Multiplex quantification of immunomodulatory proteins for cancer diagnostics

From the Paulovich Lab, Translational Science and Therapeutics Division

Immunotherapy is a type of cancer treatment that harnesses a person’s immune system to fight cancer cells. In addition to boosting certain parts of the immune system, these therapies can “teach” cells to identify specific tumor cells. Although this approach has been successful in treating some kinds of cancer, oncologists cannot fully predict how a particular patient will respond to immunotherapy.

“The goal is to try and figure out how to better deliver immune-based therapies to patients with cancers. We're seeing cures in some people who otherwise had no treatment options, but a high percentage of patients still don't respond to immune therapy,” said Dr. Amanda Paulovich, a professor in the Translational Science and Therapeutics Division and Aven Foundation Endowed Chair at Fred Hutch Cancer Center. Even in successful cases, one person might respond well to a single immunotherapeutic agent, whereas another might require a combination of multiple agents.

“There are hundreds of immunomodulatory proteins in the tumor microenvironment that are thought to shape the tumor response to these therapies, and you need to be able to measure those in order to study them and their effects,” said Dr. Paulovich. To accomplish this goal, the Paulovich lab received funding from the Beau Biden National Cancer Moonshot to develop immuno-MRM assays to measure immunomodulatory proteins in the tumor microenvironment. Immuno-MRM, a protein quantification technique developed in the Paulovich lab, couples antibody enrichment with targeted “MRM” mass spectrometry to quantify low-abundance proteins from biospecimens containing a mixture of different proteins (MRM is short for “multiple reaction monitoring”). Importantly, the assays can quantify multiple proteins from a single sample simultaneously, or in multiplex.

Researchers from the lab previously developed a multiplex immuno-MRM assay to quantify 46 immunomodulatory proteins from a single sample (the “IO-1 assay”). Now, in a new study published in Frontiers in Oncology, they describe a new “IO-2 assay” that enables the quantifications of 43 additional immunomodulatory proteins. Dr. Jeff Whiteaker, a senior staff scientist in the Paulovich lab, led both studies.

AI-generated acrylic painting of a chemistry laboratory with oversized lab equipment on the moon, with a moon-like orb in the distance.
The Paulovich lab received funding from the Beau Biden National Cancer Moonshot to develop multiplex immuno-MRM assays to measure immunomodulatory proteins in the tumor microenvironment. The IO-2 assay described in this paper is only the second in a series of a total of five IO panels that are under development in the Paulovich lab. Image generated by the author using DALL-E 2

To develop the IO-2 immuno-MRM assay, the researchers started by identifying a panel of peptides for generation of monoclonal antibodies (antibodies generated in animals to recognize unique targets – in this case, the peptides from immunomodulatory proteins). They chose the candidate peptides according to factors including detection frequency and intensity in proteomics datasets, and their physical and chemical properties. They selected a panel of 49 peptides, representing 43 proteins, for assay development.

Following the antibody panel development, the researchers characterized the performance of the multiplexed IO-2 assay in tissue and plasma matrices. This fit-for-purpose validation measured performance factors including limits of quantification and the repeatability of the assay. Then, to test the robustness of the new IO-2 assay in the real world, the researchers validated the assay in 102 tissue biopsies and 48 plasma samples obtained from patients with cancer. In the tissue biopsies, the IO-2 assay detected 46 out of the 49 peptides, corresponding to 41 out of 43 proteins. In the plasma samples, the assay detected 27 peptides, which corresponded to 25 proteins.

Next, the authors performed a proof-of-principle experiment, where they applied the new IO-2 assay and the existing IO-1 assay to two clinical trials of patients with lymphoma who were receiving an immune checkpoint inhibitor (pembrolizumab). One of the clinical sample collections consisted of matched plasma and serum from 24 patients, each collected at three timepoints in the patients’ treatments. The authors tested whether the IO assays could detect longitudinal changes in protein expression during the course of the patients’ treatments. They found 10 total peptides that showed significant expression changes across the three timepoints. In addition, two peptides from the IO-2 panel showed significant differences between patients with a complete or partial response to the treatment, and patients with stable or progressive disease. Together, these data indicate that these IO assays could be used in clinical trials to identify proteins that could be predictive biomarkers.

The monoclonal antibodies developed for this project are publicly available to the research community through the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium Assay Portal and Antibody Portal. “One of our goals, in addition to developing technologies for monitoring and measuring proteins, is to create resources for the community,” said Dr. Paulovich. “We make everything that we build available to the community through NCI portals that we helped to develop.” The Paulovich lab has developed thousands of validated, multiplexable, targeted MRM mass spec assays to quantify cancer-relevant proteins (e.g., proteins involved in the DNA damage response and the Ras/MAPK networks). These assays are run in the lab’s CLIA environment to support collaborative studies including basic, translational, and clinical research.

The IO-2 assay described in this paper is only the second in a series of a total of five IO panels that are under development in the Paulovich lab. “There are hundreds of immunomodulatory proteins in the tumor microenvironment, and we'd like to be able to characterize as many of those as possible as accurately as possible in our CLIA environment,” said Dr. Paulovich.

This work was supported by the National Cancer Institute, National Institutes of Health, NCI Beau Biden National Cancer Moonshot, NCI Clinical Proteomics Tumor Analysis Consortium, NCI Academic Industrial Partnership, NCI Research Specialist program, NCI CPTAC Antibody Characterization Program, Seattle Translational Tumor Research, Aven Foundation, and Merck Sharp & Dohme LLC.

The Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Drs. Stephen Smith, Ajay Gopal, and Amanda Paulovich contributed to this work.

Whiteaker JR, Zhao L, Schoenherr RM, Huang D, Lundeen RA, Voytovich U, Kennedy JJ, Ivey RG, Lin C, Murillo OD, Lorentzen TD, Colantonio S, Caceres TW, Roberts RR, Knotts JG, Reading JJ, Perry CD, Richardson CW, Garcia-Buntley SS, Bocik W, Hewitt SM, Chowdhury S, Vandermeer J, Smith SD, Gopal AK, Ramchurren N, Fling SP, Wang P, Paulovich AG. 2023. A multiplexed assay for quantifying immunomodulatory proteins supports correlative studies in immunotherapy clinical trials. Front Oncol. 13:1168710.