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MHC-Restricted Phosphopeptide Antigens Open access Original research MHC- restricted phosphopeptide J Immunother Cancer: first published as 10.1136/jitc-2019-000262 on 7 May 2020. Downloaded from antigens: preclinical validation and first- in- humans clinical trial in participants with high- risk melanoma 1 1 1 Victor H Engelhard , Rebecca C Obeng , Kara L Cummings, Gina R Petroni,2 Angela L Ambakhutwala,1 Kimberly A Chianese- Bullock,3 Kelly T Smith,3 Amanda Lulu,1 Nikole Varhegyi,2 Mark E Smolkin,2 Paisley Myers,4 Keira E Mahoney,4 Jeffrey Shabanowitz,4 Nico Buettner,5 Emily H Hall,6 Kathleen Haden,3 Mark Cobbold,5 Donald F Hunt,4 Geoffrey Weiss,7 Elizabeth Gaughan,7 Craig L Slingluff, Jr3 To cite: Engelhard VH, ABSTRACT pBCAR3126-134 peptide in 2/12 patients (17%, 90% CI 3% Obeng RC, Cummings KL, et al. Background Phosphorylated peptides presented by to 44%). MHC- restricted phosphopeptide MHC molecules represent a new class of neoantigens Conclusion This study supports the safety and antigens: preclinical validation expressed on cancer cells and recognized by CD8 T- immunogenicity of vaccines containing the cancer- and first- in- humans clinical associated phosphopeptides pBCAR3 and pIRS2 trial in participants with high- cells. These peptides are promising targets for cancer 126-134 1097- , and the data support continued development of risk melanoma. Journal for immunotherapy. Previous work identified an HLA- A*0201- 1105 ImmunoTherapy of Cancer restricted phosphopeptide from insulin receptor substrate immune therapy targeting phosphopeptides. Future studies 2020;8:e000262. doi:10.1136/ 2 (pIRS2) as one such target. The purpose of this study will define ways to further enhance the magnitude and jitc-2019-000262 was to characterize a second phosphopeptide, from breast durability of phosphopeptide- specific immune responses. cancer antiestrogen resistance 3 (BCAR3), and to evaluate Trial registration number NCT01846143 ► Additional material is safety and immunogenicity of a novel immunotherapic published online only. To view vaccine comprising either or both of these phosphorylated please visit the journal online peptides. BACKGROUND (http:// dx. doi. org/ 10. 1136/ jitc- Methods Phosphorylated BCAR3 protein was evaluated 2019- 000262). The development of therapeutic cancer in melanoma and breast cancer cell lines by Western vaccines based on peptides presented by http://jitc.bmj.com/ VHE, RCO, KLC, GRP, ALA and blot, and recognition by T- cells specific for HLA- A*0201- MHC- I molecules to CD8 T- cells has been restricted phosphorylated BCAR3 peptide (pBCAR3 ) KAC- B contributed equally. 126-134 pursued for many years. Studies of these was determined by 51Cr release assay and intracellular types of vaccines have recently been revital- Accepted 07 April 2020 cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 ized by the successes of checkpoint blockade and pBCAR3 peptides. For the clinical trial, participants and adoptive T-cell immunotherapies and with resected stage IIA–IV melanoma were vaccinated by interest in patient- specific mutated anti- on October 1, 2021 by guest. Protected copyright. 6 times over 12 weeks with one or both peptides in gens as vaccine targets. Peptide antigens incomplete Freund’s adjuvant and Hiltonol (poly- ICLC). tested in cancer vaccines have commonly Adverse events (AEs) were coded based on National been derived from source proteins in one Cancer Institute (NCI) Common Terminology Criteria for of three categories: tissue-specific differenti- Adverse Events (CTCAE) V.4.03, with provision for early ation proteins, cancer-germ cell proteins or study termination if dose- limiting toxicity (DLT) rates mutated proteins. The immunogenicity of exceeded 33%. The enrollment target was 12 participants antigens derived from non-mutated proteins evaluable for immune response to each peptide. T- cell may be compromised by mechanisms of self- © Author(s) (or their responses were assessed by interferon-γ ELISpot assay. tolerance.1 While antigens carrying tumor- Results pBCAR3 peptides were immunogenic in vivo in employer(s)) 2020. Re- use specific mutations may avoid some tolerance permitted under CC BY-NC. No mice, and in vitro in normal human donors, and T- cells commercial re- use. See rights concerns, identification of relevant antigens specific for pBCAR3126-134 controlled outgrowth of a tumor and permissions. Published by xenograft. The pIRS2 peptide was identified by in each patient and preparation of an appro- BMJ. 1097-1105 mass spectrometry from human hepatocellular carcinoma priate vaccine are technically challenging 2–4 For numbered affiliations see tumors. In the clinical trial, 15 participants were enrolled. and resource- intensive undertakings, end of article. All had grade 1 or 2 treatment- related AEs, but there and their therapeutic value is likely to vary Correspondence to were no grade 3–4 AEs, DLTs or deaths on study. T- cell widely. Importantly, only a small number of Dr Victor H Engelhard; responses were induced to the pIRS21097-1105 peptide in previously- identified antigens in any of these vhe@ virginia. edu 5/12 patients (42%, 90% CI 18% to 68%) and to the categories are derived from proteins that Engelhard VH, et al. J Immunother Cancer 2020;8:e000262. doi:10.1136/jitc-2019-000262 1 Open access are objectively linked to mechanisms of cellular growth METHODS J Immunother Cancer: first published as 10.1136/jitc-2019-000262 on 7 May 2020. Downloaded from control, survival or metastasis. Such antigens are particu- Preclinical studies larly appealing as immunotherapeutic targets for cancer Mice control because their alteration as a means of immune Mice expressing a chimeric class I MHC molecule escape may compromise one or more aspects of the composed of the α1 and α2 domains of human HLA- A2 malignant phenotype. We have identified peptide anti- and the α3 domain of murine H- 2Dd (AAD) have been gens of this type that are modified by intracellular phos- described.19 NOD/SCID/interleukin-2 (IL-2)Rγc-/- phorylation, naturally processed and presented by MHC- I (NSG) mice were purchased from Jackson Immunore- molecules on cancer cells and specifically recognized search Laboratories (Bar Harbor, Massachusetts, USA). by CD8 T- cells.5–7 Phosphorylation is associated with a variety of cellular control processes, some of which are Tumor cell lines dysregulated in cancer cells. The source proteins for All tumor cell lines were of human origin. Melanoma MHC- I- associated phosphopeptides identified to date are line SLM2 was obtained from the American Type Culture in large part known phosphoproteins,6 supporting the Collection (Bethesda, Maryland, USA). Melanoma line idea that the peptides are processed from folded proteins DM331 was established from a patient whose tumor was participating in signaling pathways. resected at Duke University. VMM12 and VMM18 lines One such protein is insulin receptor substrate 2 (IRS2). were established from metastatic melanoma resected IRS proteins are adapters that link signaling from upstream from patients at the University of Virginia. VMM18 also activators to multiple downstream effectors to modulate has been deposited with the ATCC. Melanoma line growth, metabolism, survival and differentiation.8 9 IRS2 1363Mel was obtained from Suzanne Topalian (Johns overexpression at the gene or protein level is evident Hopkins University). Breast cancer lines BT-549, MCF7, in many cancer types and has been demonstrated to MDAMB231, MDAMB468 and T47D were obtained from cause tumorigenesis and enhanced metastasis in vivo.8–10 Sarah Parsons (University of Virginia). Melanoma lines IRS2 phosphorylated at Ser1100 is detectable in multiple were grown at 37°C with 5% CO2 in RPMI-1640 containing cancers, and the resulting phosphopeptide (pIRS21097-1105) 10% fetal bovine serum, 15 mM HEPES, 2 mM L- gluta- is endogenously processed and presented by the MHC- I mine and 50 µg/mL gentamycin. Breast cancer lines were molecule HLA- A*0201 at levels that allow strong immune grown at 37°C with 8% CO2 in DMEM +10% FBS, 1 mM 7 recognition ( and unpublished). pIRS21097-1105- specific sodium pyruvate, 15 mM HEPES, 2 mM L-glutamine and CD8 T- cells can be generated from HLA- A2 transgenic 50 µg/mL gentamycin. mice by immunization with the phosphopeptide.6 7 These T- cells are capable of recognizing and killing human Western blot melanoma and breast tumors in vitro and controlling Cell lysates were generated as described.7 Each sample tumor growth in a xenograft tumor model system.7 was separated by sodium dodecyl sulfate-polyacr ylamide A second cancer-relevant HLA- A*0201 restricted gel electrophoresis and proteins were transferred onto phosphopeptide is derived from breast cancer anti- polyvinylidene fluoride membranes (Immobilon-FL, http://jitc.bmj.com/ estrogen resistance 3 (BCAR3). BCAR3 activates the 0.45 µm, Millipore, Bedford, Massachusetts, USA). Blots PI3K/Akt pathway and mediates both cellular migration were probed with specific primary antibodies overnight and acquisition of resistance to therapeutic antiestro- at 4°C and then with horseradish peroxidase-conjugated gens in breast cancer cells, processes that are associated secondary antibodies. Pixel densities were deter- with malignancy.11 12 A BCAR3- derived phosphopeptide mined using the AlphaEaseFC software and the densi- on October 1, 2021 by guest. Protected copyright. (pBCAR3126-134) was initially shown by us to be presented ties of BCAR3 and of BCAR3 phosphorylated at T130 by HLA- A*0201 on melanoma cells.6
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