Participant specific immune responses and outcomes are noted in table 4 with no clear associations noted in this small study. dIsCussIon Phosphopeptides function as therapeutic targets for T- cell antitumor immunity, and those that arise from cancer- associated protein phosphorylation represent an entirely new category of cancer neoantigens. We have identified hundreds of phosphopeptides from a wide range of human cancers that are collectively presented by numerous common HLA alleles. An important feature of phosphopeptides is that the phosphate can enhance binding of peptides that would otherwise bind poorly to an MHC- I molecule. 22 In addition, the phosphate creates an antigenic form that is quite distinct from that of the unphosphorylated peptide, 5–7 based on the presence of the phosphate in the T- cell receptor contact zone and the induction of a distinct MHC- bound peptide 10 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Table 3 Treatment- related adverse events Arm A pBCAR3 n=3Arm B pIRS2n=3Arm C Bothn=9Totaln=15 Category Adverse event G1 G2 G3 G1 G2 G3 G1 G2 G3 G1 G2 G3 Gastrointestinal Constipation 1 1 Diarrhea 1 2 3 Mucositis oral 1 1 Nausea 2 1 3 General andadministration siteChills 1 1 1 3 1 5 2 Edema limbs 1 1 Fatigue 2 1 2 5 1 9 2 Fever 1 1 1 3 Influenza like symptoms 1 1 1 1 Injection site reaction 3 3 9 15 Pain 2 2 Immune system Autoimmune disorder 2 1 3 Investigations Lymphocyte count decreased 1 1 Metabolism/nutrition Anorexia 1 1 Musculoskeletal/connective tissueArthralgia 1 3 4 Myalgia 2 4 6 Nervous system Dizziness 1 2 3 Headache 1 1 4 5 1 Psychiatric Agitation 1 1 Respiratory/thoracic/mediastinalCough 1 1 Sore throat 1 1 Skin/subcutaneous tissue Dry skin 1 1 Skin induration 3 1 4 5 5 8 Skin ulceration 1 1 1 1 Vascular Flushing 1 1 Hot flashes 1 1 Participant highest grade* Any 3 3 9 15 *There were no grade 3–5 treatment- related adverse events. pBCAR3, phosphopeptide from breast cancer antiestrogen resistance 3; pIRS2, phosphopeptide from insulin receptor substrate 2. conformation.22 33 As a consequence, there is negligible cross- reactivity by phosphopeptide- specific T- cells on the unphosphorylated homolog. A first challenge in selecting phosphopeptides for cancer vaccines is to define those that are uniquely presented on the surface of cancer cells, but not on normal cells. We have previously identified pIRS2 1097-1105 as a promising target based on its breadth of expres-sion, 7 and the identification of IRS2 as an oncogene that is overexpressed in multiple human cancers.8–10 The data presented here establish its prevalence in hepatocellular carcinoma. We have also provided new data on another promising phosphopeptide target, pBCAR3 126-134. BCAR3 activates the PI3K/Akt pathway and mediates both cellular migration and acquisition of resistance to therapeutic antiestrogens in breast cancer cells, processes that are associated with malignancy. 11 12 Here, we showed that BCAR3 phosphorylated at T130 is expressed in a number of malignanT- cell lines from melanoma as well as breast, that the pBCAR3126- 134 peptide is immunogenic in humans and mice, and that pBCAR3126-134 specific T- cells were able to reduce the outgrowth of of human tumor xenografts. While pBCAR3 126-134/BCAR3 phosphorylated at T130 was expressed in a high percentage of tumor cell lines, it was found infrequently among tumors surveyed to date, suggesting limited therapeutic potential. However, it but may be more prevalent on other kinds of tumors that remain to be evaluated. Based on the fact that both peptides are also presented by the widely expressed HLA- A2 molecule, we prioritized them for evaluation as cancer immunotherapeutic candidates. 11 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Table 4 IFN-γ T- cell responses, autoimmune toxicities and clinical outcomes by patient and in aggregate Patient Study armVaccine with: T- cell response detected to:Baseline* ANA/RF+On- treatmentANA+Melanoma clinical outcome October 2019 pBCAR3 pIRS2 pBCAR3 pIRS2 Either Recurrence Died 1 A + + + + 2 A + 3 B + + + 4 A + + + + + 5 B + + + + + 6 B + + 7 C + + + 8 C + + + + + + 9 C + + + 10 C + + + + 11 C + + + + + 12 C + + 13 C + + + + + + 14 C + + 15 C + + + + + Total 12 12 2 5 6 3 3 10 3 # vaccinated with that peptide 12 12 15 % T- cell response to peptide 17% 42% 40% 90% CI T- cell responses 3% to 44% 18% to 68% 19% to 64% ANA, antinuclear antibody; IFN-γ, interferon-γ; pBCAR3, phosphopeptide from breast cancer antiestrogen resistance 3; pIRS2, phosphopeptide from insulin receptor substrate 2; RF , rheumatoid factor . 12 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Figure 4 Vaccine- associated increases in immune reactivity to phosphopeptides. Immune responses to phosphopeptides were determined by ELISpot assay of 14 days in vitro stimulated peripheral blood mononuclear cells as outlined in the Methods section. Values are shown for the six patients with immune responses to either or both peptides. Time points with data that meet criteria for an immune response to pBCAR3 or pIRS2 are denoted by asterisks. Peptide vaccines have generally been found to be very safe,29 but the novelty of phosphopeptides, and the lack of reagents to test for their expression across all normal human tissues, makes it important to assess their safety in humans. Thus, we have performed a first- in- humans evaluation of a vaccine preparation comprised of these two phosphopeptides. Our findings demonstrate safety of vaccination with pIRS2 1097-1105 and pBCAR3126-134 phospho- peptides administered with IFA plus polyICLC. Adverse events were mild and comparable to those observed with other vaccines containing IFA. T- cell responses to both peptides met criteria for immunogenicity worthy of further investigation. Limitations of this pBCAR3/pIRS2 vaccine were that the immune responses were not detected ex vivo and they were usually transient, most being detected only at a single time point. Cancer vaccines with other antigens have also induced transient immune responses, 34 but a goal of vaccines is to induce more durable responses. We designed the trial such that immune response rates with the upper bound of the 90% CI exceeded 35%. The fraction of patients responding to pIRS2 1097-1105 was 42% (90% CI 18% to 68%), but the fraction responding to pBCAR3126-134 was lower at 17% (90% CI 3% to 44%). In two earlier melanoma vaccine trials testing the immunogenicity of 12 peptides that had been previously identified as valid T- cell targets, the range of immune response rates based on ex vivo ELISpot assays for each peptide ranged from 0% to 78% and 7% to 50%, respectively, with the median immune response rates across the 12 peptides of 21% and 13%, respec-tively. 15 17 In two trials with data based on ELIspot assays after one in vitro stimulation, those rates ranged from 0% to 75% (median 62%) and 0% to 90% (median 68%), respectively. 14 15 In other work, we have established that both pIRS21097-1105 and pBCAR3126-134 can be targets of pre- existing immune memory in normal individuals, but the frequency of responders to pIRS2 1097-1105 is higher (Lulu, Cummings and Engelhard, unpublished). That prior finding is supported by evidence of pre- existing immunity to those same peptides in one patient each, as well as pre- existing immunity to a third peptide (not included in the vaccine), from phospho- beta- catenin. While the natural exposure leading to pre- existing immune memory to phosphopeptides is not known, these results could indi-cate that pIRS2 1097-1105 is inherently more immunogenic. On the other hand, the magnitude of vaccine- induced 13 Engelhard VH, et al.