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148 Characterizing cancer’s dark matter, short-lived proteins, and defective ribosomal products, presented by cancer and contained in the DPV-001 cancer vaccine
  1. Ryan Meng1,
  2. Tarsem Moudgil2,
  3. Noriko Iwamoto3,
  4. Yuriko Minegishi4,
  5. Yoshinobu Koguchi2,
  6. Venkatesh Rajamanickam5,
  7. Tanisha Christie2,
  8. William L Redmond2,
  9. Carlo Bifulco2,
  10. Traci Hilton6,
  11. Alexa Dowdell2,
  12. Shawn M Jensen2,
  13. Rachel E Sanborn2,
  14. Hong-Ming Hu2,
  15. R Bryan Bell2,
  16. Walter J Urba2,
  17. Rom S Leidner2,
  18. Koji Ueda4,
  19. Takashi Shimada3,
  20. Brian Piening2 and
  21. Bernard A Fox2
  1. 1Providence Cancer Institute, Portland, OR, USA
  2. 2Earle A. Chiles Research Institute, Providence Cancer Institute, Portland, OR, USA
  3. 3Shimadzu Corporation, Kyoto, Japan
  4. 4Japanese Foundation for Cancer Research, Ariake, Tokyo, Japan
  5. 5Providence Health, Portland, OR, USA
  6. 6UbiVac, Portland, OR, USA

Abstract

Background Short-lived proteins (SLiPs), defective ribosomal products (DRiPs), and Cancer’s dark matter/non-canonical peptides (NCP) are unstable and rapidly degraded, loaded onto MHC and represent a large proportion of the epitopes presented by cancer cells. Recent data suggests that NCP may play a role in molecular control of some malignant processes, further strengthening the rationale for characterizing their presence and developing pharmacologic and immunologic approaches to impact their effect.1 Immunologic approaches seem particularly promising as NCP, that are not expressed in the thymus, represent potential shared alternative cancer neoantigens.2 Our group developed a vaccine strategy that concentrates SLiPs, DRiPs, and NCP in dendritic cell-targeted microvesicles.3 Preclinical combination immunotherapy studies documented efficacy in difficult to treat animal models4 5 and an off-the-shelf human vaccine was developed and has entered clinical trials. The current studies were undertaken to characterize the breadth of peptides presented by head and neck squamous cell cancer (HNSCC) and non-small cell lung cancer (NSCLC), as well as the proteins contained in the DPV-001 vaccine.

Methods Cancer cells were lysed and recovered lysates were treated with detergent in the presence of protease inhibitor. HLA peptides were collected from HLA complexes purified by anti-HLA-I antibody (w6/32). Recovered HLA peptides were analyzed by the microLC-QTOF MS (LCMS-9030. Shimadzu Corporation). The two components of DPV-001, UbiLT3 and UbiLT6, were assessed via deep proteomic profiling on an Orbitrap mass spectrometer (ORBITRAP FUSION LUMOS with FAIMS-Pro interface (Thermofisher Scientific)). Spectra from whole proteome tryptic digests were acquired in data-dependent acquisition (DDA) mode, and Peptide-Prism was used to identify canonical and noncanonical peptides/NCPs.

Results The approaches outlined above provide evidence of canonical and NCP, derived from noncoding or out-of-frame translations, presented by HLA of HNSCC cell lines and contained within DPV-001. These peptides provide a starting point for immunological studies.

Conclusions These results support our efforts to identify shared canonical and NCP that are targets of therapeutic immune responses. The discovery of NCP derived from supposed non-coding regions that are not expressed in the thymus, represent a novel class of potentially shared alternative cancer neoantigens. Their association with malignant processes may provide them properties similar to that of driver mutations, and increase their relevance as cancer vaccine targets.

Acknowledgements Support: Shimadzu Corporation, Providence Medical Foundation, The Harder Family, Nancy Lematta, The Chiles Foundation, Robert W. Franz, and Elsie Franz Finley

References

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  2. Cieri N, Wu CJ. Splice it up: Atypical transcripts to boost leukemia immunotherapy. Immunity 2021; 54:608–610. 10.1016/j.immuni.2021.03.016.

  3. Page DB, Hulett TW, Hilton TL, Hu H-M, Urba WJ, Fox BA. Glimpse into the future: harnessing autophagy to promote anti-tumor immunity with the DRibbles vaccine. Journal for ImmunoTherapy of Cancer 2016; 4:10.1186/s40425–016-0130–4.

  4. Yu G, Li Y, Cui Z, Morris NP, Weinberg AD, Fox BA, Urba WJ, Wang L, Hu H-M. Combinational Immunotherapy with Allo-DRibble Vaccines and Anti-OX40 Co-Stimulation Leads to Generation of Cross-Reactive Effector T Cells and Tumor Regression. Scientific Reports 2016; 6:10.1038/srep37558.

  5. Patel JM, Cui Z, Wen Z-F, Dinh CT, Hu H-M. Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism. J Immunother Cancer 2019; 7:311. 10.1186/s40425–019-0786–7.

Ethics Approval PSJH IRB# 06–108

http://creativecommons.org/licenses/by-nc/4.0/

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