8 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Table 1 Detection of pIRS21097-1105 in human hepatocellular carcinoma tissues by mass spectrometry of MHC- associated peptide extracts Tumor tissue sample Normal adjacent tissue sample HCC1 HCC2 HCC3 HCC4 HCC5 NAT2 NAT3 NAT4 NAT5 ++* +++ ++ +++ ND ND ND + ND *Copies per cell: ND: not detected, +: <1, ++: 1–5, +++: 6–20. pIRS2, phosphopeptide from insulin receptor substrate 2. Figure 3 Phosphopeptide specific T- cells mediate tumor control. Control of subcutaneous SLM2 tumors growing in NOD/ SCID- IL- 2Rγc-/- mice by pBCAR3126-134– or pS33βcat30-39- specific murine T- cells was determined as outlined in Methods. T- cells were injected as indicated by arrows. Estimated mean for each group is shown in red. Data are representative of two independent experiments (n=5 per group per experiment).approved evaluation of both pIRS21097-1105 and pBCAR3126- 134 in humans. Clinical presentation of patients vaccinated with phosphopeptidesHaving identified pIRS2 1097-1105 and pBCAR3126-134 phospho- peptides as attractive targets for cancer immunotherapy, we designed a clinical trial to test their safety and immu-nogenicity in cancer patients (figure 1). Vaccines were formulated as phosphopeptides together with a tetanus helper peptide emulsified in IFA (Montanide ISA-51) and coadministered with poly- ICLC. To avoid risk of off- target immune responses, the native pBCAR3 126-134 phosphopep- tide sequence was used. While awaiting FDA approval to accrue to pIRS2 1097-1105 peptide (Arm B), the first partici- pant was accrued to pBCAR3126-134 (Arm A), after which accrual was randomly assigned 1:1 to arm A and Arm B until cohort conditions were satisfied. Sequential accrual to Arm C occurred after the initial safety criteria for Arms A and B were satisfied and no additional safety bound was crossed. Fifteen eligible patients were enrolled, each of whom had undergone surgical resection of stage II, III or IV melanoma, with three patients each in Arms A and B, and nine in Arm C. Patient demographics and clinical features are summarized in table 2. Clinical toxicities Treatment- related adverse events are detailed for all 15 patients in table 3. There were no treatment- related grade 3–4 toxicities, no deaths on study and no DLTs. All patients had grade 1 and 2 adverse events, usually limited to 24–48 hours after each vaccine. All patients developed grade 2 vaccine injection site reactions, which were more persistent and included induration in 13 patients and skin ulceration in 2 patients. Other common treatment- related grade 1 and 2 toxicities were fatigue, chills, head-ache, myalgias, arthralgias, autoimmune disorders, fever, nausea and diarrhea. Meaningful differences in adverse events among study arms were not evident. Autoim-mune toxicities were observed in three patients: two in arm A and one in arm B, and all were grade 1. These were all treatment- associated asymptomatic elevations of serum antinuclear antibody (ANA), and were identified in patients 1A (day 183), 3B (day 85) and 4A (day 85). Three patients had pretreatment elevations of rheuma-toid factor (10C) or ANA (7C, 15C). No patients devel-oped treatment- related vitiligo. Immune responses to phosphopeptides in vaccinated patientsAll 15 eligible patients were evaluable for immune responses in PBMC. Initial ex vivo ELISpot assays to detect IFNγ production in response to peptide antigen, performed on four patients representing arms A (2A), B (3B) and C (7C, 8C), were negative. Thus, subsequent ELISpot assays were performed after one in vitro sensiti-zation for all patients. Two patients had evidence of pre- existing immune responses to phosphopeptides based on a greater than twofold increase, and increase of at least 100 spots/100 000 CD8 cells, compared with the maximum negative control at baseline. Specifically, the ratios and spot counts per 100 000 CD8 cells at baseline were as follows: patient 7, for pIRS2 (2.8x and 373 spots differ - ence); patient 8, for pBCAR3 (2.8x and 137 spots differ - ence) and for pbeta- catenin (which was not in the vaccine, 2.9x and 139 spots difference). Responses to vaccine required increases compared with any pre- existing 9 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Table 2 Patient demographics by treatment group PeptideArm A Arm B Arm C Total pBCAR3 pIRS2 Both N 3 3 9 15 Gender (female) 1 1 4 6 (40%) Caucasian, non- Hispanic 2 3 9 11 (92%) Hispanic 1 0 0 1 (7%) Age—median (range) 64 (40–67) 63 (50–85) 52 (30–75) 56 (30–85) Performance status 0 2 3 8 13 (87%) Stage at registration II–IIIA 0 0 4 4 (27%) IIIB, IIIC 1 1 5 7 (47%) IV 2 2 0 4 (27%) Distant metastatic sites None 1 1 9 11 Nodes 1 0 0 1 Soft tissue 0 2 0 2 Lung 0 1 0 1 Brain 1 0 0 1 Other visceral 0 1 0 1 Clinically NED at study entry 3 3 9 15 (100%) NED, no evidence of disease; pBCAR3, phosphopeptide from breast cancer antiestrogen resistance 3; pIRS2, phosphopeptide from insulin receptor substrate 2. responses (table 4, figure 4 and online supplementary figure 2). T- cell responses to vaccine peptides were iden- tified against pBCAR3126-134 in 2/12 patients (17%, Arms A+C) and against pIRS21097-1105 in 5/12 patients (42%, Arms B+C) (figure 4, table 4). Overall 6 of 15 patients (40%) had a T- cell response to either or both of these peptides. The majority of immune responses were detected at a single time point, typically at weeks 3, 5 or 8. One patient (#11) also showed an additional immune response at week 26. No responses against the negative control phosphopeptide, pS33-βcat 30-39, were observed at any time. In addition, responses in arm A and B patients were specific for the vaccinating peptide only. Also, there were indications of subthreshold responses in several patients at other time points that were specific for the same peptide (4A, 8C, 15C). Associations of immune response with autoimmune toxicities were not evident (table 4). These results give confidence that the signif-icant responses observed are a consequence of vaccina-tion, despite their transience. The study design set a goal for immunogenicity based on an upper limit of a 90% CI for the observed immune response rate of 35%. The values calculated for pBCAR3 126-134 and pIRS21097-1105 were 44% and 68%, respectively, for each peptide (table 4). These both exceeded the protocol specified upper limit immunogenicity criteria target of >35% supporting further study and development. The prespecified target for immune response rate coincided with 2 or more responses in 12 evaluable patients for each peptide, with greater interest in further development with higher immune response rates.Clinical outcomesOnly three patients have died from melanoma with a median follow- up of 4.9 years for those still alive. Esti- mated survival and disease- free survival curves are displayed in figure 5A. Four- year survival is estimated at 80%. New melanoma metastases were identified in 10 patients, with a median disease- free survival of just over 1.0 year and 4 years disease- free survival of 33% (95% CI 15% to 53%) (figure 5B). The early phase study was not powered to assess association of immune response with clinical outcome with reliable inference, as is reflected by the width of the confidence bounds.