-CAR.CD28ζ T cells showed a higher anti-HCC potency but also a higher level of T cell exhaustion. While MET-CAR.CD28ζ is preferred for further development, overcoming the exhaustion of MET-CAR-T cells is necessary to improve their therapeutic efficacy in vivo.

ROR-1 specific CAR-T cells with CRISPR/CAS9 mediated glucocorticoid receptor-knockout exert potent antitumor efficacy in advanced adrenocortical carcinoma

ROR-1 specific CAR-T cells with CRISPR/CAS9 mediated glucocorticoid receptor-knockout exert potent antitumor efficacy in advanced adrenocortical carcinoma

P06.15.B IMMUNOTHERAPEUTIC TARGETING OF FIBROBLAST ACTIVATION PROTEIN (FAP) IN TREATMENT REFRACTORY GLIOBLASTOMA USING NOVEL CAR-T CELL THERAPY

Abstract BACKGROUND Glioblastoma (GBM) is the most common malignant primary adult brain tumor, characterized by extensive cellular and genetic heterogeneity. The Standard of Care (SOC) therapy and clinical trials have not been successful in improving patients’ survival which underscores an urgent need for developing new effective therapies for this treatment refractory disease. MATERIAL AND METHODS In silico and in vitro analysis have revealed high level of Fibroblast Activation Protein (FAP) expression in GBM tissue in multiple cell populations including endothelial cells, fibroblasts, stromal cells and tumor cells, but not on normal brain tissue, making FAP a safe and suitable therapeutic target as it allows for destruction of tumor cells as well as effective elimination of their supporting microenvironment. As Chimeric antigen receptor (CAR) expressing T cells have shown promising results against multiple cancers in the past few years, we aimed to target FAP in GBM by FAP specific CAR-T cells generated using a novel GMP-ready FAP-CAR construct produced based on a virus-free CAR gene transfer using advanced sleeping beauty transposon technology and preclinically test their anti-tumor efficacy against GBM at in vitro and in vivo level. RESULTS Our data revealed that co-culturing CAR-T cells with FAP+ GBM cells (U87) results in increased surface expression of T-cell activation markers. Moreover, FAP CAR-T mediated cytotoxicity against FAP+ GBM cells was also observed in the co-culture of GBM and CAR-T cells. For further validation, the effect of FAP CAR-T cells will be tested on patient-derived GBM organoids. The in vivo efficacy of FAP CAR-T cells is currently under investigation and we anticipate that the treatment of GBM tumor-bearing mice with FAP CAR-Ts results in extended survival and reductions of tumor burden. CONCLUSION These rigorously obtained data show high level of efficacy for FAP-specific CAR-T cells against GBM suggesting that clinical development of this therapeutic modality can provide a novel therapeutic strategy for GBM patients that can be used in combination with SOC or other immunotherapeutics. Therefore, upon successful completion of the preclinical study, we plan to conduct a phase I dose-escalation trial of autologous FAP-CAR T cells for patients with recurrent Glioblastoma.

Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer

BackgroundChimeric antigen receptor (CAR) -T cell therapy is an efficient therapeutic strategy for specific hematologic malignancies. However, positive outcomes of this novel therapy in treating solid tumors are curtailed by the immunosuppressive tumor microenvironment (TME), wherein signaling of the checkpoint programmed death-1 (PD-1)/PD-L1 directly inhibits T-cell responses. Although checkpoint-targeted immunotherapy succeeds in increasing the number of T cells produced to control tumor growth, the desired effect is mitigated by the action of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in the TME. Previous studies have confirmed that targeting triggering-receptor-expressed on myeloid cells 2 (TREM2) on TAMs and MDSCs enhances the outcomes of anti-PD-1 immunotherapy.MethodsWe constructed carcinoembryonic antigen (CEA)-specific CAR-T cells for colorectal cancer (CRC)-specific antigens with an autocrine PD-1-TREM2 single-chain variable fragment (scFv) to target the PD-1/PD-L1 pathway, MDSCs and TAMs.ResultsWe found that the PD-1-TREM2-targeting scFv inhibited the activation of the PD-1/PD-L1 pathway. In addition, these secreted scFvs blocked the binding of ligands to TREM2 receptors present on MDSCs and TAMs, reduced the proportion of MDSCs and TAMs, and enhanced T-cell effector function, thereby mitigating immune resistance in the TME. PD-1-TREM2 scFv-secreting CAR-T cells resulted in highly effective elimination of tumors compared to that achieved with PD-1 scFv-secreting CAR-T therapy in a subcutaneous CRC mouse model. Moreover, the PD-1-TREM2 scFv secreted by CAR-T cells remained localized within tumors and exhibited an extended half-life.ConclusionsTogether, these results indicate that PD-1-TREM2 scFv-secreting CAR-T cells have strong potential as an effective therapy for CRC.

Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.

Abstract 1770: CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK)

Abstract CD28-derived co-stimulation modules are frequently used as part of the signaling domain of chimeric antigen receptors (CAR). These modules contain well characterized tyrosine-containing moieties (YMNM and PYAP) that become phosphorylated upon CAR activation, and result in IL-2 secretion and T cell expansion. Previous work by our group has shown that an additional tyrosine residue, located in the C-terminus of CD28 (Y218), was also phosphorylated upon CAR activation. The kinase responsible for this phosphorylation remains unknown, but in silico studies point towards the IL-2-inducible T-cell kinase (ITK), the protein-tyrosine kinase TEC and the bone marrow tyrosine kinase (BMX). In this work we sought to elucidate the molecular mechanism and functional relevance of CD28-Y218 phosphorylation in CAR-T cells.To analyze the kinetics of Y218 phosphorylation, prostate stem cell antigen (PSCA)-specific second-generation CAR-T cells were stimulated through co-culture with HPAC pancreatic cancer cells for 0, 1, 10, 30 and 60 minutes. Phosphorylation of CD28 Y218, Zap70, PLCγ and Vav1 was analyzed by Western blotting. To determine the role of ITK in CD28-218 phosphorylation: 1) PSCA-specific CAR-T cells were treated with ITK inhibitors 10 μM (BMS-509744 or Ibrutinib) for 24h and 2) ITK-deficient Jurkat cells expressing a PSCA-specific CAR were cocultured with HPAC cells for 0 or 10 min. To determine the functional relevance of this phosphorylation event, we generated mutant CARs where the tyrosine residue was replaced with a non-phosphorylatable amino acid (Y218F) and evaluated its effects in vivo and in vitro. CAR-T cell cytotoxicity was monitored using the xCELLigence Real Time Cytotoxicity Assay (RTCA) and cytokine production was assessed following overnight co-culture with target cells using the ELLA system (Biotechne, CA, USA). To evaluate the in vivo antitumor efficacy of PSCA-specific CAR-T cells, NSG mice were injected (s.c.) with 1 HPAC cells and infused 14 days later with 5 CAR-T cells (i.v.). Tumor growth was monitored for 35 days.Our results show that phosphorylation of CD28-Y218 occurs after the phosphorylation of early TCR signaling mediators, reaching maximum levels around 10-30 min post-stimulation. Pharmacological inhibition and genomic ablation of ITK impairs CD28-Y218 phosphorylation, suggesting an essential role of ITK in this signaling event. We observed that mutation of the Y218 residue did not affect CAR expression, in vitro cytotoxicity, or INFγ production. However, this mutation was associated with reduced IL-2 and TNFα secretion. In vivo, this mutation completely abrogated the therapeutic effect of CAR-T cells in a pancreatic cancer model. In conclusion, these results indicate that ITK is required for CD28-Y218 phosphorylation, a signaling event that plays an important role in CAR-T cell function, with direct impact on IL-2 secretion and antitumor efficacy. Citation Format: Elena Martinez-Planes, Miguel G. Fontela, Cecilia Ramello, Daniel Abate-Daga. CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1770.

Immuno(T)herapy for age-related diseases.

During the last decade, the stimulation of T-cell function by the blockage of immunosuppressive checkpoints has experienced an outstanding impact in the treatment of cancer. Development of the chimeric antigen receptor T-cell technology has also emerged as a powerful alternative for patients suffering from oncological processes, especially those affected by hematological neoplasms. Recent evidence suggest that the use of immunotherapy could be extended to non-oncological diseases and could be especially relevant for age-associated disorders, opening exciting therapeutic options for a wide range of diseases of the elderly. Here we comment on the emergence of T-cell-based immunotherapies as feasible approaches that could revolutionize the future of GeroScience.

CXCR6 expressing T cells: Functions and role in the control of tumors

CXCR6 is a receptor for the chemokine CXCL16, which exists as a membrane or soluble form. CXCR6 is a marker for resident memory T (TRM) cells that plays a role in immunosurveillance through their interaction with epithelial cells. The interaction of CXCR6 with CXCL16 expressed at the membrane of certain subpopulations of intratumor dendritic cells (DC) called DC3, ideally positions these CXCR6+ T cells to receive a proliferation signal from IL-15 also presented by DC3. Mice deficient in cxcr6 or blocking the interaction of CXCR6 with its ligand, experience a poorer control of tumor proliferation by CD8+ T cells, but also by NKT cells especially in the liver. Intranasal vaccination induces CXCL16 production in the lungs and is associated with infiltration by TRM expressing CXCR6, which are then required for the efficacy of anti-tumor vaccination. Therapeutically, the addition of CXCR6 to specific CAR-T cells enhances their intratumoral accumulation and prolongs survival in animal models of pancreatic, ovarian and lung cancer. Finally, CXCR6 is part of immunological signatures that predict response to immunotherapy based on anti-PD-(L)1 in various cancers. In contrast, a protumoral role of CXCR6+T cells has also been reported mainly in Non-alcoholic steatohepatitis (NASH) due to a non-antigen specific mechanism. The targeting and amplification of antigen-specific TRM expressing CXCR6 and its potential use as a biomarker of response to immunotherapy opens new perspectives in cancer treatment.

Co-expression IL-15 receptor alpha with IL-15 reduces toxicity via limiting IL-15 systemic exposure during CAR-T immunotherapy

BackgroundChimeric antigen receptor (CAR)-T cell therapy is a powerful adoptive immunotherapy against both B-cell malignancies and some types of solid tumors. Interleukin (IL) -15 is an important immune stimulator that may provide ideal long-term persistent CAR-T cells. However, higher base line or peak serum IL-15 levels are also related to severe toxicity, such as cytokine release syndrome (CRS), graft-versus-host disease (GVHD), and neurotoxicity.MethodsWe successfully constructed CD19 specific armored CAR-T cells overexpressing IL-I5 and IL-15 receptor alpha (IL-15Ra). In vitro cell differentiation and viability were monitored by flow cytometry, and an in vivo xenograft mouse models was used to evaluate the anti-tumor efficiency and liver damage of CAR-T cells.ResultsCAR-T cells overexpressing IL-15 alone demonstrated enhanced viability, retarded exhaustion in vitro and superior tumor-inhibitory effects in vivo. However, these tumor-free mice had lower survival rates, with serious liver injuries, as a possible result of toxicity. As expected, CAR-T cells overexpressing IL-15 combined with IL-15Ra had reduced CD132 expression and released fewer cytokines (IFNγ, IL-2 and IL-15) in vitro, as well as had the tendency to improve mouse survival via repressing the growth of tumor cells and keeping livers healthier compared to CAR-IL-15 T cells.ConclusionsThese results indicated the importance of IL-15 in enhancing T cells persistence and IL-15Ra in reducing the adverse effects of IL-15, with superior tumor retardation during CAR-T therapy. This study paves the way for the rapid exploitation of IL-15 in adoptive cell therapy in the future.