Abstract 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.

Abstract 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<0.01). We have also been developing strategies to translate insights and technologies from this study to treatment of solid tumors. In a preclinical genetically engineered mouse model (KPC mice) of pancreatic cancer that faithfully replicates most aspects of human disease, we demonstrated (Stromnes et al., Cancer Cell 2015) that CD8 T cells engineered with a high-affinity TCR specific for mesothelin (Msln) can infiltrate pancreatic tumors, mediate antitumor activity, and provide therapeutic benefit. However, since the T cells are ultimately rendered dysfunctional in the tumor microenvironment (TME), prolonging survival has required repeated infusions of T cells to sustain antitumor activity. We have now isolated and validated a human high-affinity TCR specific for Msln for use in a planned clinical trial modeled after the approach successful in KPC mice. However, we would like to both enhance and sustain antitumor activity without requiring repeated infusions. In-depth analyses of the T cells, tumors, and the TME in treated KPC mice have illuminated strategies to potentially overcome the obstacles to tumor eradication, and we have been exploring molecular engineering approaches to achieve this. One approach has been to create synthetic immunomodulatory fusion proteins (IFPs) that have an ectodomain composed of the receptor for an inhibitory ligand encountered in the TME but, rather than the natural cytoplasmic tail that would deliver an inhibitory signal, the receptor has the tail of a costimulatory receptor and delivers an activation signal. Expression of such IFPs takes advantage of the inhibitory ligands commonly encountered in the TME by T cells by co-opting potential inhibitory signals and has resulted in enhanced T cell function, persistence/survival, and antitumor activity. Another major obstacle to sustained therapeutic activity appears to be the limited access in the TME to nutrients that effector T cells can utilize as an energy source. Analysis of the metabolites present in the TME and the transcriptional program in T cells has provided insights into genetic modifications that can be made to allow T cells to survive and function in the metabolically hostile TME. These and related studies will be discussed. 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.

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.

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.

Abstract 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.