Academic Appointments


Honors & Awards


  • Gordon Research Conference in Glycobiology; Poster Award, GRC (2019)
  • Helena Anna Henzl-Gabor Young Women in Science Travel Grant, Stanford University (2018)
  • Stanford University Mass Spectrometry Research Symposium; First Place Poster, SUMS (2018)
  • Visiting Junior Scholar Fellowship, France-Stanford Center for Interdisciplinary Studies (2018)
  • NIH F32 Ruth L. Kirschstein National Research Service Award (NRSA), NIGMS (2017-2019)
  • NIH T32 Immunology Training Grant (Declined), Stanford School of Medicine (2017-2018)
  • Helena Anna Henzl-Gabor Young Women in Science Travel Grant, Helena Anna Henzl-Gabor Young Women in Science Fund for Postdoctoral Scholars (2017)
  • Research Associateship Award (Declined), National Research Council (NRC) (2016)
  • Stanford University Mass Spectrometry Seed Grant, SUMS (2016)
  • CGS/ProQuest Distinguished Dissertation Nominee, University of Virginia (2015)
  • Post Doc Symposium Oral Presentation; Third Place, University of Virginia (2015)
  • 3 Minute Thesis; First Place, University of Virginia (2014)
  • Huskey Research Symposium Oral Presentation; First Place, University of Virginia (2014)
  • Robert J. Huskey Travel Fellowship, University of Virginia (2014)
  • Washington-Baltimore Mass Spectrometry Discussion Group Travel Award, Washington-Baltimore Mass Spectrometry Discussion Group (2014)
  • Achievement Rewards for College Scientists (ARCS) Fellowship, Danaher Scholar, Achievement Rewards for College Scientists (2013-2014)
  • Huskey Research Symposium Poster; First Place, University of Virginia (2013)
  • Pratt Fellowship, University of Virginia (2013)
  • Alpha Chi Sigma Presidential Award, Alpha Chi Sigma (2010)
  • Angell Scholar, University of Michigan (2009)
  • University of Michigan Honors, University of Michigan (2007-2009)

Boards, Advisory Committees, Professional Organizations


  • Member, Society for Glycobiology (2019 - Present)
  • Member, American Chemical Society (2016 - Present)
  • Member, American Society for Mass Spectrometry (2014 - Present)
  • Member, Alpha Chi Sigma (2007 - Present)

Patents


  • Donald F. Hunt, Jeffrey Shabanowitz, Stacy A. Malaker, Victor H. Engelhard, Angela Zarling, Kara L. Cummings, Rebecca C. Obeng, and Mark Cobbold. "United States Patent US09561266 Target peptides for immunotherapy and diagnostics"
  • Donald F. Hunt, Jeffrey Shabanowitz, Paisley T. Myers, Mark Cobbold, Nico Buttner, Stacy A. Malaker, Sarah A. Penny. "United States Patent WO02017192969-A1 Composition useful for treating hepatocellular carcinoma and esophageal cancer, comprises synthetic target peptides comprising specific amino acid sequences"
  • Donald F. Hunt, Jeffrey Shabanowitz, Stacy A. Malaker, Victor H. Engelhard, Angela Zarling, Kara L. Cummings, Rebecca C. Obeng, and Mark Cobbold. "United States Patent WO2014036562-A2, WO2014036562-A3, CA2883569-A1, AU2013308409-A1, EP2897631-A2, US2015224182-A1, HK1212237-A0, US9561266-B2, US2017333541-A1 Composition useful for treating or preventing cancer and melanoma in patient comprises synthetic target peptides"
  • Donald F. Hunt, Jeffrey Shabanowitz, Stacy A. Malaker, Victor H. Engelhard, Angela Zarling, Kara L. Cummings, Rebecca C. Obeng, and Mark Cobbold. "United States Patent WO2015034519-A1 Composition for treating a proliferative disease comprises a tetanus peptide and synthetic target peptides"
  • Donald F. Hunt, Jeffrey Shabanowitz, Stacy A. Malaker, Mark Cobbold, and Sarah A. Penny. "United States Patent WO2017027403-A1; CA2995103-A1; AU2016306304-A1 Composition for treating and/or preventing cancer, e.g. leukemia comprises Class I Major Histocompatibility Complex associated glycopeptides"

Current Research and Scholarly Interests


Aberrant glycosylation is a universal feature of cancer and contributes to the ability of malignant cells to evade the immune system. Elucidating how glycosylation can downregulate the immune system is key to designing cancer immunotherapies that would serve to re-activate the immune system to specifically target and kill cancer cells. My research research uses glycobiology and mass spectrometry to identify glycosylated targets on tumor cells that contribute to immune suppression.

All Publications


  • Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT Rumachik, N. G., Malaker, S. A., Poweleit, N., Maynard, L. H., Adams, C. M., Leib, R. D., Cirolia, G., Thomas, D., Stamnes, S., Holt, K., Sinn, P., May, A. P., Paulk, N. K. 2020; 18: 98–118
  • Targeted glycan degradation potentiates the anticancer immune response in vivo. Nature chemical biology Gray, M. A., Stanczak, M. A., Mantuano, N. R., Xiao, H., Pijnenborg, J. F., Malaker, S. A., Miller, C. L., Weidenbacher, P. A., Tanzo, J. T., Ahn, G., Woods, E. C., Laubli, H., Bertozzi, C. R. 2020

    Abstract

    Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an alphaHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.

    View details for DOI 10.1038/s41589-020-0622-x

    View details for PubMedID 32807964

  • Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation. Proceedings of the National Academy of Sciences of the United States of America Debets, M. F., Tastan, O. Y., Wisnovsky, S. P., Malaker, S. A., Angelis, N., Moeckl, L. K., Choi, J., Flynn, H., Wagner, L. J., Bineva-Todd, G., Antonopoulos, A., Cioce, A., Browne, W. M., Li, Z., Briggs, D. C., Douglas, H. L., Hess, G. T., Agbay, A. J., Roustan, C., Kjaer, S., Haslam, S. M., Snijders, A. P., Bassik, M. C., Moerner, W. E., Li, V. S., Bertozzi, C. R., Schumann, B. 2020

    Abstract

    Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

    View details for DOI 10.1073/pnas.2007297117

    View details for PubMedID 32989128

  • Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells. Molecular cell Schumann, B., Malaker, S. A., Wisnovsky, S. P., Debets, M. F., Agbay, A. J., Fernandez, D., Wagner, L. J., Lin, L., Li, Z., Choi, J., Fox, D. M., Peh, J., Gray, M. A., Pedram, K., Kohler, J. J., Mrksich, M., Bertozzi, C. R. 2020

    Abstract

    Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

    View details for DOI 10.1016/j.molcel.2020.03.030

    View details for PubMedID 32325029

  • Optimal Dissociation Methods Differ for N- and O-glycopeptides. Journal of proteome research Riley, N. M., Malaker, S. A., Driessen, M., Bertozzi, C. R. 2020

    Abstract

    Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.

    View details for DOI 10.1021/acs.jproteome.0c00218

    View details for PubMedID 32500713

  • An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Proceedings of the National Academy of Sciences of the United States of America Shon, D. J., Malaker, S. A., Pedram, K., Yang, E., Krishnan, V., Dorigo, O., Bertozzi, C. R. 2020

    Abstract

    Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.

    View details for DOI 10.1073/pnas.2012196117

    View details for PubMedID 32817557

  • Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Choi, J., Wagner, L. S., Timmermans, S. E., Malaker, S. A., Schumann, B., Gray, M. A., Debets, M. F., Takashima, M., Gehring, J., Bertozzi, C. R. 2019; 141 (34): 13442–53

    Abstract

    O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

    View details for DOI 10.1021/jacs.9b04695

    View details for Web of Science ID 000484082700023

    View details for PubMedID 31373799

  • Enzyme toolkit for selective enrichment and analysis of mucin-domain glycoproteins Malaker, S. A., Shon, J., Pedram, K., Riley, N. M., Bertozzi, C. R. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2019: S42
  • Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors Rumachik, N., Malaker, S., Poweleit, N., Maynard, L., Adams, C., Leib, R., Cirolia, G., Stamnes, S., Holt, K., Sinn, P., DeRisi, J., May, A., Paulk, N. CELL PRESS. 2019: 217
  • The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Malaker, S. A., Pedram, K., Ferracane, M. J., Bensing, B. A., Krishnan, V., Pett, C., Yu, J., Woods, E. C., Kramer, J. R., Westerlind, U., Dorigo, O., Bertozzi, C. R. 2019; 116 (15): 7278–87
  • Isolation of Major Histocompatibility Complex (MHC)-Associated Peptides by Immunoaffinity Purification. Methods in molecular biology (Clifton, N.J.) Penny, S. A., Malaker, S. A. 2019; 2024: 235–43

    Abstract

    MHC moleculeson the surface of cells are responsible for the presentation of antigenic peptides to CD8+ and CD4+ T cells. Downstream analysis of these peptides can offer insight into various disease processes with an immune component, as in autoimmune diseases and cancer. A critical step in studying MHC-associated peptides is their isolation from MHCmolecules on the surface of cells. In this chapter, we detail anti-MHC antibody conjugation to beads, immunoaffinity purification of the MHCmolecules, and peptide elution. This method can be used for analysis of unmodified and/or posttranslationally modified peptides by mass spectrometry.

    View details for DOI 10.1007/978-1-4939-9597-4_14

    View details for PubMedID 31364053

  • Mass Spectrometric Identification and Molecular Modeling of Glycopeptides Presented by MHC Class I and II Processing Pathways. Methods in molecular biology (Clifton, N.J.) Malaker, S. A., Ferracane, M. J. 2019; 2024: 269–85

    Abstract

    Aberrant glycosylation is a hallmark of cancer that contributes to the disease's ability to evade the immune system. As the MHC processing pathways communicate cellular health to circulating CD8+ and CD4+ T-cells, MHC-associated glycopeptides are likely a source of neoantigens in cancer. In fact, recent advances in mass spectrometry have allowed for the detection and sequencing of tumor-specific glycopeptides from the MHC class I and class II processing pathways. Here, we describe methods for detecting, sequencing, and modeling these MHC-associated glycopeptides.

    View details for DOI 10.1007/978-1-4939-9597-4_17

    View details for PubMedID 31364056

  • Canonic and Cross-reacrtive Binding of NK cell Inhibitory Recweptors to HLA-C Allotypes Is Dication by Peptides Bound to HLA-C FRONTIERS IN IMMUNOLOGY Sim, M. J., Malaker, S. A., Khan, A., Stowell, J. M., Shabanowitz, J., Peterson, M. E., Rajagopalan, S., Hunt, D. F., Altmann, D. M., Long, E. O., Boyton, R. J. 2017; 8

    Abstract

    Human natural killer (NK) cell activity is regulated by a family of killer cell immunoglobulin-like receptors (KIRs) that bind human leukocyte antigen (HLA) class I. Combinations of KIR and HLA genotypes are associated with disease, including susceptibility to viral infection and disorders of pregnancy. KIR2DL1 binds HLA-C alleles of group C2 (Lys(80)). KIR2DL2 and KIR2DL3 bind HLA-C alleles of group C1 (Asn(80)). However, this model cannot explain HLA-C allelic effects in disease or the impact of HLA-bound peptides. The goal of this study was to determine the extent to which the endogenous HLA-C peptide repertoire can influence the specific binding of inhibitory KIR to HLA-C allotypes.The impact of HLA-C bound peptide on inhibitory KIR binding was investigated taking advantage of the fact that HLA-C*05:01 (HLA-C group 2, C2) and HLA-C*08:02 (HLA-C group 1, C1) have identical sequences apart from the key KIR specificity determining epitope at residues 77 and 80. Endogenous peptides were eluted from HLA-C*05:01 and used to test the peptide dependence of KIR2DL1 and KIR2DL2/3 binding to HLA-C*05:01 and HLA-C*08:02 and subsequent impact on NK cell function. Specific binding of KIR2DL1 to the C2 allotype occurred with the majority of peptides tested. In contrast, KIR2DL2/3 binding to the C1 allotype occurred with only a subset of peptides. Cross-reactive binding of KIR2DL2/3 with the C2 allotype was restricted to even fewer peptides. Unexpectedly, two peptides promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. We showed that presentation of endogenous peptides or HIV Gag peptides by HLA-C can promote KIR cross-reactive binding.KIR2DL2/3 binding to C1 is more peptide selective than that of KIR2DL1 binding to C2, providing an explanation for KIR2DL3-C1 interactions appearing weaker than KIR2DL1-C2. In addition, cross-reactive binding of KIR is characterized by even higher peptide selectivity. We demonstrate a hierarchy of functional peptide selectivity of KIR-HLA-C interactions with relevance to NK cell biology and human disease associations. This selective peptide sequence-driven binding of KIR provides a potential mechanism for pathogen as well as self-peptide to modulate NK cell activation through altering levels of inhibition.

    View details for DOI 10.3389/fimmu.2017.00193

    View details for Web of Science ID 000396207100001

    View details for PubMedID 28352266

    View details for PubMedCentralID PMC5348643

  • Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses. Immunogenetics Bergmann, T., Lindvall, M., Moore, E., Moore, E., Sidney, J., Miller, D., Tallmadge, R. L., Myers, P. T., Malaker, S. A., Shabanowitz, J., Osterrieder, N., Peters, B., Hunt, D. F., Antczak, D. F., Sette, A. 2017

    Abstract

    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.

    View details for DOI 10.1007/s00251-017-0978-6

    View details for PubMedID 28315936

  • Shared peptide binding of HLA Class I and II alleles associate with cutaneous nevirapine hypersensitivity and identify novel risk alleles. Scientific reports Pavlos, R., McKinnon, E. J., Ostrov, D. A., Peters, B., Buus, S., Koelle, D., Chopra, A., Schutte, R., Rive, C., Redwood, A., Restrepo, S., Bracey, A., Kaever, T., Myers, P., Speers, E., Malaker, S. A., Shabanowitz, J., Jing, Y., Gaudieri, S., Hunt, D. F., Carrington, M., Haas, D. W., Mallal, S., Phillips, E. J. 2017; 7 (1): 8653

    Abstract

    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.

    View details for DOI 10.1038/s41598-017-08876-0

    View details for PubMedID 28819312

    View details for PubMedCentralID PMC5561238

  • Identification and Characterization of Complex Glycosylated Peptides Presented by the MHC Class II Processing Pathway in Melanoma JOURNAL OF PROTEOME RESEARCH Malaker, S. A., Ferracane, M. J., Depontieu, F. R., Zarling, A. L., Shabanowitz, J., Bai, D. L., Topalian, S. L., Engelhard, V. H., Hunt, D. F. 2017; 16 (1): 228-237
  • Identification of glycopeptides as post-translationally modified neoantigens in leukemia. Cancer immunology research Malaker, S. A., Penny, S. A., Steadman, L. G., Myers, P. T., Loke, J., Raghavan, M., Bai, D. L., Shabanowitz, J., Hunt, D., Cobbold, M. 2017

    Abstract

    Leukemias are highly immunogenic but 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 β-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 post-translationally modified neoantigens provide logical targets for cancer immunotherapy.

    View details for DOI 10.1158/2326-6066.CIR-16-0280

    View details for PubMedID 28314751

  • MHC Class I-Associated Phosphopeptides Are the Targets of Memory-like Immunity in Leukemia SCIENCE TRANSLATIONAL MEDICINE Cobbold, M., De la Pena, H., Norris, A., Polefrone, J. M., Qian, J., English, A. M., Cummings, K. L., Penny, S., Turner, J. E., Cottine, J., Abelin, J. G., Malaker, S. A., Zarling, A. L., Huang, H., Goodyear, O., Freeman, S. D., Shabanowitz, J., Pratt, G., Craddock, C., Williams, M. E., Hunt, D. F., Engelhard, V. H. 2013; 5 (203)

    Abstract

    Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8(+) T cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and human leukocyte antigen-matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust CD8(+) T cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients. This reduction correlated with clinical outcome; however, immunity was restored after allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T cell adoptive transfer immunotherapies.

    View details for DOI 10.1126/scitranslmed.3006061

    View details for Web of Science ID 000324589900004

    View details for PubMedID 24048523