Chimeric Antigen Receptor T-cell Persistence Research Articles

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.

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

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

The Fate(s) of CAR T-Cell Therapy: Navigating the Risks of CAR+ T-Cell Malignancy.

The introduction of Chimeric Antigen Receptor (CAR) T-cell therapy represents a landmark advancement in treating resistant forms of cancer such as leukemia, lymphoma, and myeloma. However, concerns about long-term safety have emerged following an FDA investigation into reports of second primary malignancies (SPM) after CAR-T cell treatment. This review offers a thorough examination of how genetically modified T-cells might transform into CAR+ SPM. It explores genetic and molecular pathways leading to T-cell lymphomagenesis, the balance between CAR T-cell persistence, stemness, and oncogenic risk, and the trade-off of T-cell exhaustion, which may limit therapy efficacy but potentially reduce lymphomagenesis risk.

Overexpressing Bcl-2 enhances murine chimeric antigen receptor T cell therapy against solid tumor.

Chimeric antigen receptor T (CART) cell therapy has demonstrated promising potential in the treatment of hematologic malignancies. However, its application to solid tumors is limited due to the restrictive nature of the tumor microenvironment, resulting in functional failure and poor persistence of CART cells. Overexpression of Bcl-2 in human CART cells (hCART) has been found to significantly enhance their anti-apoptotic effects both in vitro and in vivo. Nevertheless, the evaluation of hCART cells in preclinical studies has predominantly relied on immunodeficient mice xenograft tumor models, making it challenging to assess the impact of hCART cells on normal tissues and the immune system. We established a murine CART (mCART) that overexpresses Bcl-2 and targets the epidermal growth factor receptor variant III (EGFRvIII), named EGFRvIII·mCART-Bcl2. It demonstrated superior proliferation, cytotoxicity, and anti-apoptotic capabilities in vitro. In an immunocompetent mouse model of abdominal metastasis of colorectal cancer, EGFRvIII·mCART-Bcl2 exhibited improved survival of CART in the abdomen, increased tumor clearance, and significantly prolonged overall mouse survival. In summary, our study provides evidence that the introduction of Bcl-2 into mCART cells can enhance their therapeutic efficacy against solid tumors while ensuring safety.

Applications and Opportunities for Immune Cell CAR Engineering in Comparative Oncology.

Chimeric antigen receptor (CAR) T-adoptive cell therapy has transformed the treatment of human hematologic malignancies. However, its application for the treatment of solid tumors remains challenging. An exciting avenue for advancing this field lies in the use of pet dogs, in which cancers that recapitulate the biology, immunological features, and clinical course of human malignancies arise spontaneously. Moreover, their large size, outbred genetic background, shared environment with humans, and immunocompetency make dogs ideal for investigating and optimizing CAR therapies before human trials. Here, we will outline how challenges in early clinical trials in patients with canine lymphoma, including issues related to autologous CAR T-cell manufacturing, limited CAR T-cell persistence, and tumor antigen escape, mirrored challenges observed in human CAR T trials. We will then highlight emerging adoptive cell therapy strategies currently under investigation in dogs with hematological and solid cancers, which will provide crucial safety and efficacy data on novel CAR T regimens that can be used to support clinical trials. By drawing from ongoing studies, we will illustrate how canine patients with spontaneous cancer may serve as compelling screening platforms to establish innovative CAR therapy approaches and identify predictive biomarkers of response, with a specific emphasis on solid tumors. With increased funding for canine immunotherapy studies, multi-institutional investigations are poised to generate highly impactful clinical data that should translate into more effective human trials, ultimately benefiting both human and canine cancer patients.

Class I HDAC inhibitors enhance antitumor efficacy and persistence of CAR-T cells by activation of the Wnt pathway

Epigenetic modification shapes differentiation trajectory and regulates the exhaustion state of chimeric antigen receptor T (CAR-T) cells. Limited efficacy induced by terminal exhaustion closely ties with intrinsic transcriptional regulation. However, the comprehensive regulatory mechanisms remain largely elusive. Here, we identify class I histone deacetylase inhibitors (HDACi) as boosters of CAR-T cell function by high-throughput screening of chromatin-modifying drugs, in which M344 and chidamide enhance memory maintenance and resistance to exhaustion of CAR-T cells that induce sustained antitumor efficacy both invitro and invivo. Mechanistically, HDACi decrease HDAC1 expression and enhance H3K27ac activity. Multi-omics analyses from RNA-seq, ATAC-seq, and H3K27ac CUT&Tag-seq show that HDACi upregulate expression of TCF4, LEF1, and CTNNB1, which subsequently activate the canonical Wnt/β-catenin pathway. Collectively, our findings elucidate the functional roles of class I HDACi in enhancing CAR-T cell function, which provides the basis and therapeutic targets for synergic combination of CAR-T cell therapy and HDACi treatment.

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.

Transforming CLL management with immunotherapy: Investigating the potential of CAR T-cells and bispecific antibodies

Immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies or T-cell engagers, have revolutionized the treatment landscape for various B-cell malignancies, including B-acute lymphoblastic leukemia and many non-Hodgkin lymphomas. Despite their significant impact on these malignancies, their application in chronic lymphocytic leukemia (CLL) management is still largely under investigation. Although the initial success of CD19-directed CAR T-cell therapy was observed in 3 multiply relapsed CLL patients, with 2 of them surviving over 10 years without relapse, recent CAR T-cell therapy trials in CLL have shown reduced response rates compared to their efficacy in other B-cell malignancies. One of the challenges with using immunotherapy in CLL is the compromised T-cell fitness from persistent CLL-related antigenic stimulation, and an immunosuppressive tumor microenvironment (TME). These challenges underscore a critical gap in therapeutic options for CLL patients intolerant or resistant to current therapies, emphasizing the imperative role of effective immunotherapy. Encouragingly, innovative strategies are emerging to overcome these challenges. These include integrating synergistic agents like ibrutinib to enhance CAR T-cell function and persistence and engineering newer CAR T-cell constructs targeting diverse antigens or employing dual-targeting approaches. Bispecific antibodies are an exciting "off-the-shelf" prospect for these patients, with their investigation in CLL currently entering the realm of clinical trials. Additionally, the development of allogeneic CAR T-cells and natural killer (NK) cells from healthy donors presents a promising solution to address the diminished T-cell fitness observed in CLL patients. This comprehensive review delves into the latest insights regarding the role of immunotherapy in CLL, the complex landscape of resistance mechanisms, and a spectrum of innovative approaches to surmount therapeutic challenges.

Lymphodepletion - an essential but undervalued part of the chimeric antigen receptor T-cell therapy cycle.

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens.

Real World Experience with a Zuma -1 Cohort 4 Adopted Approach to CRS and Icans in CAR-T Recipients

Introduction: Axicabtagene ciloleucel (axi-cel) is a chimeric antigen receptor T-cell (CAR-T) therapy approved for treatment of relapsed/refractory (R/R) large B cell lymphoma (LBCL). The registrational phase I/II ZUMA-1 study Cohorts 1 and 4 addressed toxicity management in terms of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) whereby tocilizumab and corticosteroids were deployed earlier in Cohort 4.(1,2) Although Cohort 4 consisted of only 41 patients, it demonstrated that earlier intervention resulted in significantly fewer cases of grade ≥3 CRS (2%) and ICANS (17%) and a lower cumulative dose of steroids. Furthermore, efficacy as determined by overall response rate (ORR) and complete response (CR) did not appear to be negatively impacted by earlier use of tocilizumab and corticosteroids. However, given the small number of patients in Cohort 4, concern has remained that earlier intervention may have a deleterious impact on CAR-T cell expansion, persistence, and efficacy. During the COVID-19 pandemic, with overwhelmed Intensive Care Units (ICUs) across the country, we adopted a Cohort 4 guided approach to CAR-T toxicity management with the hope of decreasing the need for ICU level of care following CAR-T infusion. Herein, we present our single-institution, real-world experience since implementing an early intervention strategy with Cohort 4 and compare it with our previous Cohort 1 axi-cel recipients in terms of toxicity (CRS, ICANS, ICU utilization) and efficacy. Methods: 119 patients receiving axi-cel CAR-T therapy for R/R LBCL are included in this single-institution, retrospective study with 70 in Cohort 1 and 49 in Cohort 4. The transition from a Cohort 1 to Cohort 4 approach occurred in March 2021. Thus, for Cohort 1 the data cut-off was February 2021 and for Cohort 4 it was after April 2021. The COVID pandemic and the lack of the ICU capacity was the initial chief rationale for transitioning to Cohort 4. This study was conducted following IRB approval and in accordance with the Declaration of Helsinki. R version 1.3.1099 (R-studio) was used for all statistical modeling. All statistical tests were 2-sided and conducted at the 0.05 level of significance. Results: The median age was 62 (22-88) years-old, 35% female gender, 76% advanced stage III-IV disease, 22% with bulky ≥10cm disease, 71% with diagnosis of Diffuse LBCL, and 26% with more than three lines of previous therapy. The median follow-up time at analysis was 1.5 years. Laboratory parameters and comorbidities did not significantly differ between groups except for a higher incidence of renal insufficiency in the Cohort 4 group (28.6% vs 4.3%, p<0.001) (see Table 1). The median maximum grade CRS was significantly lower for Cohort 4 (2 vs 1, p<0.001) although the median grade of ICANS was not different. The utilization of tocilizumab and siltuximab was similar in both groups. The duration of steroid use was comparable in both; however, the total steroid use (dexamethasone equivalent) was notably lower in Cohort 4 (110 mg vs 190 mg, p=0.026). ICU admission following CAR-T infusion was much lower in Cohort 4 (16.3% vs 60%, p<0.001). Most patients (>80%) were discharged home after CAR-T therapy and did not need placement in a rehabilitation center or long-term facility. The ORR and ongoing responses were similar in both groups with nearly 90% achieving a response and 60% with an ongoing response. The cumulative incidence of relapse, PFS, and OS were not significantly different between the groups (see Figure 1). Conclusion: The use of an early toxicity intervention strategy based upon ZUMA-1 Cohort 4 in the real world results in significantly lower high-grade CRS, decreased cumulative steroid dose, and an 73% reduction in ICU utilization without significantly impacting efficacy in terms of incidence of relapse, PFS, or OS. Our real-world experience with the adoption of a Cohort 4 strategy suggests that patient morbidity may be greatly attenuated without compromising CAR-T therapy response. However, the need for longer follow-up to evaluate the impact early use of steroids has on CAR-T persistence and PFS at >5 years remains.

Long Term Durable Responses in Relapsed/Refractory (r/r) ALL, DLBCL, and FL Patients Treated with Tisagenlecleucel and Its Association with Persistence of CAR T-Cells

Introduction: Monitoring of chimeric antigen receptor (CAR) transgene levels in peripheral blood post tisagenlecleucel (tisa-cel) infusion provides information on expansion and persistence of CAR T-cells. In adult diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) patients (pts), the expansion of CAR T-cells was similar between responding and non-responding pts, while in pediatric and young adult acute lymphoblastic leukemia (ALL) pts, higher expansion in responding pts was observed. Based on long term follow up data from tisa-cel treated pts, analyses were performed to delineate the impact of CAR persistence and B cell aplasia on duration of remission (DOR). Methods: Transgene levels in blood measured by quantitative polymerase chain reaction were available from pivotal phase II studies in pts with r/r ALL (ELIANA [N=79], ENSIGN [N=64], NCT03123939 [N=69], NCT01626495 [N=60]), r/r DLBCL (JULIET [N=115]), and r/r FL (ELARA [N=97]), along with the long term follow up study (NCT02445222). The time related to loss of persistence of CAR T-cells (T loss) was defined as the time at which transgene levels first dropped below 50 transgene copies/µg DNA (approximates to lower limit of quantification (LLOQ) of the assay) after maximal expansion. Kaplan Meier analysis was performed to investigate the impact of duration of T loss and time corresponding to last quantifiable level (T last) on DOR/relapse. The impact of time to B cell recovery (>1% CD19+ B cells/WBCs or >3% CD19+ B cells/lymphocytes for ALL, and 80-616 cells/µL for DLBCL and FL), on DOR/relapse was also investigated. Results: Long term CAR persistence in tisa-cel treated pts has been observed for up to 9, 6, and 2.5 years for ALL, DLBCL, and FL pts, respectively, reflecting differing length of follow up based on initiation of trials in respective indications. Across indications, pts who lost transgene within 6 months or between 6-12 months had shorter DOR relative to pts with persistent transgene (Fig 1, ALL pts). In ALL, the median T loss was 27.4, 18.2, 9.7, and 18.0 months for ongoing complete response (CR) > 12 months, CR pts between 6-12 months, relapsed pts < 12 months, and relapsed pts > 12 months, respectively. However, of the pts who lost transgene within first 6 months, some pts maintained durable responses for ≥12 months (29%, 15%, and 50% in ALL, DLBCL, and FL, respectively). Among ALL relapsed pts, majority (17/26, 65%) of the pts with T loss < 6 months showed B cell recovery, however 6 pts had T loss at < 6 months as well as B-cell recovery but maintained response for ≥ 12 months. In ALL, Pulsipher et al.,(Blood Cancer Discov. 2022)have shown that NGS MRD within the first 6 months to 1 year post infusion may be a more reliable predictor of potential relapse than B cell recovery. The median time to B-cell recovery was 266 days in pts who relapsed/censored within 12 months but was not reached for pts with ongoing response at 12 months. On the contrary, B cell recovery seems to have no association with relapse in DLBCL or FL pts. Of the ALL pts with CAR persistence, pts with <50% morphologic blasts at any time prior to infusion of tisa-cel had longer DOR compared to pts with ≥50% blasts. This may also reflect a more resistant high risk ALL at the time of study entry or greater potential for stochastic loss of CD19 in pts with the higher disease burden. Conclusions: Long term sustained remission/response was observed in tisa-cel treated pts in the pivotal trials. The analyses demonstrate a positive association between CAR persistence and durable clinical responses across indications, however, some pts maintained durable responses despite early loss of transgene and/or early B cell recovery (< 6 months post infusion; Myers et al., J Clin Oncol. 2021). To note, in DLBCL and FL, the transgene levels in blood may not represent the levels at target sites including lymph nodes, moreover, T last and T loss are dependent on the duration of follow up, LLOQ etc. Of note, these analyses represent data from pivotal tisa-cel clinical studies with a heavily treated pt population post multiple lines of therapy including prior HSCT, and may not represent pts treated with commercial product, at earlier times post diagnosis in current studies with fewer lines of prior treatment, lower pre-infusion disease burden, and/or other CAR product. In pts with early loss of transgene, further research is needed to identify potential factors or patient characteristics resulting in durable responses.

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.

Type I Interferon Blockade Enhances Transduction Efficiency and Efficacy of Non-Activated CAR T Cells

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of advanced hematologic cancers, showing impressive response rates in pivotal trials. Despite this success, challenges in CAR T-cell adoption persist due to tumor resistance mechanisms, manufacturing failures, and limitations in CAR T-cell persistence and effector function. Strategies that promote T-cell persistence, proliferation, and cytotoxic activity are essential to improving long-term outcomes of CAR T therapy. The most common protocol for CAR T-cell manufacturing involves lentiviral vector transduction of activated T-cells followed by ex vivo culture. However, this approach may drive terminal T-cell differentiation and exhaustion, compromising therapeutic efficacy. In contrast, non-activated CAR T-cell manufacturing has shown promise in enhancing T-cell differentiation and tumor control. Yet, the low transduction efficiency of quiescent T-cells remains a challenge. In this study, we explore the role of type I interferon (IFN)-mediated innate immune responses in limiting CAR T manufacturing efficiency. We hypothesize that blocking these responses using the vaccinia virus-encoded type I interferon-binding protein can enhance transduction efficiency and promote less-differentiated CAR T-cell phenotypes, including naïve and central memory-like differentiation. Our findings demonstrate that lentiviral vector transduction triggers an innate immune response in non-activated T-cells, resulting in reduced transduction efficiency, and altered T-cell differentiation. Inhibiting the anti-viral response via anti-IFN leads to improved T-cell differentiation and exhaustion characteristics. Importantly, anti-IFN exposure supports the enrichment of naive-like T cells: a subset noted for superior clinical efficacy. Anti-IFN also reduces LAG3 expression. Collectively, these findings position anti-IFN as a novel means to enhance CAR T-cell persistence and anti-tumor function. We further investigate the influence of sustained anti-IFN exposure by designing lentiviral transfer plasmids encoding both a CAR and secreted type I interferon-binding protein. These cells showed enhanced cytolytic activity and cytokine production against target cells. Our studies in mice show promising results, suggesting that the protein secretion enhance therapeutic activity against tumors employing sustained type I interferon signaling for immune escape. Overall, targeting the type I interferon response during non-activated CAR T-cell manufacturing offers a potential solution to improve transduction efficiency and enhance CAR T-cell functionality. Given the therapeutic promise of non-activated CAR T cells, our findings have immediate translational relevance for broadening their appeal against cancer. Figure 1. Exposure to anti-IFN increases transduction efficiency and naïve-like differentiation of lentivirus-transduced non-activated T-cells A) Freshly isolated human T-cells were incubated in media with aIFN or vehicle, for one hour at room temperature. The cells were resuspended in complete media and then transduced with lentiviral vector encoding iRFP. Expression of iRFP was evaluated 96 h after transduction by flow cytometry. b, Human T-cells were isolated and prepared as above. At 96 h post-transduction, cells were stained with anti-CD45RO and anti-CCR7 antibodies and samples were evaluated by flow cytometry. Data for A and B are representative of multiple experiments with three independent donors.

Dual Split-Signaling TIM3+CLEC12a Targeting CAR T-Cells with Optimized Signaling As a Safe Potential Therapy for Acute Myeloid Leukemia

Background: The success of chimeric antigen receptor (CAR) T-cell therapies targeting B cell maturation antigen (BCMA) in multiple myeloma and CD19 in B-cell malignancies has demonstrated the immense potential of this immunotherapeutic strategy. However, the translation of CAR T-cell therapy to acute myeloid leukemia (AML) patients has encountered challenges due to the absence of surface antigens specifically or preferentially expressed on AML cells. To overcome this hurdle, we explored the feasibility and safety of simultaneously targeting the AML-associated antigens CLEC12a and TIM3 using an AND-gate strategy in which the essential stimulatory and co-stimulatory T-cell activation signals are segregated into separate CARs. To improve the safety of our strategy we also attenuated signaling through the stimulatory anti-CLEC12a CAR via affinity-optimization or through targeted mutations in its immunoreceptor tyrosine-based activation motif (ITAM) regions. Methods: To identify the most optimal dual-split CAR T-cell configuration, we designed several chimeric antigen receptor constructs in which we targeted CLEC12a with stimulatory CARs (sCARs) with high (C hi) or low (C low) affinities. In addition, the high affinity C hi stimulatory CAR was mutated in the second and third ITAM regions (denoted as C hixx) to attenuate the CD3ζ stimulatory signal. TIM3 was targeted by a costimulatory CAR (cCAR) containing both CD28 and 41BB signaling domains. The dual-split CARs were generated using two separate retroviral constructs. After the assessment of the CAR expression and specific surface markers associated with memory phenotype and exhaustion, dual-split CAR T-cells were functionally tested using an isogenic cell line model for specific lysis against dual CLEC12a/TIM3 positive or single CLEC12a or TIM3 positive isogenic target cells as well as for dual antigen-specific cytokine secretion. The dual-split CAR T-cells were also tested for their efficiency to specifically eliminate leukemic stem/progenitor cells by the colony formatting unit (CFU) assay. Moreover, anti-AML activity and persistence of the dual-split CAR T-cells was assessed using an AML PDX mouse model in which human primary AML cells expressing TIM3 and CLEC12a were injected. Results: Using the isogenic cell line model, we revealed that the dual-split CAR T-cells with the combination of the high affinity CLEC12a sCAR and TIM3 cCAR (C hi+T) show antigen-specific cytokine secretion and cytotoxicity, and that this specificity was enhanced when the CLEC12a sCAR was attenuated through ITAM mutations (C hixx+T). In CFU assays, all tested CLEC12a/TIM3 dual-split CAR T-cell combinations exhibited potent inhibition (up to 95%) of leukemia blast viability and leukemia stem/progenitor self-renewal. In vivo, although AML did not engraft in all mice, the best-performing dual-split C hixx+T1 CAR T-cells displayed potent anti-leukemic activity, as they prevented engraftment in two additional mice compared to control groups (n=6 each group). Furthermore, in contrast to control groups dual-split CAR T-cells exhibited prolonged persistence in the blood up to 20 weeks after injection. Conclusion: We successfully generated highly specific dual-targeting CAR T-cells by splitting the T-cell activation signals into two simultaneously expressed CARs, and using a novel approach of attenuating the sCAR through mutations in ITAM regions. Overall, we show that dual-split CAR T cells targeting CLEC12a and TIM3, particularly the combination C hixx+T CAR T-cells, have specific and promising anti-AML activity. Our findings underscore the potential of innovative dual-split CAR T-cell therapies as a novel and effective immunotherapeutic approach for combating AML.

Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours

Rational design of chimeric antigen receptor T (CAR-T) cells based on the recognition of antigenic epitopes capable of evoking the most potent CAR activation is an important objective in optimizing immune therapy. In solid tumors, the B7-H3 transmembrane protein is an emerging target that harbours two distinct epitope motifs, IgC and IgV, in its ectodomain. Here, we generate dromedary camel nanobodies targeting B7-H3 and demonstrate that CAR-T cells, based on the nanobodies recognizing the IgC but not IgV domain, had potent antitumour activity against large tumors in female mice. These CAR-T cells are characterized by highly activated T cell signaling and significant tumor infiltration. Single-cell transcriptome RNA sequencing coupled with functional T-cell proteomics analysis uncovers the top-upregulated genes that might be critical for the persistence of polyfunctional CAR-T cells in mice. Our results highlight the importance of the specific target antigen epitope in governing optimal CAR-T activity and provide a nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

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.

CD19/CD22 targeting with cotransduced CAR T cells to prevent antigen-negative relapse after CAR T-cell therapy for B-cell ALL

AbstractCD19-negative relapse is a leading cause of treatment failure after chimeric antigen receptor (CAR) T-cell therapy for acute lymphoblastic leukemia. We investigated a CAR T-cell product targeting CD19 and CD22 generated by lentiviral cotransduction with vectors encoding our previously described fast-off rate CD19 CAR (AUTO1) combined with a novel CD22 CAR capable of effective signaling at low antigen density. Twelve patients with advanced B-cell acute lymphoblastic leukemia were treated (CARPALL [Immunotherapy with CD19/22 CAR Redirected T Cells for High Risk/Relapsed Paediatric CD19+ and/or CD22+ Acute Lymphoblastic Leukaemia] study, NCT02443831), a third of whom had failed prior licensed CAR therapy. Toxicity was similar to that of AUTO1 alone, with no cases of severe cytokine release syndrome. Of 12 patients, 10 (83%) achieved a measurable residual disease (MRD)–negative complete remission at 2 months after infusion. Of 10 responding patients, 5 had emergence of MRD (n = 2) or relapse (n = 3) with CD19- and CD22-expressing disease associated with loss of CAR T-cell persistence. With a median follow-up of 8.7 months, there were no cases of relapse due to antigen-negative escape. Overall survival was 75% (95% confidence interval [CI], 41%-91%) at 6 and 12 months. The 6- and 12-month event-free survival rates were 75% (95% CI, 41%-91%) and 60% (95% CI, 23%-84%), respectively. These data suggest dual targeting with cotransduction may prevent antigen-negative relapse after CAR T-cell therapy.

ICOS and OX40 tandem co-stimulation enhances CAR T-cell cytotoxicity and promotes T-cell persistence phenotype.

Chimeric Antigen Receptor (CAR) T-cell therapies have emerged as an effective and potentially curative immunotherapy for patients with relapsed or refractory malignancies. Treatment with CD19 CAR T-cells has shown unprecedented results in hematological malignancies, including heavily refractory leukemia, lymphoma, and myeloma cases. Despite these encouraging results, CAR T-cell therapy faces limitations, including the lack of long-term responses in nearly 50-70% of the treated patients and low efficacy in solid tumors. Among other reasons, these restrictions are related to the lack of targetable tumor-associated antigens, limitations on the CAR design and interactions with the tumor microenvironment (TME), as well as short-term CAR T-cell persistence. Because of these reasons, we developed and tested a chimeric antigen receptor (CAR) construct with an anti-ROR1 single-chain variable-fragment cassette connected to CD3ζ by second and third-generation intracellular signaling domains including 4-1BB, CD28/4-1BB, ICOS/4-1BB or ICOS/OX40. We observed that after several successive tumor-cell in vitro challenges, ROR1.ICOS.OX40ζ continued to proliferate, produce pro-inflammatory cytokines, and induce cytotoxicity against ROR1+ cell lines in vitro with enhanced potency. Additionally, in vivo ROR1.ICOS.OX40ζ T-cells showed anti-lymphoma activity, a long-lasting central memory phenotype, improved overall survival, and evidence of long-term CAR T-cell persistence. We conclude that anti-ROR1 CAR T-cells that are activated by ICOS.OX40 tandem co-stimulation show in vitro and in vivo enhanced targeted cytotoxicity associated with a phenotype that promotes T-cell persistence.

Development of a Multiscale Mechanistic Modelling Framework Integrating Differential Cellular Kinetics of CAR-T Cell Subsets and Immunophenotypes in Cancer Patients.

Chimeric antigen receptor (CAR) T-cell subsets and immunophenotypic composition of the pre-infusion product, as well as their longitudinal changes following infusion, are expected to affect CAR-T cell expansion, persistence, and clinical outcomes. Herein, we sequentially evolved our previously described cellular kinetic-pharmacodynamic (CK-PD) model to incorporate CAR-T cell product-associated attributes by utilizing published preclinical and clinical datasets from two affinity variants (FMC63 and CAT19 scFv) anti-CD19 CAR-T cells. In step 1, a unified cell-level PD model was used to simultaneously characterize the in vitro killing datasets of two CAR-Ts against CD19+ cell lines at varying effector: target (E: T) ratios. In step 2, an augmented CK-PD model for anti-CD19 CAR-Ts was developed, by integrating CK dataset(s) from two bioanalytical measurements (qPCR and flow cytometry) in cancer patients. The model described the differential in vivo expansion properties of CAR-T cell subsets. The estimated expansion rate constant was ~1.12-fold higher for CAR+CD8+ cells in comparison to CAR+CD4+ T cells. In step 3, the model was extended to characterize the disposition of four Immunophenotypic populations of CAR-T cells, including stem-cell memory (TSCM ), central memory (TCM ), effector memory (TEM ) and effector (TEFF ) cells. The model adequately characterized the longitudinal changes in immunophenotypes post anti-CD19 CAR-T cell infusion in acute lymphocytic leukemia paediatric patients. Polyclonality in the pre-infusion product was identified as a categorical covariate influencing differentiation of immunophenotypes. In the future, this model could be leveraged a priori towards optimizing the composition of CAR-T cell infusion product, and further understand the CK-PD relationship in patients.

CRISPR/Cas9: A Powerful Strategy to Improve CAR-T Cell Persistence.

As an emerging treatment strategy for malignant tumors, chimeric antigen receptor T (CAR-T) cell therapy has been widely used in clinical practice, and its efficacy has been markedly improved in the past decade. However, the clinical effect of CAR-T therapy is not so satisfying, especially in solid tumors. Even in hematologic malignancies, a proportion of patients eventually relapse after receiving CAR-T cell infusions, owing to the poor expansion and persistence of CAR-T cells. Recently, CRISPR/Cas9 technology has provided an effective approach to promoting the proliferation and persistence of CAR-T cells in the body. This technology has been utilized in CAR-T cells to generate a memory phenotype, reduce exhaustion, and screen new targets to improve the anti-tumor potential. In this review, we aim to describe the major causes limiting the persistence of CAR-T cells in patients and discuss the application of CRISPR/Cas9 in promoting CAR-T cell persistence and its anti-tumor function. Finally, we investigate clinical trials for CRISPR/Cas9-engineered CAR-T cells for the treatment of cancer.