Second-generation CAR-T Cells Research Articles

Preclinical assessment of the efficacy of B7-H3 CAR-T in renal cell carcinoma

B7-H3 is a type I transmembrane protein that belongs to the B7 immune checkpoint protein family, is aberrantly expressed in cancer cells, but rarely expressed in normal tissues, making it an attractive target for cancer therapy. Here, we found B7-H3 is highly expressed in the renal cell carcinoma (RCC) tumor tissues and RCC cell lines, but is undetectable in normal renal tissues. Therefore, we engineered second-generation CAR-T cells targeting B7-H3, incorporating either CD28 or 4–1BB co-stimulatory domains. Both CAR-T cell variants demonstrated potent antitumor activity against RCC tumors in vitro and in metastatic and orthotopic RCC mouse models. Furthermore, the B7-H3 CAR-T cells exhibited remarkable proliferation and robust cytokine release when co-cultured with RCC cancer cells. These findings demonstrated that targeting B7-H3 by CAR-T cells potentially offering a new treatment option for RCC patients.

Intrinsic ADRB2 inhibition improves CAR-T cell therapy efficacy against prostate cancer

Chimeric antigen receptor (CAR)-T cell therapy has shown limited success in patients with solid tumors. Recent invitro and invivo data have shown that adrenoceptor beta-2 (ADRB2) is a novel checkpoint receptor that inhibits Tcell-mediated anti-tumor responses. To inhibit ADRB2-mediated inhibitory signaling, we downregulated ADRB2 in CAR-T (shβ2-CAR-T) cells via RNA interference, assessed different parameters, and compared them with conventional second-generation CAR-T cells. ADRB2 knockdown CAR-T cells exhibited enhanced cytotoxicity against prostate cancer cell lines invitro, by increasing CD69, CD107a, GzmB, IFN-γ, T-bet, and GLUT-1. In addition, ADRB2 deficiency led to improved proliferation, increased CD8/CD4 Tcell ratio, and decreased apoptosis in CAR-T cells. shβ2-CAR-T cells expressed more Bcl-2 and led to the generation of more significant proportions of T central memory cells. Finally, the ZAP-70/NF-κB signaling axis was shown to be responsible for the improved functions of novel CAR-T cells. In tumor-bearing mice, shβ2-CAR-T cells performed better than conventional CAR-T cells in eradicating prostate tumors. The study provides the basis for future clinical and translational CAR-T cell research to focus on adrenergic stress-mediated challenges in the tumor microenvironment of stressed tumors.

CXCL10 and IL15 co-expressing chimeric antigen receptor T cells enhance anti-tumor effects in gastric cancer by increasing cytotoxic effector cell accumulation and survival

ABSTRACT Chimeric antigen receptor (CAR) T cells have demonstrated outstanding therapeutic success in hematological malignancies. Yet, their efficacy against solid tumors remains constrained due to inadequate infiltration of cytotoxic T and CAR-T cells in the tumor microenvironment (TME), a factor correlated with poor prognosis in patients with solid tumors. To overcome this limitation, we engineered CAR-T cells to secrete CXCL10 and IL15 (10 × 15 CAR-T), which sustain T cell viability and enhance their recruitment, thereby amplifying the long-term cytotoxic capacity of CAR-T cells in vitro. In a xenograft model employing NUGC4-T21 cells, mice receiving 10 × 15 CAR-T cells showed superior tumor reduction and extended survival rates compared to those treated with second-generation CAR-T cells. Histopathological evaluations indicated a pronounced increase in cytotoxic T cell accumulation in the TME post 10 × 15 CAR-T cell treatment. Therefore, the synergistic secretion of CXCL10 and IL15 in these CAR-T cells enhances T cell recruitment and adaptability within tumor tissues, improving tumor control. This approach may offer a promising strategy for advancing CAR-T therapies in the treatment of solid tumors.

SYST-28 FUNCTIONAL MAPPING REVEALS WIDESPREAD REMODELLING AND A NOVEL TARGETABLE PTP4A2-ROBO1 SIGNALING AXIS AT GLIOBLASTOMA RECURRENCE

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and aberrant ROBO1 signaling axis is associated with higher tumorigenicity in GBM. ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB). Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We therefore developed and validated second-generation CAR-T cells against ROBO1, which demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death compared to untransduced cells in all the three brain cancers. These findings were further validated in vivo in rGBM, MB and BM models, where ROBO1 CAR-T cells showed significant reduction in tumor burden and increase in survival of treated mice. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence.

ROBO1 CAR-T CELL THERAPY FOR TREATMENT REFRACTORY BRAIN CANCER

Abstract BTFC travel award recipient Glioblastoma (GBM) is the most common malignant adult brain tumor, with a dismal prognosis. Despite the multimodal treatment regimen, tumor recurrence is inevitable, and no standardized treatment exists for recurrent glioblastoma (rGBM). Neurodevelopment signaling pathways are often hijacked during tumor progression. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment. Our preliminary findings implicated aberrant ROBO1 signaling axis to be associated with higher tumorigenecity in GBM, making it a functionally relevant therapeutic target. Moreover, ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB), prompting the development of an anti-ROBO1 CAR-T cell therapy. Here we report the development and validation of second-generation CAR-T cells using a single-domain antibody targeting ROBO1 expressing BTICs across three different brain tumors. We validated anti-ROBO1 CAR-T cells in-vitro and in-vivo using our established patient derived tumor models. In-vitro studies demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death with ROBO1 CAR-T cells as compared to untransduced cells (UT) in all the three cancers. These findings were further validated in-vivo using 3 different BTIC lines one each from rGBM, MB and BM. ROBO1 CAR-T showed significant reduction in tumor burden and significant increase in survival of the mice treated with ROBO1 CAR-T cells in all the three cohorts. Thus, targeting ROBO1 could be a therapeutically tractable strategy for treating rGBM as well as other brain malignancies.

Abstract 3001: Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of glioblastoma

Abstract WHO grade 4 glioblastoma multiforme (GBM) has a very poor prognosis even after standard treatment with surgery, radiotherapy, and chemotherapy. Tumor recurrence is virtually inevitable when it is fatal. Chimeric Antigen Receptor (CAR)-T cell therapy is a promising new modality of therapy for GBM patients. Even though second-generation CAR-T cells may be considered to operate autonomously because co-stimulation is inherent to CAR design, the tumor microenvironment of GBM is particularly hostile to immune cells and their normal functions. In our recently published work (Gargett et al. J Immunother Cancer 2022; 10(9): e005187), CAR-T cells specific for the glioblastoma antigen, GD2, had improved antitumor activity if the CAR-T cells co-expressed IL-15. Although IL-15 co-expression prolonged control of intracranial tumor xenografts, tumors escaped immune control in most cases. Our early data suggest that a relative lack of intratumoral dendritic cells (DCs) may be a contributory factor. DCs are central to the initiation and maintenance of adaptive immune responses. Hence, we were interested to survey and characterize the DC content of glioblastoma tissues obtained either from human surgical samples or mice bearing orthotopic xenografts and syngrafts. We have used multi-colour flow cytometry to enumerate and evaluate the maturation status of subsets of DCs together with other types of immune cells among single cell suspensions of isolated glioblastoma tissues. Our preliminary findings are: Total DCs (mean +/- SD), which were defined as lineage-negative and HLA-DR (+), were significantly reduced among total viable cells of glioblastoma tissues (0.845% +/- 0.569) and peripheral blood mononuclear cells (PBMCs) (0.755% +/- 0.601) from GBM patients (n=12) compared to PBMCs (1.77% +/- 0.611) from healthy donors (n=12).No significant differences were detected in the percentages of total DCs among splenocytes of nontumor-bearing C57BL/6 (B6) mice (n=6) compared to B6 bearing othotopic syngrafts of the GL261 (n=3) or C2TA (n=3) glioblastoma cell lines. We will present more detailed analyses from human glioblastoma samples. In addition, we will present a more extensive characterization of DCs from the orthotopic murine models of glioblastoma: we will compare DCs and other types of immune cells in tumor and in the primary and secondary lymphoid tissues of both tumor-bearing and nontumor-bearing mice. We expect that the final data will fill a current gap in knowledge and indicate possible therapeutic strategies that might correct for the quantitative and qualitative defects in dendritic cells in glioblastoma. We hypothesize that adequate correction of these defects in vivo may augment the anti-glioblastoma activity of CAR-T cell therapies. Citation Format: Michael P. Brown, Tessa Gargett, Bryan J. Gardam. Investigating the quantity and quality of dendritic cells in human glioblastoma tissues and in orthotopic murine xenograft and syngraft models of glioblastoma [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 3001.

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.

Preclinical evaluation of CD70-specific CAR T cells targeting acute myeloid leukemia.

Chimeric antigen receptor (CAR)-T cell therapy has achieved unprecedented success in treating hematopoietic malignancies. However, this cell therapy is hampered in treating acute myeloid leukemia (AML) due to lack of ideal cell surface targets that only express on AML blasts and leukemia stem cells (LSCs) but not on normal hematopoietic stem cells (HSCs). We detectedtheCD70 expression on the surfaces ofAML cell lines, primary AML cells, HSC, and peripheral blood cells and generated a second-generation CD70-specific CAR-T cells using a construct containing a humanized 41D12-based scFv and a 41BB-CD3ζ intracellular signaling domain. Cytotoxicity, cytokine release, and proliferation in antigen stimulation, CD107a assay, and CFSE assays were used to demonstrate the potent anti-leukemia activity in vitro. A Molm-13 xenograft mouse model was established to evaluate the anti-leukemic activity of CD70 CAR-T in vivo. CFU assay was explored to assess the safety of CD70 CAR-T on HSC. CD70 heterogeneously expressed on AML primary cells, including leukemia blasts, leukemic progenitor, and stem cells, but not expressed on normal HSCs and majority of blood cells. Anti-CD70 CAR-T cells exhibited potent cytotoxicity, cytokines production, and proliferation when incubated with CD70+ AML cell lines. It also displayed robust anti-leukemia activity and prolonged survival in Molm-13 xenograft mouse model. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo. Our study reveals that anti-CD70 CAR-T cells are a new potential treatment for AML. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo, suggesting that future studies aiming to generate innovative combinatorial CAR constructs or to increase CD70 expression density on leukemia cell surface to prolong the life-span of CAR-T cells in the circulation will be needed in order to optimize CAR-T cell responses for AML.

Runx3-overexpression cooperates with ex vivo AKT inhibition to generate receptor-engineered T cells with better persistence, tumor-residency, and antitumor ability

BackgroundSolid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to...

The third-generation anti-CD30 CAR T-cells specifically homing to the tumor and mediating powerful antitumor activity

CAR T-cell therapy is well tolerated and effective in patients with Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). However, even second- generation anti-CD30 CAR T-cells with CD28 (28z) costimulatory domains failed to achieve the desired rate of complete responses. In the present study, we developed second-generation (CD28z) and third-generation (CD28BBz) CAR T-cells targeting CD30 and investigated their efficacy in vitro and in vivo. Both of CD28z and CD28BBz anti-CD30 CAR T cells were similar regarding amplification, T cell subsets distribution, T cell activity, effector/memory and exhaustion. However, we found that the 28BBz anti-CD30 CAR T-cells persist long-term, specifically homing to the tumor and mediating powerful antitumor activity in tumor xenograft models. Subsequently, we also demonstrated that the third generation anti-CD30 CAR T-cells have miner side effects or potential risks of tumorigenesis. Thus, anti-CD30 CAR T-cells represent a safe and effective treatment for Hodgkin lymphoma.

Development of a TCR-like antibody and chimeric antigen receptor against NY-ESO-1/HLA-A2 for cancer immunotherapy

BackgroundThe current therapeutic antibodies and chimeric antigen receptor (CAR) T cells are capable of recognizing surface antigens, but not of intracellular proteins, thus limiting the target coverage for drug development....

Targeting of the alphav beta3 integrin complex by CAR-T cells leads to rapid regression of diffuse intrinsic pontine glioma and glioblastoma

BackgroundDiffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and generally incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success...

A Novel and Successful Patient or Donor-Derived CD7-Targeted CAR T-Cell Therapy for Relapsed or Refractory T-Cell Lymphoblastic Lymphoma (R/R T-LBL)

BackgroundT-cell lymphoblastic lymphoma is an aggressive hematological malignancy, often presenting with bulky mediastinal masses or diffuse extramedullary disease (EMD). There is evidence that T-LBL differs in various aspects from T-cell acute lymphoblastic leukemia in addition to differences in clinical presentation of diseases. To date, there are only a few clinical case reports of CAR-T therapy for T-LBL. Here, we explored the efficacy and safety of CD7-targeted CAR-T cells (CD7CAR) for R/R T-LBL in a phase I clinical trial (NCT04916860).MethodsEligible R/R T-LBL patients were enrolled between November 2020 and May 2021. Peripheral blood (PB) mononuclear cells were collected from either the donor (n=1) or patients (n=7) by leukapheresis. The CD7-CAR gene was obtained by gene synthesis and then ligated into a lentiviral vector by molecular cloning. We developed second-generation CAR-T cells with an intracellular co-stimulatory domain of 4-1BB and CD3ζ targeting CD7. Intravenous fludarabine (30 mg/m 2/d) and cyclophosphamide (300 mg/m 2/d) were given to all patients on Day -5 to Day -3 prior to CD7CAR infusion.ResultsPatient characteristics are summarized in Table 1. Eight R/R T-LBL patients were enrolled with a median age of 37 years (14-47 years) and a median of 5 prior lines of therapies (2-10 lines). Two patients had a history of central nervous system involvement. Two patients relapsed from a previous allogenic (N=1) or autologous (N=1) hematopoietic stem cell transplantation (HSCT). Four patients expressed high-risk genotypes including TP53, EZH2 and RUNX1. At enrollment, 7 patients had EMD relapse (diffuse involvement, N=5; bulky mediastinal masses, N=2). One patient had no EMD involvement at enrollment due to a prior palliative mediastinal radiotherapy, but relapsed with bone marrow (BM) blasts up to 87.27%. A total of 5/8 patients had BM blasts at enrollment with median BM blasts of 17%.Both patient- and donor-derived CD7CAR-T cells were successfully generated with a transfection efficiency of 86.55% (27%-98%). A single dose of CD7CAR-T cells was infused to patients at low dose (5x10 5 cells/kg, N=1), medium dose (1x10 6 cells/kg, N=6) or high dose (2x10 6 cells/kg, N=1). The median follow-up time was of 93 days (55-166 days) by July 18, 2021, the cutoff date.Following CD7CAR infusion, 5/5 patients who had prior BM blasts achieved minimal residual disease negative (MRD-) complete remission with incomplete hematologic recovery (CRi) on Day 28, among whom 3 had already achieved MRD- CRi on Day 14. The 3 patients who did not have BM blasts prior to CAR-T infusion maintained zero BM blasts post infusion. Of the 7 patients who had EMD involvements, 4 achieved EMD CR on Day 28, and 1 on Day 51. Of the 2 patients who had bulky mediastinal masses (~7 or 6 cm), 1 had partial response and 1 had stable disease on Day 28, respectively. Of all patients, 6 subsequently underwent allo-HSCT following CD7CAR-T infusion with a median time of 54 days (42-56 days), without relapse or progression. One patient with an allo-HSCT prior to CD7CAR infusion died after receiving a second haploidentical allo-HSCT due to acute graft-versus-host disease. The other 2 patients who did not receive a transplant were on Day 55 and 73 post CD7CAR infusion with ongoing remission by the cutoff date. Mild cytokine release syndrome (CRS, ≤Grade 2) was observed in 7/8 patients. Only 1/8 patient had Grade 3 CRS and Grade 1 neurotoxicity. The median onset of CRS was 1 day post infusion (0-15 days) with a median duration of 16 days (5-19 days).CD7CAR expansion in vivo occurred as early as 3.5 days (0-11 days) post infusion and reached a median peak of 2.07x10 5 copies/ug DNA (0.75-5.36 x10 5 copies/ug DNA) at a median of 19 days (13-28 days), and was still detectable up to the last follow-up, with a median duration of 50 days (26-120 days), as measured by qPCR (Figure.1).ConclusionOur clinical trial showed that CD7CAR-T cells derived either from the patients or the donor have a high initial efficacy and a good safety profile in R/R T-LBL patients. Initial high CR could be achieved both intramedullary and extramedullary in the majority of patients, even among those who harbored with high-risk features or had diffuse extramedullary lesions. However, patients with bulky mediastinal masses may require more than one-month time to achieve remission. Long-term observation and more patients are needed to further evaluate the safety and efficacy of CD7CAR. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CAR T-Cell Therapies in Mantle Cell Lymphoma, Hints of Cure

<h2>Introduction</h2> Therapy for mantle cell lymphoma (MCL) has evolved from chemotherapy to targeted therapy, and most recently to CAR T-cell therapies. CAR T-cell therapy began in 1993 with the first generation of CAR T-cells developed in Israel. Later, in 2002 the second-generation CAR T-cells were developed against prostate cancer cells in the laboratory. The following year, second generation CAR T-cells were tested against leukemia cells using a mouse model where CD19 proved to be an effective target. Eleven years later, FDA grants breakthrough therapy designation to CD19-directed CAR T-cells. Finally, in 2017, the FDA approved the first CAR T-cell therapy in large cell lymphoma.

Novel two-chain structure utilizing KIRS2/DAP12 domain improves the safety and efficacy of CAR-T cells in adults with r/r B-ALL

Engineered T cells that express chimeric antigen receptors (CARs) have been a promising therapy for hematologic malignancies. The optimization of CAR structure using different signaling domains can alter a wide range of CAR-T cell properties, including anti-tumor activity, long-term persistence, and safety. In this study, we developed a novel CAR structure based on KIRS2/Dap12 for B cell acute lymphoblastic leukemia (B-ALL) antigen CD19 and compared the anti-tumor efficacy and safety of this construct in transduced T cells with standard second-generation CAR-T cells targeting CD19 for B-ALL in vitro and in vivo and in adult relapsed/refractory (r/r) B-ALL patients. We discovered that KIRS2/Dap12 receptor infused with 4-1BB co-stimulation domain could enhance anti-tumor efficacy by remarkably increasing the production of pro-inflammatory interleukin-2 (IL-2), especially when co-cultured with antigen-positive tumor cells. In addition, CD19-KIRS2/Dap12-BB CAR-T cells showed the inspiring outcome that complete responses were seen in 4 of 4 (100%) patients without neurotoxicity and a high rate of severe cytokine release syndrome (CRS) after CAR-T infusion in a phase I clinical trial. Given these encouraging findings, CD19-KIRS2/Dap12-BB CAR-T cells are safe and can lead to clinical responses in adult patients with r/r B-ALL, indicating that further assessment of this therapy is warranted.

Characterization of the Therapeutic Effects of Novel Chimeric Antigen Receptor T Cells Targeting CD38 on Multiple Myeloma.

Multiple myeloma (MM) is a tumor type characterized by the unregulated proliferation of clonal plasma cells in the bone marrow. Immunotherapy based on chimeric antigen receptor T cell (CAR-T) therapy has achieved exciting success in the treatment of hematological malignant tumors. CD38 is highly and evenly expressed in MM and is an attractive target for MM treatment. Here, we successfully constructed two novel second-generation CAR-T cells targeting CD38 by retroviral vector transduction. CD38 CAR-T cells could be activated effectively after stimulation with CD38-positive tumor cells and secrete cytokines such as IFN-γ and TNF-α to promote tumor cell apoptosis in in vitro experiments. Real-time fluorescence monitoring experiments, luciferase detection experiments and flow cytometry experiments revealed the efficient and specific killing abilities of CD38 CAR-T cells against CD38-positive tumor cells. The proliferation ability of CD38 CAR-T cells in vitro was higher than that of untransduced T cells. Further antitumor experiments in vivo showed that CD38 CAR-T cells could be quickly activated to secrete IFN-γ and eliminate tumors. Thus, novel CD38-targeted second-generation CAR-T cells have efficient and specific antitumor activity and may become a novel therapy for the clinical treatment of MM.

The BCMA-Targeted Fourth-Generation CAR-T Cells Secreting IL-7 and CCL19 for Therapy of Refractory/Recurrent Multiple Myeloma.

Chimeric antigen receptor (CAR) technology has revolutionized cancer treatment, particularly in malignant hematological tumors. Currently, the BCMA-targeted second-generation CAR-T cells have showed impressive efficacy in the treatment of refractory/relapsed multiple myeloma (R/R MM), but up to 50% relapse remains to be addressed urgently. Here we constructed the BCMA-targeted fourth-generation CAR-T cells expressing IL-7 and CCL19 (i.e., BCMA-7 × 19 CAR-T cells), and demonstrated that BCMA-7 × 19 CAR-T cells exhibited superior expansion, differentiation, migration and cytotoxicity. Furthermore, we have been carrying out the first-in-human clinical trial for therapy of R/R MM by use of BCMA-7 × 19 CAR-T cells (ClinicalTrials.gov Identifier: NCT03778346), which preliminarily showed promising safety and efficacy in first two enrolled patients. The two patients achieved a CR and VGPR with Grade 1 cytokine release syndrome only 1 month after one dose of CAR-T cell infusion, and the responses lasted more than 12-month. Taken together, BCMA-7 × 19 CAR-T cells were safe and effective against refractory/relapsed multiple myeloma and thus warranted further clinical study.

A chimeric antigen receptor with antigen-independent OX40 signaling mediates potent antitumor activity.

Although chimeric antigen receptor (CAR)-modified T cells have shown great success in the treatment of B cell malignancies, this approach has limited efficacy in patients with solid tumors. Various modifications in CAR structure have been explored to improve this efficacy, including the incorporation of two costimulatory domains. Because costimulatory signals are transduced together with T cell receptor signals during T cell activation, we engineered a type of CAR-T cells with a costimulatory signal that was activated independently from the tumor antigen to recapitulate physiological stimulation. We screened 12 costimulatory receptors to identify OX40 as the most effective CAR-T function enhancer. Our data indicated that these new CAR-T cells showed superior proliferation capability compared to current second-generation CAR-T cells. OX40 signaling reduced CAR-T cell apoptosis through up-regulation of genes encoding Bcl-2 family members and enhanced proliferation through increased activation of the NF-κB (nuclear factor κB), MAPK (mitogen-activated protein kinase), and PI3K-AKT (phosphoinositide 3-kinase to the kinase AKT) pathways. OX40 signaling not only enhanced the cytotoxicity of CAR-T cells but also reduced exhaustion markers, thereby maintaining their function in immunosuppressive tumor microenvironments. In mouse tumor models and in patients with metastatic lymphoma, these CAR-T cells exhibited robust amplification and antitumor activity. Our findings provide an alternative option for CAR-T optimization with the potential to overcome the challenge of treating solid tumors.

IMMU-17. CAR T CELLS TARGETING THE INTEGRIN ALPHAVBETA3 EXHIBIT ROBUST ANTI-TUMOR RESPONSES AGAINST DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND GLIOBLASTOMA (GBM)

Effective therapies for DIPG and GBM are lacking. CD19 chimeric antigen receptor (CAR) T cells are highly effective in patients with refractory B-cell malignancies. We aim to develop novel CARs for high-grade gliomas. The integrin complex alphavbeta3 was selected as a CAR-T cell target due to its expression on gliomas and their vasculature, yet with minimal expression throughout normal tissues, vessels and organs. Indeed, a majority of DIPG and GBM cell lines express surface α vβ 3. Second-generation CAR-T cells expressing an anti-α vβ 3 scFv and either a CD28 or 4-1BB co-stimulatory domain and CD3zeta were constructed. Transduced healthy, donor-derived T cells exhibited high level CAR expression, efficient expansion, and representative populations of memory subsets including central, effector, and stem cell-like memory CAR-T cells. α vβ 3.28z and α vβ 3.BBz CAR-T cells exhibited antigen-specific in vitro cytotoxicity and cytokine production against DIPG and GBM cell lines. Both CARs mediated rapid and robust anti-tumor responses in NSG mice bearing orthotopic DIPG or GBM tumors. 5/13 α vβ 3.28z and 0/14 α vβ 3.BBz treated animals died without detectable disease within 2 weeks of infusion suggesting different toxicity profiles and is consistent with faster CAR-T cell expansion in CD28-versus 4-1BB-containing CD19 CAR-T cells seen clinically. Our results demonstrate that α vβ 3.BBz CAR-T cell therapy may be both highly effective and safe in DIPG and GBM patients. Due to the restricted nature of α vβ 3 expression in normal tissues, the robust responses seen in tumor-bearing mice, and the slower kinetics of α vβ 3.BBz CAR-T cell expansion, a first-in-human clinical trial is being planned.

Prognostic Impact of Metabolic Tumor Burden in Large B-Cell Lymphoma Patients Receiving CAR T-Cell Therapy

Introduction Chimeric antigen receptor (CAR) T-cell therapy provides long-term remissions in a substantial proportion of patients with large B-cell lymphoma (LBCL) who have relapsed or are refractory (R/R) to chemoimmunotherapy. The identification of prognostic factors to identify which patients will benefit most from this therapy is crucial to improve patient selection. Even though metabolic tumor burden assessed by 18F-fluorodeoxyglucose Positron Emission Tomography (PET) has a confirmed prognostic value in the setting of chemoimmunotherapy, its predictive role after CAR T-cell therapy is not established. Methods We conducted a single-center study including all patients with R/R LBCL who received a single infusion of CD19-targeted second-generation CAR T-cells carrying a 4-1BB costimulatory domain from July 2018 to January 2020. Adverse events were graded according to the American Society for Transplantation and Cellular Therapy (ASTCT) consensus. All patients underwent a baseline PET scan and metabolic disease evaluation after infusion. Disease response assessment was conducted according to Lugano criteria. Metabolic tumor volume (MTV) and maximum standardized uptake value (SUVmax) were measured at baseline and at 1-month after CAR T-cell infusion, and correlated with disease response and development of adverse events. To identify the optimal cut-offs for metabolic parameters we used the maximally selected log-rank statistics in the PFS analysis. Results Thirty-five consecutive patients with R/R LBCL who received CAR T-cell therapy were included in the study. Patients' baseline characteristics are summarized in Table 1. Median age at treatment was 58 years, and 74% were males. At the time of diagnosis most of them had an advanced stage of disease (86%) and were refractory to the last therapy (n=31, 88%). Best response after CAR T-cell therapy included 9 (26%) patients in complete remission (CR) and 16 (46%) in partial remission (PR). Ten (28%) patients were in progressive disease (PD) at the 1-month disease evaluation. At a median follow-up of 7.6 months, median PFS and OS were 3.4 months and 8.2 months, respectively. Regarding toxicity, eleven (31%) patients developed clinically significant CAR T-cell related toxicity, defined as grade 2 or higher CRS (n=7, 20%) and grade 2 or higher ICANS (n=6, 17%). Median baseline MTV and MTV41% were 270 cm3(IQR 87-875) and 119 cm3 (IQR 32-300), respectively. Median SUVmax was 24 (IQR 17-32). Patients who responded (CR and PR) had lower baseline MTV values compared with non-responders (median of 228 cm3 vs 645 cm3, p=0.04) (Figure 1a). No association was found between MTV41% or SUVmax and disease response. In terms of PFS, a high baseline MTV (&amp;gt;82 cm3) was associated with a lower PFS compared to patients with lower MTV values (median PFS, 2.1 months vs. 6 months; HR 3.15, p= 0.02) (Table 2 and Figure 2). Patients with high baseline MTV41% values (&amp;gt;25 cm3) also had an inferior PFS (HR 3.44, p= 0.02); no association was found between baseline SUVmax and PFS (Table 2). As per OS, there was no significant association with baseline MTV, MTV41% and SUVmax (Table 2). Regarding toxicity, there was no significant association between baseline MTV, MTV 41% and SUVmax values with grade 2 or higher CRS and ICANS events (Figure 1b). All patients underwent a 1-month post-infusion PET evaluation. Disease response at this timepoint was: CR in 8 patients (23%), PR in 17 patients (49%) and PD in 10 patients (28%). For patients in CR and PR at 1-month the probability of PFS at 6 months was 62.5% and 12.7%, respectively (HR=3.89, p=0.02). For patients in PR at the 1-month evaluation, MTV values at that timepoint were predictive for PFS; patients in PR with low (&amp;lt;35 cm3) and high 1-month MTV values had a 6m-PFS of 33% and 0%, respectively (Figure 3)(HR=4.6, p = 0.01). In these patients, SUVmax values also predicted PFS and a trend towards significance was observed for MTV41% (HR=3, p=0.07). Conclusion Metabolic tumor burden parameters measured by 18FDG-PET before and 1-month after CAR-T cell infusion identify LBCL patients who benefit most from these therapies and could aid patient selection. Disclosures Iacoboni: Novartis, Gilead, Celgene, Roche: Honoraria. Villacampa:AstraZeneca: Other: advisory role; Merck Sharp &amp; Dohme: Honoraria. Abrisqueta:AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau. Bosch:Hoffmann-La Roche: Research Funding. Barba:Novartis, Celgene, Gilead, Pfizer, Amgen, Roche: Honoraria.