Abstract
CD8
+
T lymphocytes mediate potent immune responses against tumor, but the role of human CD4
+
T cell subsets in cancer immunotherapy remains ill-defined. Herein, we exhibit that CD26 ...identifies three T helper subsets with distinct immunological properties in both healthy individuals and cancer patients. Although CD26
neg
T cells possess a regulatory phenotype, CD26
int
T cells are mainly naive and CD26
high
T cells appear terminally differentiated and exhausted. Paradoxically, CD26
high
T cells persist in and regress multiple solid tumors following adoptive cell transfer. Further analysis revealed that CD26
high
cells have a rich chemokine receptor profile (including CCR2 and CCR5), profound cytotoxicity (Granzyme B and CD107A), resistance to apoptosis (c-KIT and Bcl2), and enhanced stemness (β-catenin and Lef1). These properties license CD26
high
T cells with a natural capacity to traffic to, regress and survive in solid tumors. Collectively, these findings identify CD4
+
T cell subsets with properties critical for improving cancer immunotherapy.
BackgroundAdoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve ...the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure.MethodsWe created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies.ResultsWe found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma.ConclusionsOur aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.
Pancreatic adenocarcinoma (PDAC) remains a formidable disease that needs improved therapeutic strategies. Even though immunotherapy has revolutionized treatment for various solid tumor types, it ...remains largely ineffective in treating individuals with PDAC. This review describes how the application of genome-wide analysis is revitalizing the field of PDAC immunotherapy. Major themes include new insights into the body's immune response to the cancer, and key immunosuppressive elements that blunt that antitumor immunity. In particular, new evidence indicates that T cell-based antitumor immunity against PDAC is more common, and more easily generated, than previously thought. However, equally common are an array of cellular and molecular defenses employed by the tumor against those T cells. These discoveries have changed how current immunotherapies are deployed and have directed development of novel strategies to better treat this disease. Thus, the impact of genomic analysis has been two-fold: both in demonstrating the heterogeneity of immune targets and defenses in this disease, as well as providing a powerful tool for designing and identifying personalized therapies that exploit each tumor's unique phenotype. Such personalized treatment combinations may be the key to developing successful immunotherapies for pancreatic adenocarcinoma.
How naturally arising human CD4
T helper subsets affect cancer immunotherapy is unclear. We reported that human CD4
CD26
T cells elicit potent immunity against solid tumors. As CD26
T cells are often ...categorized as T
17 cells for their IL-17 production and high CD26 expression, we posited these populations would have similar molecular properties. Here, we reveal that CD26
T cells are epigenetically and transcriptionally distinct from T
17 cells. Of clinical importance, CD26
and T
17 cells engineered with a chimeric antigen receptor (CAR) regressed large human tumors to a greater extent than enriched T
1 or T
2 cells. Only human CD26
T cells mediated curative responses, even when redirected with a suboptimal CAR and without aid by CD8
CAR T cells. CD26
T cells cosecreted effector cytokines, produced cytotoxic molecules, and persisted long term. Collectively, our work underscores the promise of CD4
T cell populations to improve durability of solid tumor therapies.
Abstract
Background
Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for ...solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogeneous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape.
Methods
Here we have developed a novel, dual-specific, tandem CAR T (TanCART) cell with the ability to simultaneously target both EGFRvIII and IL-13Rα2, two well-characterized tumor antigens that are frequently found on the surface of GBM cells but completely absent from normal brain tissues. We employed both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogeneous tumors.
Results
Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < .05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete, and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < .05).
Conclusions
We demonstrate that TanCART is effective against heterogeneous tumors in the brain. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.
BackgroundBlockade of the vascular endothelial growth factor (VEGF) pathway represents a long-standing strategy for anti-tumor activity, with drugs employing this approach for a variety of solid ...tumor types. It is also known that VEGF directly drives T cell exhaustion and dysfunction.1 However, systemic VEGF blockade causes significant cardiovascular and renal toxicity and limits the dose of these medications, restricting their maximal theoretical benefit.In an updated analysis of our previously published single cell RNA sequencing data from infusion products of Tisagenlecleucel,2 we discovered expression of VEGF and VEGFR1 on CAR-T cell infusion products. Building on this observation, we found that our previously reported CD703 and Mesothelin CAR-T cells produce VEGF, as detected by ELISA, and upregulate VEGFR1 upon activation (p<.0001), suggesting active signaling along this pathway may impact CAR-T cell biology.MethodsTo improve the therapeutic window of VEGF blockade and mitigate putative VEGF mediated T cell dysfunction, we generated CD70 and Mesothelin targeted CAR-T cells that secrete a VEGF-blocking single chain variable fragment (scFv, 70VEGF and MesoVEGF respectively).Results70VEGF and MesoVEGF completely abrogated VEGF signaling in a HEK293 reporter assay (p<0.0001) and blocked all detectable VEGF by ELISA during CAR-T production (p<.0001) compared to CD19 scFv expressing control constructs (7019 and Meso19). 70VEGF and MesoVEGF demonstrated superior activation (by CD69 expression) and expansion in both short term plate bound antigen assays and long term repetitive stimulation assays with CD70 or Mesothelin expressing K562 targets. 70VEGF and MesoVEGF also mediated superior cytotoxicity and CAR-T expansion against RCC, AML, NSCLC, and ovarian cancer cell lines in vitro compared to 7019 and Meso19. Preliminary data suggest that neither CRISPR-Cas9 VEGFA knockout of 7019 (7019,VEGFKO) nor exogenous administration of anti-VEGF scFv alone were sufficient to phenocopy the proliferative advantage of 70VEGF when co-cultured with tumor cells; however, when exogenous anti-VEGF scFv was added to 7019,VEGFKO CAR-T cells the performance was similar to 70VEGF, arguing that a combination of intracrine and exogenous VEGF modulate CAR-T cell function. Nanostring gene expression profiling revealed distinct expression programs between MesoVEGF and Meso19 including lower apoptosis of the former. In vivo experiments with AML PDX, orthotopic RCC, and ovarian/lung cancer metastatic models are ongoing and will be reported at the conference.ConclusionsTargeted delivery of a VEGF blocking scFv is a promising therapeutic strategy that augments CAR-T cell function in a tumor and target agnostic fashion and warrants further development.Acknowledgements*These first authors contributed equallyReferencesKim C, Jang M, Kim Y. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers. Sci Immunol. 2019;4(41)Haradhvala N, Leick M, Maurer K. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nature Medicine. 2022;(9):1848–1859Leick M, Silva H, Scarfò S. Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia, Cancer Cell. 2022;40(5):494–508.Ethics ApprovalAll care and conducted experiments on mice were carried out with protocols approved by the MGH Institutional Animal Care and Use Committee.
BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid ...tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).
BackgroundThe inadequate ability of adoptively transferred T cells to eradicate solid tumors limits their use in treatments for patients afflicted with those cancers. Efforts to improve ACT for solid ...tumors aim to identify strategies that poise T cells for optimal response. We have previously identified a specific subset of CD4 T cells which express high levels of the ubiquitous ectoenzyme dipeptidyl peptidase-4 (DPP-4), also known as CD26, that produce a tremendous antitumor response in solid tumor models. We therefore sought to investigate the importance of CD26 on T cells destined for ACT.MethodsWe adoptively transferred tumor specific CD26+ T cells into melanoma tumor-bearing CD26-/- mice, and continuously blocked the CD26 enzymatic activity of the donor cells in vivo with sitagliptin, an established competitive inhibitor of CD26.ResultsTumors in sitagliptin-treated mice eventually reached study endpoint, while tumors untreated mice were regressed for 130+ days. Tumor infiltration of donor cells and host CD8 and CD4 cells was diminished with sitagliptin treatment. A 32-plex cytokine array of blood plasma revealed a diminished profile of cytokines and chemokines, indicating that the inflammatory response of the T cells was dampened with sitagliptin treatment. Further experiments characterized the ability of CD26+ T cells to respond to tumor trafficking signals with a transwell migration assay and found that sitagliptin treatment significantly impaired their migratory capacity. However, sitagliptin did not impair the ability of T cells to functionally respond to antigen.ConclusionsThese data suggest that the enzymatic activity of CD26 is important for the ability of T cells to migrate to the tumor site in order to mount an effective antitumor response. Further investigations into the mechanism behind the role of CD26 are ongoing.Ethics ApprovalThis study was approved by the Medical University of South Carolina’s IACUC, protocol #00488
BackgroundChimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, ultimately leading to its FDA approval for relapsed/refractory acute lymphoblastic ...leukemia and large cell lymphomas in 2017. Despite the success of CAR T cells in the clinic, toxicities such as cytokine release syndrome (CRS) can be severe. Attempts to mitigate these effects have primarily focused on the blockade of macrophage-derived cytokines, such as IL-6 and IL-1B. Herein, we show that the pharmaceutical blockade or genetic deletion of interferon gamma (IFNg, a CAR-T-derived cytokine that strongly correlates with CRS in the clinic, appears to be a viable target for the reduction of CAR-T-associated toxicities.MethodsPharmacologic (blocking antibody) and genetic (CRISPR/Cas9) approaches were used to block IFNg signaling and/or production by CAR T cells. In vitro CAR-T function and cytotoxicity was tested using ELISA, flow cytometry and short-/long-term killing assays prior to their assessment in vivo. NSG mice were injected with Nalm6 or JeKo-1 cancer cells prior to treatment with IFNg-modified CAR-T and tumor size and IFNg production were measured. To determine how the loss of IFNg might affect innate immune cells, CAR-T, macrophages and tumor cells were co-cultured and assessed by flow cytometry, immunofluorescence, Luminex and RNA sequencing.ResultsIFNg could be blocked using an anti-IFNg antibody or CRISPR/Cas9 editing of the CAR T cells without affecting T cell activation, proliferation or cytokine production (IL-2, TNFa, GM-CSF). Successful blockade of the IFNg signaling pathway was confirmed by reduced phosphorylation of JAK1, JAK2 and STAT1, even in the presence of exogenous IFNg. Loss of IFNg did not reduce the cytotoxic potential or persistence of CAR-T against hematologic malignancies in vitro or in vivo. When cultured with macrophages and cancer cells, IFNg knockout (IFNgKO) CAR-T yielded decreased levels of IL-1B, IL-6, IL-13, MCP1 and CXCL10, indicating a reduction in macrophage activation induced by CAR-T in the absence of IFNg. Serum from tumor-bearing mice treated with IFNgKO CAR-T elicited lower activation of macrophages in vitro compared to those treated with IFNg-producing CAR-T cells. Furthermore, IFNgKO CAR T cells co-cultured with tumor cells and macrophages demonstrated less exhaustion as shown by reduced expression of PD1, Tim3 and Lag3 and increased IFNgKO CAR-T expansion.ConclusionsCollectively, these data suggest that IFNg is not required for the efficacy of CAR-T in hematologic malignancies and can potentially be targeted to reduce toxicity and enhance CAR-T efficacy and persistence in the clinic.
ICOS costimulation generates Th17 cells with durable memory responses to tumor. Herein, we found that ICOS induces PI3K/p110δ/Akt and Wnt/β-catenin pathways in Th17 cells. Coinhibiting PI3Kδ and ...β-catenin altered the biological fate of Th17 cells. Th17 cells inhibited of both pathways expressed less RORγt, which, in turn, reduced their ability to secrete IL-17. Unexpectedly, these cells were more effective (than uninhibited cells) at regressing tumor when infused into mice, leading to long-term curative responses. PI3Kδ inhibition expanded precursor Th17 cells with a central memory phenotype that expressed nominal regulatory properties (low FoxP3), while β-catenin inhibition enhanced Th17 multifunctionality in vivo. Remarkably, upon TCR restimulation, RORγt and IL-17 rebounded in Th17 cells treated with PI3Kδ and β-catenin inhibitors. Moreover, these cells regained β-catenin, Tcf7, and Akt expression, licensing them to secrete heightened IL-2, persist, and eradicate solid tumors without help from endogenous NK and CD8 T cells. This finding shines a light on ways to repurpose FDA-approved drugs to augment T cell-based cancer immunotherapies.
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