J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access Figure 5 Clinical outcomes of vaccinated patients. Kaplan- Meier estimates of overall survival (A) and disease- free survival (B) outcomes are shown for all 15 patients as of October 2019. responses to pBCAR3126-134 was comparable to those induced to pIRS21097-1105. This raises the possibility that patient- specific factors as well as differences in inherent immunogenicity may be factors in limited responses to pBCAR3 126-134. An important goal for future investigation is to iden- tify vaccination strategies that enhance the magnitude and persistence of the immune responses. We have found that vaccination with cocktails of peptides can enhance overall immune response rate to 100% even though individual peptides may have lower immune response rates. 14 Thus, one strategy to enhance the immunologic activity of phosphopeptide vaccines is to create cocktails with additional phosphopeptide antigens, and to vacci-nate with peptides modified to enhance MHC binding (eg, pBCAR3(V) 126-134). Another approach is to optimize the vaccine adjuvants. The TLR3 agonist poly- ICLC has enhanced immune responses to other peptide vaccines, especially when combined with IFA. 35 In the present trial, the poly- ICLC was not incorporated in the peptide/IFA emulsion, but was administered by separate injection into the same site as the emulsion. In other experience where poly- ICLC enhanced immunogenicity of peptide vaccines containing IFA, poly- ICLC was incorporated in the emul- sion. 35 36 Thus, other approaches to enhance the immune responses to phosphopeptides include incorporation of TLR agonists directly in the vaccine emulsion or vaccina-tion in combination with other immune adjuvants, such as agonistic CD40 antibody. The pBCAR3 126-134 peptide may also be rendered more immunogenic if the C- ter- minal residue is modified to valine, as per the preliminary data. It is also important to establish whether the tran-sience of responses can be mitigated by the inclusion of checkpoint blockade inhibitors, or other modalities that target immunosuppressive mechanisms. While phosphopeptides represent a new class of tumor neoantigens, the vaccine strategies that employ them are also worthy of further investigation. It would be difficult to rely on endogenous kinase activity to phosphorylate proteins expressed via a genetically based vaccine, and at present we expect that phosphopeptide vaccines will remain based on synthetic peptides. It would be useful to explore whether such vaccines might benefit from the use of longer peptides, which have shown promise in other studies, 37–39 or by coupling them to various lipids or adju- vants.37–39 While we presently have no evidence that the phosphate moiety is removed by plasma phosphatases, we have found that MHC binding stabilizes the phosphate against removal by phosphatases in vitro (Obeng et al, unpublished). Thus, some consideration to the use of non- hydrolyzable phosphophate analogs is reasonable, although these have frequently been shown to be anti-genically distinct. 40 The availability of preclinical models employing HLA transgenic mice is of particular value in testing these approaches on peptides of human origin prior to incorporating them into clinical trials. ConClusIons The findings from this clinical trial highlight the safety of vaccination with phosphopeptides arising from oncogenic phosphoproteins. The pIRS2 1097-1105 and pBCAR3126-134 phosphopeptides are expressed not only by melanomas, but also by several additional kinds of cancer cells. Thus, our results open the door to vaccines using these phosphopeptides for an array of other cancers. Enhancing phosphopeptide immunogenicity with other adjuvant strategies is expected to elevate the promise for this approach, and to increase the prospect for clinical benefit. Phosphopeptides are presented by most MHC- I alleles so far examined, enabling the approach to be exploited to a broad range of patients. In addition, indi-viduals without a clinical diagnosis of cancer often have T- cells that recognize some of these phosphopeptides 30 (Lulu, Cummings and Engelhard, unpublished). An intriguing possibility is that these T- cell responses may arise as a result of successful immune surveillance of early cancers, and it is notable that patients who develop clin-ical cancers typically have diminished T- cell responses to 14 Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262Open access phosphopeptides.30 The presence of T- cell responses to phosphopeptides in otherwise healthy individuals also provides strong evidence that T- cells reactive to those peptides are safe and that induction of such responses in cancer patients will not induce significant autoimmunity to normal tissues. The present findings provide rationale for next generation phosphopeptide vaccines targeting a larger number of antigens presented by a range of MHC- I alleles, and also provide a rationale for adoptive cell ther - apies targeting these peptides. Author affiliations 1Beirne Carter Center for Immunology Research and Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA 2Department of Public Health Sciences, University of Virginia School of Medicine, Charlottesville, Virginia, USA 3Department of Surgery/Division of Surgical Oncology, University of Virginia School of Medicine, Charlottesville, Virginia, USA 4Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA 57Medical Research Council Centre for Immune Regulation and Clinical Immunology Service, University of Birmingham College of Medical and Dental Sciences, Birmingham, UK 6Office of Clinical Research, University Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA 7Medicine/Division of Hematology- Oncology, University of Virginia School of Medicine, Charlottesville, Virginia, USA Acknowledgements We acknowledge assistance of Donna Deacon for vaccine preparation and Carmel Nail and Patrice Neese for assistance in vaccine administration. We acknowledge Erin Jeffrey, Paisley Myers, Dennis Underwood and Andy Hurwitz, all of Agenus, Inc., for sharing information on expression of pBCAR3 126-134 and pIRS21097-1105 on tumor samples. Contributors RCO, KLC and AA performed the preclinical cellular immunological studies. NB and MC procured hepatocellular carcinoma samples and performed peptide extraction. PM, KM, JS and DFH conducted mass spectrometry and analyzed the data. AA, KAC- B, GRP and CLS designed the clinical trial. KH, GW, EG and CLS recruited patients, administered vaccines and assessed adverse events. EA was the primary clinical research coordinator, who also participated in patient enrollment, safety assessments, and data management and reporting. KTS, KLC, AL and NV analyzed patient immune responses. GRP and MS conducted statistical analyses of preclinical and clinical immune response data. VE, RCO, KLC, GRP and CLS summarized immunological and clinical outcomes and participated in writing the manuscript. All authors have reviewed the manuscript. Funding This study was supported by USPHS grants R01 CA134060 and CA190665 (VE), R01 AI033993 (DFH), P30 CA44579 (University of Virginia Cancer Center: Biorepository and Tissue Research Facility, Biostatistics), the Commonwealth Foundation for Cancer Research, and the Ludwig Institute for Cancer Research. RCO was supported by USPHS grant F31 CA119954, AA was a recipient of a Sidney Kimmel Scholar award, and AL was supported by USPHS grant T32 CA009109. Competing interests VE, RCO, KLC, AL, PM, JS and DFH have ownership interest in Agenus, which has licensed phosphopeptide technology and associated patent applications from the University of Virginia Licensing and Ventures Group. PM is now an employee of Agenus. VE is a consultant and the recipient of a Sponsored Research Agreement from Agenus. CLS is the recipient of research grants from GlaxoSmithKline, Merck, and Celldex, and is an inventor for patents for other peptides used in cancer vaccines, which are held by the UVA Licencing and Ventures Group; and is (or has been) a consultant/advisory board member for Immatics. Curevac, and Polynoma. MC is now an employee of AstraZeneca. Patient consent for publication Not required.