Melanoma line SLM2 was obtained from the American Type Culture Collection (Bethesda, Maryland, USA). Melanoma line DM331 was established from a patient whose tumor was resected at Duke University. VMM12 and VMM18 lines were established from metastatic melanoma resected from patients at the University of Virginia. VMM18 also has been deposited with the ATCC. Melanoma line 1363Mel was obtained from Suzanne Topalian (Johns Hopkins University). Breast cancer lines BT-549, MCF7, MDAMB231, MDAMB468 and T47D were obtained from Sarah Parsons (University of Virginia). Melanoma lines were grown at 37°C with 5% CO 2 in RPMI-1640 containing 10% fetal bovine serum, 15 mM HEPES, 2 mM L- gluta- mine and 50 µg/mL gentamycin. Breast cancer lines were grown at 37°C with 8% CO2 in DMEM +10% FBS, 1 mM sodium pyruvate, 15 mM HEPES, 2 mM L- glutamine and 50 µg/mL gentamycin. Western blot Cell lysates were generated as described.7 Each sample was separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and proteins were transferred onto polyvinylidene fluoride membranes (Immobilon- FL, 0.45 µm, Millipore, Bedford, Massachusetts, USA). Blots were probed with specific primary antibodies overnight at 4°C and then with horseradish peroxidase- conjugated secondary antibodies. Pixel densities were deter - mined using the AlphaEaseFC software and the densi-ties of BCAR3 and of BCAR3 phosphorylated at T130 (phosphoT130- BCAR3) were normalized to those from glyceraldehyde 3- phosphate dehydrogenase (GAPDH). Antihuman BCAR3 was from Bethyl labs (Montgomery, Texas, USA). Anti- GAPDH was from Santa Cruz (Santa Cruz, California, USA). A polyclonal antibody recognizing T130 phosphorylated BCAR3 protein was generated by AbboMax (San Jose, California, USA) by immunization of a rabbit with a 13mer phosphopeptide (CRHIMDR[pT]PEKLK), and sequential affinity purification on columns derivatized with the 13mer and its non- phosphorylated counterpart. Specificity was established by Western anal-ysis with or without competing 1.5 µg of phosphorylated or unphosphorylated 13mer BCAR3 peptides per 5 µg of antibody (online supplementary figure 1A). Protein lysates from cells in which expression of BCAR3 had been knocked down by RNA interference and used in a Western blot verified that the antibody recognized a band 3 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access of ~90 kDa corresponding to BCAR3 (online supplemen- tary figure 1B), and detection of this band was abrogated by treatment with lambda phosphatase (online supple-mentary figure 1C). However, this antibody also detected an unidentified phosphoprotein with a molecular weight larger than 102 kDa (online supplementary figure 1B), precluding its use in immunohistochemical analyzes. Phosphopeptide detection by mass spectrometry Tumor and normal adjacent tissues from HLA- A*0201 + hepatocellular carcinoma patients were processed and phospholigandome analysis performed as described. 20 Peptides were isolated from tissue extracts using the pan- human MHC class I antibody W6/32, desalted and concen-trated via STop And Go Extraction tips, and resuspended in formic acid. Phosphopeptides were enriched using iron- III- iminodiacetic acid immobilized metal affinity chromatography and analyzed using high- performance liquid chromatography/electrospray ionization tandem mass spectrometry. Data analysis was performed using Xcalibur software (Thermo Electron Corporation) and raw data files were searched using Byonic (Protein Metrics) against the Swissprot human protein database. Phosphopeptide sequences were confirmed by accurate mass measurement and manual interpretation of MS2 spectra. Relative abundances of detected phosphopep-tides were calculated based on internal phosphorylated and nonphosphorylated peptide standards present at fixed concentrations within each sample. Peptides for preclinical studies and HLA-A*0201 binding assays pBCAR3 126-134 (IMDR[pT]PEKL), pBCAR3(V)126-134 (IMDR[pT]PEKV), pS33-βcat30-39 (YLD[pS]GIHSGA), and their nonphosphorylated counterparts were synthe-sized by GenScript (Piscataway, New Jersey, USA). Binding of peptides to purified HLA- A*0201 was determined in an equilibrium assay by competition with a radiolabeled indicator peptide as described. 21 22 In vitro culture of human T-cell and cytotoxicity assayAutologous mature dendritic cells were pulsed with phosphopeptides of interest for 2 hours at room tempera- ture and then irradiated at 3500 rad. T- cells were enriched from donor peripheral blood mononuclear cells (PBMCs) using a human Pan T- cell isolation kit II (Miltenyi, Bergisch Gladbach, Germany) and then incubated with phosphopeptide- pulsed dendritic cells in microwells at 37°C with 5% CO 2 for 2 weeks in medium with 20 ng/mL IL-7, 10 ng/mL IL-15, 1 ng/mL IL-10 and 20 U/mL IL-2. The cultures were restimulated with phosphopeptide- pulsed irradiated autologous PBMCs every 7 days. Two weeks after in vitro culture, the T- cells were incubated with phosphopeptide- pulsed 51Cr- labeled targets at a ratio of 60:1 for 4 hours. Radioactivity was measured on a Wallac Wizard 1470 Automatic Gamma Counter. Normal-ized percent lysis was calculated as: ((radioactivity values for pulsed targets – background values for unpulsed targets)/values for positive control) ×100.Immune responses of AAD mice to phosphopeptidesBone marrow derived dendritic cells (BMDC) from AAD mice were prepared as described. 5 23 BMDC were pulsed with 10 µg/mL phosphopeptide and 10 µg/mL β2- micro- globulin for 2–3 hours, irradiated (3000 cGy), washed and injected intravenous (1.5–2.5×105) into AAD mice. Mice received intravenous booster immunizations 6 days later with phosphopeptide, CpG (type C, ODN 2395, InvivoGen (San Diego, California, USA)), and antimouse CD40 (FGK45, gift of Stephen Schoenberger, La Jolla Institute of Allergy and Immunology). CD8α + T- cells were enriched from spleens and lymph nodes 5 d later using magnetic beads (Miltenyi) and incubated for 5 hours with peptide- pulsed stimulators (C1R- AAD) in medium containing 0.5 µg/mL anti- CD107a, 5 µg/mL Brefelden A (Sigma- Aldrich) and 1 µM monensin (eBioscience). Cells were stained for CD8α (eBioscience), permeabi-lized and fixed with Cytoperm/Cytofix (BD Bioscience), and stained for intracellular interferon-γ (IFNγ) (eBiosci-ence). Samples were analyzed on a FACSCanto I or II (BD Bioscience) using FlowJo software (Tree Star, Ashland, Oregon, USA). Tumor control using pBCAR3 126-134-specific T-cell lines pBCAR3126-134- specific T- cell lines were established using enriched CD8 cells harvested from mice immunized as described above and repetitively recultured with pBCAR3(V) 126-134 peptide- pulsed irradiated AAD+ spleno- cytes in media supplemented with IL-2. Lines were estab-lished within 3–4 weeks, as documented by 70%–85% of the cells responding to peptide pulsed stimulators in the effector function assays described above. For tumor control experiments, male NSG mice aged 7–8 weeks were inoculated s.c. with 1.4×10 6 SLM2 human melanoma cells. Three days later, mice were injected intravenous with 6×10 6 pBCAR3126-134- or pS33-βcat30-39- specific T- cells. Four days later, the mice received an additional 0.3×106 T- cells. 1500U IL-2 was administered i.p. every other day for 10 days beginning on the day of T- cell transfer. Tumor size was measured with a digital caliper and calculated as L x W. Tumor growth was analyzed using repeated measure models after transforming to the log scale assuming an AR1 covariance structure. Specific comparisons between pBCAR3- or pS33-βcat- treated and control mice were made with F- tests based on contrasts and estimates were transformed back for plotting. Clinical trial PatientsPatients with resected AJCC stage IIA–IV (V.7) mela-noma 24 were eligible, as well as patients with disease who failed other approved therapies. However, all who enrolled were clinically free of disease. Inclusion criteria included: HLA- A2 expression, ages 18 years and above, Eastern Cooperative Oncology Group performance status 0–1, adequate liver and renal function, and ability to give informed consent. Exclusion criteria included: preg-nancy; cytotoxic chemotherapy, IFN, immunomodulatory 4 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Figure 1 Design for clinical trial to evaluate safety and immunogenicity of phosphopeptide- based vaccine in melanoma patients. Participants were eligible for all arms with stage IIIB- IV melanoma, with measurable disease, or after surgical resection. Eligibility for arm C was extended to stage II–IIIA patients, after surgical resection. The study was also opened to participants with other solid tumors, but only participants with melanoma enrolled. IFA, incomplete Freund’s adjuvant; PBMC, peripheral blood mononuclear cell; pIRS2, phosphopeptide from insulin receptor substrate 2; tet, tetanus helper peptide.therapies, or radiation within the preceding month; known or suspected allergies to vaccine components; multiple brain metastases; use of steroids; Class III–IV heart disease; systemic autoimmune disease with visceral involvement or uncontrolled diabetes (hemoglobin A1C ≥7%). Vaccine preparation and administration pBCAR3 126-134 (IMDR(pT)PEKL) and pIRS21097-1105 (RVA(pS)PTSGV) were synthesized and purified (>95%) by PolyPeptide Laboratories (San Diego, California, USA), solubilized in aqueous solution, sterile- filtered, vialed in sterile single- use vials, lyophilized and stored at −80°C. The tetanus helper peptide AQYIKANSKFIG-ITEL (P2 830-844, modified by adding alanine to the N- ter- minus),25 26 was prepared, sterile filtered, vialed and lyophilized under Good Manufacturing Practice condi-tions as described. 17 25 Vials were submitted for quality- assurance studies including sterility, identity, purity, concentration, general safety, pyrogenicity and stability in accordance with Code of Federal Regulations guide- lines and BB- IND #15 134. Polyinosinic- polycytidylic acid stabilized with poly- L- lysine and carboxymethylcellulose (poly- ICLC, Hiltonol; Oncovir, Washington, D.C., USA) was provided by the Ludwig Institute (New York, New York, USA) as a clinical grade reagent. Each vaccination consisted of 100 µg of either or both phosphopeptides and 200 µg of the tetanus peptide in a water- in- oil emul- sion with an equal volume of an incomplete Freund’s adju- vant (IFA) (Montanide ISA-51 VG adjuvant, Seppic, Paris, France). Vaccines were administered subcutaneously (0.5 mL) and intradermally (0.5 mL) at one skin puncture site. One mg of poly- ICLC was administered immediately thereafter by a separate 0.5 mL injection (0.25 mL subcu- taneously and 0.25 mL intradermally) into the precise sites where the peptide emulsion was given. Vaccine sites were rotated to different skin regions on each vaccination date, using a minimum of two separate extremities. Vaccines were administered in two treatment cycles (figure 1). 5 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Clinical trial design This was an open- label, pilot, proof- of- concept study to assess phosphopeptide vaccine safety and immuno-genicity. It was designed with three arms to assess each phosphopeptide individually before assessing the combi-nation: Arm A, pBCAR3 126-134 only; Arm B, pIRS21097-1105 only; and Arm C, pBCAR3126-134 + pIRS21097-1105. Arms A and B were restricted to patients with stage IIIB–IV mela- noma (at original presentation or at recurrence, with or without measurable disease). Once safety was evident for each peptide in these higher- risk patients, Arm C was open for stage IIIB–IV patients and also for patients with lower risk (stage IIA–IIIA) melanoma rendered clinically free of disease by surgery, other therapy or spontaneous remission. Accrual to arm C occurred only after continu-ation was deemed appropriate in the initial safety phase of arms A and B. Maximum target accrual was set at 21 eligible participants who received a vaccination. Provided safety rules were met, immunogenicity would be assessed for each phosphopeptide in up to 12 evaluable partici- pants total. Toxicity assessment The study was monitored continuously for treatment- related adverse events.