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1040-B Identifying tumor-associated antigens in different stages of multiple myeloma using low input HLA immunopeptidomics
  1. Gabrielle M Hernandez1,
  2. Hannah Taylor1,
  3. Eva K Verzani1,
  4. Claudia Ctortecka1,
  5. David A Berrios Nolasco2,
  6. Karl R Clauser1,
  7. Namrata D Udeshi1,
  8. Jennifer G Abelin1,
  9. Elisabet Manasanch2 and
  10. Steven A Carr1
  1. 1The Broad Institute of MIT and Harvard, Cambridge, MA, USA
  2. 2The University of Texas MD Anderson Cancer Center, Houston, TX, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Multiple myeloma (MM) is a rare cancer that leads to malignant plasma cells. There are efforts to uncover how MM develops from precursor disease states, such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Characterizing the evolution of MM allows for earlier prognosis and intervention. Limited availability of tumor (CD138+) cells from patients poses challenges in studying MM stages. Immunopeptidomics typically requires a minimum of 50–100 million cells, exceeding what can typically be obtained from blood cancers. We optimized our HLA immunopurification protocol and paired it with ultra-sensitive mass spectrometry to enable analysis of sample inputs as low as 1 million cells. With this sensitive low input workflow, we characterized the immunopeptidomes of n=35 MM patients, across different stages of the disease.

Methods We optimized our low input HLA immunopurification protocol by reducing lysis buffer volume, antibody concentration, protein G bead volume, bead wash volume, and desalting sorbent.1 2 Using HLA-I/II-expressing A375 cells, we refined the protocol with a range of cell inputs (0.5, 1, 2, 5, and 10 million cells). To confirm the method utility, we profiled a cohort of CD138+ MM samples (≤2 million cells) from a range of disease stages (MGUS; n=7, SMM; n=9, newly diagnosed multiple myeloma: NDMM; n=10, and relapsed/refractory multiple myeloma: RDMM; n=9). We used the Bruker trapped ion mobility spectrometry time of flight (timsTOF) single cell proteomics (SCP) instrument for low input HLA-I/II immunopeptidomics, interpreting spectra with Spectrum Mill against a database containing a reference human proteome and ribosome-sequencing-derived novel or unannotated open reading frames (nuORFs).3

Results Optimized low input immunopurification and ultra-sensitive mass spectrometry achieved ~700 HLA-I and ~650 HLA-II peptides on average across the cohort. In some samples, we identified up to ~4000 HLA-I and ~2000 HLA-II peptides. Given the depth, we found HLA-I nuORF-derived peptides, TAAs (e.g., BCMA, MMSET), and CTAs (e.g., MAGE family).

Conclusions Highly sensitive, low input immunopeptidome analysis enables the discovery of HLA peptides presented by primary MM tumor cells. We detected nuORF-derived peptides in the HLA-I and HLA-II immunopeptidome from MM samples for the first time. This optimized workflow and the resulting data may yield disease state biomarkers and immunotherapeutic targets.


  1. Abelin JG, Bergstrom EJ, Rivera KD, Taylor HB, Klaeger S, Xu C, Verzani EK, Jackson White C, Woldemichael HB, Virshup M, Olive ME, Maynard M, Vartany SA, Allen JD, Phulphagar K, Harry Kane M, Rachimi S, Mani DR, Gillette MA, Satpathy S, Clauser KR, Udeshi ND, Carr SA. Workflow enabling deepscale immunopeptidome, proteome, ubiquitylome, phosphoproteome, and acetylome analyses of sample-limited tissues. Nat Commun. 2023 Apr 3;14(1):1851. doi: 10.1038/s41467-023-37547-0. PMID: 37012232; PMCID: PMC10070353.

  2. Phulphagar KM, Ctortecka C, Jacome ASV, Klaeger S, Verzani EK, Hernandez GM, Udeshi ND, Clauser KR, Abelin JG, Carr SA. Sensitive, high-throughput HLA-I and HLA-II immunopeptidomics using parallel accumulation-serial fragmentation mass spectrometry. Mol Cell Proteomics. 2023 May 2:100563. doi: 10.1016/j.mcpro.2023.100563. Epub ahead of print. PMID: 37142057.

  3. Ouspenskaia T, Law T, Clauser KR, Klaeger S, Sarkizova S, Aguet F, Li B, Christian E, Knisbacher BA, Le PM, Hartigan CR, Keshishian H, Apffel A, Oliveira G, Zhang W, Chen S, Chow YT, Ji Z, Jungreis I, Shukla SA, Justesen S, Bachireddy P, Kellis M, Getz G, Hacohen N, Keskin DB, Carr SA, Wu CJ, Regev A. Unannotated proteins expand the MHC-I-restricted immunopeptidome in cancer. Nat Biotechnol. 2022 Feb;40(2):209–217. doi: 10.1038/s41587-021-01021-3. Epub 2021 Oct 18. PMID: 34663921.

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