Chimeric Antigen Receptor T-cell Activity Research Articles

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFβ-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma

AbstractHematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies and is particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we analyzed a cohort of 48 patients who were treated with B-cell maturation agent (BCMA) CAR-Ts to characterize the kinetics of cytopenia, identify predictive factors, and determine potential mechanism that underlie these toxicities. The overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, and was still present after 1 year in 4 and 3 patients, respectively. The presence of cytopenia at baseline and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms that underpin cytopenias, we evaluated the paracrine effect of BCMA CAR-Ts on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-Ts (spCARs) halted HSPC differentiation, thereby promoting more immature phenotypes with a reduction in the expression of granulocytic, monocytic, and erythroid markers, which could be prevented with a combination of interferon gamma, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with the early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). Our results suggest that CAR-T activation negatively influences hematopoietic differentiation through paracrine effects, thereby inducing HSPCs maturation arrest. Moreover, our study contributes to the understanding of severe cytopenia observed after CAR-T therapy in MM and provides potential treatments to prevent or decrease its severity.

T-cell dysfunction in CLL is mediated through expression of Siglec-10 ligands CD24 and CD52 on CLL cells

AbstractAutologous T-cell–based therapies, such as chimeric antigen receptor (CAR) T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) and correlate with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aimed to investigate the mechanisms through which CLL cells suppress CAR T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion, and specifically that restimulation of CAR T cells in the presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, coculture of T cells with CD40-activated CLL cells did not lead to T-cell dysfunction, and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40 signaling cascade revealed that the Spare Respiratory Capacity (SRC) kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52, which was prevented by dasatinib, suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced CAR T-cell dysfunction upon coculture with resting CLL cells. These results demonstrated that T cells derived from CLL patients can be reinvigorated by manipulating CLL–T-cell interactions. Targeting CD24- and CD52-mediated CLL–T-cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL.

Tuning CAR T-cell therapies for efficacy and reduced toxicity

Chimeric antigen receptor (CAR) T-cell therapies are a standard of care for certain relapsed or refractory B-cell cancers. However, many patients do not respond to CAR T-cell therapy or relapse later, short- and long-term toxicities are common, and current CAR T-cell therapies have limited efficacy for solid cancers. The gene engineering inherent in CAR T-cell manufacture offers an unprecedented opportunity to control cellular characteristics and design products that may overcome these limitations. This review summarises available methods to “tune” CAR T-cells for optimal efficacy and safety. The components of a typical CAR, and the modifications that can influence CAR T-cell function are discussed. Methods of engineering passive, inducible or autonomous control mechanisms into CAR T-cells, allowing selective limitation or enhancement of CAR T-cell activity are reviewed. The impact of manufacturing processes on CAR T-cell function are considered, including methods of limiting CAR T-cell terminal differentiation and exhaustion, and the use of specific T-cell subsets as the CAR T starting material. We discuss the use of multicistronic transgenes and multiplexed gene editing. Finally, we highlight the need for innovative clinical trial designs if we are to make the most of the opportunities offered by CAR T-cell therapies.

CD28 costimulation augments CAR signaling in NK cells via the LCK/CD3Z/ZAP70 signaling axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3Z creates a platform that recruits critical kinases, such as LCK and ZAP70, initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy.

EPEN-02. CAR T-CELLS TARGETED TO B7-H3 OR HER2 FUNCTION INDEPENDENTLY OF ANTIGEN DENSITY IN XENOGRAFT MODELS OF EPENDYMOMA

Abstract BACKGROUND Targeted treatment options are desperately needed for ependymomas (EPN). Chimeric antigen receptor (CAR) T-cells have immense potential to positively transform treatment outcomes; however, little is known regarding antitumor efficacy of CAR T-cells against EPNs. Specifically, antigen density has emerged as a major influencer of CAR T-cell potency. However, the specific high and low antigen density thresholds governing CAR T-cell efficacy for EPN are unknown. We hypothesize that antigen density heterogeneity governs CAR T-cell potency against EPN. METHODS Using quantitative flow cytometry, we determined antigen density of B7-H3 and HER2 (the two CAR targets currently in pediatric EPN clinical trials) on EPN patient derived cell lines (1425, ST1, CPITT, ST2, EPI, L46SJ). We generated second generation B7-H3 and HER2-CAR T-cells with CD28z signaling domain and compared long-term cytolytic activity, expansion, and cytokine production against EPNs. RESULTS We found that all EPN cell lines express remarkably high yet variable density of B7-H3 (6.3e4 – 3.4e5 molecules/ cell). In contrast, surface density of HER2 was consistently and significantly lower (860 – 2.5e4 molecules/ cell). In repeated-stimulation assays, B7-H3-CAR T-cells killed 7-10 times and expanded up to 325,360-fold when cultured against EPN. However, we observed no significant difference in total expansion or correlation with antigen density against EPNs. Surprisingly, expansion of HER2-CAR T-cells against EPNs was drastically higher (up to over 100-billion-fold) and greatly exceeded that of B7-H3-CAR-T cells against the same EPN cells despite much lower antigen density. CONCLUSIONS Our studies reveal significant antigen density heterogeneity of B7-H3 and HER2 in EPN, which may lead to target-specific impacts on CAR T-cell persistence. Indeed, results suggest that lower antigen density of HER2 may be superior for sustained CAR T-cell activity. Future work will validate whether HER-CAR T-cells have superior antitumor efficacy in vivo to further delineate role of antigen density thresholds in CAR T-cell potency.

MiR-34a promotes the immunosuppressive function of multiple myeloma-associated macrophages by dampening the TLR-9 signaling.

Promising outcomes have been observed in multiple myeloma (MM) with the use of immunotherapies, specifically chimeric antigen receptor T (CAR-T) cell therapy. However, a portion of MM patients do not respond to CAR-T therapy, and the reasons for this lack of response remain unclear. The objective of this study was to investigate the impact of miR-34a on the immunosuppressive polarization of macrophages obtained from MM patients. The levels of miR-34a and TLR9 (Toll-like receptor 9) were examined in macrophages obtained from both healthy individuals and patients with MM. ELISA was employed to investigate the cytokine profiles of the macrophage samples. Co-culture experiments were conducted to evaluate the immunomodulatory impact of MM-associated macrophages on CAR-T cells. There was an observed suppressed activation of macrophages and CD4+ T lymphocytes in the blood samples of MM patients. Overexpression of miR-34a in MM-associated macrophages dampened the TLR9 expression and impaired the inflammatory polarization. In both the co-culture system and an animal model, MM-associated macrophages suppressed the activity and tumoricidal effect of CAR-T cells in a miR-34a-dependent manner. The findings imply that targeting the macrophage miR-34a/TLR9 axis could potentially alleviate the immunosuppression associated with CAR-T therapy in MM patients.

EVEREST-2: A seamless phase 1/2 study of A2B694, a mesothelin (MSLN) logic-gated Tmod CAR T-cell therapy, in patients with solid tumors that show MSLN expression and human leukocyte antigen (HLA)-A*02 loss of heterozygosity (LOH).

TPS2699 Background: Despite the success in hematologic cancers, chimeric antigen receptor (CAR) T-cell therapies are challenging to implement in solid tumors owing to a lack of tumor-specific targets that discriminate cancer from normal cells. MSLN expression normally is limited to the mesothelium of major body cavities but can be upregulated in diverse solid tumor types (TCGA 2022), making it a potential target for cancer therapy. MSLN-targeted cell approaches, including CAR T-cell and T-cell receptor fusion therapies, have shown promising clinical activity; however, on-target, off-tumor toxicity including fatal events have occurred (1-3). A2B694 is an autologous logic-gated, MSLN-targeted Tmod CAR T-cell therapy that addresses the challenges of on-target, off-tumor toxicity by combining 2 CARs: an activating and blocking receptor. The activator recognizes MSLN present on the surface of both tumor and normal cells; the blocker binds HLA-A*02 and prevents CAR T-cell activity. Thus, in patients with both germline HLA-A*02 and tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (4). Through this unique discriminatory mechanism, A2B694 may provide a therapeutic window to treat patients with MSLN-expressing solid tumors exhibiting HLA-A*02 LOH. Methods: EVEREST-2 (NCT06051695) is a first-in-human, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B694 in adults with recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression, including non-small cell lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, mesothelioma, or other solid tumors with MSLN expression. Eligible patients should have received ≥1 line of prior therapy such as checkpoint inhibitor, molecular targeted, or chemotherapy. Enrollment to EVEREST-2 occurs through the prescreening study BASECAMP-1 (NCT04981119), which identifies patients with tumor-associated HLA-A*02 LOH via next-generation sequencing (Tempus AI, Inc.). Eligible patients enroll in the BASECAMP-1 study and undergo leukapheresis. A2B694 is manufactured from cryopreserved T cells when clinically appropriate for patients. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B694 and identify the recommended phase 2 dose (RP2D). The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B694. Phase 2 will assess overall response rate per RECIST v1.1. 1. Beatty, et al. Gastroenterology. 2018. 2. Haas, et al. Mol Ther. 2023. 3. Hong, et al. ESMO 2021. Abstract 9590. 4. Hamburger, et al. Mol Immunol. 2020. Clinical trial information: NCT06051695 .

Prolonged cytopenias after immune effector cell therapy and lymphodepletion in patients with leukemia, lymphoma and solid tumors

Background aimsThe success of chimeric antigen receptor (CAR) T-cell therapy in treating B-cell malignancies has led to the evaluation of CAR T-cells targeting a variety of other malignancies. Although the efficacy of CAR T-cells is enhanced when administered post-lymphodepleting chemotherapy, this can trigger bone marrow suppression and sustained cytopenia after CD19.CAR T-cell therapy. Additionally, systemic inflammation associated with CAR T-cell activity may contribute to myelosuppression. Cytopenias, such as neutropenia and thrombocytopenia, elevate the risk of severe infections and bleeding, respectively. However, data on the incidence of prolonged cytopenias after immune effector therapy in the solid tumor context remain limited. ObjectiveWe compared the incidence of prolonged cytopenias after immune effector therapy including genetically modified T-cells, virus-specific T-cells (VSTs) and NKT-cells, as well non-gene-modified VSTs for leukemia, lymphoma, and solid tumors (ST) to identify associated risk factors. MethodsA retrospective analysis was conducted of 112 pediatric and adult patients with relapsed and/or refractory cancers who received lymphodepleting chemotherapy followed by immune effector therapy. Patients treated with 13 distinct immune effector cell therapies through 11 single-center clinical trials and 2 commercial products over a 6-year period were categorized into 3 types of malignancies: leukemia, lymphoma and ST. We obtained baseline patient characteristics and adverse events data for each participant, and tracked neutrophil and platelet counts following lymphodepletion. ResultsOf 112 patients, 104 (92.9%) experienced cytopenias and 88 (79%) experienced severe cytopenias. Patients with leukemia experienced significantly longer durations of severe neutropenia (median duration of 14 days) compared with patients with lymphoma (7 days) or ST (11 days) (P = 0.002). Patients with leukemia also had a higher incidence of severe thrombocytopenia (74.1%), compared with lymphoma (46%, P = 0.03) and ST (14.3%, P < 0.0001). Prolonged cytopenias were significantly associated with disease type (63% of patients with leukemia, 44% of patients with lymphoma, and 22.9% of patients with ST, P = 0.006), prior hematopoietic stem cell transplant (HSCT) (66.7% with prior HSCT versus 38.3% without prior HSCT, P = 0.039), and development of immune effector cell-associated neurotoxicity syndrome (ICANS) (75% with ICANS versus 38% without ICANS, P = 0.027). There was no significant association between prolonged cytopenias and cytokine release syndrome. ConclusionsImmune effector recipients often experience significant cytopenias due to marrow suppression following lymphodepletion regardless of disease, but prolonged severe cytopenias are significantly less common after treatment of patients with lymphoma and solid tumors.

Abstract 4002: Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as a promising approach for treating hematological malignancies, particularly B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to delve into the activation dynamics and therapeutic efficacy of CD19 CAR-T cells in a disseminated NALM6-luc human B-ALL murine model. Experimental design: NALM6-Luc-mCh-Puro cells were implanted intravenously in female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Animals were treated with CD19 CAR-T cells alone and in combination with ibrutinib. The anti-tumor activity was assessed, and the persistence and activation of CAR-T cells in the blood were evaluated using flow cytometry. Additionally, ddPCR analysis was employed to quantify CAR copy numbers, providing insights into the T-cell persistence over time. Results: Treatment with CD19 CAR-T cells exhibited notable anti-cancer activity and that activity was enhanced in combination with ibrutinib, in line with other reports. The increased time to progression was noted as 69.2% for the CAR-T group and 138.4% for the combination of CAR-T and ibrutinib compared to control. Flow cytometry analysis showed that CAR-T cells persisted in the blood throughout the study. The mean CAR-T absolute cell count for both CAR-T and CAR-T plus ibrutinib treated groups increased from 2 cell/µL blood on Day 0 to 11 and 51 cells/µL of blood, respectively, by Day 39. The progressive increase of PD-1+ and LAG-3+ percentage cells correlates positively with tumor burden, indicating a dynamic link between biomarker upregulation and the presence of tumor cells in mice. ddPCR analysis revealed an initial CAR copy decrease in all groups, followed by gradual increase in tumor-bearing mice treated with CD19 CAR-T cells, with a dramatic increase observed by Day 39. Results from CAR-T treated and CAR-T plus ibrutinib treated groups were comparable, showing mean copy numbers per ng of DNA increasing from less than 1 on day 4 to 17.9 and 23.3, respectively, on day 39. Importantly, no changes were noted in non-tumor implanted mice, emphasizing the tumor dependency for CAR-T cell expansion. Conclusion: This study emphasizes the marked anti-cancer activity of CD19 CAR-T cells in the NALM6-Luc-mCh-Puro B-ALL murine model. Comprehensive analysis of CAR-T cell persistence and activation, including insights from ddPCR, provides a robust understanding of therapeutic response dynamics. The observed in vivo anti-cancer efficacy aligns with the persistence of CAR-T cells revealed by flow cytometry and the dynamic expansion captured through ddPCR analysis. Our results indicate ddPCR and flow cytometry as complementary and independent methods for assessing the systemic CAR-T cell persistence, contributing to optimizing therapy for B-cell acute lymphoblastic leukemia. Citation Format: Prashant Pandey, David Draper, Sheri Barnes, Yewei Xing, Derrik Germain, Lauren Kucharczyk, Roys Stacey. Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model [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 4002.

Abstract 3993: A tunable safety switch for solid tumor CAR T-cell therapy

Abstract On-target off-tumor toxicity, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS) are severe immune-related adverse events (irAEs) that are frequently associated with Chimeric Antigen Receptor (CAR) T-cell therapy. Current efforts to manage such therapy-related toxicities involve incorporation of an inducible suicide agent within CAR constructs, such as iCaspase-9 or herpes simplex virus type 1 thymidine kinase that can be selectively activated to produce toxic effects within CAR T cells and attenuate their activity. However, while activation of these agents helps to mitigate or overcome such unwarranted toxicities, the therapeutic benefit of CAR T cells anti-tumor activity is also compromised. Therefore, to continue maintenance of CAR T cells’ therapeutic function while minimizing irAEs, an ideal safety switch should 1) rapidly inhibit the activation and proliferation of CAR T cells exposed to the target antigen, 2) reversibly inhibit activity without inducing CAR T cell elimination and 3) be clinically translatable for safe application in patients. Our laboratory investigated one such safety switch to inhibit CAR T-cell activity while maintaining their therapeutic function. We showed that incorporating a mutant variant of c-KIT D816V (KITv) in the intracellular domain of mesothelin-targeting second-generation CAR T cells (M28z-KITv) improved efficacy in solid tumors with low antigen, or an immunosuppressive environment. Herein, we evaluate the use of Dasatinib, a clinically available multitarget (BCR, SRC, c-KIT) tyrosine kinase inhibitor (TKI), as a tunable safety switch to reversibly inhibit M28z-KITv CAR T-cell functional activity. In cohorts of mice established with lung adenocarcinoma, daily administration of Dasatinib starting on day 1 or day 3 after CAR T-cell administration stabilized tumor growth, which otherwise continued to regress in untreated mice, indicating inhibition of CAR T-cell functional activity. Upon discontinuation of Dasatinib, tumors regressed, indicating reversal of CAR T-cell functional activity. In an experiment conducted to investigate functional persistence of CAR T cells upon long-term exposure to Dasatinib (1 month), we noted uninhibited activity of CAR T cells to rechallenged tumors. Dasatinib, thus may act as a tunable safety switch to regulate M28z-KITv CAR T-cell activity without compromising its therapeutic function. Citation Format: Kyohei Misawa, Meriem Taleb, Srijita Banerjee, William-Ray Vista, Navin K. Chintala, Prasad S. Adusumilli. A tunable safety switch for solid tumor 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 3993.

Efficient Combinatorial Adaptor-Mediated Targeting of Acute Myeloid Leukemia with CAR T-Cells

Introduction: Chimeric Antigen Receptor (CAR) T-cell therapies are remarkably efficient in treating B- and plasma-cell malignancies by targeting cell-of-origin antigens, eradicating both tumor cells as well as healthy B- and plasma-cell counterparts. Their loss can be compensated by immunoglobulin substitution until they regenerate from hematopoietic stem and progenitor cells (HSPCs). However, similar on-target, off-tumor cell-of-origin elimination would be detrimental in the context of HSPC-derived malignancies, such as acute myeloid leukemia (AML) or myelodysplastic neoplasia (MDS), as the CAR T-cell activity might lead to terminal ablation of hematopoiesis. In principle, CAR T-cells could be used to abrogate both, AML cells as well as HSPCs, followed by HSPC transplantation, ensuring hematopoietic recovery after termination of CAR T-cell activity and possibly without genotoxic preconditioning. This approach could be particularly beneficial for patients unable to tolerate radiation and high-dose chemotherapy. In order to address this challenge, we developed adaptor-mediated CAR T-cells displaying the single-chain variable fragment E2, AdFITC(E2), that binds to fluorescein conjugated to antigen-binding adaptors in diabody format (Db-FM). Such tagged adaptors, used as single linkers or in combination, would then act as safety switch, modulating CAR T-cell activation, proliferation, and lytic activity. Results: We generated second-generation AdFITC(E2)-CAR T-cells and multiple diabody-based adaptors against selected AML antigens. We then tested the diabodies targeting CD33 and CD117 with respect to their ability to mediate CAR T-cell biocidal activity as single adaptors or in combination. Combinatorial staining with CD117 Db-FM and CD33 Db-FM, each added at saturating concentration for the cognate antigen, resulted in higher fluorescein decoration of target cells, with more than two-fold increased fluorescence intensity detected by flow cytometry upon combined staining, thus enhancing AdFITC(E2)-CAR T-cell target density. In vitro cytotoxicity assays of healthy-donor-derived AdFITC(E2)-CAR T-cells, both against cell lines (MOLM14-CD117 +GFP +Luc +) as well as patient-derived AML blasts, revealed that dual adaptor use significantly improved tumor cell lysis compared to equimolar concentration of single adaptors. To further investigate the basis for increased killing by dual targeting, we performed live-cell imaging of effector-target cell interactions in microwells. The addition of both CD117 and CD33 Db-FM to the co-culture of MOLM14-CD117 +GFP +Luc + and AdFITC(E2)-CAR T-cells resulted in faster tumor cell lysis (measured by influx of PI) which correlated with a more rapid, durable engagement time of CAR T-cells with target cells. We next tested CAR T-cell biocidal activity in therapeutic xenogeneic mouse models engrafted with MOLM14-CD117 +GFP +Luc + where AdFITC(E2)-CAR T-cells in combination with CD117 or CD33 Db-FM administered as single adaptors were as efficient as direct CAR T-cells against the same antigens. Injection of AdFITC(E2)-CAR T-cells and both CD117 and CD33 Db-FM effectively inhibited tumor growth, outperforming monotherapies, and leading in some cases to full AML elimination in bone marrow and blood as shown by bioluminescence and terminal flow cytometry analysis (Figure 1). Conclusions: We here tested an adaptor CAR T-cell approach using multiple adaptors to on-off modulate and enhance CAR T-cell activity, focusing on AML as a relevant disease model with high clinical need for therapeutic improvement. The high heterogeneity in antigen expression on AML cells supports a combinatorial targeting strategy, individualized based on the respective AML immunophenotype. Due to their relatively small molecular weight (~55kDa), diabody-based adaptors are rapidly cleared from the body via the kidneys allowing for rapid control over AdFITC-CAR T-cell on-off activity prior to HSPC transplantation. We envision that this approach has the clinical potential to improve CAR T-cell safety profiles by enhancing the specificity towards target cells, while ameliorating the side-effects against healthy tissues expressing only one antigen of the combination.

Determinants of Response to Anti CD-19 CAR-T Cells for Diffuse Large B-Cell Lymphoma in Pre-Treatment Peripheral Blood Mononuclear Cells Using Single-Cell RNA-Seq

Background: Chimeric antigen receptor T (CAR-T) cell therapy has become the standard of care in patients with relapsed/refractory diffuse large cell lymphoma (R/R DLBCL), providing durable remission in 40% of patients. Lack of response is likely to be attributed to inter- and intra-patient variability, including PBMC heterogeneity and apheresis material composition, CAR-T infusion product, in-vivo expansion and activity of CAR-T cells and the development of tumor-intrinsic mechanisms. Current methods for evaluating CAR-T cell activation and potency have limited predictive value for clinical outcomes. To address this challenge, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from patients with R/R LBCL patients at the time of apheresis for the manufacturing of anti-CD19 CAR-T cell therapy. Methods: Between 10/2020 and 03/2022, 31 patients who underwent lymphopheresis were enrolled. Samples were obtained - 1) At day of lymphopheresis from peripheral blood; 2) On the day of infusion of CAR-T from the product; and 3) On day +7 after infusion from peripheral blood. All patients had day +30 PET-CT. In addition, PBMCs samples from healthy donors were used as control to analyze baseline characteristics compared to the DLBCL patients. PBMCs from patients of healthy donors were purified from frozen blood samples by density gradient separation, stained using human CD45 antibody and loaded onto a 10x Chromium system. libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit. The data was processed and analyzed using the Seurat R package. The study was approved by the local Ethic committee. Results: Patient characteristics: 31 RR DLBCL patients (42% females; median age 72, range 47-81 years), all treated with CAR-T cells therapy as third line of therapy, participated in the study. All patients underwent lymphopheresis, followed by CAR-T production. Disease status at lymphodepletion was complete remission (CR, n=4, 13%), partial remission (n=6, 19%), and progressive disease (n=21, 68%) and patients received either tisagenlecleucel (n=20, 65%) or axicabtagene ciloleucel (n=11, 35%) . PET scan performed on day 30 post treatment revealed that 68% and 32% obtained CR, and PD, respectively. scRNA analysis revealed differences in PBMC composition between healthy donors and LBCL patients (Figure 1A), with lower levels of naïve and memory T cells and elevated levels of activated monocytes in LBCL patients, suggesting a shift towards an inflamed and pre-dysfunctional immune state. Categorization of LBCL patients into responders (PR/CR on day +30 PET-CT) and non-responders (PD on day +30 PET-CT) revealed differences in immune cell subsets between responders and non-responders (Figure 2A), with enriched inflammation-related pathways in non-responders, In contrast, responders exhibited a distinct healthy-like immune profiles, particularly high levels of naive T cells, similar to that obtained in their age matched healthy controls. Of note, non-responders showed alterations in immune cell subsets, including increased levels of T regulatory cells and effector NK cells. Furthermore, non-responders demonstrated a prominent inflammatory immune profile, characterized by gene pathways related to inflammation and activation, such as type I and III IFN-G, and TNFb response (Figure 1B). Notably, an IFN-G score was significantly higher in non-responder's monocytes subpopulations, compared to responders. Conclusions: These findings indicate that the success of CAR-T therapy could be determined by the molecular characteristics and subpopulation distribution of each patient's PBMC. The diverse immune profiles of DLBCL patients might impact the effectiveness of their immune cells in generating successful CAR-T products or providing a conducive environment for CAR T cells to combat cancer. Furthermore, these findings emphasize the potential of scRNA-seq in evaluating PBMC from CAR-T patients prior to treatment, allowing the potential calculation of an “immune potency score” associated with the probability of a positive outcome from CAR-T treatment of DLBCL patients. This score could potentially be used by oncologists in decision-making processes to determine the appropriate candidates for CAR-T therapy.

Advancing CAR-T Therapy in Acute Lymphoblastic Leukemia: Multi-Omic Analyses of CD19-Directed CAR-T Cells Enabled By an Ex Vivo Co-Culture Platform

Background: Acute Lymphoblastic Leukemia (ALL) has witnessed remarkable progress in the treatment of patients with Chimeric Antigen Receptor T-cell (CAR-T) therapies, leading to high response rates and durable remissions. However, challenges persist for patients who do not respond to CAR-T treatment, experience relapse, or encounter adverse events such as cytokine release syndrome. Overcoming these challenges necessitates a deeper understanding of mechanisms and the development of advanced technologies and assays. This study focuses on harnessing the potential of an innovative microfluidic platform for high-throughput cell-based assays, enabling multi-omics and functional analysis using a minimal amount of primary patient cells. By utilizing this platform, we aim to gain insight into the dynamics of primary patient material by evaluating responses to aid in selection of promising CAR-T enhancements or combination therapies designed to overcome mechanisms of CAR-T failure or relapse to improve clinical outcomes. Methods: For the cytotoxicity assay, we utilized autologous or allogeneic pairs of CD19-directed CAR-T cells and T-cell negative fractions from ALL patients' blood apheresis samples, containing CD19-expressing cancer cells. A negative control was established using untransduced T-cells. To ensure high cell viability and purity, the cells underwent careful processing and were labeled with long-lived dyes for tracking. Multiple cell suspensions with precise Effector to Target to Negative Fraction ratios were prepared. Only a few thousand cells from each coculture suspension were seeded in microfluidic culture chambers, which required no fluidic control system. Experiments were conducted for up to 96 hours with media replacement every 24 hours. At each endpoint, media was collected for cytokine quantification, and cells were stained for live/dead counting as well as quantification of intracellular or surface marker expression by imaging. After image acquisition, cells were collected for RNA extraction and gene expression analysis. Results: Results demonstrated that the developed co-culture platform allows precise control over culture conditions, ensuring culture of accurate ratios of highly complex cell populations. The viability of primary cells in the device guaranteed functional relevance of the cytotoxicity assays for up to a 96-hour timepoint. Strong CAR-T-mediated cell killing was observed, with a dose-dependent effect depending on the co-culture's relative CAR-T content. Less than 50% cell survival was observed in cocultures with Effector to Target ratio 1:1, as compared to the untransduced T-cell control. Furthermore, strong CAR-T activation was observed with over 60% of CART expressing CD25 and CD69 surface markers, as measured by imaging. A multi-color staining panel allowed for precise monitoring of changes over time within the assay of immune cell populations, including cancer cells, monocytes/macrophages, and NK cells, among others. Cytokine analysis revealed an increased secretion of IL-2, TNFa, IFNg, and Granzyme B in coculture with CAR-T cells compared to untransduced T-cells, with a dose-dependent effect based on the CAR-T cell ratio. Finally, RNA isolated from remaining cells at the end of the assay was of high quality with yields sufficient to enable transcriptome analysis for each treatment condition. Discussion: This study contributes to the development of innovative multiplexed high throughput strategies for in vitro functionality assessments in cell therapy, including combination studies and deeper mechanistic understandings. The platform's ability to generate fully integrated, multi-omics readings from complex allogeneic and autologous cocultures of CAR-T and patient cells offers a new frontier in early discovery and translational cancer research. This opens new possibilities for drug discovery, drug screening, and disease modeling, ultimately advancing our understanding of complex biological processes and accelerating therapeutic development. Future directions include exploring mechanisms of resistance and sensitivity, including donor-specific effector cell functionality, combination studies, impacts of immunosuppressive cell types, patient heterogeneity of target antigen expression or other co-stimulatory molecules, and evaluation of product potency or fitness of incoming apheresis material.

A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy

Chimeric antigen receptor (CAR) Tcell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR Tcells undergo dynamic expansion and contraction invivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and Tcell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression invivo and demonstrate imaging of the cells with TMP radiotracers. Invitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Exploiting the CD200-CD200R immune checkpoint axis in multiple myeloma to enhance CAR T-cell therapy

AbstractPatients with multiple myeloma (MM) treated with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells usually relapse with BCMA+ disease, indicative of CAR T-cell suppression. CD200 is an immune checkpoint that is overexpressed on aberrant plasma cells (aPCs) in MM and is an independent negative prognostic factor for survival. However, CD200 is not present on MM cell lines, a potential limitation of current preclinical models. We engineered MM cell lines to express CD200 at levels equivalent to those found on aPCs in MM and show that these are sufficient to suppress clinical-stage CAR T-cells targeting BCMA or the Tn glycoform of mucin 1 (TnMUC1), costimulated by 4-1BB and CD2, respectively. To prevent CD200-mediated suppression of CAR T cells, we compared CRISPR-Cas9–mediated knockout of the CD200 receptor (CD200RKO), to coexpression of versions of the CD200 receptor that were nonsignaling, that is, dominant negative (CD200RDN), or that leveraged the CD200 signal to provide CD28 costimulation (CD200R-CD28 switch). We found that the CD200R-CD28 switch potently enhanced the polyfunctionality of CAR T cells, and improved cytotoxicity, proliferative capacity, CAR T-cell metabolism, and performance in a chronic antigen exposure assay. CD200RDN provided modest benefits, but surprisingly, the CD200RKO was detrimental to CAR T-cell activity, adversely affecting CAR T-cell metabolism. These patterns held up in murine xenograft models of plasmacytoma, and disseminated bone marrow predominant disease. Our findings underscore the importance of CD200-mediated immune suppression in CAR T-cell therapy of MM, and highlight a promising approach to enhance such therapies by leveraging CD200 expression on aPCs to provide costimulation via a CD200R-CD28 switch.

Endogenous H3.3K27M derived peptide restricted to HLA-A∗02:01 is insufficient for immune-targeting in diffuse midline glioma

Diffuse midline glioma (DMG) is a childhood brain tumor with an extremely poor prognosis. Chimeric antigen receptor (CAR) Tcell therapy has recently demonstrated some success in DMG, but there may a need to target multiple tumor-specific targets to avoid antigen escape. We developed a second-generation CAR targeting an HLA-A∗02:01 restricted histone 3K27M epitope in DMG, the target of previous peptide vaccination and Tcell receptor-mimics. These CAR Tcells demonstrated specific, titratable, binding to cells pulsed with the H3.3K27M peptide. However, we were unable to observe scFv binding, CAR Tcell activation, or cytotoxic function against H3.3K27M+ patient-derived models. Despite using sensitive immunopeptidomics, we could not detect the H3.3K27M26-35-HLA-A∗02:01 peptide on these patient-derived models. Interestingly, other non-mutated peptides from DMG were detected bound to HLA-A∗02:01 and other class I molecules, including a novel HLA-A3-restricted peptide encompassing the K27M mutation and overlapping with the H3 K27M26-35-HLA-A∗02:01 peptide. These results suggest that targeting the H3 K27M26-35 mutation in context of HLA-A∗02:01 may not be a feasible immunotherapy strategy because of its lack of presentation. These findings should inform future investigations and clinical trials in DMG.

Mechanisms and strategies for safe chimeric antigen receptor T-cell activity control.

The clinical application of chimeric antigen receptor (CAR) T-cell therapy has rapidly changed the treatment options for terminally ill patients with defined blood-borne cancer types. However, CAR T-cell therapy can lead to severe therapy-associated toxicities including CAR-related hematotoxicity, ON-target OFF-tumor toxicity, cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Just as CAR T-cell therapy has evolved regarding receptor design, gene transfer systems and production protocols, the management of side effects has also improved. However, because of measures taken to abrogate adverse events, CAR T-cell viability and persistence might be impaired before complete remission can be achieved. This has fueled efforts for the development of extrinsic and intrinsic strategies for better control of CAR T-cell activity. These approaches can mediate a reversible resting state or irreversible T-cell elimination, depending on the route chosen. Control can be passive or active. By combination of CAR T-cells with T-cell inhibiting compounds, pharmacologic control, mostly independent of the CAR construct design used, can be achieved. Other strategies involve the genetic modification of T-cells or further development of the CAR construct by integration of molecular ON/OFF switches such as suicide genes. Alternatively, CAR T-cell activity can be regulated intracellularly through a self-regulation function or extracellularly through titration of a CAR adaptor or of a priming small molecule. In this work, we review the current strategies and mechanisms to control activity of CAR T-cells reversibly or irreversibly for preventing and for managing therapy-associated toxicities.

Enhancement of CAR-T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins.

Existing treatments for cholangiocarcinoma have poor efficacy. However, chimeric antigen receptor-T (CAR-T) cells are emerging as a potential therapeutic strategy. Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR-T cell infiltration and function. This study aimed to improve the function of CAR-T cells through knock down immune checkpoints and immunosuppressive molecular receptors. We evaluated the expression of epidermal growth factor receptor (EGFR) and B7 homolog 3 protein (B7H3) antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry. Subsequently, we engineered CAR-T cells targeting EGFR and B7H3 antigens. We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR-T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR-T cells for antitumor activity both in vitro, using tumor cell lines and cholangiocarcinoma organoid models, and in vivo, using humanized mouse models. We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues. EGFR-CAR-T and B7H3-CAR-T cells demonstrated specific anti-tumor activity. We found an abundance of programmed cell death protein 1 (PD-1), T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and T cell immunoglobulin and ITIM domain (Tigit) on infiltrated CD8+ T cells in the cholangiocarcinoma microenvironment. We then decreased the expression of these 3 proteins on the surface of CAR-T cells, named PTG-scFV-CAR-T cells. Furthermore, we knocked-down the expression of transforming growth factor beta receptor (TGFβR), interleukin-10 receptor (IL-10R), and interleukin-6 receptor (IL-6R) of PTG-scFV-CAR-T cells. Those cells, named PTG-T16R-scFV-CAR-T cells, potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoid model. Finally, the PTG-T16R-scFv-CAR-T cells showed greater inhibitory effect on tumor growth in vivo, and were superior in prolonging the survival of mice. Our results revealed that PTG-T16R-scFV-CAR-T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long-term efficacy both in vitro and in vivo. This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.

TGF-β blocking combined with photothermal therapy promote tumor targeted migration and long-term antitumor activity of CAR-T cells.

TGF-β is widely existed in tumor microenvironment, taking part in tumorigenesis process including angiogenesis, cancer associated fibroblast (CAF) proliferation, and immunosuppression. It inhibited the activation, proliferation, migration and differentiation of T cells, in which way caused a limited therapeutic effects of chimeric antigen receptor T (CAR-T) towards solid tumor such as lymphoma. To targeted block TGF-β at tumor site, we take advantages of nano-techniques to deliver TGF-β inhibitors LY2157299 (LY) towards the tumor sites, in order to help achieve a improved and long-term functions of CAR-T towards lymphoma. Based on amphipathic hydroxyethyl starch-polycaprolactone (HES-PCL), LY and photosensitizer indocyanine green (ICG) were co-loaded in HES-PCL to achieve LY/ICG@HES-PCL nanoparticle. The enhanced function of CAR-T benefited from LY/ICG@HES-PCL were verified through lymphoma Raji cells in vitro and Nod scid gamma mice engrafted with the Raji cells in vivo. LY was targeted transported to tumor site and accelerated release by mild ICG photothermal. Chemokines CXCL9/10/11​at the tumor site relevant to CAR-T migration and chemokines receptor CXCR3 of CAR-T could be up-regulated by LY, thus facilitated the enhanced accumulation of CAR-T at lymphoma site. T effector memory cells differentiation could also be accelerated by LY/ICG@HES-PCL. Combined therapy of LY/ICG@HES-PCL and CAR-T achieved 2.4 times higher antitumor activity and 2.7 times higher relapse inhibiting rates than CAR-T alone within 15 days and 11 days, respectively. The results suggested that LY/ICG@HES-PCL facilitated the enhanced therapeutic index of CAR-T cells towards lymphoma simply and safely, it may be further potentiated applied for other solid tumors.