Identification of the Targets of T-cell Receptor Therapeutic Agents and Cells by Use of a High-Throughput Genetic Platform.

T-cell receptor (TCR)-based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high-throughput genetic platform (termed "PresentER") that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

ALK and RET Inhibitors Promote HLA Class I Antigen Presentation and Unmask New Antigens within the Tumor Immunopeptidome.

T-cell immunotherapies are often thwarted by the limited presentation of tumor-specific antigens abetted by the downregulation of human leukocyte antigen (HLA). We showed that drugs inhibiting ALK and RET produced dose-related increases in cell-surface HLA in tumor cells bearing these mutated kinases in vitro and in vivo, as well as elevated transcript and protein expression of HLA and other antigen-processing machinery. Subsequent analysis of HLA-presented peptides after ALK and RET inhibitor treatment identified large changes in the immunopeptidome with the appearance of hundreds of new antigens, including T-cell epitopes associated with impaired peptide processing (TEIPP) peptides. ALK inhibition additionally decreased PD-L1 levels by 75%. Therefore, these oncogenes may enhance cancer formation by allowing tumors to evade the immune system by downregulating HLA expression. Altogether, RET and ALK inhibitors could enhance T-cell-based immunotherapies by upregulating HLA, decreasing checkpoint blockade ligands, and revealing new, immunogenic, cancer-associated antigens.

Depleting T regulatory cells by targeting intracellular Foxp3 with a TCR mimic antibody.

Depletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date. We have generated a T cell receptor mimic antibody, "Foxp3-#32," recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs.

An immunogenic WT1-derived peptide that induces T cell response in the context of HLA-A*02:01 and HLA-A*24:02 molecules.

The Wilms' tumor oncogene protein (WT1) is a highly validated tumor antigen for immunotherapy. WT1-targeted immunotherapy has been extensively explored in multiple human trials in various cancers. However, clinical investigations using WT1 epitopes have generally focused on two peptides, HLA-restricted to HLA-A*02:01 or HLA-A*24:02. The goal of this study was to identify new epitopes derived from WT1, to expand the potential use of WT1 as a target of immunotherapy. Using computer-based MHC-binding algorithms and in vitro validation of the T cell responses specific for the identified peptides, we found that a recently identified HLA-A*24:02-binding epitope (239-247), NQMNLGATL (NQM), was also a strong CD8+ T cell epitope for HLA-A*02:01 molecule. A peptide second position Q240L substitution (NLM) or Q240Y substitution (NYM), further enhanced the T cell responses in both HLA-A*02:01 positive and HLA-A*24:02 positive healthy donors. Importantly, T cells stimulated with the new analog peptides displayed heteroclitic cross-reactivity with the native NQM sequence and were able to kill HLA-matched WT1-positive tumor cell lines and primary leukemia blasts. In addition, longer native and heteroclitic HLA-DR.B1-binding peptides, comprising the nine amino acid NQM or NLM sequences, could induce T cell response that recognized the CD8+ epitope NQM, suggesting the processing and the presentation by HLA-A*02:01 molecules of the CD8+ T cell epitope embedded within it. Our studies suggest that the analog peptides NLM and NYM could be potential candidates for future immunotherapy targeting WT1 positive cancers in the context of HLA-A*02:01 and A*24:02 positive populations.

Kinase Regulation of Human MHC Class I Molecule Expression on Cancer Cells.

The major histocompatibility complex I (MHC-1) presents antigenic peptides to tumor-specific CD8+ T cells. The regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. Regulators of cell-surface HLA amounts were discovered using a pooled human kinome shRNA interference-based approach. Hits scoring highly were subsequently validated by additional RNAi and pharmacologic inhibitors. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of MHC-I expression and antigen presentation machinery in multiple cancer types, acting through an ERK output-dependent mechanism; the pathways responsible for increased MHC-I upon kinase inhibition were mapped. Activated MAPK signaling in mouse tumors in vivo suppressed components of MHC-I and the antigen presentation machinery. Pharmacologic inhibition of MAPK signaling also led to improved peptide/MHC target recognition and killing by T cells and TCR-mimic antibodies. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Cancer Immunol Res; 4(11); 936-47. ©2016 AACR.

Opportunities and challenges for TCR mimic antibodies in cancer therapy.

INTRODUCTION: Monoclonal antibodies (mAbs) are potent cancer therapeutic agents, but exclusively recognize cell-surface targets whereas most cancer-associated proteins are found intracellularly. Hence, potential cancer therapy targets such as over expressed self-proteins, activated oncogenes, mutated tumor suppressors, and translocated gene products are not accessible to traditional mAb therapy. An emerging approach to target these epitopes is the use of TCR mimic mAbs (TCRm) that recognize epitopes similar to those of T cell receptors (TCR). AREAS COVERED: TCRm antigens are composed of a linear peptide sequence derived from degraded proteins and presented in the context of cell-surface MHC molecules. We discuss how the nature of the TCRm epitopes provides both advantages (absolute tumor specificity and access to a new universe of important targets) and disadvantages (low density, MHC restriction, MHC down-regulation, and cross-reactive linear epitopes) to conventional mAb therapy. We will also discuss potential solutions to these obstacles. EXPERT OPINION: TCRm combine the specificity of TCR recognition with the potency, pharmacologic properties, and versatility of mAbs. The structure and presentation of a TCRm epitope has important consequences related to the choice of targets, mAb design, available peptides and MHC subtype restrictions, possible cross-reactivity, and therapeutic activity.

T cell receptor mimic antibodies for cancer therapy.

The major hurdle to the creation of cancer-specific monoclonal antibodies (mAb) exhibiting limited cross-reactivity with healthy human cells is the paucity of known tumor-specific or mutated protein epitopes expressed on the cancer cell surface. Mutated and overexpressed oncoproteins are typically cytoplasmic or nuclear. Cells can present peptides from these distinguishing proteins on their cell surface in the context of human leukocyte antigen (HLA). T cell receptor mimic (TCRm) mAb can be discovered that react specifically to these complexes, allowing for selective targeting of cancer cells. The state-of-the-art for TCRm and the challenges and opportunities are discussed. Several such TCRm are moving toward clinical trials now.