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Figure S2: Engineered murine T cells exhibit effector function in response to cognate peptide.

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Adoptive T-cell therapy using high-affinity T-cell receptors (TCR) to target tumor antigens has potential for improving outcomes in high-grade serous ovarian cancer (HGSOC) patients. Ovarian tumors develop a hostile, multicomponent tumor microenvironment containing suppressive cells, inhibitory ligands, and soluble factors that facilitate evasion of antitumor immune responses. Developing and validating an immunocompetent mouse model of metastatic ovarian cancer that shares antigenic and immunosuppressive qualities of human disease would facilitate establishing effective T-cell therapies. We used deep transcriptome profiling and IHC analysis of human HGSOC tumors and disseminated mouse ID8VEGF tumors to compare immunologic features. We then evaluated the ability of CD8 T cells engineered to express a high-affinity TCR specific for mesothelin, an ovarian cancer antigen, to infiltrate advanced ID8VEGF murine ovarian tumors and control tumor growth. Human CD8 T cells engineered to target mesothelin were also evaluated for ability to kill HLA-A2+ HGSOC lines. IHC and gene-expression profiling revealed striking similarities between tumors of both species, including processing/presentation of a leading candidate target antigen, suppressive immune cell infiltration, and expression of molecules that inhibit T-cell function. Engineered T cells targeting mesothelin infiltrated mouse tumors but became progressively dysfunctional and failed to persist. Treatment with repeated doses of T cells maintained functional activity, significantly prolonging survival of mice harboring late-stage disease at treatment onset. Human CD8 T cells engineered to target mesothelin were tumoricidal for three HGSOC lines. Treatment with engineered T cells may have clinical applicability in patients with advanced-stage HGSOC.

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BackgroundIn the U.S., more than 50% of ovarian cancer patients die within 5 years of diagnosis, highlighting the need for innovations such as engineered T cell therapies. Mesothelin (Msln) is an attractive immunotherapy target for this cancer, as it is overexpressed by the tumor and contributes to malignant and invasive phenotypes, making antigen loss disadvantageous to the tumor. We previously showed that adoptively transferred T cells engineered to be Msln-specific (TCR1045) preferentially accumulate within established ovarian tumors, delay tumor growth and significantly prolong survival in the ID8VEGF mouse model. However, T cell persistence and anti-tumor activity were not sustained, and we and others have previously detected FasL in the tumor vasculature and the tumor microenvironment (TME) of human and murine ovarian cancers, which can induce apoptosis in infiltrating lymphocytes expressing Fas receptor (Fas).MethodsTo concurrently overcome this mechanism for potential immune evasion and enhance T cell responses, we generated an immunomodulatory fusion protein (IFP) containing the Fas extracellular binding domain fused to a 4-1BB co-stimulatory domain, rather than the natural death domain. T cells engineered to express TCR1045 alone or in combination with the IFP were transferred into ID8VEGF-tumor bearing mice and evaluated for persistence, proliferation, anti-tumor cytokine production, and therapeutic efficacy.ResultsRelative to T cells modified only to express TCR1045, T cells engineered to express both TCR1045 and a Fas IFP preferentially persisted in the TME of tumor-bearing mice due to improved T cell proliferation and survival. Moreover, adoptive immunotherapy with IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCR1045 T cells lacking the IFP.ConclusionsFas/FasL signaling can mediate T cell death in the ovarian cancer microenvironment, as well as induce activation-induced cell death, an apoptotic mechanism responsible for regulating T cell expansion. Upregulation of FasL by tumor cells and tumor vasculature represents a mechanism for protecting growing tumors from attack by tumor-infiltrating lymphocytes. As many solid tumors overexpress FasL, an IFP that converts the Fas-mediated death signal into pro-survival and proliferative signals may provide an opportunity to enhance engineered adoptive T cell therapy against many malignancies.

Adoptive T-cell therapy using high-affinity T-cell receptors (TCR) to target tumor antigens has potential for improving outcomes in high-grade serous ovarian cancer (HGSOC) patients. Ovarian tumors develop a hostile, multicomponent tumor microenvironment containing suppressive cells, inhibitory ligands, and soluble factors that facilitate evasion of antitumor immune responses. Developing and validating an immunocompetent mouse model of metastatic ovarian cancer that shares antigenic and immunosuppressive qualities of human disease would facilitate establishing effective T-cell therapies. We used deep transcriptome profiling and IHC analysis of human HGSOC tumors and disseminated mouse ID8VEGF tumors to compare immunologic features. We then evaluated the ability of CD8 T cells engineered to express a high-affinity TCR specific for mesothelin, an ovarian cancer antigen, to infiltrate advanced ID8VEGF murine ovarian tumors and control tumor growth. Human CD8 T cells engineered to target mesothelin were also evaluated for ability to kill HLA-A2+ HGSOC lines. IHC and gene-expression profiling revealed striking similarities between tumors of both species, including processing/presentation of a leading candidate target antigen, suppressive immune cell infiltration, and expression of molecules that inhibit T-cell function. Engineered T cells targeting mesothelin infiltrated mouse tumors but became progressively dysfunctional and failed to persist. Treatment with repeated doses of T cells maintained functional activity, significantly prolonging survival of mice harboring late-stage disease at treatment onset. Human CD8 T cells engineered to target mesothelin were tumoricidal for three HGSOC lines. Treatment with engineered T cells may have clinical applicability in patients with advanced-stage HGSOC.

Background: Less than half of ovarian cancer patients survive five years after diagnosis. This rate has changed little in the last 30 years, highlighting the need for novel therapies. A promising new strategy with the potential to control tumor growth without toxicity to healthy tissues employs immune T cells engineered to target proteins uniquely overexpressed in tumors. Mesothelin (Msln) is overexpressed in high grade serous ovarian cancer, contributes to the malignant and invasive phenotype, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Methods: The ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill murine ovarian tumor cells in vitro and in vivo. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies administered alone or in combination, ultimately allowing targeting up to three inhibitory receptors simultaneously. Single cell RNA sequencing was used to profile the impact of combination checkpoint blockade on both the engineered T cells and the tumor microenvironment. Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that elements in the tumor microenvironment (TME) limited engineered T cell persistence and ability to kill cancer cells. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased effector cytokine production by intratumoral TCRMsln T cells. Single cell RNA-sequencing revealed gene expression changes in engineered T cells and myeloid cells in the TME consistent with activation and inflammation. Moreover, combining adoptive immunotherapy with triple checkpoint blockade prolonged survival in the cohort of treated tumor-bearing mice, relative to TCRMsln with or without anti-PD1, or double-agent treatments. Conclusions: Inhibitory receptor/ligand interactions within the tumor microenvironment can dramatically reduce T cell function, suggesting tumor cells may increase expression of the ligands for PD-1, Tim-3 and Lag-3 for protection from tumor-infiltrating lymphocytes. In a model of advanced ovarian cancer, triple checkpoint blockade significantly improved the anti-tumor function of transferred engineered T cells and improved outcomes in mice in a setting in which single checkpoint blockade had no significant activity. These results suggest that disrupting multiple inhibitory signaling pathways simultaneously, which can be more safely pursued in a cell intrinsic form through genetic engineering, may be necessary for improved efficacy in patients. Citation Format: Kristin G. Anderson, Yapeng Su, Madison G. Burnett, Breanna M. Bates, Magdalia L. Rodgers Suarez, Susan L. Ruskin, Aesha Vakil, Valentin Voillet, Raphael Gottardo, Philip Greenberg. Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer [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 3608.

BackgroundIn the USA, more than 50% of patients with ovarian cancer die within 5 years of diagnosis, highlighting the need for therapeutic innovations. Mesothelin (MSLN) is a candidate immunotherapy target; it is overexpressed by ovarian tumors and contributes to malignant/invasive phenotypes, making tumor antigen loss disadvantageous. We previously showed that MSLN-specific T cell receptor (TCR)-engineered T cells preferentially accumulate within established tumors, delay tumor growth, and significantly prolong survival in the ID8VEGF mouse model that replicates many aspects of human disease. However, T cell persistence and antitumor activity were not sustained. We therefore focused on Fas/FasL signaling that can induce activation-induced cell death, an apoptotic mechanism that regulates T cell expansion. Upregulation of FasL by tumor cells and tumor vasculature has been detected in the tumor microenvironment (TME) of human and murine ovarian cancers, can induce apoptosis in infiltrating, Fas (CD95) receptor-expressing lymphocytes, and can protect ovarian cancers from tumor-infiltrating lymphocytes.MethodsTo overcome potential FasL-mediated immune evasion and enhance T cell responses, we generated an immunomodulatory fusion protein (IFP) containing the Fas extracellular binding domain fused to a 4-1BB co-stimulatory domain, rather than the natural death domain. Murine T cells were engineered to express an MSLN-specific TCR (TCR1045), alone or with the IFP, transferred into ID8VEGF tumor-bearing mice and evaluated for persistence, proliferation, cytokine production and efficacy. Human T cells were similarly engineered to express an MSLN-specific TCR (TCR530) alone or with a truncated Fas receptor or a Fas-4-1BB IFP and evaluated for cytokine production and tumor lysis.ResultsRelative to murine T cells expressing only TCR1045, T cells expressing both TCR1045 and a Fas-4-1BB IFP preferentially persisted in the TME of tumor-bearing mice, with improved T cell proliferation and survival. Moreover, TCR1045/IFP+ T cells significantly prolonged survival in tumor-bearing mice, compared with TCR1045-only T cells. Human T cells expressing TCR530 and a Fas-4-1BB IFP exhibit enhanced functional activity and viability compared with cells with only TCR530.ConclusionsAs many ovarian tumors overexpress FasL, an IFP that converts the Fas-mediated death signal into pro-survival and proliferative signals may be used to enhance engineered adoptive T cell therapy for patients.

Background Adoptive cell therapy (ACT) with genetically-modified T cells has shown impressive results against some hematologic cancers, but limited efficacy against tumors with restrictive tumor microenvironments (TMEs). FasL is a particular obstacle for ACT;1 it is expressed in many tumors and TMEs,1 including AML,2 ovarian3 and pancreatic cancers,4 and upregulated on activated T cells, where it can mediate activation-induced cell death (AICD).5 Methods We engineered T cells to boost function with novel immunomodulatory fusion proteins (IFPs) that combine an inhibitory ectodomain with a costimulatory endodomain. Like current checkpoint-blocking therapies, IFPs can abrogate an inhibitory signal, but also provide an often absent costimulatory signal. Additionally, IFP-driven signals are delivered only to the T cells concurrently engineered to be tumor-specific, thereby avoiding systemic T cell activation. For FasL-expressing TMEs, we developed an IFP that replaces the Fas intracellular tail with costimulatory 4-1BB. We tested the the Fas-4-1BB IFP in primary human T cells and in immunocompetent murine models of leukemia and pancreatic cancer. Results Fas-4-1BB IFP expression enhanced primary human T cell function and enhanced lysis of Panc1 pancreatic tumor cells in vitro. Fas-4-1BB IFP-engineered murine T cells exhibited increased pro-survival signaling, proliferation, antitumor function and altered metabolism in vitro. Notably, the Fas ectodomain is trimeric5 and the 4-1BB intracellular domain requires trimerization to signal.6 In contrast, the CD28 domain is dimeric and did not enhance function when paired with 4-1BB.In vivo, Fas-4-1BB increased T cell persistence and function, and Fas-4-1BB T cell ACT significantly improved survival in a murine AML model. When delivered with a mesothelin-specific TCR, Fas-4-1BB T cells prolonged survival in the autochthonous KPC pancreatic cancer model, increasing median survival to 65 from 37 days (with TCR-only, **P=0.0042). Single-cell RNA sequencing revealed differences in the endogenous tumor-infiltrating immune cells, included changes in cell frequency and programming. Conclusions We developed an engineering approach to enhance the in vivo persistence and antitumor efficacy of transferred T cells. Our targeted, two-hit strategy uses a single fusion protein to overcome a death signal prevalent in the TME of many cancers and on activated T cells, and to provide a pro-survival costimulatory signal to T cells. Our results suggest that this fusion protein can increase T cell function when combined with murine or human TCRs, and can significantly improve therapeutic efficacy in liquid and solid tumors, supporting clinical translation. References Yamamoto, T.N., et al., T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy. J Clin Invest 2019. Contini P, et al., In vivo apoptosis of CD8(+) lymphocytes in acute myeloid leukemia patients: involvement of soluble HLA-I and Fas ligand. Leukemia 2007;21(2):p. 253–60. Motz GT, et al., Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors. Nat Med 2014;20(6):p. 607–15. Kornmann M, et al., Fas and Fas-ligand expression in human pancreatic cancer. Ann Surg 2000. 231(3): p. 368–79. Villa-Morales M and J Fernandez-Piqueras, Targeting the Fas/FasL signaling pathway in cancer therapy. Expert Opin Ther Targets 2012;16(1):p. 85–101. Wyzgol, A., et al., Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand. J Immunol 2009;183(3):p. 1851–61.

A Fas-4-1BB immunomodulatory fusion protein converts a pro-death to a pro-survival signal, enhancing T cell function and improving the efficacy of adoptive T cell therapy in immunocompetent murine models of liquid and solid tumors., Adoptive T cell therapy (ACT) with genetically modified T cells has shown impressive results against some hematologic cancers, but efficacy in solid tumors can be limited by restrictive tumor microenvironments (TMEs). For example, Fas ligand is commonly overexpressed in TMEs and induces apoptosis in tumor-infiltrating, Fas receptor–positive lymphocytes. We engineered immunomodulatory fusion proteins (IFPs) to enhance ACT efficacy, combining an inhibitory receptor ectodomain with a costimulatory endodomain to convert negative into positive signals. We developed a Fas-4-1BB IFP that replaces the Fas intracellular tail with costimulatory 4-1BB. Fas-4-1BB IFP-engineered murine T cells exhibited increased pro-survival signaling, proliferation, antitumor function, and altered metabolism in vitro. In vivo, Fas-4-1BB ACT eradicated leukemia and significantly improved survival in the aggressive KPC pancreatic cancer model. Fas-4-1BB IFP expression also enhanced primary human T cell function in vitro. Thus, Fas-4-1BB IFP expression is a novel strategy to improve multiple T cell functions and enhance ACT against solid tumors and hematologic malignancies., Graphical Abstract

Objectives Mouse models of ovarian cancer commonly transfer large numbers of tumor cells into the peritoneal cavity to establish experimental metastatic disease, which may not adequately model early metastatic spread from a primary tumor site. We hypothesized we could develop an ovarian cancer model that predictably represents micro-metastatic disease. Methods Murine ID8VEGF ovarian cancer cells were transduced to express enhanced luciferase (eLuc) to enable intravital detection of microscopic disease burden and injected beneath the ovarian bursa of C57Bl/6 mice. At 6 or 10 weeks after orthotopic injection, when mice had detectable metastases, hysterectomy and bilateral salpingo-oophorectomy was performed to remove all macroscopic disease, and survival monitored. Immunohistochemistry and gene expression profiling were performed on primary and metastatic tumors. Results eLuc-transduced ID8VEGF cells were brighter than cells transduced with standard luciferase, enabling in vivo visualization of microscopic intra-abdominal metastases developing after orthotopic injection. Primary surgical cytoreduction removed the primary tumor mass but left minimal residual disease in all mice. Metastatic sites that developed following orthotopic injection were similar to metastatic human ovarian cancer sites. Gene expression and immune infiltration were similar between primary and metastatic mouse tumors. Surgical cytoreduction prolonged survival compared to no surgery, with earlier cytoreduction more beneficial than delayed, despite micro-metastatic disease in both settings. Conclusions Mice with primary ovarian tumors established through orthotopic injection develop progressively fatal metastatic ovarian cancer, and benefit from surgical cytoreduction to remove bulky disease. This model enables the analysis of therapeutic regimens designed to target and potentially eradicate established minimal residual disease.

Metabolic constrains induce transcriptional deregulation of CD8+ T cells in pancreatic tumor microenvironment, driving progressive dysfunction. Here, metabolic reprogramming through enforced very-long-chain acyl-CoA dehydrogenase expression enhances intratumor T cells survival and persistence, overcoming a major hurdle to immunotherapy for PDA., CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.

Staining conditions for murine IHC.

Cell culture information (media conditions) for the Kuramochi, TYKNU, OVKATE, OVSAHO, COV318, COV362, OVCAR3, and OAW28 human ovarian cancer cells lines.

Staining conditions for human IHC.

Immune checkpoint inhibitors (ICIs) have improved overall survival for cancer patients, however, optimal duration of ICI therapy has yet to be defined. Given ICIs were first used to treat patients with metastatic melanoma, a condition that at the time was incurable, little attention was initially paid to how much therapy would be needed for a durable response. As the early immunotherapy trials have matured past 10 years, a significant per cent of patients have demonstrated durable responses; it is now time to determine whether patients have been overtreated, and if durable remissions can still be achieved with less therapy, limiting the physical and financial toxicity associated with years of treatment. Well-designed trials are needed to identify optimal duration of therapy, and to define biomarkers to predict who would benefit from shorter courses of immunotherapy. Here, we outline key questions related to health, financial and societal toxicities of over treating with ICI and present four unique clinical trials aimed at exposing criteria for early cessation of ICI. Taken together, there is a serious liability to overtreating patients with ICI and future work is warranted to determine when it is safe to stop ICI.

Though anger and anxiety are related, putative explanations for this association remain unclear. Beliefs about one's state of uncertainty may be a pathway-the belief that one's uncertain state is unavoidable might lead to anxiety, whereas the belief that one's uncertain state is avoidable might lead to both anxiety and anger. To test this hypothesis, participants experienced an uncertainty induction and were then assigned to the avoidable uncertainty condition (experimental group) or the unavoidable uncertainty condition (control group). State anger and anxiety were assessed at baseline, following the uncertainty induction, and following the "avoidableness" manipulation. The uncertainty induction was successful; participants reported higher levels of anxiety at post-induction compared to baseline. As expected, the experimental group reported increases in anger from post-induction to post-manipulation whereas the control group reported decreases in anger. These findings suggest that when one's state of uncertainty is avoidable, anger is experienced alongside anxiety.

Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy, leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this, short-boosted Ag-specific CD8 T cells continue to contract gradually over time, which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant, functional Ag-specific CD8 T cells that are poised for immediate protection; however, this is at the expense of forming stable long-term memory.

Over half of patients diagnosed with high grade serous ovarian cancer will die within five years, highlighting the need for therapy innovation. Engineering T cells to target proteins uniquely overexpressed in tumors has the potential to limit tumor growth without toxicity—mesothelin (Msln) is a rational immunotherapy target as it contributes to the malignant/invasive phenotype and has limited expression in healthy cells. T cells were engineered to express a high-affinity Msln-specific T cell receptor (TCRMsln) and adoptively transferred into a disseminated ID8VEGF murine model, which recapitulates many features of human ovarian cancer. TCRMsln+ T cells preferentially accumulated within established tumors, delayed tumor growth, and significantly prolonged survival. However, T cell persistence and anti-cancer activity were limited by elements in the tumor microenvironment (TME), including Fas Ligand (FasL) that can induce apoptosis in infiltrating lymphocytes expressing Fas receptor (Fas). To overcome this potential T cell evasion mechanism, we generated a panel of immunomodulatory fusion proteins (IFPs) containing the Fas extracellular binding domain fused to a co-stimulatory domain, replacing the natural death domain. Relative to T cells modified with only TCRMsln, T cells that expressed both TCRMsln and a Fas IFP preferentially infiltrated tumors and expanded/persisted in the TME of tumor-bearing mice. Moreover, adoptive immunotherapy with TCRMsln+IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCRMsln+ only T cells. As many solid tumors overexpress FasL, IFPs may provide an opportunity to enhance engineered adoptive T cell therapy against many malignancies.

We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T-cell responses by transfer of genetically engineered T cells. Our largest clinical experience has been in treating human acute myelogenous leukemia (AML), in which we have utilized a high-affinity TCR specific for WT1, a protein associated with promoting leukemic transformation that is overexpressed in human leukemic stem cells, to genetically engineer CD8 T cells. We recently reported a study (Chapuis et al., Nat Med 2019) in which we treated leukemia patients at high risk of relapse (after hematopoietic cell transplant) that demonstrated all treated patients remain alive and relapse free at a median of 48 months, compared to a relapse rate of ~35% in the concurrent matched cohort (p Citation Format: Philip D. Greenberg, Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Luigi Nezi, Teresa Manzo, Shannon K. Oda, Kelly G. Paulson, Rachel Perret, Leah Schmidt, Tom M. Schmitt, Ingunn M. Stromnes, Aude G. Chapuis. Targeting pancreatic cancer with TCR-engineered T cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I11.

Generalized anxiety disorder (GAD) symptoms are associated with elevated anger; however the nature of these associations is unclear. We tested the hypothesis that anger perpetuates GAD symptoms and associated cognitive vulnerabilities by examining the effects of laboratory-induced anger on worry, negative interpretative style, and negative beliefs about uncertainty. Participants were randomized to an anger induction (n = 43) or a control condition (n = 34). An interpretation bias task, questionnaire items assessing beliefs about uncertainty, and a worry task were administered following the manipulation. Participants in the anger condition reported greater increases in negative interpretative style and in the belief that uncertainty is unfair and spoils everything than those in the control condition; however no group differences were found related to worry. Results provide partial support for the notion that anger contributes to cognitive vulnerabilities underlying GAD, namely negative interpretative style and specific beliefs about uncertainty.

Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5-30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses.

Over 20,000 women are diagnosed with ovarian cancer in the United States annually, and over half will die within 5 years. Outcomes have changed little in the last 20 years, highlighting the need for therapy innovation. One promising new strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors; such T cell immunotherapies have the potential to control tumor growth without toxicity to healthy tissues. In considering candidate immunotherapy targets, we focused on mesothelin (MSLN), which contributes to invasive progression and malignancy in ovarian cancer but has limited expression in healthy cells. We showed that T cells engineered to express a human or mouse MSLN-specific high-affinity T cell receptor (TCRMSLN) can kill human patient-derived ovarian cancer cell lines or the murine ID8 cell line, respectively. In a disseminated ID8 tumor model, adoptively transferred TCRMSLN T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that the ovarian tumor microenvironment (TME) limits engineered T cell persistence and cancer cell killing. To identify immunosuppressive features active in both the human and murine ovarian TME, we performed gene expression analyses. Deep transcriptome profiling confirmed similar gene expression signatures in human cancers and in the preclinical ID8 model. Among these, RNA sequencing detected PD-L1, Galectin-9 and Galectin-3, ligands for CD8 T cell-expressed PD-1, Tim-3 and Lag-3 ‘checkpoint’ receptors, respectively. We also measured PD-L1, Galectin-9 and Galectin-3 expression in human and mouse ovarian cancers by flow cytometry and immunohistochemistry, and multiplex immunohistochemistry of human ovarian tumors confirmed the presence of endogenous CD8 T cells expressing one, two or all three inhibitory receptors. Moreover, flow cytometry revealed that TCRMSLN-transduced T cells increase expression of the inhibitory receptors in ID8 tumors relative to cells in the spleen as early as three weeks after transfer, in association with decreased production of anti-tumor cytokines. Based on our results, we hypothesized that we could overcome engineered T cell suppression via inhibitory receptor ligation. We treated tumor-bearing mice with TCRMSLN T cells plus anti-PD-1, anti-Tim-3 and/or anti-Lag-3 checkpoint-blocking antibodies, targeting up to three inhibitory receptors simultaneously. Triple checkpoint blockade dramatically increased anti-tumor cytokine production by intratumoral TCRMSLN T cells. As many solid tumors both overexpress MSLN as well as PD-1, Tim-3 and Lag-3, the use of multi-checkpoint blockade with engineered T cells has real potential to also enhance the efficacy of engineered adoptive T cell therapy against other malignancies. Citation Format: Kristin G. Anderson, Madison G. Burnett, Valentin Voillet, Edison Y. Chiu, Breanna M. Bates, Nicolas M. Garcia, Raphael Gottado, Philip D. Greenberg. Using T cell engineering plus triple checkpoint blockade to enhance the efficacy of adoptive immunotherapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3208.

We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. Our largest clinical experience is in treating human Acute Myelogenous Leukemia (AML). After identifying that WT1, a gene associated with promoting leukemic transformation, is over-expressed in human leukemic stem cells, and demonstrating in a clinical trial that in vitro expanded WT1-specific CD8 T cell clones can be safely transferred, exhibit anti-leukemic activity, and provide therapeutic benefit to AML patients, we extensively screened normal human repertoires and isolated a high affinity TCR specific for WT1 for genetically engineering CD8 T cells to reproducibly create cells with high avidity for leukemic cells. We have initially pursued this strategy in a 2 Arm trial for leukemia patients either at high risk of relapse (Arm 1) after hematopoietic cell transplant (HCT) or who have already relapsed after HCT (Arm 2). The prophylactic arm is now completed, with very encouraging results- all patients treated with engineered T cells remain alive and relapse free at a median of 38 months, compared to a relapse rate of ~50% in a concurrent matched cohort. Results in relapsed patients (Arm 2) have been less effective, and we have used high-dimensional analyses including single cell RNAseq both to elucidate the reasons for failure to eradicate the leukemia and to design strategies to overcome these obstacles. Our results have identified several mechanisms by which the leukemia escapes, and we have been testing approaches that employ further genetic modification of the T cells to enhance efficacy. The predominant reason for leukemia progression despite targeted therapy with T cells is the inability of the T cells to persist and maintain function in the context of encountering a rapidly proliferating myeloid leukemia. This reflects both engagement of pathways inhibitory to T cells by the leukemic cells, and apoptosis of the T cells from repetitive stimulation. We are addressing this issue by creating immuno-modulatory fusion proteins (IFPs) that have the ectodomain of an inhibitory or death receptor fused to a survival costimulatory domain. Results with two such IFPs will be discussed, a CD200R/CD28 fusion that binds the inhibitory ligand CD200 commonly expressed on leukemic cells but provides a CD28 costimulatory signal and a Fas/4-1BB fusion that binds FasL but rather than induce death promotes proliferation and survival. A more uncommon reason explaining progression is loss of expression of the WT1 epitope being targeted. This has occurred in 2 patients, but for distinct reasons. In one patient this reflected loss of a component of the immunoproteasome, and we have now isolated a TCR that recognizes an epitope not dependent on the immunoproteasome. In a second patient the level of WT1 expression declined- interestingly, this patient was treated with Vidaza, which can increase expression of WT1, and post-Vidaza the transferred T cells persisting in the patient’s bone marrow recognized and responded in vivo to the relapsing leukemia. The approaches and technologies we are developing and testing in leukemia are also applicable to solid tumors, and preclinical studies in pancreatic and ovarian cancers will be discussed. Citation Format: Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Kelly Paulson, Rachel Perret, Leah Schmidt, Thomas Schmitt, Ingunn Stromnes, Aude Chapuis, Philip D. Greenberg. Utilizing synthetic biology and high-dimensional probing to address therapeutic obstacles and empower engineered T cells with the capacity to eradicate tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA17.

Effective cellular therapy for human malignancies requires first identifying and validating an appropriate antigenic target, and then establishing in each patient a tumor-reactive T cell response of high avidity and high magnitude that is safe and can infiltrate and retain function in the tumor microenvironment. We have been exploring in preclinical models and clinical trials methods to reproducibly provide such responses by transfer of genetically engineered T cells that acquire target specificity by virtue of an introduced high affinity TCR. To identify candidate antigens in leukema, we examined purified human leukemic stem cells for over-expression of genes based on comparisons to purified human hematopoietic stem cells as well as normal somatic tissues. Our analysis revealed that WT1, a gene known to be associated with promoting leukemic transformation, is expressed in comparative abundance in human leukemic stem cells. Preclinical studies were then performed in a mouse model, and revealed that CD8 T cells specific for this oncogene with even higher avidity than can be detected in normal repertoires could be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. For our initial clinical trial, poor prognosis leukemia patients who relapsed after hematopoietic cell transplant (HCT) were treated with transfer of WT1-specific CD8 T cells clones isolated and expanded in vitro from the HCT donor. This study demonstrated that such T cells were safe, mediated in vivo anti-leukemic activity, and were associated with maintenance of long-term remissions in some patients, but generating sufficient numbers of WT1-specific CD8 T cells with high avidity for the target in each patient represented a substantive problem. Therefore, to create a more predictably effective standardized reagent for treatment of patients with a tumor that expresses the target antigen and shares the associated MHC restricting allele, we pursued methods to genetically engineer patient T cells to acquire high avidity for the tumor target. This requires identifying a high affinity TCR and producing a vector that can achieve high-level expression of the genes encoding the Vα and Vβ genes of a TCR demonstrated to have high affinity for the target epitope. Therefore, we screened a large number of normal repertoires for the presence of high avidity WT1-specific CD8 T cells, and selected the T cell clone expressing the highest affinity TCR. We then incorporated changes in the TCR genes such as codon optimization to enhance expression, and introduced a point mutation in each chain to create a disulfide bond that minimizes the potential problem of mispairing of the introduced TCR chains with the endogenous TCR chains. We have now have now initiated a trial in which this high affinity, WT1-specific, HLA-A2-restricted TCR is being introduced into patient CD8 T cells with a lentiviral vector and the transduced cells are being infused into the patient. The early results from this trial appear promising in terms of both evidence of antileukemic activity and the capacity for the transferred cells to persist in patients, and we plan to begin very shortly another trial in patients with non-small cell lung cancer (NSCLC) utilizing this same TCR, as WT1 is also commonly overexpressed in NSCLC as well as many other malignancies. For many candidate target antigens that are also normal self-antigens, isolating high affinity TCRs may not be readily achieved from normal repertoires. Therefore, we have developed strategies to enhance the affinity of isolated TCRs with retention of specificity, including saturation mutagenesis of CDR3 regions and an in vitro thymic selection system that allows for capture of a more diverse set of high affinity specific TCR genes during TCR gene rearrangement. These approaches induce modifications to the TCR region that predominantly makes contacts with the peptide epitope rather than MHC, which is necessary to minimize the risk of off-target toxicity from promiscuous peptide/MHC recognition. However, it remains essential that such modified TCRs do not induce unanticipated tissue damage, and we are using bioinformatics as well as modeling in the mouse to uncover any potential for off-target toxicity. Unfortunately, providing a high avidity T cell response does not necessarily result in tumor eradication, as there are other substantive obstacles that can preclude even a T cell expressing a high affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies, particularly in a pancreatic ductal adenocarcinoma (PDA) model, already appear very promising, as we have demonstrated that T cells expressing a high affinity TCR targeting a tumor antigen expressed by PDA cells can infiltrate the tumor, mediate tumor lysis, modify the tumor stroma, and provide therapeutic benefit. We have already identified high affinity human TCRs specific for this tumor antigen, and plan to use the insights derived from these studies to initiate within the next 1-2 years clinical trials in human pancreatic and ovarian cancers. The genetically-engineered mouse models of spontaneously developing tumors we are using, which recapitulate many aspects of the analogous human cancer, are also making it possible to assess strategies to improve the efficacy of T cell therapy. These models have helped elucidate the importance of not only cell extrinsic mechanisms of regulation and dysfunction that render T cells unresponsive, particularly via inhibitory cells commonly present in the tumor microenvironment that interfere with an effector response such as the accumulation of regulatory CD4 T cells (Treg), myeloid derived suppressor cells (MDSC), and tumor-associated macrophages (TAM), but also the cell intrinsic mechanisms that derive in large part from persistent stimulation by the tumor antigen and ultimately can render T cells progressively dysfunctional, leading to epigenetic modifications that eventually result in non-responsive cells that cannot be readily rescued. These cumulative mechanisms highlight the difficulties eliciting and/or sustaining responses to tumor antigens. Strategies to disrupt inhibitory pathways by systemic administration of mAbs or cytokines are currently being pursued clinically, but such reagents globally disrupt inhibitory pathways which can have significant toxicity to the host. Therefore, we are evaluating strategies to sustain function and anti-tumor activity by genetically modifying T cells to enhance function and to be resistant to obstacles that prevent tumor eradication. As different tumor types exhibit unique characteristics and are capable of engaging distinct inhibitory pathways, improved understanding of the immunobiology of the tumor type to be treated will likely prove essential for designing effective therapies. However, the relatively straightforward means to use synthetic biology to genetically engineer T cells to acquire novel capacities to overcome inhibitory signals and function in the tumor microenvironment suggests that cancer therapy with engineered T cells will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Tom M. Schmitt, Andrea Schietinger, Ingunn M. Stromnes, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Kristin G. Anderson, Merav Bar, Aude G. Chapuis. Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY31-03. doi:10.1158/1538-7445.AM2015-SY31-03

Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (T(RM) cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of T(RM) cells is unknown. We found that CD8(+) T(RM) cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P1, a receptor for sphingosine 1-phosphate). Forced expression of S1P1 prevented the establishment of T(RM) cells. Cytokines that induced a T(RM) cell phenotype (including transforming growth factor-β (TGF-β), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P1 provides a switch that dictates whether CD8(+) T cells commit to recirculating or tissue-resident memory populations.

Nonlymphoid T cell populations control local infections and contribute to inflammatory diseases, thus driving efforts to understand the regulation of their migration, differentiation, and maintenance. Numerous observations indicate that T cell trafficking and differentiation within the lung are starkly different from what has been described in most nonlymphoid tissues, including intestine and skin. After systemic infection, we found that >95% of memory CD8 T cells isolated from mouse lung via standard methods were actually confined to the pulmonary vasculature, despite perfusion. A respiratory route of challenge increased virus-specific T cell localization within lung tissue, although only transiently. Removing blood-borne cells from analysis by the simple technique of intravascular staining revealed distinct phenotypic signatures and chemokine-dependent trafficking restricted to Ag-experienced T cells. These results precipitate a revised model for pulmonary T cell trafficking and differentiation and a re-evaluation of studies examining the contributions of pulmonary T cells to protection and disease.

The purpose of this study was to extend previous work examining publication rates for the anxiety disorders and publication topics for generalized anxiety disorder (GAD). Specifically, we examined anxiety disorder publication rates in MEDLINE and PsycINFO from 1998 to 2008. The results show: (1) that with the exception of panic disorder, there was a significant increase in the annual rate of publications for every anxiety disorder; (2) that GAD had the second lowest annual rate of publications in every year - with no more than 8% of anxiety disorder publications devoted to GAD in any given year; and (3) that GAD publications focused more often on treatment (44%) than on descriptive issues (26%), process issues (22%), and general reviews (8%). Given that citation analysis appears to be a valid indicator of research progress, the current findings suggest that research on GAD continues to lag behind research on most other anxiety disorders.

Unlike cancer vaccines and immune modulators such as checkpoint inhibitors that seek to harness patient immune responses, adoptive therapy with genetically engineered T cells seeks to create responses that don’t exist in the patient’s immune system. Molecular technologies now make it feasible to not only create T cells with specificity for the tumor by introduction of a selected antigen-specific receptor, but also with qualities not naturally found, including improved function and resistance to immunosuppression. We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. For human acute myelogenous leukemia (AML), we have pursued targeting WT1, a gene overexpressed in human leukemic stem cells that is associated with promoting leukemic transformation. Preclinical studies performed in a mouse model demonstrated that CD8 T cells expressing a high affinity TCR specific for this oncogene can be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. We have advanced this approach to a clinical trial in leukemia patients with poor prognostic factors that place them at high risk of relapse after hematopoietic cell transplant (HCT), using a high-affinity human TCR specific for WT1 to transduce CD8 cells and reproducibly create high-avidity T cells that recognize leukemic cells. Our clinical results demonstrate that such T cells can prevent leukemic relapse and sustain long-term remissions, and can mediate antileukemic activity in patients who have relapsed. This therapy is now being tested in AML patients who have minimal residual disease after induction therapy and are not candidates for HCT, as well as in solid tumors that similarly overexpress WT1. Unfortunately, there are substantive obstacles in targeting established tumors that can preclude even a T cell expressing a high-affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of hematologic and solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies reveal promising antitumor activity, but demonstrate that repeated infusions of functional T cells are required to sustain a therapeutic response in the context of the immunosuppressive tumor microenvironment, and we are engineering T cells to overcome these inhibitory signals and enhance efficacy. In place of current strategies that disrupt inhibitory pathways by systemic administration of blocking mAbs, which globally disrupt immune regulation and thus can have significant toxicity to the host, we are creating synthetic immunomodulatory fusion proteins that take advantage of the expression of inhibitory ligands by tumors by still binding the inhibitory ligand but alternatively delivering a costimulatory rather than inhibitory signal. Additionally, as the antitumor activity of CD8 T cells is enhanced by a concurrent CD4 T cell response, we are engineering CD4 T cells as well as CD8 T cells to create an orchestrated antitumor response. The results suggest that cancer therapy with engineered T cells can provide effective antitumor responses and will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Tom M. Schmitt, Ingunn M. Stromnes, Leah Schmidt, Aude G. Chapuis. Engineering T cells to eradicate tumors in the age of synthetic biology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA02.

Ovarian cancer is the most lethal gynecologic cancer, with more than 20,000 new diagnoses and 15,000 cancer-related deaths annually. Current standard therapy for advanced ovarian cancer incorporates upfront surgical cytoreduction followed by cytotoxic chemotherapy. But even with advances in surgical technique, chemotherapeutics and targeted delivery methods, the overall five-year survival rate remains ~50%, highlighting the need for novel treatment strategies. We have focused our efforts on immunotherapy by adoptive transfer of engineered T cells that can target proteins uniquely overexpressed in ovarian cancer. Recent work has identified mesothelin (MSLN) as a potential target, as this protein is overexpressed in malignant ovarian tissue, promotes the oncogenic phenotype, and has limited expression in healthy cells. Preclinical studies have demonstrated that mouse and human T cells engineered to express T cell receptors (TCR) with high-affinity for MSLN kill respectively murine and human ovarian tumor cells in vitro. Furthermore, in vivo studies in mice treating metastatic disease developing after intraperitoneal introduction of mouse ID8 ovarian cancer cells have demonstrated that engineered, adoptively transferred T cells preferentially accumulate in ID8 ovarian cancers, reduce tumor burden and prolong survival. However, our results also revealed that the tumor microenvironment (TME) can limit the persistence and killing capacity of engineered T cells. Immunosuppressive cells, inhibitory ligands that reduce T cell function, and cell death-inducing ligands are abundant within ID8 tumors, and cellular and molecular analyses of human ovarian cancer specimens showed that human T cell therapy will face similar TME-mediated obstacles. The ovarian cancer TME is also a nutrient- and oxygen-deprived milieu, and the adaptive metabolic responses required for survival by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell responses to inhibitory signals, including metabolic pathways, have the potential to influence activity in the TME, and to enhance T cell function and improve anti-tumor efficacy. Ongoing studies exploring strategies to overcome elements common to the human and murine TME, including both direct modulation of the microenvironment and engineering of T cells to overcome critical components of immune evasion by solid tumors and to promote T cell survival and function, will be discussed. Building upon this work, we have been developing a novel model that more directly reflects the clinical setting requiring intervention. Following orthotopic injection of ID8 ovarian cancer cells directly beneath the ovarian bursa, tumorigenesis occurs initially within the ovary/fallopian tube microenvironment, and is followed by development of small volume metastatic disease. This orthotopic model, which results in formation of intraperitoneal metastatic implants originating from the ovary, replicates the development and patterns of metastatic spread of human ovarian cancer. Following intervention with primary surgical cytoreduction that includes hysterectomy and bilateral salpingo-oophorectomy, mice are being treated by adoptive transfer of antigen-specific T cells to target minimal residual disease in the peritoneal cavity, which models the clinical situation of patients requiring treatment after optimal surgical cytoreduction. Ongoing studies will be discussed that have been designed to modulate the function and persistence of transferred, MSLN-specific T cells and assess the impact on in vivo tumor development and survival in this clinically relevant model of ovarian cancer treatment. Citation Format: Christopher B. Morse, Kristin G. Anderson, Breanna M. Bates, Edison Y. Chiu, Nicolas M. Garcia, Philip D. Greenberg. Adoptive T cell therapy for ovarian cancer: Application of a surgically relevant model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A57.

Fas/FasL signaling plays a significant role in the generation and persistence of tumors. We and others have detected FasL expression in the tumor microenvironment (TME) of human ovarian, pancreatic, and other cancers, where it can function to protect tumor cells from tumor-infiltrating lymphocytes. Adoptive immunotherapy, a promising treatment option, uses genetically modified T cells to eliminate tumors. However, efficacy of T cell immunotherapy is dampened by limited costimulation as well as increased inhibitory and death signals in the TME. We previously showed enhanced therapeutic efficacy by engineering T cells to express an immunomodulatory fusion protein (IFP) to convert the inhibitory CD200R signal to a costimulatory CD28 signal and questioned if IFPs could use a 4-1BB signal to enhance in vivo persistence and memory formation. 4-1BB forms a trimer when binding its ligand, that is requisite for an effective costimulatory signal. We and others engineered T cells to express 4-1BB with an ectodomain that dimerizes, however this IFP did not improve T cell function. We hypothesized a Fas IFP with a 4-1BB signal would effectively replace Fas signaling and provide costimulation, as Fas also trimerizes when engaged by ligand. T cells transduced with the Fas-4-1BB IFP exhibited enhanced accumulation and function in vitro. In a model of leukemia, Fas-4-1BB-transduced T cells eradicated otherwise lethal disease and exhibited increased persistence. Preliminary data in the autochthonous KPC pancreatic and ID8 ovarian cancer models support increased efficacy. We describe for the first time that an IFP can generate an effective 4-1BB signal to overcome obstacles in the TME and enhance T cell immunotherapy of solid and liquid malignancies.

Over 20,000 women are diagnosed with ovarian cancer annually—more than half will die within 5 years and this rate has changed very little in the last 20 years, highlighting the need for innovative therapies. Reprogramming immune system cells to target and kill cancer cells represents a promising new treatment strategy. Immune T cells have the potential to control tumor growth without toxicity to healthy tissues when engineered to target proteins uniquely overexpressed in tumors. Recent technological advances have helped identify and validate Wilms' Tumor Antigen 1 (WT1) and mesothelin (MSLN) as valid antigen targets for ovarian cancer; these proteins contribute to malignant and invasive phenotypes and have limited expression in healthy cells. In preclinical studies using either patient-derived cell lines or the mouse ID8 ovarian tumor model, we show that T cells engineered to express either a WT1- or MSLN- specific high-affinity T cell receptor (TCR) can kill human and murine ovarian tumor cells in vitro. Moreover, in a disseminated in vivo murine model, adoptively transferred TCRengineered T cells preferentially accumulated within established ID8 tumors, delayed ovarian tumor growth, and prolonged mouse survival. However, our data also reveal that the tumor microenvironment (TME) limits the persistence and killing capacity of the engineered T cells. Cellular and molecular analyses of human tumor specimens show human therapy will face similar obstacles posed by the TME. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function. Citation Format: Kristin G. Anderson, Breanna M. Bates, Edison Y. Chiu, Philip D. Greenberg. ENGINEERING ADOPTIVE T CELL THERAPY FOR EFFICACY IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-078.

Over 20,000 women are diagnosed with ovarian cancer annually; more than half will die within 5 years, which has changed little over the last 20 years. One promising new treatment strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors, with the potential to control tumor growth without toxicity to healthy tissues. Recently, mesothelin (MSLN) has been identified as a valid antigen target in ovarian cancer; minimally expressed in healthy cells, MSLN contributes to malignant and invasive phenotypes. In preclinical studies using patient-derived cell lines or the mouse ID8 ovarian tumor model, we found that T cells engineered to express a high-affinity MSLN-specific T cell receptor (TCR) can kill ovarian tumor cells in vitro. In vivo, adoptively transferred TCR-engineered T cells preferentially accumulate within disseminated ID8 tumors, delay tumor growth and prolong mouse survival, but our data also show the tumor microenvironment (TME) can limit engineered T cell persistence and function. Other analyses show human therapy will face similar TME-mediated obstacles. The ovarian cancer TME is a nutrient- and oxygen-deprived milieu, and adaptive metabolic responses by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell metabolic pathways, and thereby influence activity in the TME, might enhance T cell function and improve anti-tumor efficacy by overcoming a critical component of immune evasion by solid tumors. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function.

Effective cellular therapy for human malignancies requires first identifying and validating an appropriate antigenic target, and then establishing in each patient a tumor-reactive T cell response of high avidity and high magnitude that is not only safe but can infiltrate and retain function in the tumor microenvironment. We have used molecular expression profiling to detect antigens selectively or markedly over-expressed by tumors, and then used these antigens as stimuli to generate T cells from normal repertoires. We have developed a high throughput technology to identify those T cells that express high affinity TCRs, and to then isolate from these T cells the TCR genes, place them either directly or after affinity enhancement into shuttle vectors, and use these reagents to create recipient T cells with high avidity for tumor targets that can be administered in vivo. We have utilized in silico, in vitro, and preclinical mouse models to assess the safety and potential efficacy of T cells expressing such TCRs. We are currently pursuing targeting of 3 antigens that are expressed in both murine and human tumors and are pro-oncogenic, contributing to the malignant phenotype. This includes ongoing clinical trials that will be discussed in acute myelogenous leukemia and in non-small cell lung cancer or mesothelioma targeting WT1 with T cells transduced to express a high affinity TCR specific for WT1, as well as trials being designed to target Mesothelin (MSLN) with T cells transduced to express a high affinity TCR specific for MSLN in pancreatic and ovarian cancer that are anticipated to begin within 8-12 months. Our clinical results in treatment of AML and preclinical results in mouse models of pancreatic and ovarian cancer appear very promising. However, the composite clinical data, as well as the results in the preclinical mouse models that drive our clinical trials, demonstrate substantial obstacles to sustaining T cell function in vivo after transfer, particularly in the context of solid tumors. Engineered T cells with specificity for tumor antigens appear to have the capacity to infiltrate and accumulate in solid tumors, and to initially mediate anti-tumor activity, but frequently become dysfunctional in the tumor microenvironment. Studies are being pursued to identify the critical obstacles to maintaining T cell function and achieving more reproducible tumor eradication, including modulating the tumor microenvironment and engineering T cells to express immunomodulatory fusion proteins (IFP) that recognize ligands for inhibitory signals but deliver an activation/costimulatory signal. Our data suggest that engineering T cells to acquire novel properties not naturally found in unmanipulated T cells has the potential to create effective therapies for human cancers. Citation Format: Philip D. Greenberg, Aude Chapuis, Dan Egan, Ingunn Stromnes, Sunil Hingorani, Shannon Oda, Rachel Perret, Kristin Anderson, Tom Schmitt. Building better T cells for targeting and eliminating tumors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA18.

Vaccines are desired that maintain abundant memory T cells at nonlymphoid sites of microbial exposure, where they may be anatomically positioned for immediate pathogen interception. Here, we test the impact of antigen persistence on mouse CD8 and CD4 T cell distribution and differentiation by comparing responses to infections with different strains of LCMV that cause either acute or chronic infections. We used in vivo labeling techniques that discriminate between T cells present within tissues and abundant populations that fail to be removed from vascular compartments, despite perfusion. LCMV persistence caused up to ∼30-fold more virus-specific CD8 T cells to distribute to the lung compared with acute infection. Persistent infection also maintained mucosal-homing α4β7 integrin expression, higher granzyme B expression, alterations in the expression of the TRM markers CD69 and CD103, and greater accumulation of virus-specific CD8 T cells in the large intestine, liver, kidney, and female reproductive tract. Persistent infection also increased LCMV-specific CD4 T cell quantity in mucosal tissues and induced maintenance of CXCR4, an HIV coreceptor. This study clarifies the relationship between viral persistence and CD4 and CD8 T cell distribution and mucosal phenotype, indicating that chronic LCMV infection magnifies T cell migration to nonlymphoid tissues.

Tumor progression occurs through the modulation of a number of physiological parameters, including the development of immunosuppressive mechanisms to prevent immune detection and response. Among these immune evasion mechanisms, the mobilization of myeloid-derived suppressor cells (MDSC) is a major contributor to the suppression of antitumor T-cell immunity. Patients with renal cell carcinoma (RCC) show increased MDSC, and methods are being explored clinically to reduce the prevalence of MDSC and/or inhibit their function. In the present study, we investigated the relationship between MDSC and the therapeutic potential of a TRAIL-encoding recombinant adenovirus (Ad5-TRAIL) in combination with CpG-containing oligodeoxynucleotides (Ad5-TRAIL/CpG) in an orthotopic mouse model of RCC. This immunotherapy effectively clears renal (Renca) tumors and enhances survival, despite the presence of a high frequency of MDSC in the spleens and primary tumor-bearing kidneys at the time of treatment. Subsequent analyses revealed that the CpG component of the immunotherapy was responsible for decreasing the frequency of MDSC in Renca-bearing mice; further, treatment with CpG modulated the phenotype and function of MDSC that remained after immunotherapy and correlated with an increased T-cell response. Interestingly, the CpG-dependent alterations in MDSC frequency and function did not occur in tumor-bearing mice complicated with diet-induced obesity. Collectively, these data suggest that in addition to its adjuvant properties, CpG also enhances antitumor responses by altering the number and function of MDSC.

We have entered a new and exciting era in cancer therapy, in which immunotherapeutic strategies are achieving unprecedented successes and are increasingly becoming incorporated into standard of care regimens. Checkpoint blockade is dependent on inducing and/or reactivating or sustaining responses to the tumor by T cells already in the patient. Similarly, vaccines attempt to generate and/or expand responses of T cells naturally present in the normal repertoire. However, these strategies require that functional tumor-reactive T cells exist in the patient's repertoire and that the method pursued can harness those T cells to create a potent response that will function in the tumor microenvironment, which limits the settings in which these approaches will prove effective. Adoptive T cell therapy, in which patient T cells can be expanded to large numbers ex vivo before infusion, provides a means to bypass or overcome these obstacles, particularly with the advent of genetic engineering that now makes it possible to create T cells not only with specificity for the tumor but also with qualities not naturally found, including improved function and resistance to immunosuppression. We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. The first issue is to identify tumor antigens that can be safely, effectively, and reproducibly targeted. We have used analyses of differential gene expression to search for antigenic targets that are either uniquely expressed in a tumor or are differentially expressed at high levels in the tumor with much lower and limited expression in normal tissues, and that preferentially are associated with the malignant phenotype to reduce the risk of antigen loss by the tumor. In our search for targets in acute myelogenous leukemia (AML), we found that WT1, a gene known to be associated with promoting leukemic transformation, is expressed in comparative abundance in human leukemic stem cells. The next step is to generate T cells specific for the target antigen that can recognize and eliminate malignant cells expressing the antigen. Extensive screening of normal human repertoires revealed a high affinity TCR specific for WT1 that can recognize leukemic cells, and that could be inserted into CD8 T cells to reproducibly produce high avidity T cells for use in therapy. Preclinical studies performed in a mouse model demonstrated that CD8 T cells specific for this oncogene expressing a high affinity TCR can be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. We have advanced this approach targeting WT1 to an initial clinical trial in leukemia patients with poor prognostic factors that make them at high risk of relapse after hematopoietic cell transplant (HCT). The Vα and Vβ genes of the human WT-1 specific TCR were codon optimized to enhance expression, modified by a point mutation in each chain to create an interchain disulfide bond that minimizes the potential problem of mispairing of the introduced TCR chains with the endogenous TCR chains, and inserted these TCR genes into a lentiviral vector. Preliminary results of this trial, which has provided evidence that such T cells can prevent leukemic relapse and sustain long-term remissions, will be discussed. This therapy is now being advanced for use in AML patients who are not HCT candidates. We have also now initiated additional trials with this TCR for treatment of patients with non-small cell lung cancer (NSCLC) or mesothelioma, as WT1 is commonly overexpressed in NSCLC as well as many other malignancies. For many candidate target antigens that are also normal self-antigens, isolation of high affinity TCRs may not be readily achieved from normal repertoires. However, it is now feasible to engineer TCRs that have higher affinities than normally exist for their antigen target. We have developed strategies to enhance the affinity of isolated TCRs with retention of specificity, including saturation mutagenesis of CDR3 regions and an in vitro thymic selection system that allows for capture of a more diverse set of high affinity specific TCRs during TCR gene rearrangement. These approaches induce modifications to the TCR region that predominantly makes contacts with the peptide epitope rather than MHC, which is necessary to minimize the risk of off-target toxicity from promiscuous peptide/MHC recognition. However, it remains essential that such modified TCRs do not induce unanticipated tissue damage, and we are using bioinformatics, functional screening, and modeling in the mouse to uncover any potential for off-target toxicity. Unfortunately, providing a high avidity T cell response does not necessarily result in tumor eradication, as there are other substantive obstacles that can preclude even a T cell expressing a high affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies, particularly in a pancreatic ductal adenocarcinoma (PDA) model, already appear very promising, as we have demonstrated that T cells expressing a high affinity TCR targeting a tumor antigen expressed by PDA cells can infiltrate the tumor, mediate tumor lysis, modify the tumor stroma, and provide therapeutic benefit. We have now identified high affinity human TCRs specific for this tumor antigen, and plan to use the insights derived from these studies to initiate within the next year clinical trials in human pancreatic and ovarian cancers. The genetically-engineered mouse models of spontaneously developing tumors we are using, which recapitulate many aspects of the analogous human cancer, are also making it possible to assess strategies to improve the efficacy of T cell therapy. These models have helped elucidate the importance of not only cell extrinsic mechanisms of regulation and dysfunction that render T cells unresponsive, particularly via inhibitory cells commonly present in the tumor microenvironment that interfere with an effector response, such as the accumulation of regulatory CD4 T cells (Treg), myeloid derived suppressor cells (MDSC), and tumor-associated macrophages (TAM), but also the cell intrinsic mechanisms that derive in large part from persistent stimulation by the tumor antigen and ultimately can render T cells progressively dysfunctional, leading to epigenetic modifications that eventuallly result in non-responsive cells that cannot be readily rescued. These cumulative mechanisms highlight the difficulties eliciting and/or sustaining responses to tumor antigens. Strategies to disrupt inhibitory pathways by systemic administration of mAbs or cytokines are currently being pursued clinically, but such reagents globally disrupt inhibitory pathways and thus can have significant toxicity to the host. Therefore, we are evaluating strategies to sustain function and anti-tumor activity by genetically modifying T cells to enhance function and to be resistant to obstacles that prevent tumor eradication. As different tumor types exhibit unique characteristics and are capable of engaging distinct inhibitory pathways, improved understanding of the immunobiology of the tumor type to be treated will likely prove essential for designing effective therapies. However, the relatively straightforward means to use synthetic biology to genetically engineer T cells to acquire novel capacities to overcome inhibitory signals and function in the tumor microenvironment suggests that cancer therapy with engineered T cells will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Andrea Schietinger, Tom M. Schmitt, Ingunn M. Stromnes, Alec Wilkens, Aude G. Chapuis. Engineering T cell responses to tumors: Taking the immune system where no responses have gone before [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA01.

Over 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed very little in the last 20 years, highlighting the need for innovative therapies. Reprogramming immune system cells to target and kill ovarian cancer cells represents a potentially promising new treatment strategy. Immune T cells have the potential to control tumor growth without toxicity to healthy tissues if engineered to target proteins uniquely overexpressed in tumors. Recent technological advances have helped identify and validate Wilms’ Tumor Antigen 1 (WT1) and mesothelin (MSLN) as promising ovarian cancer antigen targets; these proteins contribute to malignant and invasive phenotypes and have limited expression in healthy cells. Using patient-derived cell lines and the murine ID8 tumor, we show that T cells engineered to express a high-affinity T cell receptor (TCR) targeting either WT1 or MSLN can kill human and murine ovarian tumor cells in vitro. Moreover, in an in vivo model of established disease with disseminated ID8 tumor, adoptively transferred TCR-engineered T cells infiltrated and preferentially accumulated within ovarian tumors, delayed tumor growth and prolonged survival. However, our data revealed that the tumor microenvironment (TME) limits the survival and killing capacity of these engineered T cells. Cellular and molecular analyses of human tumor specimens have revealed human therapy will face similar obstacles. Therefore, ongoing studies will be discussed that are further exploring strategies to overcome these common elements of the immunosuppressive TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function.

Interpretations of negative, positive, and ambiguous situations were examined in individuals with generalized anxiety disorder (GAD), other anxiety disorders (ANX), and no psychiatric condition (CTRL). Additionally, relationships between specific beliefs about uncertainty (Uncertainty Has Negative Behavioral and Self-Referent Implications [IUS-NI], and Uncertainty Is Unfair and Spoils Everything [IUS-US]) and interpretations were explored. The first hypothesis (that the clinical groups would report more concern for negative, positive, and ambiguous situations than would the CTRL group) was supported. The second hypothesis (that the GAD group would report more concern for ambiguous situations than would the ANX group) was not supported; both groups reported similar levels of concern for ambiguous situations. Exploratory analyses revealed no differences between the GAD and ANX groups in their interpretations of positive and negative situations. Finally, the IUS-US predicted interpretations of negative and ambiguous situations in the full sample, whereas the IUS-NI did not. Clinical implications are discussed.

The ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) has been suggested as a potential biomarker for breast cancer risk. We evaluated within-person variability of this biomarker in ten healthy Caucasian women aged 23-58 years. Each study participant was asked to provide an overnight fasting morning urine sample once a week for an average of 8 weeks. These urine samples were assayed for 2-OHE1 and 16alpha-OHE1 by using competitive enzyme immunoassay kits purchased from the ImmunaCare Corporation. The coefficients of variation for urinary 2-OHE1/16alpha-OHE1 over the study period ranged from 13.7 to 59.6% (mean, 33.3%) in our study participants. There was a good correlation between the level of the urinary 2-OHE1/16alpha-OHE1 ratio in any single urine sample and the average ratio over the 8-week study period from the same woman, with the mean correlation coefficient of 0.85. These results indicated that the within-person variation of the 2-OHE1 to 16alpha-OHE1 ratio for most women was moderate and the level of this ratio in a single urine sample, in general, reflects reasonably well the level of this biomarker over a 2-month period.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk-factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data, and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific autoantibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies., By correlating patient symptoms with in-depth profiling of blood cells and plasma components throughout COVID-19 infection, this study identifies factors that may predict sustained disease.