Sensitive detection of cancer antigens enabled by user-defined peptide libraries

Kasidet Manakongtreecheep^#^{1

2

3}, Claudia Ctortecka^#¹, Luis O Correa-Medero¹, Timothy Zhu^{1

3}, Isabelle Lippincott¹, Griffin M Lawrence¹, Alexis Howard³, Gabrielle M Hernandez¹, Cleo Forman³, Emma C Duggan¹, Marta A Wilbrink¹, Eva K Verzani¹, Alexander B Afeyan^{2

3}, Jiaxun Li³, Alexey I Nesvizhskii⁴, Giacomo Oliveira³, Derin B Keskin^{1

2

3

5

6}, Patrick A Ott^{1

2

3}, Karl R Clauser¹, Matthew Bakalar¹, Siranush Sarkizova¹, Nir Hacohen^{7

8

9}, Steven A Carr¹⁰, Jennifer G Abelin^{11

12}, Catherine J Wu^{13

14

15}

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
² Harvard Medical School, Boston, MA, USA.
³ Dana Farber Cancer Institute, Boston, MA, USA.
⁴ Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
⁵ Department of Computer Science, Metropolitan College, Boston University, Boston, MA, USA.
⁶ Technical University of Denmark, Lyngby, Denmark.
⁷ Broad Institute of MIT and Harvard, Cambridge, MA, USA. nhacohen@broadinstitute.org.
⁸ Harvard Medical School, Boston, MA, USA. nhacohen@broadinstitute.org.
⁹ Massachusetts General Hospital, Krantz Family Center for Cancer Research, Boston, MA, USA. nhacohen@broadinstitute.org.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA, USA. scarr@broad.mit.edu.
¹¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA. jabelin@broadinstitute.org.
¹² Dana Farber Cancer Institute, Boston, MA, USA. jabelin@broadinstitute.org.
¹³ Broad Institute of MIT and Harvard, Cambridge, MA, USA. catherine_wu@dfci.harvard.edu.
¹⁴ Harvard Medical School, Boston, MA, USA. catherine_wu@dfci.harvard.edu.
¹⁵ Dana Farber Cancer Institute, Boston, MA, USA. catherine_wu@dfci.harvard.edu.

^# Contributed equally.

PMID: 41663542
DOI: 10.1038/s41587-026-03003-9

Sensitive detection of cancer antigens enabled by user-defined peptide libraries

Kasidet Manakongtreecheep et al. Nat Biotechnol. 2026.

. 2026 Feb 9.

doi: 10.1038/s41587-026-03003-9. Online ahead of print.

Authors

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
² Harvard Medical School, Boston, MA, USA.
³ Dana Farber Cancer Institute, Boston, MA, USA.
⁴ Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
⁵ Department of Computer Science, Metropolitan College, Boston University, Boston, MA, USA.
⁶ Technical University of Denmark, Lyngby, Denmark.
⁷ Broad Institute of MIT and Harvard, Cambridge, MA, USA. nhacohen@broadinstitute.org.
⁸ Harvard Medical School, Boston, MA, USA. nhacohen@broadinstitute.org.
⁹ Massachusetts General Hospital, Krantz Family Center for Cancer Research, Boston, MA, USA. nhacohen@broadinstitute.org.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA, USA. scarr@broad.mit.edu.
¹¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA. jabelin@broadinstitute.org.
¹² Dana Farber Cancer Institute, Boston, MA, USA. jabelin@broadinstitute.org.
¹³ Broad Institute of MIT and Harvard, Cambridge, MA, USA. catherine_wu@dfci.harvard.edu.
¹⁴ Harvard Medical School, Boston, MA, USA. catherine_wu@dfci.harvard.edu.
¹⁵ Dana Farber Cancer Institute, Boston, MA, USA. catherine_wu@dfci.harvard.edu.

^# Contributed equally.

PMID: 41663542
DOI: 10.1038/s41587-026-03003-9

Abstract

Human leukocyte antigen (HLA)-bound tumor peptides can be routinely isolated from cancer samples and identified using mass spectrometry (MS). However, MS approaches can be stochastic or rely on spectral libraries, which are not customarily available for individual-specific peptides, thus limiting the ability to discover novel peptides. Here, we introduce Pepyrus, which generates user-defined, individual-specific or disease-specific peptide libraries in Escherichia coli to improve the sensitivity and confidence of MS peptide identification, including lowly abundant neoantigens. Using Pepyrus-generated peptide libraries paired with an HLA-specific data-independent acquisition strategy, we recover >75% of the expected sequences per single injection for libraries of >10,000 peptides and identify 0.1 fmol of spiked-in peptides in a complex background. We apply Pepyrus to create personalized libraries, facilitating identification of clinically relevant HLA peptides, including several novel peptides from cell lines derived from persons with melanoma and renal cell carcinoma. Pepyrus enables identification of rare HLA-bound peptides and provides the ability to generate large training datasets to improve spectra, retention time and ion mobility prediction tools.

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Conflict of interest statement

Competing interests: P.A.O. has received research funding from and/or has advised Agenus, Arcturus, Amgen, Armo BioSciences, Array, AstraZeneca/MedImmune, Bristol-Meyers Squibb, Celldex, CytomX, Evaxion, Immunetune, Imunon, LGChem, Merck, Neon Therapeutics (now BioNTechUS), Novartis, Pharmajet, Phio, Pfizer, Oncorus, Roche/Genentech, Servier and Xencor. J.G.A. is a paid consultant for Moderna. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer, PrognomIQ and Mobilion. C.J.W. is an equity holder of BioNtech, is a member of the scientific advisory board of Adventris, Aethon Therapeutics, Nature’s Toolbox and Repertoire and receives research funding from Pharmacyclics. D.B.K. is a scientific advisor for Immunitrack, a wholly owned subsidiary of Eli Lilly and Company and Breakbio. D.B.K. owns equity in Affimed, Agenus, Armata Pharmaceuticals, Beam Therapeutics, Breakbio, BioMarin Pharmaceutical, Celldex Therapeutics, Editas Medicine, Immunitybio, Lexicon Pharmaceuticals, Moderna, Prime Medicine, Sana Biotechnology and Summit Therapeutics. N.H. holds equity in and advises Danger Bio/Related Sciences, Repertoire Immune Medicines and CytoReason, owns equity and has licensed patents to BioNtech and receives research funding from Bristol Myers Squibb, Moderna, ResolveM/JJDC, Takeda and Calico Life Sciences. A.I.N. is the founder of Fragmatics, serves on the scientific advisory boards of Protai Bio, Infinitopes and Mobilion Systems and has financial interest because of the licensing of MSFragger to commercial entities. G.O. is a consultant for Bicycle Therapeutics. The other authors declare no competing interests.

References

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Sensitive detection of cancer antigens enabled by user-defined peptide libraries

Affiliations

Sensitive detection of cancer antigens enabled by user-defined peptide libraries

Authors

Affiliations

Abstract

Conflict of interest statement

References

Grants and funding

LinkOut - more resources

Full Text Sources

Research Materials