Mice 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 Immunoresearch Laboratories (Bar Harbor, Massachusetts, USA).

All tumor cell lines were of human origin. 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% CO₂ in RPMI-1640 containing 10% fetal bovine serum, 15 mM HEPES, 2 mM L-glutamine 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.

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 determined using the AlphaEaseFC software and the densities 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 analysis 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 of ~90 kDa corresponding to BCAR3 (online supplementary figure 1B), and detection of this band was abrogated by treatment with lambda phosphatase (online supplementary 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.

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 concentrated 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 phosphopeptides were calculated based on internal phosphorylated and nonphosphorylated peptide standards present at fixed concentrations within each sample.

pBCAR3_126-134 (IMDR[pT]PEKL), pBCAR3(V)_126-134 (IMDR[pT]PEKV), pS33-βcat_30-39 (YLD[pS]GIHSGA), and their nonphosphorylated counterparts were synthesized 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

Autologous mature dendritic cells were pulsed with phosphopeptides of interest for 2 hours at room temperature 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% CO₂ 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. Normalized percent lysis was calculated as: ((radioactivity values for pulsed targets – background values for unpulsed targets)/values for positive control) ×100.

Bone 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-microglobulin 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), permeabilized and fixed with Cytoperm/Cytofix (BD Bioscience), and stained for intracellular interferon-γ (IFNγ) (eBioscience). Samples were analyzed on a FACSCanto I or II (BD Bioscience) using FlowJo software (Tree Star, Ashland, Oregon, USA).

pBCAR3_126-134-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⁺ splenocytes in media supplemented with IL-2. Lines were established 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 1.4×10⁶ SLM2 human melanoma cells. Three days later, mice were injected intravenous with 6×10⁶ pBCAR3_126-134- or pS33-βcat_30-39-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.

Patients with resected AJCC stage IIA–IV (V.7) melanoma24 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: pregnancy; cytotoxic chemotherapy, IFN, immunomodulatory 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%).

pBCAR3_126-134 (IMDR(pT)PEKL) and pIRS2_1097-1105 (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 AQYIKANSKFIGITEL (P2_830-844, modified by adding alanine to the N-terminus),25 26 was prepared, sterile filtered, vialed and lyophilized under Good Manufacturing Practice conditions 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 guidelines 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 emulsion with an equal volume of an incomplete Freund’s adjuvant (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 subcutaneously 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).

This was an open-label, pilot, proof-of-concept study to assess phosphopeptide vaccine safety and immunogenicity. It was designed with three arms to assess each phosphopeptide individually before assessing the combination: Arm A, pBCAR3_126-134 only; Arm B, pIRS2_1097-1105 only; and Arm C, pBCAR3_126-134 + pIRS2_1097-1105. Arms A and B were restricted to patients with stage IIIB–IV melanoma (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 continuation 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 participants total.

The study was monitored continuously for treatment-related adverse events. Adverse events were described and coded based on NCI CTCAE v4.03. A dose-limiting toxicity (DLT) was defined as any unexpected adverse event that is possibly, probably or definitely related and: (1) ocular ≥grade 1, (2) non-hematologic/non-metabolic ≥Grade 3, or (3) hematologic/metabolic ≥Grade 3. Toxicities with known or likely autoimmune features and affecting vital organs, including colitis, hepatitis, hypophysitis, uveitis and adrenal insufficiency, were recorded and assessed with special interest. These were considered DLTs if ≥grade 2 and probably or definitely related. Grade 2 autoimmune toxicities involving these organs/tissues were not considered DLTs if only possibly related. Vitiligo was not considered a DLT, but was recorded. Hyperthyroidism or hypothyroidism was considered a DLT if ≥grade 3. All vaccinated participants were followed for a minimum of 4 weeks for assessment of DLTs.

The number of vaccinated participants who experienced DLTs guided decisions about safety. In the initial safety phase for arm A or Arm B, one participant was accrued. If that subject did not experience a DLT within 4 weeks, 2 additional participants were accrued. If 0/3 DLTs were observed, then the initial safety criteria for the arm were satisfied; if ≥2/3 participants experienced a DLT, then accrual to that arm would be halted permanently; otherwise three additional participants would be accrued to the arm to assess safety. Accrual to arm C occurred only after the initial safety criteria for arms A and B were satisfied. A maximum of nine participants were accrued in sequential cohorts of 3. Accrual continued as long as no more than 33% of participants experienced a DLT.

Blood (100–140 mL) was drawn on days indicated in figure 1. PBMCs were isolated from 80 mL (except 120 mL week 0) using Ficoll gradient centrifugation and cryopreserved in 10% dimethyl sulfoxide/90% serum. Thawed PBMCs were incubated 2 hours at 37°C with 40 µg/mL each of pBCAR3_126-134, pIRS2_1097-1105 and pS33-βcat_30-39 (control), washed to remove unbound peptide, and resuspended in medium containing 10 ng/mL recombinant human IL-7 (Peprotech, Rocky Hill, New Jersey, USA) for 3 days. New medium containing recombinant human IL-7 plus 10 ng/mL IL-15 (Peprotech) was added on days 3, 7 and 10. Cells were assayed on day 14 for IFNγ production by ELISpot assay using methods and criteria described previously.14 15 Briefly, T2-B7 cells were pulsed with each of the three phosphopeptides for each assay date. Negative controls included no peptide, irrelevant peptide (HIV gag peptide restricted by HLA-A2; Atlantic Peptides, LLC, Lewisburg, Pennsylvania, USA), and positive controls included PMA-ionomycin, PHA. Responses were corrected for the fraction of CD8 T-cells in PBMC as determined by flow cytometry of individual samples at culture initiation. T-cell responses were calculated using the following definitions: Nvax=number T-cells responding to vaccine peptide; Nneg=number T-cells responding to maximum negative control; Rvax=Nvax/Nneg. A patient was considered to have a T-cell response to vaccination (binary yes/no), only if all of the following criteria were met: (1) Nvax exceeded Nneg by at least 100 spots/100 000 CD8 cells, (2) (Nvax – 1 SD) ≥ (Nneg +1 SD) and (3) Rvax after vaccination ≥2 x Rvax prevaccine. Prevaccine Rvax values less than one (eg, control counts exceed number of responding T-cells) were set equal to one to indicate no response and to prevent overinflating adjusted fold-increases due to prevaccine ratios less than one or division by zero. Interassay coefficients of variation (CVs) were calculated for the response of 2 normal donors to a mixture of viral peptides (CEF peptide pool, Proimmune, Sarasota, Florida, USA): for the high responder, mean number of spots per 1 00 000 cells was 309, and CV was 29%.

Since this was a first-in-humans clinical trial with phosphopeptide antigens for cancer, a first goal was to assess safety with a small study. The biological primary endpoint was evidence of immunogenicity. The prespecified level of interest was two or more patients with T-cell responses. Beyond that, CI for the observed proportions would determine how much the data supported immunogenicity. Assuming that the response to one phosphopeptide is not affected by the presence of the other for those in arm C, the immunogenicity of each phosphopeptide was based on the total number of participants vaccinated with the specific phosphopeptide, alone or in combination with the other (ie, Arm A+Arm C for pBCAR3_126-134; Arm B+Arm C for pIRS2_1097-1105). Based on the upper limit of a one-sided 90% CI, if the upper limit of the observed bound was >35% to any single phosphopeptide, we would conclude that the phosphopeptides was immunogenic and worthy of further study. To define the CD8 T-cell response rate with higher precision would require a much larger sample size, which was not indicated in this first-in-humans trial.

All participants were clinically free of disease at enrollment. Disease-free survival (DFS) is defined as the time from on-study until the earliest documentation of a new metastasis or death from any cause, or censored at the date of last follow-up for those without a documented event. Overall survival is defined as the time from on-study to death from any cause, or censored at the date of last follow-up for those still alive.