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.

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.