Analysis of exploratory endpoint All participants were clinically free of disease at enroll-ment. 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. resultsphosphot 130-bCAr3 is expressed in cancer cell lines but not normal melanocytesWe previously found that pIRS2 1097-1105 is naturally presented by HLA- A*0201 on cancer cells with high levels of protein, and that it serves as a target for immune mediated tumor control. 7 Using mass spectrometry, we identified another HLA- A*0201- associated peptide on melanoma cells comprising residues 126–134 of the BCAR3 protein, which was phosphorylated at T130 (pBCAR3 126-134).6 We evaluated additional melanoma and breast cancer cell lines for expression of the phos-phorylated source protein using a commercially available polyclonal BCAR3 antibody and a validated phosphoT 130- BCAR3 antibody (described in Methods and online supplementary figure 1). BCAR3 protein expression was quite heterogenous in both melanoma and breast cancer cells (figure 2A,B). Nonetheless, phosphoT 130- BCAR3 was detected in most of the melanoma and breast cancer cell lines (figure 2A–C). Because of a strong cross- reactive band evident in Western blots, the phosphoT 130- BCAR3 antibody could not be used for immunohistochemical analysis of human tumor specimens. A survey by mass spectrometry of presented peptides extracted from several HLA- A*0201 + primary tumor samples failed to identify pBCAR3126-134 (Dennis Underwood, Agenus, personal communication). However, the pIRS21097-1105 peptide was directly identified by mass spectrometry in naturally presented peptides extracted from 4/5 hepatocellular carcinoma (HCC) samples at 1–5 (two tumors) and 6–20 (two tumors) copies per cell (table 1). It was found at less than one copy per cell on 1/4 normal adjacent tissue samples. In addition, a phosphoproteome analysis identi-fied phosphoT 130- BCAR3 in 3/105 primary breast cancer samples, while 22/105 of these same samples expressed phosphoS 1100- IRS2.27 28 PhosphoS1100- IRS2 has also been identified by phosphoproteome analysis in an unspecified fraction of high- grade serous ovarian 28 and non- small- cell lung carcinomas,29 2/4 chronic lymphocytic leukemia isolates and 1/1 primary mantle cell lymphoma.30 These results suggest that the expression of pBCAR3126- 134 on HLA- A*0201+ tumors may be limited, although a more comprehensive survey of additional tumor types is warranted, while demonstrating that pIRS21097-1105 is expressed broadly and frequently on multiple tumor types. pbCAr3 126-134 is immunogenic and enables immune control of tumor outgrowth in a preclinical model The unphosphorylated BCAR3126-134 peptide is predicted to bind weakly to HLA- A*0201 by the artificial neural network (ANN) algorithm31 and the stabilized matrix method32 (IEDB analysis resource, http://www. immuneepitope. org), and this was confirmed by direct binding assays (figure 2D). pBCAR3126-134 bound to HLA- A*0201 somewhat better than the unphosphorylated peptide, in keeping with the role of phosphate as a surface anchor residue, 22 but still bound with a significantly lower affinity than the IP30 peptide that we routinely use as a high affinity positive control 21 (figure 2D). A pBCAR3 peptide modified by substitution of V for L at residue 134, the P9 anchor position, (pBCAR3(V) 126-134) was predicted to have higher affinity for HLA- A*0201 by ANN anal- ysis, but showed no difference in direct binding assays. Both peptides induced antigen specific human T- cells in vitro (figure 2E), while only pBCAR3(V) 126-134 was immu- nogenic in vivo in HLA- A2 transgenic mice (figure 2F). Nonetheless, all T- cells generated by immunization with pBCAR3(V)126-134 showed complete cross- reactivity with pBCAR3126-134 as assessed by quantifying the fraction of IFNγ+ T- cells after a 5- hour incubation with stimulators pulsed with either the modified or the natural phospho-peptides, and this was maintained over graded doses of the phosphopeptides (figure 2G). We used two T- cell lines generated from pBCAR3(V) 126-134– immunized mice to assess the expression of pBCAR3126-134 on human cancer cell lines. Melanoma and breast cancer cell lines that express the phosphoT 130- BCAR3 protein by Western blot were recognized well by both T- cell lines, and the extent of T- cell recognition varied among the cells, suggesting that the epitope is displayed at varied levels on the tumor cells (figure 2H). However, the level of T- cell recognition did not correlate with phosphoT 130- BCAR3 protein levels by Western blot, suggesting variability in the efficiency of Ag processing and presentation, MHC levels or expression of molecules supporting T- cell recogni- tion might vary among these cells. We also evaluated the ability of pBCAR3 126-134- specific T- cell line 1, and murine T- cells specific for another phosphopeptide derived from β-catenin, pS33-βcat30-39, to control growth of SLM2 tumors in a xenograft model. Both T- cell populations reduced tumor growth in vivo (figure 3). These results supported the evaluation of both pBCAR3 126-134 and pS33- βcat30-39 as candidates for cancer immunotherapy. Food and Drug Administration (FDA) concerns about possible expression of pS33-βcat 30-39 in normal tissues precluded inclusion in a human clinical trial. However, the FDA 7 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Figure 2 Expression of phosphoT130- BCAR3 protein and recognition of pBCAR3126-134 phosphopeptide in melanoma and breast cancer cells. (A–C) Expression of phosphoT130- BCAR3 and BCAR3 in melanoma (A) and breast cancer (B) cells was evaluated by Western blot using the validated phosphoT130- BCAR3 antibody and a commercially available BCAR3 antibody. GAPDH expression served as a loading control, and DM331 melanoma cells were included in both panels for comparison. Data are representative of two (melanoma) and three (breast) independent experiments. (C) Relative expression of phosphoT130- BCAR3 in melanoma and breast cancer cells normalized to the respective GAPDH levels. (D) pBCAR3 126-134, pBCAR3(V)126-134, the non- phosphorylated forms and a control peptide (IP30) were tested for their ability to compete with a radiolabeled peptide derived from hepatitis B core protein for binding to purified HLA- A*0201. The dose required to reduce binding of the radiolabeled peptide by 50% (dotted line) corresponds to the IC50 value. Data are representative of three independent experiments. (E) Human T- cells from one donor were cultured in 10 microwells containing irradiated stimulators pulsed with pBCAR3126-134 or pBCAR3(V)126-134 and evaluated for pBCAR3- or pBCAR3(V)- specific responses in a 51Cr- release assay after 2 weeks using targets pulsed with either the phosphorylated or unphosphorylated forms of the peptide. Data points represent individual microcultures from one donor. (F) AAD transgenic mice were immunized with BMDC pulsed with pBCAR3 126-134 or pBCAR3(V)126-134. Six days later, mice received a boost of phosphopeptide, CpG, and anti- CD40. T- cells were harvested from the spleens and lymph nodes and evaluated for cytokine production after 5 hour incubation with pulsed stimulators. Dot plots are gated on CD8 cells. (G) Mice were immunized with pBCAR3(V)126-134, and the percentages of CD8 T- cells isolated from them that recognized the immunogen or the parental pBCAR3126-134 were determined by intracellular staining for IFNγ. Data are representative of two experiments. (H) Melanoma and breast cancer cells expressing endogenous phosphoT130- BCAR3 were incubated separately with two independently derived murine pBCAR3126-134 specific T- cell lines and the fraction that produced IFNγ quantified by flow cytometry. Data are normalized to % of maximal response that was quantified using stimulators pulsed with 10 µg/mL of pBCAR3126-134 (range of maximal response was 70%–85%). Data are representative of three independent experiments (F–H).