Dominant Negative TGF-β Receptor Research Articles

Abstract CT170: Trial in progress: A phase 1, first in human clinical trial for T-Plex, a multiplexed, enhanced T cell receptor-engineered T cell therapy (TCR-T) for solid tumors

Abstract Checkpoint immunotherapies have revolutionized solid tumor treatment yet durably benefit a minority of patients, as they rely on endogenous anti-tumor T cells. A potential solution for patients lacking functional endogenous anti-tumor T cells is engineering their T cells with exogenous T cell receptors (TCRs) to target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) targeted single tumor antigens on single HLA and produced partial, short-lasting responses. Solid tumors are notoriously heterogenous with highly variable antigen expression. Recent discoveries also identified HLA loss of heterozygosity in up to 40% of solid tumors, allowing tumor cells to evade T cell attack. To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed cell therapy comprising 2-3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens on different HLA types with confirmed tumor expression. To deepen clinical responses, TCR-T cells are engineered to express CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to have >100-fold improved cytotoxicity and cytokine secretion over CD4+ cells expressing the TCR alone. Finally, to allow T cell persistence despite immunosuppressive TGF-β in the solid tumor microenvironment, TCR-T cells also express the dominant negative TGF-βreceptor, enabling ~10-fold improved proliferation in the presence of TGF- β compared to T cells expressing the TCR and CD8α/β co-receptors alone. A proprietary transposon vector with larger cargo limit enables the inclusion of these additional genes. The Phase 1 study utilizes a separate screening protocol to pre-identify patients with head and neck, cervical, anogenital cancers, NSCLC, and melanoma, any time during standard clinical care, enabling rapid enrollment into the treatment protocol upon disease progression. Screening comprises germline HLA testing, and archival tumor testing for antigen expression and exclusion of HLA loss. Treatment includes standard non-myeloablative lymphodepletion followed by one or 2 doses of T-Plex infused 28 days apart. Dose escalation under the interval 3+3 design starts with testing single TCR-Ts in dose levels 1 and 2. Thereafter, TCR-Ts are combined and escalated in dose levels 3 and 4. TCR-Ts currently in the master protocol target PRAME, MAGE-A1 or HPV16 on HLA-A*02:01 or MAGE-A1 on HLA-C*07:02. Additional TCR-Ts added to the ImmunoBank and master protocol go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety, feasibility and identifying the recommended Phase 2 dose. Secondary endpoints are rates and durations of response and exploratory endpoints measure T cell activation and persistence. Two additional TCR-Ts are on track to be added to the ImmunoBank, which could allow 50-80% of common solid tumor patients to qualify for multiplexed TCR-T therapy. Citation Format: Justin Moser, Brian Pico, Brian Henick, Rom Leidner, Jared Weiss, James Isaacs, Jaspreet Grewal, Michael Hurwitz, Jim Murray, Marlyane Motta, Yun Wang, Shrikanta Chattopadhyay, Debora Barton, Gavin MacBeath, Sajeve Thomas. Trial in progress: A phase 1, first in human clinical trial for T-Plex, a multiplexed, enhanced T cell receptor-engineered T cell therapy (TCR-T) for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT170.

A novel GPI-anchored dominant-negative TGF-β receptor II renders T cells unresponsive to TGF-β signaling

Transforming growth factor β (TGF-β) is a pleiotropic cytokine expressed by a wide range of cell types and is known for hampering the effectiveness of cancer immune cell therapeutic approaches. We have designed a novel construct containing the extracellular domain of the TGF-β receptor II linked to a glycosylphosphatidylinositol (GPI) anchor (GPI-ecto-TβRII) lacking the transmembrane and cytoplasmic signaling domain of TGF-β receptor II (TβRII). Tcells transduced with lentivirus expressing the GPI-ecto-TβRII construct show 5 to 15 times higher membrane expression compared with a previously established dominant-negative receptor carrying a truncated signaling domain. GPI-ecto-TβRII expression renders Tcells unresponsive to TGF-β-induced signaling seen by a lack of SMAD phosphorylation upon exogeneous TGF-β treatment. Transduced Tcells continue to express high levels of IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF), among other cytokines, in the presence of TGF-β while cytokine expression in untransduced Tcells is being markedly suppressed. Furthermore, Tcells expressing GPI-ecto-TβRII constructs have been shown to efficiently capture and inactivate TGF-β from their environment. These results indicate the potential benefits of GPI-ecto-TβRII expressing cytotoxic Tcells (CTLs) in future cell therapies.

Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer

BackgroundThe immune checkpoint molecules, Transforming growth factor beta receptor II (TGFβRII) and T cell immunoglobulin and mucin domain 3 (TIM3), have been identified as contributors to T cell immune suppression in prostate cancer. The objective of this investigation was to improve the tumor killing capability of prostate-specific membrane antigen (PSMA)-chimeric antigen receptor T (CAR-T) cells by targeting TIM3 and TGFβRII simultaneously. MethodsTo generate dnTGFβRII-trTIM3-PSMA-CAR-T (DT-PSMA-CAR-T) cells, the surface of PSMA-CAR-T cells was overexpressed with dominant negative TGFβRII (dnTGFβRII) and truncated extracellular TIM3 (trTIM3). The efficacy of DT-PSMA-CAR-T cells was assessed through in vitro killing experiments and animal experiments. ResultsThe DT-PSMA-CAR-T cells demonstrated the ability to eradicate PSMA-positive prostate cancer cells, even in the presence of exogenous TGF-β and/or TIM3 activating antibodies. In addition, the cells demonstrated the ability to eliminate tumor tissue in an immunodeficient mouse model transplanted with GAL9-PSMA-PC3 cells in vitro, prolonging survival without significant toxic side effects. ConclusionsThis study emphasizes that upregulating dnTGFβRII and trTIM3 on the surface of T cells can potentially diminish the inhibitory effects of TGFβRII and TIM3.

Product characteristics and clinical trial design for T-Plex: Multiplexed, enhanced T cell receptor engineered T cell therapy for solid tumors.

2554 Background: Checkpoint immunotherapies have revolutionized the treatment of solid tumors yet durably benefit a minority of patients because they rely on endogenous anti-tumor T cells. In patients lacking functional anti-tumor T cells, a solution is engineering their T cells with exogenous T cell receptors (TCRs) to efficiently target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) only produced partial, short-lasting responses because they targeted single tumor antigens. Solid tumors are notoriously heterogenous not only with highly variable antigen expression levels but also with recently identified HLA gene losses occurring in up to 40% of solid tumors, enabling tumor cells to evade T cell attack. Methods: To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed therapy comprising 2-3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens presented on different HLA types with confirmed tumor expression. Results: To deepen clinical responses, TCR-T cells are engineered to express the CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to become >100-fold more cytotoxic and secrete cytokines at >100-fold lower target antigen levels. Finally, to enable T cell persistence despite immunosuppressive TGF-β in the tumor microenvironment, engineered TCR-T cells express the dominant negative TGF-β receptor allowing ~10-fold improved proliferation and ~2-fold improved cytokine production in the presence of TGF-β. The inclusion of these additional genes is enabled by a transposon manufacturing system with no cargo limit. Patient selection for the Phase 1 study uses a separate screening protocol to identify patients any time during standard clinical care so they can rapidly enroll into the treatment protocol upon progression. Screening comprises germline HLA testing, then archival tumor testing for antigen expression and HLA loss. Treatment includes standard lymphodepletion followed by 2 doses of T-Plex infused 28 days apart. T-Plex initially starts with 2 TCR-Ts targeting different MAGE-A1 antigens presented either on HLA-A*02:01 or HLA-C*07:02. Dose escalation starts with testing the single TCR-Ts individually in dose levels 1 and 2. The two TCR-Ts will then be combined and escalated in dose levels 3 and 4. Additional TCR-Ts added to the ImmunoBank will go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety and feasibility, secondary endpoints are response rates and duration of response and exploratory endpoints measure T cell persistence. Conclusions: Four additional TCR-Ts are on track to be added to the ImmunoBank in 2023 which makes 50-80% of patients with common solid tumors eligible for multiplexed TCR-T therapy.

Abstract LB097: Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma

Abstract Introduction: We have completed two CAR T cell clinical trials for glioblastoma (GBM) and have identified several key challenges to therapeutic efficacy, including the inherently heterogenous genomic landscape and the immunosuppressive tumor microenvironment (TME) found in GBM. Our previous study showed that EGFR variant III (EGFRvIII)-targeting monovalent CAR T cells reduced target-positive tumor cell populations, but tumor recurrence resulted from target-negative tumor cells, highlighting the limitation of single-target approaches in heterogenous tumors. With regards to the highly immunosuppressive TME in GBM, we found that transforming growth factor-β (TGFβ) was present in the GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFb is consistently highly expressed in both GBM tumor cell lines and patient tumor tissues. Methods: We used two parallel scFv constructs, independently targeting both IL13Rα2 and EGFRvIII, in combination with a truncated dominant negative (dn) TGFβ receptor II. This trivalent construct was designed to explore possible additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome the immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFb construct broadened the targeted tumor cell repertoire, blocked TGFβ signaling, and served as a sink for free TGFβ in the GBM TME to overcome the suppressive function of TGFβ. Results: The tri-modular CAR T construct had an enhanced proliferative response when compared with the CART-EGFR-IL13Rα2 construct, in vitro. In co-culture assays, this construct led to reduced PD-1 expression and increased central memory phenotype, when compared to the bicistronic CAR T construct, which suggested a lower fraction of exhausted T cells. Tri-modular CAR T cells blocked the suppressive pSmad2/3 signaling pathway, leading to the increased tumor killing activity in co-culture experiments with both adherent and suspension GBM cell lines. In an immunodeficient mouse model, tri-modular CAR T cells eradicated tumor cells efficiently and mice had a longer median survival when compared those treated with the bicistronic CART-EGFR-IL13Rα2 cells, lacking the dnTGFb receptor II. Conclusion: Overcoming the adaptive changes in the local TME and addressing antigen heterogeneity will be required to improve the clinical efficacy of CAR T-directed strategies. Our combination work showed that bicistronic CART constructs cooperate with truncated TGFβ receptor II efficiently. In summary, the dominant-negative TGFβ RII CART-EGFR-IL13Rα2 structure is a promising strategy to address the clinical challenges of antigenic heterogeneity and the immunosuppressive TME in GBM we have observed in our two GBM CART cell trials at UPenn. Citation Format: Nannan Li, Jesse Rodriguez, Zev Binder, Donald O’Rourke. Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB097.

Abstract CT097: First report of preliminary safety, efficacy, and pharmacokinetics of C-CAR031 (GPC3-specific TGFβRIIDN CAR-T) in patients with advanced HCC

Abstract Introduction: Chimeric antigen receptor (CAR) T cells can mediate deep and durable responses in hematologic malignancies, however, achieving success in solid tumors has been so far limited largely by lack of suitable solid tumor-associated antigens and the immunosuppressive tumor microenvironment (TME). GPC3 is a surface antigen overexpressed in hepatocellular cancer (HCC) and virtually absent on healthy tissues. In this first-in-human (FIH) study, we investigated the feasibility, safety and initial anti- HCC efficacy of C-CAR031. C-CAR031 is an autologous, GPC3-directed armored CAR-T with affinity-tuned scFv to enhance the safety profile, and a 4-1BB and CD3ζ signaling domain. The C-CAR031 transgene includes a T2A viral self-cleaving peptide and a dominant negative TGF-β receptor II (TGFβRIIDN). The expression of TGFβRIIDN protects the cells against the immunosuppressive HCC TME and the T2A peptide allows for equimolar expression of the two transgene products. Methods: This FIH, open-label dose escalation trial employs an accelerated titration plus i3+3 design. Histologically confirmed GPC3+ advanced HCC patients (pts) who failed systemic treatments received a single-dose i.v. infusion of C-CAR031 following standard lymphodepletion. The primary objective was to assess the safety and tolerability. Adverse events (AEs) were graded using CTCAE 5.0, and cytokine release syndrome (CRS)/immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT 2019 criteria. Results: As of Dec. 31st 2022, 7 pts received two dose levels (DL1, n=1; DL2, n=6) of C-CAR031. The median number of prior lines of therapies was 4 (range 1-6). The median follow-up was 77 (40-213) days. Six pts with ≥28 days’ follow-up were eligible for safety evaluation. The only ≥Gr3 non-hematologic product-related AE observed was transient Gr3 AST elevation in two pts. Five of 6 pts experienced Gr1/2 CRS, with median time to onset and duration of 3 (range 2-7) and 4 (4-6) days. No DLT or ICANS was observed. Of the 5 pts evaluable for preliminary efficacy, 4 pts had unconfirmed PR, which are currently pending confirmation. AFP was also stabilized or reduced in all 4 patients with uPR. All 5 pts had reduction in tumor burden, with a median change of -31.2% (range -3.4- -60.6%)/-41.4% (-3.4- -56.6%) per RECIST1.1/mRECIST. C-CAR031showed a robust cellular kinetic profile. In DL2, the median Tmax, Cmax and AUC0-28Day were 15 days, 772,014 copies/μg gDNA and 7,747,054 days*copies/μg gDNA, respectively. CAR-T cells were detectable in blood of all pts in the last follow-up. Conclusions: In this FIH study, C-CAR031 is well tolerated and shows promising anti- tumor activity. Enrollment is ongoing to confirm initial results. Citation Format: Qi Zhang, Qihan Fu, Wanyue Cao, Xingyuan Xu, Ao Xia, Jiaqi Huang, Andy Zou, Judy Zhu, Fei Wang, Yi Hong, Hui Wan, Yihong Yao, Nina Chu, Ryan Gilbreth, Maria Letizia Giardino Torchia, John Stone, Attilio Bondanza, Benny Farsaci, Gordon Moody, Mark Cobbold, Tingbo Liang. First report of preliminary safety, efficacy, and pharmacokinetics of C-CAR031 (GPC3-specific TGFβRIIDN CAR-T) in patients with advanced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT097.

Abstract 548: EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC

Abstract The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 548.

TGF-β1 mediates pathologic changes of secondary lymphedema by promoting fibrosis and inflammation.

BackgroundSecondary lymphedema is a common complication of cancer treatment, and previous studies have shown that the expression of transforming growth factor‐beta 1 (TGF‐β1), a pro‐fibrotic and anti‐lymphangiogenic growth factor, is increased in this disease. Inhibition of TGF‐β1 decreases the severity of the disease in mouse models; however, the mechanisms that regulate this improvement remain unknown.MethodsExpression of TGF‐β1 and extracellular matrix molecules (ECM) was assessed in biopsy specimens from patients with unilateral breast cancer‐related lymphedema (BCRL). The effects of TGF‐β1 inhibition using neutralizing antibodies or a topical formulation of pirfenidone (PFD) were analyzed in mouse models of lymphedema. We also assessed the direct effects of TGF‐β1 on lymphatic endothelial cells (LECs) using transgenic mice that expressed a dominant‐negative TGF‐β receptor selectively on LECs (LECDN‐RII).ResultsThe expression of TGF‐β1 and ECM molecules is significantly increased in BCRL skin biopsies. Inhibition of TGF‐β1 in mouse models of lymphedema using neutralizing antibodies or with topical PFD decreased ECM deposition, increased the formation of collateral lymphatics, and inhibited infiltration of T cells. In vitro studies showed that TGF‐β1 in lymphedematous tissues increases fibroblast, lymphatic endothelial cell (LEC), and lymphatic smooth muscle cell stiffness. Knockdown of TGF‐β1 responsiveness in LECDN‐RII resulted in increased lymphangiogenesis and collateral lymphatic formation; however, ECM deposition and fibrosis persisted, and the severity of lymphedema was indistinguishable from controls.ConclusionsOur results show that TGF‐β1 is an essential regulator of ECM deposition in secondary lymphedema and that inhibition of this response is a promising means of treating lymphedema.

Single-Cell Characterization of Hepatic CD8+ T Cells in a Murine Model of Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC), an organ-specific autoimmune disease, is characterized by injury to small bile ducts, inflammatory cell infiltrates within the liver, progressive cholestasis, and in some cases, cirrhosis with unclear pathogenesis. We aimed to clarify the importance role of hepatic immunce cells in the pathogenesis of human and experimental PBC.The dominant-negative TGFβ receptor type II transgenic (dnTGFβRII) mice, a well-studied and established murine model of PBC were used to identify changes of immune cells, especially the pathogenic CD8+ T cells. The high-throughput single-cell RNA sequencing technology were applied and found functional heterogeneity among the hepatic CD8+ T cells subsets in dnTGFβRII mice. CD8+ T cells were confirmed the key cells leading to the pathogenesis of PBC in dnTGFβRII mice, and identified the terminally differentiated CD8αα T cells and CD8αβ T cell subsets in the liver of dnTGFβRII mice. While terminally differentiated CD8αα T cells have higher cytokine production ability and cytotoxicity, the terminally differentiated CD8αβ T cells retain their proliferative profile. Our work suggests that there are developmental and differentiated trajectories of pathogenic CD8+ T cell subsets in the pathogenesis of PBC. A further clarification of their roles would be helpful to our understanding of the pathogenesis of PBC and may potentially lead to identifying novel therapeutic modalities.

CAR T Cells with a Dominant-Negative TGFβ Receptor Are Safe and Feasible.

PSMA-targeting CAR T cells with a dominant-negative TGFβR show safety and feasibility in prostate cancer.

Dominant negative TGF-β receptor type II in T lymphocytes promotes anti-tumor immunity by modulating T cell subsets and enhancing CTL responses

Transforming growth factor-β (TGF-β) is a multifunctional regulatory cytokine that maintains tolerance in the immune system by regulating the proliferation, differentiation and survival of lymphocytes. TGF-β blockade therapy for cancer has achieved some results but shows limited efficacy and side effects because these drugs are not selective and act on various types of cells throughout the body. We demonstrate here that dominant negative TGF-β receptor type II specifically targeting T cells decreases tumor load in tumor-bearing mice. In addition, the dominant negative TGF-β receptor type II promotes the proliferation and differentiation of T cells and increases the expression of T-bet, which in turn promotes the secretion of granzyme A, granzyme B, perforin and IFN-γ secreted by T cells, and enhances the cytotoxicity and anti-tumor effects of T cells. Moreover, we also found that dominant negative TGF-β receptor type II reduces the proportion of regulatory T cells (Tregs) in tumor tissue and spleen of tumor-bearing mice. Co-culture experiments with T cells and tumor cells revealed that dominant negative TGF-β receptor type II inhibited tumor cell proliferation and increased apoptosis. Our results indicate that specifically inhibiting TGF-β receptor type II in T cells increases anti-tumor immunity and has a strong therapeutic potential.

Enoxacin Up-Regulates MicroRNA Biogenesis and Down-Regulates Cytotoxic CD8 T-Cell Function in Autoimmune Cholangitis.

Primary biliary cholangitis (PBC) is a prototypical organ-specific autoimmune disease that is mediated by autoreactive T-cell attack and destruction of cholangiocytes. Despite the clear role of autoimmunity in PBC, immune-directed therapies have failed to halt PBC, including biologic therapies effective in other autoimmune diseases. MicroRNA (miRNA) dysregulation is implicated in the pathogenesis (PBC). In the dominant-negative TGF-β receptor type II (dnTGFβRII) mouse model of PBC, autoreactive CD8 T cells play a major pathogenic role and demonstrate a striking pattern of miRNA down-regulation. Enoxacin is a small molecule fluoroquinolone that enhances miRNA biogenesis, partly by stabilizing the interaction of transactivation response RNA-binding protein with Argonaute (Ago) 2. We hypothesized that correcting aberrant T-cell miRNA expression with enoxacin in dnTGFβRII mice could modulate autoreactive T-cell function and prevent PBC. Here, we show that liver-infiltrating dnTGFβRII CD8 T cells have significantly decreased levels of the miRNA biogenesis molecules prolyl 4-hydroxylase subunit alpha 1 (P4HA1) and Ago2 along with significantly increased levels of granzyme B and perforin. Enoxacin treatment significantly up-regulated miRNAs in dnTGFβRII CD8 T cells and effectively treated autoimmune cholangitis in dnTGFβRII mice. Enoxacin treatment directly altered T cells both ex vivo and in vitro, resulting in altered memory subset numbers, decreased proliferation, and decreased interferon-γ production. Enoxacin significantly decreased CD8 T-cell expression of the transcription factor, Runx3, and significantly decreased perforin expression at both the mRNA and protein levels. Enoxacin increases miRNA expression in dnTGFβRII CD8 T cells, reduces CD8 T-cell pathogenicity, and effectively halted progression of autoimmune biliary disease. Targeting the miRNA pathway is a therapeutic approach to autoimmunity that corrects pathological miRNA abnormalities in autoreactive T cells.

CAR T-cell therapy for solid tumours

Chimeric antigen receptor (CAR) T-cell therapy has fundamentally changed the therapeutic landscape for haematological malignancies. CAR T-cell therapy involves the collection of a patient's T cells, ex-vivo genetic modification of the cells to encode a synthetic receptor that binds a specific tumour antigen, and then re-infusion of these cells back into the patient. The clinical success of CAR T-cell therapy in blood cancers has generated enthusiasm for testing the technology in solid tumours. However, the biology of solid tumours is more complex than that of haematological malignancies. Unfortunately, CAR T-cell therapy for solid tumours suffered a setback on June 2, 2021, when Tmunity Therapeutics stopped its phase 1 clinical trial in men with prostate cancer because two patients died from immune effector cell-associated neurotoxicity syndrome (ICANS). Tmunity Therapeutics had been developing prostate-specific membrane antigen (PSMA)-directed, TGF-β-insensitive CAR T cells. Prostate cancer cells secrete TGF-β as an immunosuppressive factor, so the investigators hypothesised that using a PSMA-directed CAR T cell with coexpression of a dominant-negative TGF-β receptor would enhance antitumour immunity in patients with prostate cancer. Early trial results had shown no dose-limiting toxicities, and only one, reversible, case of cytokine release syndrome (CRS). ICANS, however, although seen less frequently than CRS, has been reported in up to half of all patients receiving CAR T-cell therapy for various haematological cancers and thus was also a risk in solid tumour trials. ICANS needs prompt diagnosis and appropriate management to avoid rapid clinical deterioration. Unlike CRS, the precise underlying pathophysiology of ICANS remains poorly understood, and although the acute symptoms of CRS and ICANS are generally reversible with supportive care, steroids, and immunosuppressive drugs, they can be associated with substantial morbidity, with some patients requiring admission to an intensive care unit. In addition to CRS and ICANS, the development of CAR T-cell therapy for solid tumours faces many fundamental challenges, the most difficult being the identification of an ideal target antigen. Haematological cancers typically express a single, specific, tumour-associated antigen, whereas solid tumours have substantial antigen heterogeneity. Of concern, many antigens associated with solid tumours are also expressed on healthy tissue, raising the risk of off-target adverse events. Genetic modification technologies can help to circumvent this risk, but they are not a failsafe solution. Other major challenges include how to traffic the CAR T cells to the tumour bed because of a lack of appropriate cytokines in the tumour milieu; how to prevent CAR T cells from losing their effectiveness over time (so-called T-cell fitness); how to lymphodeplete the tumour to allow for CAR T-cell expansion in the absence of effective lymphodepleting chemotherapies; and, finally, how to overcome the hostile immunosuppressive conditions of the tumour microenvironment. A recent development might help to overcome two of these challenges. Using a new CAR T-cell technology called synthetic Notch (or synNotch), CAR T cells can be made to target specific cancers very precisely. Researchers at the Children's Hospital Los Angeles (Los Angeles, CA, USA) have developed a modified version of a CAR T cell for treating neuroblastoma. The synNotch protein on the surface of the T cell is designed to recognise an antigen called GD2. When it does, the synNotch protein then instructs the T cell to activate its CAR T-cell properties, enabling it to recognise a second antigen called B7H3. This gating property is key to minimising toxicity, as healthy cells sometimes express one of these antigens, but rarely both. Neuroblastoma, however, has both GD2 and B7H3 antigens. Interestingly, synNotch CAR T cells are metabolically more stable than regular CAR T cells, enabling them to target a tumour over a longer timeframe and overcome the problems associated with CAR T-cell fitness. The use of CAR T-cell therapy has yielded excellent early-to-mid-term results in patients with haematological cancers, but the transfer of this technology to solid cancers faces more substantive biological barriers and risks to patients. Despite many ongoing clinical trials, very few have reported any results and all are at early stages. Consequently, many unknowns remain. The potential is evident, but it will only be realised through carefully designed trials incorporating thorough patient monitoring and stringent stopping rules coupled to detailed preclinical translational research.

A Novel TGF-β2/Interleukin Receptor Signal Conversion Platform That Protects CAR/TCR T Cells from TGF-β2-Mediated Immune Suppression and Induces T Cell Supportive Signaling Networks

Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder the efficacy of adoptively transferred T cells. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces hetero-dimerization of these receptors and phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. In several previous reports, a dominant negative receptor (DNR) version of TGFBR2, that does not contain signaling domains, was shown to protect T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs.Herein, we report the development of a novel TGF-β signal conversion platform that provides a T cell supportive signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of a T cell stimulating interleukin receptor. The signaling domains of interleukin receptors that were chosen for this platform are naturally activated via cytokine-induced hetero-dimerization and induce STAT transcription factor signaling pathways beneficial to effector T cell function and persistence. Based on the ability of lentiviral vectors to express large and complex gene expression cassettes, we constructed single vectors encoding both chimeric TGF-β receptors (CTBR) in the context of either a CAR or transgenic TCR. This required expression of four unique proteins from a single lentiviral vector when CTBR was combined with a transgenic TCR consisting of unique alpha and beta chains.To demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on multiple functional attributes of CTBR expressing T cells, including gene expression, cytokine production, cytotoxicity, and metabolic function. As anticipated, the STAT signaling signature induced by a particular CTBR was predictive of the functional consequence of exposure to TGF-β. In the case of a CTBR based on IL-12 signaling domains (CTBR12) that strongly induced STAT4 phosphorylation, CTBR12 expressing T cells secreted significantly greater amounts of IFNγ than control T cells following activation in the presence of TGF-β. This result was consistent with IL-12-mediated STAT4 signaling, which induces IFNγ production in effector T cells. We next evaluated CTBR expressing T cells in a stringent serial re-stimulation assay in the presence of TGF-β but absence of exogenous IL-2 cytokine support. Antigen driven T cell expansion was severely limited by exposure to TGF-β in this assay, and either CAR or transgenic TCR expressing cells ultimately lost cytotoxic activity over time. T cells overexpressing the dominant negative TGF-β receptor failed to expand and clear tumor cells following multiple rounds of antigen-driven stimulation in the presence of TGF-β, recapitulating the impact of tumor-derived TGF-β on infiltrating lymphocyte function. On the other hand, CTBR expressing cells maintained their ability to expand and kill tumor targets in the presence of TGF-β.Together, these data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into a STAT signal that supports T cell effector function. The specific impact on T cell function is dependent on the nature of the signaling domains utilized. This platform is relevant in the context of both CAR T cells and transgenic TCR T cells, and has the potential to produce superior T cell responses in the immunosuppressive tumor microenvironment. DisclosuresBoyerinas:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Murray:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio: Employment, Equity Ownership. Parsons:bluebird bio: Employment, Equity Ownership. Seidl:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership, Patents & Royalties.

Combined tumor-directed recruitment and protection from immune suppression enable CAR T cell efficacy in solid tumors.

CAR T cell therapy remains ineffective in solid tumors, due largely to poor infiltration and T cell suppression at the tumor site. T regulatory (Treg) cells suppress the immune response via inhibitory factors such as transforming growth factor-β (TGF-β). Treg cells expressing the C-C chemokine receptor 8 (CCR8) have been associated with poor prognosis in solid tumors. We postulated that CCR8 could be exploited to redirect effector T cells to the tumor site while a dominant-negative TGF-β receptor 2 (DNR) can simultaneously shield them from TGF-β. We identified that CCL1 from activated T cells potentiates a feedback loop for CCR8+ T cell recruitment to the tumor site. This sustained and improved infiltration of engineered T cells synergized with TGF-β shielding for improved therapeutic efficacy. Our results demonstrate that addition of CCR8 and DNR into CAR T cells can render them effective in solid tumors.

Gut microbiota contributes to sexual dimorphism in murine autoimmune cholangitis.

The data demonstrated that a transgenic murine model of primary biliary cholangitis (PBC), expressing dominant negative TGF-β receptor Ⅱ (dnTGFβRⅡ) under the CD4 promoter, showed similarity to PBC patients that is female-dominant. Female dnTGFβRII mice developed more severe lymphocytic infiltration in the liver and had higher levels of inflammatory cytokines, including IFN-γ and TNF-α, than the male mice. Interestingly, elimination of testosterone through gonadectomy in male dnTGFβRII mice did not influence disease severity, supporting that testosterone is an unessential factor in sustaining liver immune homeostasis. Meanwhile, it was observed that treating dnTGFβRII mice with oral antibiotics markedly reduced the differences in the levels of lymphocytic infiltration and cytokines between males and females, suggesting that the commensal gut microbiome plays a role in determining the observed sexual differences in dnTGFβRII mice. Furthermore, the diversity of gut microbiota composition and their metabolic functions in the male and female groups through metagenomic sequencing analysis were identified. The results revealed a testosterone-independent and commensal gut microbiota-mediated female bias in PBC.

A phase I clinical trial of PSMA-directed/TGFβ-insensitive CAR-T cells in metastatic castration-resistant prostate cancer.

125 Background: Prostate specific membrane antigen (PSMA) is a highly expressed tumor-associated antigen potentially amenable to chimeric antigen receptor-modified T (CAR-T) cell therapy for castration-resistant prostate cancer (CRPC). However, a primary challenge to the success of CAR-T therapy in CRPC is the immunosuppressive microenvironment, characterized by high levels of TGFβ. The immunosuppressive functions of TGFβ can be inhibited in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. Methods: We conducted a first-in-human phase 1 clinical trial to evaluate the feasibility, safety and preliminary efficacy of PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in patients with metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting (LD) chemotherapy. In Cohort 3, one patient received 1-3 x 108/m2 CART-PSMA-TGFβRdn cells following a LD chemotherapy regimen of cyclophosphamide and fludarabine (Cy/Flu). In Cohort -3, three patients received 1-3 x 107/m2 CART-PSMA-TGFβRdn cells following Cy/Flu. Patients underwent metastatic tumor biopsies at baseline and on day 10 following treatment. Quantitative PCR of CART-PSMA-TGFβRdn DNA was performed at serial timepoints to evaluate for CAR-T expansion and persistence in peripheral blood and trafficking to target tissues. Multiplex cytokine analysis assessed CART-PSMA-TGFβRdn bioactivity. Results: Ten patients received CART-PSMA-TGFβRdn therapy across dose-level cohorts. All CART-PSMA-TGFβRdn infusion products met target transduction efficiency. Evaluation of CAR-T cellular kinetics demonstrated dose-dependent peripheral blood T cell expansion, as well as tumor tissue trafficking in post-treatment tumor biopsies. At Cohort 2 and above, 5 of 7 treated patients developed grade ≥2 cytokine release syndrome (CRS). Marked increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) correlated with high-grade CRS events. One grade 5 adverse event (sepsis) occurred in Cohort 3. PSA decline was observed in 6 of 10 patients (median decline -33.2%, range -11.6% to -98.3%), and PSA30 response occurred in 4 of 10 patients (including one patient achieving PSA < 0.1 ng/mL). Conclusions: Adoptive cellular therapy with CART-PSMA-TGFβRdn is safe and feasible in patients with metastatic CRPC. A dose-dependent and lymphodepletion chemotherapy-dependent relationship was observed with CART-PSMA-TGFβRdn cell expansion, cytokine expression, CRS, and anti-tumor effect. Correlative cell trafficking and paired tumor Nanostring analyses will be presented. Future clinical investigations seek to enhance anti-tumor efficacy, while optimizing the therapeutic window. Clinical trial information: NCT03089203.

Dominant Negative TGF-β Receptor Type II in T Lymphocytes Promotes Anti-Tumor Immunity by Inducing the Proliferation and Differentiation of T Cells and Enhancing CTL Responses

Dominant Negative TGF-β Receptor Type II in T Lymphocytes Promotes Anti-Tumor Immunity by Inducing the Proliferation and Differentiation of T Cells and Enhancing CTL Responses

Abstract B36: TGFβ blockade and epigenetic modulation for cancer treatment: Efficient breast cancer targeted therapy with TCR-T cell transfer

Abstract Introduction: The cancer testis (CT) antigens, such as NY-ESO1, are considered ideal targets for immunotherapy as they are highly immunogenic and their restricted expression malignancies and not normal tissue except testis. However, the expression of CT antigen is downregulated in many tumor types, including breast cancer due to DNA methylation. DNA methylation is a common epigenetic mechanism to regulate gene expression and phenotype and maintain DNA integrity and stability. DNA methylation inhibition can induce NY-ESO1 expression on tumor cells and sensitize them to NY-ESO1 specific T cells. NY-ESO1 targeted T cell receptors (TCR) have been successfully used to engineer T cells for adoptive T cell therapy (ACT). We hypothesize that incorporating a dominant-negative TGFβ receptor II (dnTGFβRII) into the NY-ESO-1 TCR construct to transduce T cells combined with epigenetic modulator treatment may improve T cell effector function in immunosuppressive tumor microenvironment. Methods: Seven different human breast cancer cell lines were treated with epigenetic modulators at different doses and analyzed by flow cytometry to check the increase in expression of NY-ESO1 antigen and HLA-A2.1. Eight-weeks or older NSG mice were inoculated with HLA-A2.1+, NY-ESO negative at baseline breast cancer cells subcutaneously. PBMCs from healthy donors were transduced with a HLA-A2.1 NY-ESO1 TCR with/without dnTGFβRII two day after activation with OKT3 antibody plus IL-2. The transduction efficiency was confirmed by flow cytometry prior to ACT. Equal numbers of TCR+ cells were injected 10 days after tumor inoculation. Mice were then monitored for tumor growth till endpoint. The spleens and tumors were collected for further analysis of immunophenotype and function. Results: The expression of NY-ESO1 was inducible with epigenetic modulation in the majority of breast cancer cell-lines tested (5/7). More than 60% of Vβ+ (marker for surface expression of specific TCR) cells were observed after retroviral transduction to PBMCs. The transduced NY-ESO1 TCR/dnTGFβRII cells with epigenetic modulator were successfully able to infiltrate into tumor microenvironment in higher numbers and control cancer growth of inoculated tumor significantly (p<0.01) in comparison to control tumor and control NY-ESO1 TCR only groups. The tumor infiltrated lymphocytes (TILs) from NY-ESO1 TCR/dnTGFβRII with treatment showed that regulatory T cells and cytotoxic T cells were significantly lower and higher, respectively. Conclusions: Epigenetic modification enhances the cytotoxicity of TCR engineered T cells with dnTGFβRII. Our results demonstrate the feasibility of combined epigenetic modulation and TCR-T treatment for breast cancer patients. Citation Format: Satoko Matsueda, Kunle Odunsi, Richard C. Koya. TGFβ blockade and epigenetic modulation for cancer treatment: Efficient breast cancer targeted therapy with TCR-T cell transfer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B36.

A phase I clinical trial of PSMA-directed/TGFβ-insensitive CAR-T cells in metastatic castration-resistant prostate cancer.

TPS269 Background: Adoptive immunotherapy with Chimeric Antigen Receptor (CAR)-T cells is a novel approach for the treatment of prostate cancer. However, the prostate cancer immunosuppressive microenvironment, including high levels of TGFβ, may limit the therapeutic potential of re-directed T cells upon tumor infiltration. The inhibition of TGFβ signaling via co-expression of a dominant negative TGFβ receptor (TGFβRdn) can enhance antitumor immunity. Co-expression of TGFβRdn on PSMA-redirected CAR-T cells in in vivo disseminated tumor models led to increased T cell proliferation, enhanced cytokine secretion, resistance to exhaustion, long-term persistence, and greater induction of tumor eradication. Methods: We are conducting a first-in-human phase 1 clinical trial evaluating the safety and preliminary efficacy of lentivirally-transduced PSMA-redirected/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, patients received a single dose of 1-3 x 107/m2 (Cohort 1) or 1-3 x 108/m2 (Cohort 2) CART-PSMA-TGFβRdn cells without lymphodepleting chemotherapy. In Cohort 3, 1-3 x 108/m2 CART-PSMA-TGFβRdn cells are administered following a lymphodepleting chemotherapy regimen of cyclophosphamide and fludarabine (cy/flu). A currently accruing modified protocol seeks to optimize the therapeutic window with CART-PSMA-TGFβRdn (CAR-T dose of 1-3 x 107/m2 following lymphodepleting cy/flu). Eight patients have received a single dose of CART-PSMA-TGFβRdn. CAR-T expansion and persistence in peripheral blood and trafficking to target tissues is evaluated via quantitative PCR of CART-PSMA-TGFβRdn DNA. Bioactivity of CAR-T cells in peripheral blood is evaluated via multiplex immunoassays. Additional correlative analyses will interrogate the therapeutic contribution of TGFβRdn, as well as early markers of response and resistance to CART-PSMA-TGFβRdn therapy. Clinical trial information: NCT03089203.