TP53 (tumor protein P53), a tumor suppressor gene, is the most commonly mutated cancer-driver gene. Nevertheless, drugs that target mutant p53, the protein product, remain unavailable today, decades after the discovery of its critical role in cancer. Although drugs that inactivate proteins encoded by mutant oncogenes, such as epidermal growth factor receptor (EGFR) or BRAF, are available, proteins encoded by tumor suppressor genes are already inactivated through mutation. Reactivating such proteins by use of pharmacologic agents is very challenging. Thus, new approaches to target such inactivated proteins, including those encoded by TP53, are actively being sought.

We attempted to develop an immunotherapeutic approach to target proteins encoded by a mutant TP53 gene. p53 is an intracellular protein, primarily located within the nucleus, thus out of reach for conventional antibody-based therapies. However, proteins are degraded into peptides by the proteasome, and a fraction of these peptides can be presented by the human leukocyte antigen (HLA) on the cell surface. This in principle makes it possible for suitably designed proteins to recognize peptide fragments of intracellular proteins when bound to HLA on the cell surface. The arginine-to-histidine substitution at codon 175 (R175H) is the most common TP53 mutation and is the most frequent mutation in any tumor suppressor gene. The peptide HMTEVVRHC (mutant amino acid underlined), derived from the p53^R175H mutation, can bind to a particular HLA allele (HLA-A*02:01) and form a peptide-HLA complex on the cell surface. HLA-A*02:01 is the most frequent HLA-A type in the U.S. population. Thus, the p53^R175H/HLA-A*02:01 complex is a particularly attractive therapeutic target, shared among many cancer patients. However, such neoantigen peptide-HLA complexes typically exist at low density on cell surfaces, and a potent therapeutic format will be required to achieve meaningful therapeutic effects. T cells can be activated in the presence of a very low number of antigens. We therefore attempted to generate a T cell–based therapy that links T cells to cancer cells through a newly developed antibody that specifically binds to the p53^R175H peptide-HLA complex.

Using a large phage library that displays diverse antibody variable fragments, we identified H2, an antibody fragment that binds with high affinity to the p53^R175H peptide-HLA complex but not to its wild-type counterpart. We converted H2 into a T cell–based immunotherapeutic agent—a bispecific single-chain diabody—by fusing it with an antibody fragment that binds to the T cell receptor–CD3 complex on T cells. This bispecific antibody binds to the p53^R175H peptide-HLA complex with an affinity [dissociation constant (K_d) = 86 nM] higher than that typical for T cell receptors and redirects T cells to recognize cancer cells that express the complex. Despite a very low density of the peptide-HLA complex on the cell surface, as quantified with mass spectrometry, the bispecific antibody effectively activated T cells to secrete cytokines and kill target cancer cells. This killing was dependent on the expression of both the cognate HLA and particular TP53 mutation. The bispecific antibody also resulted in regression of human xenograft tumors in mice, both when treatment was initiated soon after tumor engraftment and when the tumors were already well established. Structure of the H2 antibody fragment with the p53^R175H peptide-HLA complex showed that H2 formed a cage-like structure around the mutant amino acid (His¹⁷⁵) and one adjacent amino acid (Arg¹⁷⁴). The stability …