Efficacy Of Chimeric Antigen Receptor Research Articles

CAR-macrophage versus CAR-T for solid tumors: The race between a rising star and a superstar.

Adoptive cell therapy (ACT) has been demonstrated to be one of the most promising cancer immunotherapy strategies due to its active antitumor capabilities in vivo. Engineering T cells to overexpress chimeric antigen receptors (CARs), for example, has shown potent efficacy in the therapy of some hematologic malignancies. However, the efficacy of chimeric antigen receptor T cell (CAR-T) therapy against solid tumors is still limited due to the immunosuppressive tumor microenvironment (TME) of solid tumors, difficulty in infiltrating tumor sites, lack of tumor-specific antigens, antigen escape, and severe side effects. In contrast, macrophages expressing CARs (CAR-macrophages) have emerged as another promising candidate in immunotherapy, particularly for solid tumors. Now at its nascent stage (with only one clinical trial progressing), CAR-macrophage still shows inspiring potential advantages over CAR-T in treating solid tumors, including more abundant antitumor mechanisms and better infiltration into tumors. In this review, we discuss the relationships and differences between CAR-T and CAR-macrophage therapies in terms of their CAR structures, antitumor mechanisms, challenges faced in treating solid tumors, and insights gleaned from clinical trials and practice for solid tumors. We especially highlight the potential advantages of CAR-macrophage therapy over CAR-T for solid tumors. Understanding these relationships and differences provides new insight into possible optimization strategies of both these two therapies in solid tumor treatment.

BCKDK modification enhances the anticancer efficacy of CAR-T cells by reprogramming branched chain amino acid metabolism

Altered Branched Chain Amino Acids (BCAA), including leucine, isoleucine and valine, are frequently observed in patients with advanced cancer. We evaluated the efficacy of Chimeric Antigen Receptor (CAR) T cell-mediated cancer cell lysis potential in the immune microenvironment of BCAA supplementation and deletion. BCAA supplementation increased cancer cell killing percentage, while accelerating BCAA catabolism and deceasing BCAA transporter decreased cancer cell lysis efficacy. We thus designed BCKDK engineering CAR T cells for the reprogramming of BCAA metabolism in the tumor microenvironment based on the genotype and phenotype modification. BCKDK overexpression (OE) in CAR-T cells significantly improved cancer cell lysis, while BCKDK knockout (KO) resulted in inferior lysis potential. In an in vivo experiment, BCKDK-OE CAR-T cells treatment significantly prolonged the survival of mice bearing NALM6-GL cancer cells, with the differentiation of central memory cells and the increasing proportion of CAR-T cells in peripheral circulation. BCKDK-KO CAR-T cells treatment resulted in shorter survival and decreasing percentage of CAR-T cells in peripheral circulation. In conclusion, BCKDK engineered CAR-T cells exert distinct phenotype for the superior anticancer efficiency.

Development of a chimeric cytokine receptor that captures IL-6 and enhances the antitumor response of CAR-T cells

The efficacy of chimeric antigen receptor (CAR)-engineered Tcell therapy is suboptimal in most cancers, necessitating further improvement in their therapeutic actions. However, enhancing antitumor Tcell response inevitably confers an increased risk of cytokine release syndrome associated with monocyte-derived interleukin-6 (IL-6). Thus, an approach to simultaneously enhance therapeutic efficacy and safety is warranted. Here, we develop a chimeric cytokine receptor composed of the extracellular domains of GP130 and IL6RA linked to the transmembrane and cytoplasmic domain of IL-7R mutant that constitutively activates the JAK-STAT pathway (G6/7R or G6/7R-M452L). CAR-T cells with G6/7R efficiently absorb and degrade monocyte-derived IL-6 invitro. The G6/7R-expressing CAR-T cells show superior expansion and persistence invivo, resulting in durable antitumor response in both liquid and solid tumor mouse models. Our strategy can be widely applicable to CAR-T cell therapy to enhance its efficacy and safety, irrespective of the target antigen.

Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment.

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Induction of immortal-like and functional CAR T cells by defined factors.

Long-term antitumor efficacy of chimeric antigen receptor (CAR) T cells depends on their functional persistence in vivo. T cells with stem-like properties show better persistence, but factors conferring bona fide stemness to T cells remain to be determined. Here, we demonstrate the induction of CAR T cells into an immortal-like and functional state, termed TIF. The induction of CARTIF cells depends on the repression of two factors, BCOR and ZC3H12A, and requires antigen or CAR tonic signaling. Reprogrammed CARTIF cells possess almost infinite stemness, similar to induced pluripotent stem cells while retaining the functionality of mature T cells, resulting in superior antitumor effects. Following the elimination of target cells, CARTIF cells enter a metabolically dormant state, persisting in vivo with a saturable niche and providing memory protection. TIF represents a novel state of T cells with unprecedented stemness, which confers long-term functional persistence of CAR T cells in vivo and holds broad potential in T cell therapies.

Abstract 4003: TBK1 inhibition enhances CAR-T cell efficacy against patient-derived organotypic tumor spheroids

Abstract Novel therapeutic strategies are needed to improve the efficacy of chimeric antigen receptor (CAR)-T cells in the treatment of solid tumors. Here, we set out to evaluate novel therapeutic strategies to improve the efficacy of B7-H3 (CD276) directed (B7-H3.CAR-T) using 3D microfluidic cultures of patient-derived organotypic tumor spheroids (PDOTS). PDOTS are generated from fresh patient tumor tissue and contain tumor cells, autologous immune cells and stromal cells, therefore recapitulating key features of the tumor microenvironment (TME), enabling the study of tumor-immune dynamics. We confirmed activity of B7-H3.CAR-T in PDOTS, although a strong correlation between B7-H3 expression and CAR-T efficacy was not observed. Mechanistic studies subsequently demonstrated dynamic upregulation of co-inhibitory receptors on CAR-T cells following target cell encounter leading to CAR-T cell dysfunction and limiting CAR-T efficacy in B7-H3 expressing tumors. PD-1 blockade restored CAR-T activity in monotypic and organotypic tumor spheroids with improved tumor control and upregulation of effector cytokines. Given the emerging role of TANK-binding kinase 1 (TBK1) as an immune evasion gene, we examined the effect of TBK1 inhibition on CAR-T efficacy. Similar to PD-1 blockade, TBK1 inhibition restored CAR-T activity in monotypic and organotypic tumor spheroids, prevented CAR-T cell dysfunction, and enhanced T cell proliferation. Inhibition or deletion of TBK1 also enhanced sensitivity of cancer cells to immune-mediated killing. Taken together, our results demonstrate the feasibility and utility of ex vivo profiling of CAR-T cells using PDOTS, and suggest that targeting TBK1 is a novel strategy to enhance CAR-T efficacy by overcoming tumor-intrinsic and -extrinsic resistance mechanisms. Citation Format: Luke Maggs, Yi Sun, Giulia Cattaneo, Marco Ventin, Feng Chen, Martin Q. Rasmussen, Xinhui Wang, Cristina R. Ferrone, Soldano Ferrone, Russell W. Jenkins. TBK1 inhibition enhances CAR-T cell efficacy against patient-derived organotypic tumor spheroids [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 4003.

Abstract 3997: PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer

Abstract Background: The efficacy of chimeric antigen receptor (CAR) T cell therapy remains modest for the treatment of solid tumors due to restricted T cell infiltration and immunosuppressive tumor microenvironment (TME). Leveraging the window of opportunity between leukapheresis and infusion using bridging therapy might improve TME and potentiate CAR-T cell therapy. Here, we assessed the potential of poly (ADP-ribose) polymerase (PARP) inhibitors plus anlotinib as bridging therapy for CAR-T cells with resistance to TGFβ and capability of rapid expansion by bait-and-switch strategy of dual-targeting mesothelin (MSLN) and CD19 in preclinical models of advanced ovarian cancer. Methods: Immunocompetent mice bearing ID8 tumors were treated with niraparib (21 days) plus anlotinib (14 days) and euthanized at different timepoints (0, 7, or 14 days since discontinuation) to quantify tumor-infiltrating T cells and profile T cell functions. CAR-T cells transfected with dominant-negative TGFβRII dual-targeting MSLN and CD19 were produced by IASO Biotherapeutics. NSG mice bearing MSLN-positive cell- or patient-derived xenografts (CDX or PDX) of ovarian cancer received CAR-T cells alone or after bridging therapy (niraparib plus anlotinib followed by a wash-out period of 7 days). Tumor volume was balanced before CAR-T cell infusion. Replication-deficient NALM6 cells pre-treated with mitomycin C were injected to provide CD19 antigen. A PET scan that visualized granzyme B release was used to track CAR-T cells and assess immune-mediated tumor killing. Results: Niraparib combined with anlotinib increased tumor-infiltrating T cells without impairing T cell functionality. The effects of promoting T cell infiltration remained significant at 14 days after discontinuation, which might associate with lasting activation of cGAS-STING pathway and secretion of downstream chemokines (CXCL10 and CCL5), and normalized tumor vasculature featured by better perfusion and reduced leakage. The engineered CAR-T cells could resist TGFβ and displayed improved proliferation, cytotoxicity, cytokine release, and in vivo antitumor activities upon CD19 stimulation. Importantly, bridging therapy significantly increased CAR-T cell infiltration, curbed tumor growth, and prolonged survival in mice bearing SKOV3 CDX, HRD-negative PDX, and another multidrug-resistant PDX. PET imaging showed that bridging therapy increased tumor-infiltrating CAR-T cells and boosted tumor-killing capability. Conclusions: PARP inhibitors plus anlotinib as bridging therapy facilitated CAR-T cell infiltration and enhanced antitumor activities in multiple preclinical models of advanced ovarian cancer, and an early phase I trial (NCT05141253) is ongoing to evaluate PARP inhibitors plus anlotinib as bridging therapy for CAR-T cell therapy in patients with refractory MSLN-positive ovarian cancer. Citation Format: Huayi Li, Minghua Xiang, Jiahao Liu, Wei Mu, Li Zhu, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer [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 3997.

Abstract 3841: Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies

Abstract Despite the remarkable efficacy of chimeric antigen receptor T -cell therapy (CART) for the treatment of B-cell malignancies, durable remission remains limited often due to tumor relapse. We have previously demonstrated that tumor-derived extracellular vesicles (EVs) induced CART cell failure in preclinical models. To characterize the impact of tumor EV induced CART cell dysfunction, we assessed microRNA (miRNA) profiles contained within tumor EVs from two independent patient cohorts and studied how they contributed to CART dysfunction. Our first cohort included patients with chronic lymphocytic leukemia (n=50). We previously showed that CLL patient derived EVs induce a state of CART cell dysfunction. To understand this dysfunction, we interrogated the miRNA cargo significantly enriched in CLL EVs and how their coculture impacts CART signaling by RNAseq. Here, we found a significant enrichment in miR-125a within CLL EVs as compared to healthy donor controls (p=.0001). CART cocultured with CLL EVs showed alterations in T cell activation pathways, where miR-125a targets TNFAIP3 and NFkB pathways were activated. These findings implicate miR-125a from CLL EVs in CART dysfunction. Next, we aimed to validate the impact of miRNA on CART cell outcomes in the clinic. We interrogated miRNA profiles from patients with diffuse large B cell lymphoma (DLBCL) treated with CART cell therapy (CART19-28ζ, axicabtagene ciloleucel). EV miRNA cargo was compared between responders (complete remission at 6 months, n=10) and non-responders (no response 1 month post CART19, n=10) to identify differentially enriched miRNAs across groups. miR-125a was again significantly upregulated in EVs of non-responders as compared to responders to CART19 treatment (fold change=3.28, padj=.018). To study the direct effect of miR-125a on CART cell functions, we generated and studied the effector functions of CART19-28z following transfection with miR-125a or negative miR control. CART19 cells transfected with miR-125a showed a dose dependent decrease in killing of lymphoma cells as compared to miR-control (p=.0001). miR-125a also significantly increased the rate of apoptosis in CART19 cells (fold change=1.95, p=.024) and decreased the level of CART proliferation (fold change=.71, p=.013) as compared to controls. Notably, miR-125a did not impact tumor cell growth, further verifying a direct CART effect. In summary, our studies demonstrate the impact of tumor derived miRNAs on CART cell outcomes. They identify that miR-125a is 1) associated with CLL EV induced CART dysfunction, 2) significantly elevated in non-responders to CART therapy in the clinic, and 3) able to decrease CART19 efficacy through reduced CART killing/proliferation and increased apoptosis. These results highlight a novel mechanism of CART19 failure induced through miRNA cargo delivery by tumor-derived EVs and the role of miR-125a in inducing CART19 dysfunction. Citation Format: Olivia Sirpilla, R. Leo Sakemura, Ismail Can, Truc N. Huynh, James H. GIrsch, Claudia Manriquez Roman, Carli M. Stewart, Kun Yun, Ekene J. Ogbodo, Long Mai, Omar Gutierrez Ruiz, Makena Rodriguez, Brooke Kimball, Hong Xia, Jose C. Villasboas, Stephen Ansell, Nora Bennani, Patrick B. Johnston, Jonas Paludo, Januario Castro, Mohamed Kharfandabaja, Sameer Parikh, Neil Kay, Yi Lin, Elizabeth Siegler, Saad Kenderian. Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies [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 3841.

Successful Treatment of Refractory Orbital Plasmacytoma with Chimeric Antigen Receptor T Cell Therapy: A Case Report and Review of the Literature.

Orbital plasmacytoma is a rare plasma cell tumor that may arise as an aggressive form of extramedullary multiple myeloma. Treatment modalities include surgical excision, radiation, and chemotherapy. Chimeric antigen receptor T cell therapy is currently reserved for refractory disease. The authors present a case of a 69-year-old woman with an extensive orbital plasmacytoma refractory to multimodal therapy who was treated with idecabtagene vicleucel chimeric antigen receptor T cell therapy. Four days after infusion, the patient exhibited grade 1 cytokine release syndrome, which resolved with tocilizumab. The orbital plasmacytoma significantly decreased in size 1 month after treatment and demonstrated complete serological response and sustained tumor burden reduction at 10-month follow-up. This case highlights the efficacy of chimeric antigen receptor T cell therapy for refractory orbital plasmacytoma and calls attention to potential inflammatory toxicities.

Biomarkers for predicting efficacy of chimeric antigen receptor T cell therapy and their detection methods

AbstractCancer immunotherapy has emerged as the fourth most prevalent approach to tumor treatment, alongside surgery, radiotherapy, and chemotherapy. After several decades of development, chimeric antigen receptor T (CAR‐T) cell therapy, a promising branch of adoptive T‐cell therapy, has demonstrated superior efficacy and safety in comparison to other cell therapies in the treatment of cancer. At present, CAR‐T cells are predominantly used to treat hematological malignancies, although their application in solid tumors is being readily investigated. Although numerous studies have examined the biomarkers associated with the safety of CAR‐T cell therapy, few have evaluated predictors of CAR‐T cell therapeutic efficacy. Thus, the primary objective of this review article was to provide a comprehensive overview of the factors predicting the efficacy of CAR‐T cell therapy, with a particular focus on biomarkers and their detection methods.

Engineering Improved CAR T Cell Products with A Multi-Cytokine Particle Platform for Hematologic and Solid Tumors.

Despite the remarkable clinical efficacy of chimeric antigen receptor (CAR) T cells in hematological malignancies, only a subset of patients achieves a durable complete response (dCR). DCR has been correlated with CAR T cell products enriched with T cells memory phenotypes. Therefore, reagents that consistently promote memory phenotypes during the manufacturing of CAR T cells have the potential to significantly improve clinical outcomes. A novel modular multi-cytokine particle (MCP) platform is developed that combines the signals necessary for activation, costimulation, and cytokine support into a single "all-in-one" stimulation reagent for CAR T cell manufacturing. This platform allows for the assembly and screening of compositionally diverse MCP libraries to identify formulations tailored to promote specific phenotypes with a high degree of flexibility. The approachis leveraged to identify unique MCP formulations that manufacture CAR T cellproducts from diffuse large B cell patients with increased proportions of memory-like phenotypes MCP-manufactured CAR T cells demonstrate superior anti-tumor efficacy in mouse models of lymphoma and ovarian cancer through enhanced persistence. These findings serve as a proof-of-principle of the powerful utility of the MCP platform to identify "all-in-one" stimulation reagents that can improve the effectiveness of cell therapy products through optimal manufacturing.

Mitochondrial isocitrate dehydrogenase impedes CAR T cell function by restraining antioxidant metabolism and histone acetylation

The efficacy of chimeric antigen receptor (CAR) Tcell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Long-lived memory CAR Tcells are critical for improving tumor clearance and long-term protection. However, during rapid exvivo expansion or invivo tumor eradication, metabolic shifts and inhibitory signals lead to terminal differentiation and exhaustion of CAR Tcells. Through a mitochondria-related compound screening, we find that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances memory CAR Tcell formation and sustains anti-leukemic cytotoxicity invivo. Mechanistically, IDH2 impedes metabolic fitness of CAR Tcells by restraining glucose utilization via the pentose phosphate pathway, which alleviates oxidative stress, particularly in nutrient-restricted conditions. In addition, IDH2 limits cytosolic acetyl-CoA levels to prevent histone acetylation that promotes memory cell formation. In combination with pharmacological IDH2 inhibition, CAR Tcell therapy is demonstrated to have superior efficacy in a pre-clinical model.

CD177 drives the transendothelial migration of Treg cells enriched in human colorectal cancer.

Regulatory T (Treg) cells regulate immunity in autoimmune diseases and cancers. However, immunotherapies that target tumor-infiltrating Treg cells often induce unwanted immune responses and tissue inflammation. Our research focussed on exploring the expression pattern of CD177 in tumor-infiltrating Treg cells with the aim of identifying a potential target that can enhance immunotherapy effectiveness. Single-cell RNA sequencing (scRNA-seq) data and survival data were obtained from public databases. Twenty-one colorectal cancer patient samples, including fresh tumor tissues, peritumoral tissues and peripheral blood mononuclear cells (PBMCs), were analysed using flow cytometry. The transendothelial activity of CD177+ Treg cells was substantiated using in vitro experiments. ScRNA-seq and flow cytometry results indicated that CD177 was exclusively expressed in intratumoral Treg cells. CD177+ Treg cells exhibited greater activation status and expressed elevated Treg cell canonical markers and immune checkpoint molecules than CD177- Treg cells. We further discovered that both intratumoral CD177+ Treg cells and CD177-overexpressing induced Treg (iTreg) cells had lower levels of PD-1 than their CD177- counterparts. Moreover, CD177 overexpression significantly enhanced the transendothelial migration of Treg cells in vitro. These results demonstrated that Treg cells with higher CD177 levels exhibited an enhanced activation status and transendothelial migration capacity. Our findings suggest that CD177 may serve as an immunotherapeutic target and that overexpression of CD177 may improve the efficacy of chimeric antigen receptor T (CAR-T) cell therapy.

CAR-T Cell Therapy in B-Cell Acute Lymphoblastic Leukemia.

Treatment of refractory and relapsed (R/R) B acute lymphoblastic leukemia (B-ALL) is an unmet medical need in both children and adults. Studies carried out in the last two decades have shown that autologous T cells engineered to express a chimeric antigen receptor (CAR-T) represent an effective technique for treating these patients. Antigens expressed on B-cells, such as CD19, CD20, and CD22, represent targets suitable for treating patients with R/R B-ALL. CD19 CAR-T cells induce a high rate (80-90%) of complete remissions in both pediatric and adult R/R B-ALL patients. However, despite this impressive rate of responses, about half of responding patients relapse within 1-2 years after CAR-T cell therapy. Allo-HSCT after CAR-T cell therapy might consolidate the therapeutic efficacy of CAR-T and increase long-term outcomes; however, not all the studies that have adopted allo-HSCT as a consolidative treatment strategy have shown a benefit deriving from transplantation. For B-ALL patients who relapse early after allo-HSCT or those with insufficient T-cell numbers for an autologous approach, using T cells from the original stem cell donor offers the opportunity for the successful generation of CAR-T cells and for an effective therapeutic approach. Finally, recent studies have introduced allogeneic CAR-T cells generated from healthy donors or unmatched, which are opportunely manipulated with gene editing to reduce the risk of immunological incompatibility, with promising therapeutic effects.

Immunotherapy targeting B cells and long-lived plasma cells effectively eliminates pre-existing donor-specific allo-antibodies

Pre-existing anti-human leukocyte antigen (HLA) allo-antibodies constitute a major barrier to transplantation. Current desensitization approaches fail due to ineffective depletion of allo-specific memory B cells (Bmems) and long-lived plasma cells (LLPCs). We evaluate the efficacy of chimeric antigen receptor (CAR) Tcells targeting CD19 and B cell maturation antigen (BCMA) to eliminate allo-antibodies in a skin pre-sensitized murine model of isletallo-transplantation. We find that treatment of allo-sensitized hosts with CAR Tcells targeting Bmems and LLPCs eliminates donor-specific allo-antibodies (DSAs) and mitigates hyperacute rejection of subsequent isletallografts. We then assess the clinical efficacy of the CAR T therapy for desensitization in patients with multiple myeloma (MM) with pre-existing HLA allo-antibodies who were treated with the combination of CART-BCMA and CART-19 (ClinicalTrials.gov: NCT03549442) and observe clinically meaningful allo-antibody reduction. These findings provide logical rationale for clinical evaluation of CAR T-based immunotherapy in highly sensitized candidates to promote successful transplantation.

Efficacy and Safety of Chimeric Antigen Receptor T Cells Therapy Combined with Zanubrutinib in the Treatment of Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Background: While complete response rate of CD19 CAR-T in diffuse large B-cell lymphoma (DLBCL) reached 40-60%, about 30%-50% of patients still face relapse, strategies that can enhance the therapeutic effects of CAR-T cells infusion should be actively explored. Bruton's tyrosine kinase (BTK) is an essential component of multiple signaling pathways including B-cell receptor signaling that regulate B cell proliferation, survival, and functions. These receptor signaling has been implicated in the survival of malignant B-cells making it a promising therapeutic target for various B-cell malignancies and inflammatory diseases. Different types of BTKi regulate T cell and B cell function through different signal pathways. Research has proved that CD19 CAR-T Cells in combination with ibrutinib for the treatment of relapsed/refractory aggressive B-cell lymphomas shows a high response rate and low level of adverse events, while zanubrutinib has not been reported. Therefore, our center explored the efficacy and safety of CD19 CAR-T Cells in combination with zanubrutinib for the strategy of relapsed/refractory DLBCL. Patients and Methods: The study included 6 r/r DLBCL patients, 4 patients have previously received 3-line or above treatment, and 2 patients began CAR-T treatment after 1-line chemotherapy. All patients received fludarabine (FLU) and cyclophosphamide (CTX) conditioning chemotherapy (FLU 30mg/m 2. d1-3; CTX 500mg/m 2, d1-3) before CAR-T infusions. T cells were apheresis collected and transduced with a lentiviral CD19 scFv CAR fused with intracellular signaling domains: CD28/CD27/CD3z-iCasp9. CAR-T cells were infused at dose of 2.0x10 6 cells/kg. The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR-T infusion dose were quantitatively scored and documented. All patients began to combine with zanubrutinib after one month of CAR-T cells infusion. Results: After one month of CAR-T cells infusion, total of 50% (3/6) of patients achieved complete response (CR), while 50%(3/6) of patients were evaluated as partial response (PR), with an overall response rate (ORR) of 100%. Within 6 months after combined with zanubrutinib, all patients of PR achieved CR. In the evaluation of safety, 83% (5/6) of patients occurred grade 2 cytokine release syndrome (CRS), and 33.3% (2/6) patient occurred grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS), no case of severe CRS defined as≥grade 3 occurred, 66.7% (4/6) of patients occurred grade 2 hematotoxicity and 33.3% (2/6) of patients occurred grade 4 hematotoxicity. After a median follow-up of 19.5 months, 6 patients maintained CR, with an duration of response (DOR) of 100%, median DFS and OS were not reached. All patients can be detected CAR-T cells sustain in peripheral blood mononuclear cells continuously until last follow-up date(Fig.2). Continued follow-up will confirm whether the CAR-T cells therapy combined with zanubrutinib can obtain long term overall survival in these study. Conclusions: Our study proved that CD19 CAR-T Cells combined with zanubrutinib for the strategy of relapsed/refractory DLBCL and as maintenance treatment showed strong efficacy with acceptable toxicity, the current results already support that zanubrutinib in the treatment of r/r DLBCL is expected to reduce the risk of relapsed, thereby prolonging the disease-free survival of patients, without increasing the incidence of adverse effects. More follow-up and clinical trials are needed to clarify the efficacy of Chimeric Antigen Receptor T Cells Therapy combined with zanubrutinib in the treatment of r/r DLBCL.

Mitochondrial Isocitrate Dehydrogenase Inhibition Enhances CAR T-Cell Function By Restraining Antioxidant Metabolism and Histone Acetylation

Introduction: The efficacy of chimeric antigen receptor (CAR) T-cell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Mitochondria are vital for the regulation of memory T-cell formation or exhaustion. A mitochondria-related compound screening was performed and we found that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances long-lived memory CAR T-cell formation, and improves tumor clearance in vivo. IDH2 as the key component of the tricarboxylic acid (TCA) cycle, we hypothesized that IDH2 inhibition might promote CAR T-cell persistence by reprogramming the metabolism. The study aims to systematically evaluate the role and the mechanism of IDH2 inhibition on CAR T cells, and explore the application of enasidenib in CAR T-cell therapy. Methods and Results: To identify mitochondrial components that affect the long-term efficacy of CAR T-cells, we performed an in vitro screening using a mitochondria-related compound library based on the enrichment of the CD62L + CAR T-cell subset, which mainly contains T memory stem cells (T SCM) and central memory T cells (T CM). Among several candidate compounds that promoted memory CAR T-cell formation, enasidenib (ENA), an inhibitor of both wild-type and mutant IDH2 enzymes, was the most effective one. The proportion of CD62L has increased by over 20%. To determine the effect of ENA on exhaustion induced by tonic CAR signaling and tumor antigen stimulation, we measured the expression of inhibitory receptors PD-1, TIM-3, and LAG-3 and the level of apoptosis in freshly expanded CAR T cells and B-ALL encountered CAR T cells. CAR T cells treated with ENA exhibited reduced surface levels of those inhibitory receptors. We measured the production of granzyme B and IFNγ, and tumor rechallenge assay in CAR T cells after ENA treatment to assess the effector function, ENA treated CAR T cells exerted enhanced and sustained cytotoxicity in a tumor rechallenge assay at an extreme E:T ratio of 1:10. These results were observed not only in CD19-41BBζ CAR T cells, but also in CD19-28ζ and GD2-28ζ CAR T cells. CAR T cells expanded in the presence of ENA significantly prolonged survival of recipients after Nalm-6 infusion (p < 0.0001), extended in vivo ENA treatment further enhanced CAR T-cell expansion and tumor suppression. The median survival was extended from 45 to 67.5 days. Consistent with the effect of ENA treatment, IDH2 knockdown increased the proportions of the T scm/T n and T cm subsets, and alleviated CAR T-cell exhaustion. IDH2-knockdown CAR T cells had sustained cytotoxicity and higher CD62L expression after rounds of killing B-ALL cells in vitro. To systematically explore the effects of IDH2 inhibition on the metabolism of CAR T cells, relative metabolite amounts was measured using mass spectrometry (MS). IDH2-inhibited CAR T cells exhibited a substantial reduction of metabolites in the TCA cycle, including succinate, fumarate, and malate, and in the glycolysis pathway, including lactate, phosphoenolpyruvate. Mechanistically, IDH2 inhibition reprogram central carbon metabolism of CAR T cells by redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway. In addition, IDH2 limits cytosolic acetyl-CoA level to prevent histone acetylation that promotes memory cell formation. Significance: Our study indicates that metabolic intervention in CAR T cells with the FDA-approved IDH2 inhibitor enasidenib can advance current treatment, with better tumor eradication and CAR T-cell persistence.

Senza5 TM CART5: An Autologous CD5-Deleted Anti-CD5 CART Product with Enhanced Anti-T-Cell Lymphoma Activity

Introduction: Treatment with adoptive T cell therapies expressing Chimeric Antigen Receptors (CART) produces unprecedented clinical responses in relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma. Despite the transformative potential of these therapies for cancer, most patients treated with CART therapy experience incomplete responses or eventual relapse. Moreover, CART therapy is not yet effective for solid cancers or other hematological malignancies such as T-cell neoplasms. Thus, there is a dire need to enhance CART efficacy and extend this approach to more cancer types. We previously presented preliminary preclinical results of CD5-directed CAR-T cells (CART5) that had CD5 knocked out (KO) [ Blood (2020) 136 (Supplement 1): 51-52]. CD5 KO not only prevented CART fratricide but also dramatically enhanced CART function in multiple in vivo models. Enhanced CART function reflected disinhibition of early T-cell activation by abrogating CD5 signaling. Here, we combine the functional advantage of CD5 KO CART5 with a clinical-grade “rapid” 5-day manufacturing process, characterize the resulting drug product, and evaluate its in vivo antitumor efficacy in a human xenograft model of T-cell lymphoma/leukemia. Methods and Results: Using normal healthy donor T cells, we developed a proprietary, “rapid” 5-day GMP-validated manufacturing process for CD5 KO CART5, completing the process in 5 days (CART5 rapid) instead of the conventional 14+ days (CART5 conv). At the end of manufacturing, the two products possessed similar ratios of CD8 + and CD4 + T cells, CD5 editing, and CAR5 expression. The CART5 rapid product did not exhibit any genetic or functional abnormalities characteristic of tumorigenic risk, demonstrated by low off-target gene editing in low-risk genes, no in vitro soft agar colony formation, and normal karyotypes. Importantly, the CART5 rapid manufacturing process did not skew TCRβ profiles, indicating that the CART5 rapid product retains a diverse T-cell repertoire. CD5 KO CART5 rapid cells possessed high specificity for T-cell leukemia/lymphoma, reflected by the absence of recognition of ex vivo normal human cells (cardiomyocytes, hepatocytes, neurons, monocytes, etc). Interestingly, CD5 KO CART5 rapid cells possessed a more central memory-like phenotype than CD5 KO CART5 conv cells which has been correlated with improved clinical efficacy. To compare their antitumor efficacy, we performed an in vivo experiment with CD5 + Jurkat cells utilizing a suboptimal dose of CART5 cells. Immunodeficient NSG mice were engrafted with 10 6 Jurkat cells on day -7 and treated intravenously on day 0 with a suboptimal dose (0.5x10 6 CAR + cells) of CD5 KO CART5 rapid or CD5 KO CART5 conv cells. Controls received an equivalent total number of Mock KO UTD conv. Mice treated with CD5 KO CART5 rapid cells demonstrated superior antitumor responses compared to CD5 KO CART5 conv. Improved tumor control correlated with dramatically increased overall survival of mice treated with CD5 KO CART5 rapid (median overall survival Mock KO UTD = 21 days; CD5 KO CART5 conv = 44 days vs. CD5 KO CART5 rapid = not reached, p=0.0019, Mantel-Cox; Figure 1). Conclusion: This novel CD5 KO anti-CD5 CART product manufactured in a 5-day GMP process shows high specificity and significantly enhanced antitumor efficacy resulting from the synergistic effect of improved early T-cell activation due to CD5 deletion and preservation of T-cell memory phenotypes conferred by a novel, 5-day manufacturing processes. Autologous Senza5 TM CART5 for the treatment of CD5 + nodal T-cell lymphomas is expected to enter the clinic in a Phase I study later this year.

CD8+ chimeric antigen receptor T cells manufactured in absence of CD4+ cells exhibit hypofunctional phenotype

BackgroundCell culture conditions during manufacturing can impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products. Production methods have not been standardized because the optimal approach remains unknown....

Recombinant oncolytic adenovirus armed with CCL5, IL-12, and IFN-γ promotes CAR-T infiltration and proliferation in vivo to eradicate local and distal tumors

The efficacy of chimeric antigen receptor T (CAR-T) cells for solid tumors remains unsatisfactory due to the limited tumor infiltration and immunosuppressive microenvironment. To overcome these limitations, the genetically engineered recombinant oncolytic adenoviruses (OAVs) that conditionally replicate in tumor cells were developed to modify the tumor microenvironment (TME) to facilitate CAR-T-mediated tumor eradication. Here in the present study, a novel recombinant OAV carrying CCL5, IL12, and IFN-γ controlled by Ki67 promoter was constructed (named AdKi67-C3). The antitumor activity of AdKi67-C3 was tested in vitro and in vivo by using mono administration or combing with CAR-T cells targeting B7H3. It proved that CCL5 expressed by AdKi67-C3 indeed induced more CAR-T migration in vitro and CAR-T infiltration in tumor mass in vivo. Meanwhile, cytokines of IFN-γ and IL12 secreted by AdKi67-C3-infected tumor cells significantly promoted proliferation and persistence of CAR-T cells in vitro and in vivo. In tumor-bearing xenograft mouse models of kidney, prostate or pancreatic cancer, local pretreatment with AdKi67-C3 dramatically enhanced CAR-T cell efficacy and eliminated local and distant tumors. More importantly, mice achieving complete tumor regression resisted to re-challenge with the same tumor cells, suggesting establishment of long-term antitumor immune response. Therefore, OAVs armored with cytokines could be developed as a bioenhancer to defeat the immunosuppressive microenvironment and improve therapeutic efficacy of CAR-T in solid tumors.