Abstract SY31-03: Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies
Authors: Greenberg Philip D. , Schmitt Tom M. , Schietinger Andrea , Stromnes Ingunn M. , Hingorani Sunil R. , Oda Shannon K. , Perret Rachel , Anderson Kristin G. , Bar Merav , Chapuis Aude G. 
Published: August 2015

Abstract
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