Comprehensive mutagenesis identifies the peptide repertoire of a p53 T-cell receptor mimic antibody that displays no toxicity in mice transgenic for human HLA-A*0201.

T-cell receptor mimic (TCRm) antibodies have expanded the repertoire of antigens targetable by monoclonal antibodies, to include peptides derived from intracellular proteins that are presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface. We have previously used this approach to target p53, which represents a valuable target for cancer immunotherapy because of the high frequency of its deregulation by mutation or other mechanisms. The T1-116C TCRm antibody targets the wild type p5365-73 peptide (RMPEAAPPV) presented by HLA-A*0201 (HLA-A2) and exhibited in vivo efficacy against triple receptor negative breast cancer xenografts. Here we report a comprehensive mutational analysis of the p53 RMPEAAPPV peptide to assess the T1-116C epitope and its peptide specificity. Antibody binding absolutely required the N-terminal arginine residue, while amino acids in the center of the peptide contributed little to specificity. Data mining the immune epitope database with the T1-116C binding consensus and validation of peptide recognition using the T2 stabilization assay identified additional tumor antigens targeted by T1-116C, including WT1, gp100, Tyrosinase and NY-ESO-1. Most peptides recognized by T1-116C were conserved in mice and human HLA-A2 transgenic mice showed no toxicity when treated with T1-116C in vivo. We conclude that comprehensive validation of TCRm antibody target specificity is critical for assessing their safety profile.

Therapeutic Antibodies against Intracellular Tumor Antigens.

Monoclonal antibodies are among the most clinically effective drugs used to treat cancer. However, their target repertoire is limited as there are relatively few tumor-specific or tumor-associated cell surface or soluble antigens. Intracellular molecules represent nearly half of the human proteome and provide an untapped reservoir of potential therapeutic targets. Antibodies have been developed to target externalized antigens, have also been engineered to enter into cells or may be expressed intracellularly with the aim of binding intracellular antigens. Furthermore, intracellular proteins can be degraded by the proteasome into short, commonly 8-10 amino acid long, peptides that are presented on the cell surface in the context of major histocompatibility complex class I (MHC-I) molecules. These tumor-associated peptide-MHC-I complexes can then be targeted by antibodies known as T-cell receptor mimic (TCRm) or T-cell receptor (TCR)-like antibodies, which recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. Advances in the production of TCRm antibodies have enabled the generation of multiple TCRm antibodies, which have been tested in vitro and in vivo, expanding our understanding of their mechanisms of action and the importance of target epitope selection and expression. This review will summarize multiple approaches to targeting intracellular antigens with therapeutic antibodies, in particular describing the production and characterization of TCRm antibodies, the factors influencing their target identification, their advantages and disadvantages in the context of TCR therapies, and the potential to advance TCRm-based therapies into the clinic.

Development of a T-cell Receptor Mimic Antibody against Wild-Type p53 for Cancer Immunotherapy.

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Because of its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anticancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of MHC I and can be bound by T-cell receptors (TCR). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201-presented wild-type p53 T-cell epitope, p5365-73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitroIn vivo, the antibody targets p5365-73 peptide-expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy. Cancer Res; 77(10); 2699-711. ©2017 AACR.