Activation-induced Cell Death Research Articles

Hu8F4-CAR T cells with mutated Fc spacer segment improve target-specificity and mediate anti-leukemia activity in vivo

Hu8F4 is a T cell receptor (TCR)-like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is comprised of the Hu8F4 scFv, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain, and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from AML patients in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor (FcγR)-expressing cells. We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with FcγR-expressing cells in vivo. The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor-binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

Overexpression of an Engineered SerpinB9 Enhances Allogeneic T-Cell Persistence and Efficacy.

Allogeneic chimeric antigen receptor (CAR)-expressing T cells offer many advantages over autologous therapies, but their benefits are curtailed by graft-versus-host disease (GvHD) and elimination by recipient immune cells. Moreover, just as with autologous therapies, allogeneic CAR T cells are susceptible to activation-induced cell death (AICD) caused by chronic antigen exposure (CAE). Granzyme B (GzmB) and Fas/FasL-initiated, caspase-mediated apoptosis are key mechanisms of T-cell death caused by T/NK cell-mediated allorejection or CAE. We explored a protective strategy of engineering CAR T cells to overexpress variants of the GzmB-specific serine protease inhibitor, SerpinB9 (SB9), to improve allogeneic T-cell persistence and antitumor efficacy. We showed that the overexpression of an SB9 variant with broadened caspase specificity, SB9(CAS), not only significantly reduced rejection of allogeneic CAR T cells, but also increased their resistance to AICD and enabled them to thrive better under CAE, thus improving allogeneic T-cell persistence and antitumor activity in vitro and in vivo. In addition, while SB9(CAS)-overexpression improved the efficacy of allogeneic CAR T-cell therapy by conferring protection to cell death, we did not observe any autonomous growth and the engineered CAR T cells were still susceptible to an inducible suicide switch. Hence, SB9(CAS)-overexpression is a promising strategy that can strengthen current development of cell therapies, broadening their applications to address unmet medical needs.

Role of 14-3-3? in the Activation-Induced Cell Death

Role of 14-3-3? in the Activation-Induced Cell Death

Immunoinhibitory effects of anti-tuberculosis therapy induce the host vulnerability to tuberculosis recurrence.

The host immune responses play a pivotal role in the establishment of long-term memory responses, which effectively aids in infection clearance. However, the prevailing anti-tuberculosis therapy, while aiming to combat tuberculosis (TB), also debilitates innate and adaptive immune components of the host. In this study, we explored how the front-line anti-TB drugs impact the host immune cells by modulating multiple signaling pathways and subsequently leading to disease relapse. Administration of these drugs led to a reduction in innate immune activation and also the cytokines required to trigger protective T cell responses. Moreover, these drugs led to activation-induced cell death in the mycobacterial-specific T cell leading to a reduced killing capacity. Furthermore, these drugs stalled the T cell differentiation into memory subsets by modulating the activation of STAT3, STAT4, FOXO1, and NFκB transcription factors and hampering the Th1 and Th17-mediated long-term host protective memory responses. These findings suggest the urgent need to augment directly observed treatment, short-course (DOTS) therapy with immunomodulatory agents to mitigate the adverse effects linked to the treatment.IMPORTANCEAs a central component of TB eradication initiatives, directly observed treatment, short-course (DOTS) therapy imparts immune-dampening effects during the course of treatment. This approach undermines the host immune system by delaying the activation process and lowering the immune response. In our investigation, we have unveiled the impact of DOTS on specific immune cell populations. Notably, the signaling pathways involving STAT3 and STAT4 critical for memory responses and NFκβ associated with pro-inflammation were substantially declined due to the therapy. Consequently, these drugs exhibit limited effectiveness in preventing recurrence of the disease. These observations highlight the imperative integration of immunomodulators to manage TB infection.

Dysfunction of type 1 and type 2 immune cells: a lesson from exhausted-like ILC2s and their activation-induced cell death.

The concept of immune cell exhaustion/dysfunction has developed mainly to understand impaired type 1 immune responses especially by CD8 T cells against tumors or virus-infected cells and has been applied to other lymphocytes. Natural killer (NK) cells and CD4 T cells support the efficient activation of CD8 T cells but exhibit a dysfunctional phenotype in tumor microenvironments and in chronic virus infections. In contrast, the concept of type 2 immune cell exhaustion/dysfunction is poorly established. Group 2 innate lymphoid cells (ILC2s) and T-helper 2 (Th2) cells are the major lymphocyte subsets that initiate and expand type 2 immune responses for antiparasitic immunity or allergy. In mouse models of chronic parasitic worm infections, Th2 cells display impaired type 2 immune responses. Chronic airway allergy induces exhausted-like ILC2s that quickly fall into activation-induced cell death to suppress exaggerated inflammation. Thus, the modes of exhaustion/dysfunction are quite diverse and rely on the types of inflammation and the cells. In this review, we summarize current knowledge of lymphocyte exhaustion/dysfunction in the context of type 1 and type 2 immune responses and discuss ILC2-specific regulatory mechanisms during chronic allergy.

Incorporating IL7 receptor alpha signaling in the endodomain of B7H3-targeting chimeric antigen receptor T cells mediates antitumor activity in glioblastoma.

CAR-T-cell therapy has shown promise in treating hematological malignancies but faces challenges in treating solid tumors due to impaired T-cell function in the tumor microenvironment. To provide optimal T-cell activation, we developed a B7 homolog 3 protein (B7H3)-targeting CAR construct consisting of three activation signals: CD3ζ (signal 1), 41BB (signal 2), and the interleukin 7 receptor alpha (IL7Rα) cytoplasmic domain (signal 3). We generated B7H3 CAR-T cells with different lengths of the IL7Rα cytoplasmic domain, including the full length (IL7R-L), intermediate length (IL7R-M), and short length (IL7R-S) domains, and evaluated their functionality in vitro and in vivo. All the B7H3-IL7Rα CAR-T cells exhibited a less differentiated phenotype and effectively eliminated B7H3-positive glioblastoma in vitro. Superiority was found in B7H3 CAR-T cells contained the short length of the IL7Rα cytoplasmic domain. Integration of the IL7R-S cytoplasmic domain maintained pSTAT5 activation and increased T-cell proliferation while reducing activation-induced cell death. Moreover, RNA-sequencing analysis of B7H3-IL7R-S CAR-T cells after coculture with a glioblastoma cell line revealed downregulation of proapoptotic genes and upregulation of genes associated with T-cell proliferation compared with those in 2nd generation B7H3 CAR-T cells. In animal models, compared with conventional CAR-T cells, B7H3-IL7R-S CAR-T cells suppressed tumor growth and prolonged overall survival. Our study demonstrated the therapeutic potential of IL7Rα-incorporating CAR-T cells for glioblastoma treatment, suggesting a promising strategy for augmenting the effectiveness of CAR-T cell therapy.

Abstract 3739: Different combination regimens for OX40 agonists and PD-1 inhibitors exert different antitumor effects in OX40 humanized mice

Abstract OX40, also known as CD134 or TNFRSF4, is a member of the tumor necrosis factor receptor (TNFR) superfamily which mainly expressed on the surface of activated T cells including Treg cells. Engagement of OX40 with its ligand, TNFSF4, leads to effector T cell expansion and cytokine release, inhibits activation-induced cell death (AICD) and promotes the survival of antigen-specific memory T cells. In contrast to the massive hit of immune checkpoint inhibitors like PD-1 and PD-L1, the benefit of agonistic antibodies in clinical trials has not been completely demonstrated to date. On one hand, higher doses could leads to T cell exhaustion or AICD, paradoxically, with no efficacy improvement. Another hand, the limited understanding of the biology of immune checkpoints underlying “accelerator and brake” should account for the slow clinical progress of OX40-targeted biologics. It seems that the dosing sequence or timing of anti-OX40 and anti-PD-1 is an important consideration in combination therapies. Given this, we firstly evaluated the antitumor effects of two different anti-human OX40 agonists in syngeneic MC38 and Pan02 tumor models, respectively, in hOX40 knockin mice. We observed tumor regressions upon treatment of OX40 agonistic Ab1 in a dose-dependence way in the homozygous hOX40 knockin mice engrafted with MC38 tumors. The anti-hOX40 Ab1 at high-dose (10 mg/kg) led to ~60% TGI, but no response at very low dose (0.1 mg/kg). Furthermore, the homozygous hOX40 knockin mice inoculated with Pan02 pancreatic tumors were treated with a novel strong anti-hOX40 agonist (Ab2) and surrogate anti-mPD-1. Notably, Anit-hOX40 (Ab2) alone at low dose (1 mg/kg, Q3D*9 doses) exerted strong anti-tumor potency (TGI=59.3%, p< 0.005) and no statistical significant antitumor difference was observed between high-dose group (at 5mg/kg) and low-dose group (at 1 mpk) as monotherapy, though anti-hOX40 (Ab2) at 5mg/kg showed the best therapeutic response (TGI=72%, p=0.0002) among all treatment groups. In contrast, the second therapeutic response was observed in the combo group treated with first dosing of 5mg/kg anti-hOX40 (Ab2) combining delayed dosing of 10mg/kg anti-mPD-1 (TGI=64%, p< 0.005) and the concurrent dosing group of Ab2 (5 mg/kg, Q3D × 9 doses) plus anti-mPD-1 (2 mg/kg, Q3D × 9 doses) (TGI=62.3%, p= 0.001). In conclusion, different human-specific OX40 agonists displayed different anti-tumor activity as monotherapy, and different dose led to different antitumor potency for the same agonist. In terms of different combination regimens, although no advanced benefit was observed for the combination of anti-mPD-1/anti-OX40 (Ab2) in comparison to strong anti-OX40 agonist (Ab2) alone, it demonstrated that the dosing timing and sequence of either anti-PD-1 or anti-hOX40 did has impact on the therapeutic outcome. Citation Format: Yi Li, Yanlei Zhang, Xiaolei Qiu, Dongxiao Feng, Ruilin Sun, Daniel X. He. Different combination regimens for OX40 agonists and PD-1 inhibitors exert different antitumor effects in OX40 humanized mice [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 3739.

Abstract 5227: Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues

Abstract Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies, offering hope to patients with limited treatment options. However, extending this therapy to myeloid malignancies and solid tumors has proven challenging due to co-expression of targetable antigens on hematopoietic progenitors and healthy tissues. In this context, γδ T cells show promise, as they can directly identify and eliminate malignant cells via recognition of multiple tumor-associated stress antigens rarely expressed on normal tissues. We leveraged the tumor-sensing capabilities of γδ T cells with enhanced tumor localization by employing a non-signaling CAR (nsCAR) that excludes the CD3ζ domain, facilitating targeted tumor cell killing while sparing healthy tissues. We designed second-generation lentiviral constructs for anti-CD33-scfv for in vitro evaluation against acute myeloid leukemia (AML) lines HL-60, KG-1a, and MOLM-13 and the chronic myeloid leukemia (CML) line K-562 Additionally, we tested CD33/CD123 dual-targeting nsCAR constructs (ns-dCAR) to determine if the addition of CD123 targeting enhanced the therapeutic index. We also tested whether a secreted IL-15 (sIL-15) would enhance CAR-T cell fitness. We observed significant upregulation of CD69 in Jurkat cells transduced with signaling CARs in co-culture with KG-1a cells but not in those with nsCAR constructs. We also noticed a time-dependent reduction (43%) in the CD3ζ+ CD33CAR+ population over a 7-day extended coculture while the nsCAR+ Jurkat population remained stable, suggesting potential mitigation of activation-induced cell death (AICD). Expanded and activated γδ T cells from healthy donors were then transduced with nsCAR lentiviral vectors. The ns33CAR+ γδ T cells exhibited enhanced killing capability against HL-60 (up to 1.3x) and K-562 (up to 1.6x) compared to unmodified γδ T cells (UTD) in a 24-hour cytotoxicity assay. Incorporation of sIL-15 into the nsCD33 CAR construct also increased killing across all 4 cell lines (up to 1.8x for HL-60, up to 2.6x for KG-1a, up to 2.0x for MOLM-13 and up to 2.0x for K-562). Minimal cytotoxicity (<10%) was observed for nsCARs or UTDs against normal CD33+/CD123+ cells from healthy donor PBMC or CD34+ HPSCs. The ns-dCAR constructs did not improve in vitro AML killing compared to ns33CAR alone, although ns-dCARs generally exhibited lower transduction efficiency. In summary, our findings suggest that combining nsCAR constructs with γδ T cells may widen the therapeutic window to expand the reach of CAR-T therapy to cancers with limited target antigen expression on critical healthy tissues. Further optimization to improve integration and incorporation of membrane-bound IL-15 co-expression holds the potential to enhance both the efficacy and safety profiles of next-generation adoptive cell therapies against a broader spectrum of cancers. Citation Format: Lei Ding, Yanjie Li, Jiexue Li, Mariska Ter Haak, Lawrence Lamb. Gamma-delta (γδ) CAR-T cells lacking the CD3ζ signaling domain enhance targeted killing of AML cells and preserve healthy tissues [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 5227.

Abstract 3245: Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia

Abstract Large granular lymphocyte (LGL) leukemia is a rare heterogeneous blood cancer that is characterized by the clonal lymphoproliferation of CD8+ T cells (T-LGL leukemia) or, less frequently, natural killer cells (NK-LGL leukemia). These cells evade activation induced cell death, allowing them to accumulate as highly cytotoxic lymphocytes that contribute to autoimmune attacks, neutropenia, and anemia. Frontline treatments are limited to repurposed, nonspecific immunosuppressive agents with limited efficacy and incomplete response rates. As there are no FDA-approved antineoplastic agents for LGL leukemia, our research explores novel therapeutic avenues for this orphan disease. Somatic activating mutations in the transcription factor STAT3 are the most common molecular alteration found in >50% of patients, and STAT3 signaling is dysregulated in almost all LGL leukemia patients. In the largest molecularly profiled LGL leukemia patient cohort to date, we found that STAT3 mutations frequently co-occur with mutations in chromatin and epigenetic modifying genes, suggesting crosstalk and cooperativity between STAT3 activation and epigenetic dysregulation. Moreover, TET2, which promotes DNA demethylation, is frequently inactivated in LGL leukemia via loss-of-function mutations and promoter hypermethylation. As DNA hypermethylation is an emerging hallmark of LGL leukemia, we evaluated the therapeutic potential of two epigenetic agents, azacitidine (AZA) and cladribine (CLAD), that directly and indirectly inhibit DNA hypermethylation, respectively. We hypothesize that AZA and CLAD will 1) exhibit cell intrinsic efficacy, 2) reprogram the epigenome of LGL leukemia through DNA hypomethylation to normalize aberrant gene expression, and 3) induce cytokine-mediated immune responses. AZA and CLAD cytotoxicity was assessed by the Cell Titer Glo viability assay in human LGL leukemia cell lines. Each exhibited potent anti-leukemic effects with IC50 values in the low micromolar and nanomolar ranges. Western blotting analysis demonstrated the decrease in protein expression of DNA methyltransferases and phosphorylated STAT3 levels with treatment in a time- and dose-dependent manner. We also explored the immunomodulatory effects of these hypomethylating agents since LGL leukemia is a pro-inflammatory malignancy with cytotoxic lymphocytes. Preliminary findings using the Isoplexis platform suggest that AZA and CLAD induce production of anti-inflammatory cytokines in LGL leukemia cells. Ongoing studies will identify differential methylation, chromatin accessibility, and gene expression using RRBS, ATAC-seq, and RNA-seq of AZA or CLAD treated samples to define the mechanisms and consequences of DNA demethylation. Uncovering the reprogramming capabilities of AZA and CLAD could allow for the potential repurposing of these inhibitors for treatment of LGL leukemia patients. Citation Format: Ariana Sabzevari, Ipsita Pal, Tess Deddens, Todd Fox, Aakrosh Ratan, David J. Feith, Thomas P. Loughran. Use of hypomethylating agents to reprogram the epigenome in large granular lymphocyte leukemia [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 3245.

Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness

Genes limiting Tcell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to Tcell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 Tcells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling Tcell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic Tcell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional Tcell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting Tcell antitumor activity.

Mutant KRAS-activated circATXN7 fosters tumor immunoescape by sensitizing tumor-specific T cells to activation-induced cell death

Mutant KRAS (KRASMUT) is often exploited by cancers to shape tumor immunity, but the underlying mechanisms are not fully understood. Here we report that tumor-specific cytotoxic T lymphocytes (CTLs) from KRASMUT cancers are sensitive to activation-induced cell death (AICD). circATXN7, an NF-κB-interacting circular RNA, governs T cell sensitivity to AICD by inactivating NF-κB. Mechanistically, histone lactylation derived from KRASMUT tumor cell-produced lactic acid directly activates transcription of circATXN7, which binds to NF-κB p65 subunit and masks the p65 nuclear localization signal motif, thereby sequestering it in the cytoplasm. Clinically, circATXN7 upregulation in tumor-specific CTLs correlates with adverse clinical outcomes and immunotherapeutic resistance. Genetic ablation of circAtxn7 in CD8+ T cells leads to mutant-selective tumor inhibition, while also increases anti-PD1 efficacy in multiple tumor models in female mice. Furthermore, targeting circATXN7 in adoptively transferred tumor-reactive CTLs improves their antitumor activities. These findings provide insight into how lymphocyte-expressed circRNAs contribute to T-cell fate decisions and anticancer immunotherapies.

Activation-Induced Senescent Cell Death based on Chiral CoHAu Nanoassemblies with Enantioselective Cascade-Catalytic Ability.

Chirality is common in nature, which determines the high enantioselectivity of living systems. Selecting suitable chiral configurations is of great meaning for nanostructures to function better in biological systems. In this study, chiral Co3O4-H2TPPS-Au (CoHAu) nanoassemblies are constructed to accelerate the production ∙OH by consuming D-glucose (D-Glu, widely spread in nature) based on their outstanding enantioselective cascade-catalytic abilities. In particular, D-CoHAu nanoassemblies are more effective in the catalytic conversion of D-Glu than L-CoHAu nanoassemblies. This phenomenon is due to the stronger binding affinity of D-CoHAu nanoassemblies indicated by the lower Km value. Moreover, D-CoHAu nanoassemblies display excellent consumption-ability of D-Glu and production of ∙OH in living cells, which can eliminate senescent cells effectively based on their intracellular enantioselective cascade-catalysis. This research establishes the foundation for bio-mimicking nanostructures with unique functionalities to regulate abnormal biological activities better.

Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study

BackgroundCommercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy.MethodsWe developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL.ResultsPreclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia.ConclusionsWe developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL.Trial registrationNCT05338931; Date: 2022–04-01.

Combining Apoptotic Resistance and Cytokine Signaling to Improve Persistence and Anti-Tumor Activity of CAR Vδ2 T Cells In Vivo

Vγ9Vδ2 T cells (Vδ2 T cells hereafter) are an attractive platform for allogeneic chimeric antigen receptor (CAR)-modified cell therapy, as their invariant TCR minimizes their risk of graft-versus-host disease. However, the susceptibility of Vδ2 T cells to activation-induced cell death (AICD) and dependency on exogenous cytokine supplementation for prolonged persistence limit their in vivo efficacy. Addressing these limitations in would increase therapeutic potential of engineered Vδ2 T cells. Fas signaling is critical for AICD induction in Vδ2 T cells. To attenuate AICD, we generated a truncated Fas receptor lacking a signaling endodomain (tFas), to prevent downstream activation of apoptosis. Incubation with soluble FasL for 24h induced apoptosis in unmodified Vδ2 T cells and CD19.CAR-expressing Vδ2 T cells, while tFas-armed CD19.CAR-Vδ2 T cells resisted FasL-induced cell death. In extended (9-day) coculture experiments with CD19(+) NALM6 target cells at a 1:2 E:T ratio without exogenous cytokines, tFas-armed CD19.CAR-Vδ2 T cells produced enhanced anti-tumor control at early time points, compared to unarmed CD19.CAR-Vδ2 T cells which exhibited only transient activity, resulting in tumor regrowth. However, the lack of cytokine support limited long-term expansion of tFas/CD19.CAR-Vδ2 T cells resulting in tumor regrowth after 6 days of coculture. IL-18 signaling via MyD88 stimulates Vδ2 T cells, promoting their growth. Hence, we hypothesized combining IL-18/MyD88 signaling with AICD suppression would synergize to improve anti-tumor function of Vδ2 T cells. We generated a chimeric receptor consisting of the extracellular domain of Fas linked with intracellular MyD88 (Fas88) aiming to both prevent AICD and transmit MyD88 signaling upon engagement with FasL. Fas88-armed CD19.CAR-Vδ2 T cells resisted FasL-induced apoptosis similar to those armed with a tFas construct. Furthermore, in cocultures with NALM6 cells, Fas88/CD19.CAR-Vδ2 T cells resisted AICD and, in contrast to tFas/CD19.CAR-Vδ2 T cells, expanded over 9 days in the absence of exogenous cytokines and mediated a significantly superior tumor control. Next, we investigated the activity of armed CD19.CAR-Vδ2 T cells in a xenograft mouse model, where NALM6 cells (0.5 x 10 6 cells/mouse) were intravenously infused into NSG mice followed 5 days later by a single injection of Vδ2 T cells (4 x 10 6 cells/mouse). Both tumor and T cell populations were tracked via dual bioluminescent imaging. Unmodified CD19.CAR-Vδ2 T cells extended median survival from 18 days (control group) to 30 days but demonstrated limited persistence. tFas-armed CD19.CAR-Vδ2 T cells produced comparable tumor control, with a median survival of 31.5 days, suggesting that AICD suppression alone is insufficient for the long-term activity of CD19.CAR-Vδ2 T cells in vivo. In contrast, Fas88/CD19.CAR-Vδ2 T cells persisted and expanded in tumor-bearing mice improving the median survival to 47 days (p = 0.0478), indicating the ability of Fas88 to sustain functional persistence of CD19.CAR-Vδ2 T cells. This data shows that combining AICD suppression with MyD88 signaling improves the therapeutic potency of CAR-engineered Vδ2 T cells, demonstrating a means for exploiting Vδ2 T cells for allogeneic adoptive immunotherapy. Current work is focused on evaluating this approach for clinical translation.

Gamma-Delta (γδ) CAR-T Cells Lacking the CD3z Signaling Domain Enhance Targeted Killing of Tumor Cells and Preserve Healthy Tissues

Introduction: Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies for patients who have limited treatment options. Successful advancement of CAR-T therapy to myeloid hematopoietic and solid malignancies, however, has been limited by the potential of life-threatening on-target, off-tumor toxicities against healthy tissues, the natural tumor heterogeneity and consequent selection pressure leading to relapse. Addressing these limitations to widen the therapeutic index is crucial as we seek safer and more effective CAR-T therapies. In this regard, γδ T cells as first responders of immunity hold promise as they can directly identify and eliminate malignant cells through the recognition of multiple tumor-associated stress antigens not commonly expressed on normal tissues. We sought to combine the tumor-sensing capabilities of γδ T cells and the localization enhancement of CAR-T by excluding the CD3ζ domain in a non-signaling CAR (nsCAR) to allow the targeted killing against tumors, while sparing healthy tissues. Methods: Standard and nsCAR constructs against CD19, CD33 and CD123 were cloned into separate second-generation lentiviral vectors. The CAR constructs contained either a scFv targeting the respective antigen (anti-CD19/anti-CD33) or an IL-3 zetakine (IL-3z) targeting the CD123 followed by a flag-tag, a hinge, a transmembrane domain and a costimulatory domain + CD3ζ. IL-15 co-expression was incorporated to enhance γδ T cell fitness and persistence. CAR activation was determined by Jurkat T cells co-cultured with target ALL or AML cell lines followed by lentivector transduction of activated and expanded Vδ2 + γδ T cells. Cellular cytotoxicity was assessed at multiple E:T ratios against respective leukemia cell lines and healthy peripheral blood cells expressing the target antigen. Results: We observed significant upregulation of CD69 surface expression indicating activation of CD3ζ+ CD19CAR/CD33CAR/IL-3z Jurkat cells (3x, 20x, and 2x respectively) following culture with Nalm6 (CD19) and KG-1 (CD33 & IL-3z). CD3ζ- ns19CAR/ns33CAR/nsIL-3z Jurkat cells did not upregulate CD69 in parallel cultures. We also observed a time-dependent reduction of the CD3ζ+ CD33 CAR + population over 7 days (43%) in Jurkat cells following extended coculture with KG-1 cells, while the nsCAR + Jurkat population remained unchanged, indicating the nsCAR may mitigate activation induced cell death (AICD). Ex vivo activated γδ T cells from healthy donors (N=4), were transduced with nsCAR lentiviral vectors with high efficiency (up to 80%). The ns19CAR cells effectively killed CD19 + Nalm6 cells and demonstrated enhanced (>1.5x) cytotoxicity compared to untransduced γδ T cells (UTD). After 48hr co-culture (E:T=2:1), the ns19CAR cells killed 79.7%+6.6% Nalm6 cells compared to 46.1%+7.2%) with UTD. Minimal cytotoxicity was observed for ns19CAR and UTD γδ T cells against B cells from healthy donor PBMC 5.2%+6.6% for ns19CAR vs. to -4.6%+7.7% with UTD. No significant difference in cytotoxicity against the CD19- K562 cells between the UTD or ns19CAR cells (73.5% vs. 71.5%, E:T=2:1) suggesting the enhanced cytotoxicity is ns19CAR directed. Similarly, ns33CAR and nsIL-3z cells, although requiring higher E:T ratios for similar killing efficiency, continued to demonstrate enhanced (up to 2.0x) killing against AML cells (HL-60, KG-1 and MOLM13) and CML cells K-562 compared to UTD in cytotoxicity assay with 24hr co-culture. Meantime, minimal cytotoxicity (<10%) was observed for ns33CAR, nsIL-3z, or UTD γδ T cells against CD33+ cells isolated from healthy donor PBMC. Conclusions: Ex vivo activated nsCAR cells efficiently recognize and kill leukemia cell lines while sparing peripheral blood cells bearing the same target antigen. The nsCAR cells also show increased cytotoxicity against leukemias over unmodified activated γδ T cells suggesting improvement in tropism and/or binding efficiency. In summary, our findings showed that the combination of nsCAR on γδ T cells may increase the therapeutic index to allow expansion of CAR-T therapy to cancers with unacceptable target expression on critical healthy cell populations. Further optimization of the nsCAR constructs may potentially enhance both the efficacy and safety profile of the next generation adoptive cell therapies against wider selection of cancers.

Combination of Ibrutinib with CD19 Chimeric Antigen Receptor (CAR) T Cells Suggests Greater Contribution to CAR T-Cell Efficacy Than Other Approved BTK Inhibitors in the Preclinical Setting

Background: CD19-directed CAR T-cell therapy has demonstrated remarkable clinical benefits for patients with B-cell malignancies, but impaired CAR T-cell fitness can result in lack of response or relapse. Rational combinations that enhance T-cell fitness may overcome primary and secondary resistance. Bruton tyrosine kinase inhibitors (BTKi), such as Ibrutinib (Ibr) have been shown to enhance CAR T-cell efficacy (Fraietta et al, Blood 2016;Liu et al, Cancer Sci 2020), but the precise mechanism remains unclear. In addition to BTK, Ibr potently inhibits IL-2-inducible tyrosine kinase (ITK), an important mediator of T-cell signaling. Ibr has been shown to enhance Th1 polarization through suppression of the Th2 differentiation pathway, thereby potentiating a cytotoxic effector response (Qin et al, J Immunother 2020). Consistent with this finding, the Th1 phenotype was more frequent in peripheral blood mononuclear cells of Ibr-exposed patients than in acalabrutinib (Acala)-exposed patients (Scarfò et al, Blood 2021). Therefore, we aimed to assess the potential impact of ITK inhibition (ITKi) on CD19 CAR T-cell function by comparing Ibr to other approved BTKi that display low or no ITKi activity (Estupiñán et al, Front Cell Dev Biol 2021). Methods: CD19-41bb CAR T cells (CAR19) were generated by lentiviral transduction of healthy donor T cells. BTKi additivity to CAR19 function was evaluated in a Luciferase (Luc)+ Ramos, BTKi resistant systemic tumor model in NSG mice co-treated with covalent BTKis Ibr, Acala, zanubrutinib (Zanu), and CAR T cells. Next, to evaluate the impact of ITKi on CAR19 fitness and cytotoxicity, repeated stimulation in an in vitro cytotoxicity assay was performed at low CAR19 effector-to-tumor ratio (1:20) to mimic higher tumor burden stress conditions in the presence of each BTKi, including non-covalent BTKi pirtobrutinib (Pirto) (500 nM). Tumor cells were replenished every 3 days and cytotoxicity was measured on day 7 by luciferase assay. For phenotypic characterization, CAR T cells were repeatedly stimulated by plate-bound anti-Whitlow linker every 3 days. Viability and proliferation were measured by cell counter. Flow cytometry (FACS) was performed on surface and intracellular T-cell phenotypic, activation, and exhaustion markers. Metabolic fitness (glycolysis and ATP production) was characterized using Seahorse (Van der Windt, 2012). Impact of Ibr on signal strength was evaluated in CD19-expressing Jurkat reporter cells (CAR-J19) with knock-in of mKate in frame with Nur77. Ibr was added to coculture of CAR-J19 and tumor cells and upregulation of Nur77 was measured by FACS. Downstream ITK signaling pathway was measured by Western blot. Results: Preliminary in vivo experiments demonstrated that co-administration of each BTKi with CD19 CAR T cells improved tumor control at day 26 (N=5 per group; P≤0.0004 vs CAR T cells alone). In serial killing experiments, addition of Ibr to the coculture increased proliferation (n=2; P<0.05), viability (n=2; P=0.01), and the cytotoxicity of CAR T cells against Ramos cells ( P=0.02 vs CAR19 alone) to a greater extent than other BTKis tested (Fig. 1A). Prolonged stimulation of CAR19 with Ibr led to downregulation of activation and inhibitory markers on CAR19 and maintenance of a naive T-cell phenotype (Fig. 1B) that was confirmed by increased metabolic fitness (n=5; P<0.05). Addition of Ibr to CAR-J19 resulted in a 72% reduction in CAR activation and reduced phosphorylation of ITK, PLCG, and Zap70. Additional data including the impact of ITK knockout on CAR-T cells will be presented. Conclusions: Our study expands on the hypothesized mechanism of action underlying the clinical benefit observed with Ibr in CD19 CAR T-cell-treated patients with chronic lymphocytic leukemia or mantle cell lymphoma (Liu et al, Front Immunol 2022; Wang et al, NEJM 2020). Our data suggest that Ibr enhances CAR T-cell fitness to a greater extent than both covalent and noncovalent BTKi that display reduced or no ITKi activity. Through ITKi, Ibr tempers CAR T-cell activation induced cell death, maintains a naive state and prevents premature exhaustion, thereby enabling more efficacious tumor cell killing over time. Together, these data support the further exploration of Ibr to enhance CD19 CAR T-cell efficacy.

A First-in-Human Phase I Study of AT101, a Novel Anti-CD19 Chimeric Antigen Receptor T Cell Product Targeting a Membrane-Proximal Domain of CD19 in Adults with Relapsed or Refractory B Cell Non-Hodgkin Lymphoma

Introduction: Anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are highly efficacious against advanced B cell non-Hodgkin lymphoma (NHL) but the majority of patients (pts) ultimately fail. Several mechanisms contribute to CART19 failure, including CD19-neg escape and CART dysfunction. Notably, all four commercial CART19 products utilize the FMC63 single chain variable fragment (scFv) with high-avidity specificity to a CD19 membrane-distal epitope. Interestingly, loss of the FMC63-recognized epitope due to CD19 mutations ( Zhang Z, JITC, 2020) or epitope-masking (CAR19:CD19) ( Ruella M, Nat Med, 2018) has been described. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 with low avidity could: 1. mitigate CD19 epitope loss; but also 2. enhance CART functions ( Fig. 1A) . Methods and results: We developed an autologous, CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218) specific to a membrane-proximal epitope of CD19 (amino acids T51,S53, E55,K59 and K63). We previously reported initial preclinical data ( Patel R., ASH, 2021 #2798) that demonstrated h1218-CART19 recognize and kill B-cell leukemia that aberrantly expresses the FMC63-CAR19. More recently, we demonstrated that differently than FMC63-based CART, h1218-CART19 could respond to and kill tumor cells expressing point mutations of CD19 (L174V and R163L). Of note, h1218-CART19 had enhanced control of CD19+ B-cell neoplasms as compared to FMC63-CART19 both in vitro and in vivo. Mechanistically, thanks to the lower avidity and faster on/off rate, h1218-CART19 had decreased T cell exhaustion and activation-induced cell death. Given the promising preclinical results, we tested the clinical h1218-CART19 product (AT101) in a first-in-human, multi-center, phase I/II, dose-escalation clinical trial for pts with histologically confirmed relapsed or refractory B cell NHL (NCT05338931). For the phase I study, pts were treated with a single intravenous infusion of AT101 (day 0) in three dose levels (DL) based on a standard 3+3 design: DL1 (0.2 x 10 6 cells/kg), DL2 (1.0 x 10 6 cells/kg), or DL3 (5.0 x 10 6 cells/kg). All pts received lymphodepletion with intravenous fludarabine (250 mg/m 2) and cyclophosphamide (25 mg/m 2) on days -4, -3, and -2. The primary objective was to determine the maximum tolerated dose (MTD) of AT101. The secondary objectives were to evaluate AT101 pharmacokinetics and preliminary efficacy. At the July 3, 2023 data cut, 12 pts (median age 62.5 years (range 39-84); diffuse large B cell lymphoma (DLBCL, n=7, 58.3%), follicular lymphoma (FL, n=3, 25.0%), mantle cell lymphoma (MCL, n=1, 8.3%), and marginal zone lymphoma (MZL, n=1, 8.3%)) received AT101 ( Fig. 1B). The median follow-up after AT101 was 6.5 months (1.5-13.7 months), the median number of previous lines of treatment 3 (range 1-8); no prior cellular therapies. Five pts (41.7%) had previous autologous stem cell transplantation. Two pts (16.7%) had refractory disease and two pts (16.7%) received bridging therapy. Three pts (2 pts at DL2 and 1 pt at DL3) developed a grade (G) 1 cytokine-release syndrome (CRS) and one pt at DL3 developed a G3 CRS that resolved within 24 hours. Three pts (1 pt at each DL) developed immune-cell-related neurotoxicity syndrome (ICANS), of which one G4 dose-limiting toxicity (DLT) was reported at DL1 that corrected within 6 days. One DL3 pt experienced G3 sepsis that promptly resolved; the same pt ultimately developed fatal neutropenic septic shock outside the DLT period. Four additional patients experienced ≥G3 neutropenia without evidence of infection. Complete response (CR) occurred in 8/12 pts (66.6%) and overall response (CR+partial response) in 83.3% (95%CI, 51.6-97.9) at day 28 post-AT101 infusion ( Fig. 1B). Remarkably, in DL2 and DL3, the CR rate was 100.0%. Of the 8 pts in CR none has relapsed (median follow-up 6.0 months). AT101 cells expanded (peak in DL3 113.6 ± 6.50 x10 4 CAR gene copies/ug DNA) and persisted in patients' blood. Conclusion: In this first-in-human phase I trial, AT101 was tolerable with limited and manageable toxicities. Notable preliminary efficacy was observed at the RP2D with no evidence of disease relapse after achieving a CR. These results warrant the pursuit of the planned phase II expansion cohort.

TNF-α protects from exacerbated myocarditis and cardiac death by suppressing expansion of activated heart-reactive CD4+ T cells.

Tumour necrosis factor α (TNF-α) represents a classical pro-inflammatory cytokine, and its increased levels positively correlate with the severity of many cardiovascular diseases. Surprisingly, some heart failure patients receiving high doses of anti-TNF-α antibodies showed serious health worsening. This work aimed to examine the role of TNF-α signalling on the development and progression of myocarditis and heart-specific autoimmunity. Mice with genetic deletion of TNF-α (Tnf+/- and Tnf-/-) and littermate controls (Tnf+/+) were used to study myocarditis in the inducible and the transgenic T cell receptor (TCRM) models. Tnf+/- and Tnf-/- mice immunized with α-myosin heavy chain peptide (αMyHC) showed reduced myocarditis incidence, but the susceptible animals developed extensive inflammation in the heart. In the TCRM model, defective TNF-α production was associated with increased mortality at a young age due to cardiomyopathy and cardiac fibrosis. We could confirm that TNF-α as well as the secretome of antigen-activated heart-reactive effector CD4+ T (Teff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to Teff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4+ T lymphocytes from both models of myocarditis showed a strongly increased fraction of Teff cells in hearts, spleens, and in the blood of Tnf+/- and Tnf-/- mice. Indeed, antigen-activated Tnf-/- Teff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive Teff. TNF-α signalling promotes myocarditis development by activating cardiac endothelial cells. However, in the case of established disease, TNF-α protects from exacerbating cardiac inflammation by inducing activation-induced cell death of heart-reactive Teff. These data might explain the lack of success of standard anti-TNF-α therapy in heart failure patients and open perspectives for T cell-targeted approaches.

Regulation of human and mouse bystander T cell activation responses by PD-1.

Bystander activation of memory T cells occurs via cytokine signaling alone in the absence of T cell receptor (TCR) signaling and provides a means of amplifying T cell effector responses in an antigen-nonspecific manner. While the role of Programmed Cell Death Protein 1 (PD-1) on antigen-specific T cell responses is extensively characterized, its role in bystander T cell responses is less clear. We examined the role of the PD-1 pathway during human and mouse non-antigen-specific memory T cell bystander activation and observed that PD-1+ T cells demonstrated less activation and proliferation than activated PD-1- populations in vitro. Higher activation and proliferative responses were also observed in the PD-1- memory population in both mice and patients with cancer receiving high-dose IL-2, mirroring the in vitro phenotypes. This inhibitory effect of PD-1 could be reversed by PD-1 blockade in vivo or observed using memory T cells from PD-1-/- mice. Interestingly, increased activation through abrogation of PD-1 signaling in bystander-activated T cells also resulted in increased apoptosis due to activation-induced cell death (AICD) and eventual T cell loss in vivo. These results demonstrate that the PD-1/PD-Ligand 1 (PD-L1) pathway inhibited bystander-activated memory T cell responses but also protected cells from AICD.

Reconciling intrinsic properties of activating TNF receptors by native ligands versus synthetic agonists.

The extracellular domain of tumor necrosis factor receptors (TNFR) generally require assembly into a homotrimeric quaternary structure as a prerequisite for initiation of signaling via the cytoplasmic domains. TNF receptor homotrimers are natively activated by similarly homo-trimerized TNF ligands, but can also be activated by synthetic agonists including engineered antibodies and Fc-ligand fusion proteins. A large body of literature from pre-clinical models supports the hypothesis that synthetic agonists targeting a diverse range of TNF receptors (including 4-1BB, CD40, OX40, GITR, DR5, TNFRSF25, HVEM, LTβR, CD27, and CD30) could amplify immune responses to provide clinical benefit in patients with infectious diseases or cancer. Unfortunately, however, the pre-clinical attributes of synthetic TNF receptor agonists have not translated well in human clinical studies, and have instead raised fundamental questions regarding the intrinsic biology of TNF receptors. Clinical observations of bell-shaped dose response curves have led some to hypothesize that TNF receptor overstimulation is possible and can lead to anergy and/or activation induced cell death of target cells. Safety issues including liver toxicity and cytokine release syndrome have also been observed in humans, raising questions as to whether those toxicities are driven by overstimulation of the targeted TNF receptor, a non-TNF receptor related attribute of the synthetic agonist, or both. Together, these clinical findings have limited the development of many TNF receptor agonists, and may have prevented generation of clinical data which reflects the full potential of TNF receptor agonism. A number of recent studies have provided structural insights into how different TNF receptor agonists bind and cluster TNF receptors, and these insights aid in deconvoluting the intrinsic biology of TNF receptors with the mechanistic underpinnings of synthetic TNF receptor agonist therapeutics.