See rights and permissions. Published by BMJ.AbstrACt background Phosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T- cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA- A*0201- restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides.Methods Phosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T- cells specific for HLA- A*0201- restricted phosphorylated BCAR3 peptide (pBCAR3 126-134) was determined by 51Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA–IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund’s adjuvant and Hiltonol (poly- ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose- limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T- cell responses were assessed by interferon-γ ELISpot assay.results pBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T- cells specific for pBCAR3 126-134 controlled outgrowth of a tumor xenograft. The pIRS21097-1105 peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment- related AEs, but there were no grade 3–4 AEs, DLTs or deaths on study. T- cell responses were induced to the pIRS2 1097-1105 peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3126-134 peptide in 2/12 patients (17%, 90% CI 3% to 44%). Conclusion This study supports the safety and immunogenicity of vaccines containing the cancer- associated phosphopeptides pBCAR3 126-134 and pIRS21097- 1105, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide- specific immune responses. trial registration number NCT01846143 bACkground The development of therapeutic cancer vaccines based on peptides presented by MHC- I molecules to CD8 T- cells has been pursued for many years. Studies of these types of vaccines have recently been revital-ized by the successes of checkpoint blockade and adoptive T- cell immunotherapies and by interest in patient- specific mutated anti- gens as vaccine targets. Peptide antigens tested in cancer vaccines have commonly been derived from source proteins in one of three categories: tissue- specific differenti- ation proteins, cancer- germ cell proteins or mutated proteins. The immunogenicity of antigens derived from non- mutated proteins may be compromised by mechanisms of self- tolerance. 1 While antigens carrying tumor- specific mutations may avoid some tolerance concerns, identification of relevant antigens in each patient and preparation of an appro-priate vaccine are technically challenging and resource- intensive undertakings, 2–4 and their therapeutic value is likely to vary widely. Importantly, only a small number of previously- identified antigens in any of these categories are derived from proteins that 2 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access are objectively linked to mechanisms of cellular growth control, survival or metastasis. Such antigens are particu-larly appealing as immunotherapeutic targets for cancer control because their alteration as a means of immune escape may compromise one or more aspects of the malignant phenotype. We have identified peptide anti-gens of this type that are modified by intracellular phos-phorylation, naturally processed and presented by MHC- I molecules on cancer cells and specifically recognized by CD8 T- cells. 5–7 Phosphorylation is associated with a variety of cellular control processes, some of which are dysregulated in cancer cells. The source proteins for MHC- I- associated phosphopeptides identified to date are in large part known phosphoproteins, 6 supporting the idea that the peptides are processed from folded proteins participating in signaling pathways. One such protein is insulin receptor substrate 2 (IRS2). IRS proteins are adapters that link signaling from upstream activators to multiple downstream effectors to modulate growth, metabolism, survival and differentiation. 8 9 IRS2 overexpression at the gene or protein level is evident in many cancer types and has been demonstrated to cause tumorigenesis and enhanced metastasis in vivo. 8–10 IRS2 phosphorylated at Ser1100 is detectable in multiple cancers, and the resulting phosphopeptide (pIRS21097-1105) is endogenously processed and presented by the MHC- I molecule HLA- A*0201 at levels that allow strong immune recognition (7 and unpublished). pIRS21097-1105- specific CD8 T- cells can be generated from HLA- A2 transgenic mice by immunization with the phosphopeptide.6 7 These T- cells are capable of recognizing and killing human melanoma and breast tumors in vitro and controlling tumor growth in a xenograft tumor model system. 7 A second cancer- relevant HLA- A*0201 restricted phosphopeptide is derived from breast cancer anti-estrogen resistance 3 (BCAR3). BCAR3 activates the PI3K/Akt pathway and mediates both cellular migration and acquisition of resistance to therapeutic antiestro-gens in breast cancer cells, processes that are associated with malignancy. 11 12 A BCAR3- derived phosphopeptide (pBCAR3126-134) was initially shown by us to be presented by HLA- A*0201 on melanoma cells.6 Here, we show that it also is immunogenic in HLA- A2 transgenic mice and that the resulting CD8 T- cells can directly recognize mela- noma and breast cancer tumor cell lines. Several reports, including our own, have established the safety and immunogenicity of multipeptide vaccines derived from non- mutated tissue- specific proteins. 13–18 In the clinical trial reported here, we now evaluate the safety and immunogenicity of a vaccine consisting of these two oncologically and immunologically relevant phosphory-lated peptides, pBCAR3 126-134 and pIRS21097-1105.MethodsPreclinical studiesMiceMice expressing a chimeric class I MHC molecule composed of the α1 and α2 domains of human HLA- A2 and the α3 domain of murine H- 2D d (AAD) have been described.19 NOD/SCID/interleukin-2 (IL-2)Rγc-/- (NSG) mice were purchased from Jackson Immunore-search Laboratories (Bar Harbor, Massachusetts, USA). Tumor cell lines All tumor cell lines were of human origin.