Tumor Infiltrating Lymphocytes Target HLA-I Phosphopeptides Derived From Cancer Signaling in Colorectal Cancer.

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients' tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients' tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.

Characteristics of Immune Memory and Effector Activity to Cancer-Expressed MHC Class I Phosphopeptides Differ in Healthy Donors and Ovarian Cancer Patients.

Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.

MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma.

BACKGROUND: Phosphorylated peptides presented by MHC molecules represent a new class of neoantigens expressed on cancer cells and recognized by CD8 T-cells. These peptides are promising targets for cancer immunotherapy. Previous work identified an HLA-A*0201-restricted phosphopeptide from insulin receptor substrate 2 (pIRS2) as one such target. The purpose of this study was to characterize a second phosphopeptide, from breast cancer antiestrogen resistance 3 (BCAR3), and to evaluate safety and immunogenicity of a novel immunotherapic vaccine comprising either or both of these phosphorylated peptides. METHODS: Phosphorylated BCAR3 protein was evaluated in melanoma and breast cancer cell lines by Western blot, and recognition by T-cells specific for HLA-A*0201-restricted phosphorylated BCAR3 peptide (pBCAR3126-134) was determined by 51Cr release assay and intracellular cytokine staining. Human tumor explants were also evaluated by mass spectrometry for presentation of pIRS2 and pBCAR3 peptides. For the clinical trial, participants with resected stage IIA-IV melanoma were vaccinated 6 times over 12 weeks with one or both peptides in incomplete Freund's adjuvant and Hiltonol (poly-ICLC). Adverse events (AEs) were coded based on National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) V.4.03, with provision for early study termination if dose-limiting toxicity (DLT) rates exceeded 33%. The enrollment target was 12 participants evaluable for immune response to each peptide. T-cell responses were assessed by interferon-γ ELISpot assay. RESULTS: pBCAR3 peptides were immunogenic in vivo in mice, and in vitro in normal human donors, and T-cells specific for pBCAR3126-134 controlled outgrowth of a tumor xenograft. The pIRS21097-1105 peptide was identified by mass spectrometry from human hepatocellular carcinoma tumors. In the clinical trial, 15 participants were enrolled. All had grade 1 or 2 treatment-related AEs, but there were no grade 3-4 AEs, DLTs or deaths on study. T-cell responses were induced to the pIRS21097-1105 peptide in 5/12 patients (42%, 90% CI 18% to 68%) and to the pBCAR3126-134 peptide in 2/12 patients (17%, 90% CI 3% to 44%). CONCLUSION: This study supports the safety and immunogenicity of vaccines containing the cancer-associated phosphopeptides pBCAR3126-134 and pIRS21097-1105, and the data support continued development of immune therapy targeting phosphopeptides. Future studies will define ways to further enhance the magnitude and durability of phosphopeptide-specific immune responses. TRIAL REGISTRATION NUMBER: NCT01846143.

Evaluation of the regulatory model for Ni2+ sensing by Nur from Streptomyces coelicolor.

Streptomyces coelicolor is a soil-dwelling bacterium that is medically important due to its ability to produce several antibiotics, and nickel accumulation within this organism has been shown to prevent the production of the antibiotic undecylprodigiosin. The transcriptional repressor important in regulation of nickel uptake is the homodimeric Nur, a member of the Fur family. Nur contains two metal-binding sites per monomer: the M-site and the Ni-site. The work described here seeks to determine the roles of each of the metal-binding sites to establish a model of Nur activity through mutational studies, metal titrations, and fluorescence anisotropy. Through these studies, a model of Nur activity is proposed in which femtomolar metal binding to one M-site of Nur prompts DNA-binding, and metal binding to the second M-site fully activates the protein. Evidence is provided that shows cooperative metal binding to the Ni-site, but this process dampens affinity for promoter DNA.

Murine xenograft bioreactors for human immunopeptidome discovery.

The study of peptides presented by MHC class I and class II molecules is limited by the need for relatively large cell numbers, especially when studying post-translationally modified or otherwise rare peptide species. To overcome this problem, we pose the hypothesis that human cells grown as xenografts in immunodeficient mice should produce equivalent immunopeptidomes as cultured cells. Comparing human cell lines grown either in vitro or as murine xenografts, we show that the immunopeptidome is substantially preserved. Numerous features are shared across both sample types, including peptides and proteins featured, length distributions, and HLA-binding motifs. Peptides well-represented in both groups were from more abundant proteins, or those with stronger predicted HLA binding affinities. Samples grown in vivo also recapitulated a similar phospho-immunopeptidome, with common sequences being those found at high copy number on the cell surface. These data indicate that xenografts are indeed a viable methodology for the production of cells for immunopeptidomic discovery.

Shared peptide binding of HLA Class I and II alleles associate with cutaneous nevirapine hypersensitivity and identify novel risk alleles.

Genes of the human leukocyte antigen (HLA) system encode cell-surface proteins involved in regulation of immune responses, and the way drugs interact with the HLA peptide binding groove is important in the immunopathogenesis of T-cell mediated drug hypersensitivity syndromes. Nevirapine (NVP), is an HIV-1 antiretroviral with treatment-limiting hypersensitivity reactions (HSRs) associated with multiple class I and II HLA alleles. Here we utilize a novel analytical approach to explore these multi-allelic associations by systematically examining HLA molecules for similarities in peptide binding specificities and binding pocket structure. We demonstrate that primary predisposition to cutaneous NVP HSR, seen across ancestral groups, can be attributed to a cluster of HLA-C alleles sharing a common binding groove F pocket with HLA-C*04:01. An independent association with a group of class II alleles which share the HLA-DRB1-P4 pocket is also observed. In contrast, NVP HSR protection is afforded by a cluster of HLA-B alleles defined by a characteristic peptide binding groove B pocket. The results suggest drug-specific interactions within the antigen binding cleft can be shared across HLA molecules with similar binding pockets. We thereby provide an explanation for multiple HLA associations with cutaneous NVP HSR and advance insight into its pathogenic mechanisms.

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia.

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I-associated peptide antigens with O-linked ß-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376-84. ©2017 AACR.