Abstract IA01: Engineering T cell responses to tumors: Taking the immune system where no responses have gone before
Authors: Greenberg Philip D. , Anderson Kristin G. , Egan Dan , Hingorani Sunil R. , Oda Shannon K. , Perret Rachel , Schietinger Andrea , Schmitt Tom M. , Stromnes Ingunn M. , Wilkens Alec , Chapuis Aude G. 
Published: November 2016

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.