Multimodal blood based profiling reveals insights into mechanisms of immunotherapy resistance

Abstract Many cancer patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses. Circulating biomarkers have the potential to identify patients with primary resistance or early progression on therapy to alter treatment course and avoid unnecessary toxicity. Unbiased multimodal proteomic profiling in blood has been underexplored due to the previously limited scalability of multiplexing technologies or cohorts lacking time-series sampling. To address this, we performed plasma proteomic profiling of >2,900 proteins and high-dimensional mass cytometry of peripheral blood lymphocytes across serial time points in 250 metastatic melanoma patients on ICB treatment. We further obtained 92 patient-matched tumor samples, which were processed for single-cell and/or bulk RNA sequencing. Proteins upregulated post-ICB were associated with inflammatory pathways involving the activation of effector immune functions. Expression of genes corresponding to these proteins was higher in immune cells involved in recruitment and tumor reactivity. Expression of genes corresponding to plasma proteins more abundant in non-responders was highest in suppressive myeloid subsets and malignant cells. We further posit the involvement of these non-responder genes in immunosuppressive and pro-tumor interactions, which we confirmed using publicly available spatial transcriptomic data. We also found that epithelial-specific proteins in the circulation of responders post-ICB correlate with patient toxicity and likely originate from healthy tissues. Together, these data represent one of the deepest peripheral biomarker studies using paired blood and tumor samples in melanoma patients treated with ICB, and begin to elucidate the complex interplay between tumors and the systemic immune response within the host. Competing Interest Statement RWJ is a member of the advisory board for and has a financial interest in Xsphera Biosciences Inc., a company focused on using ex vivo profiling technology to deliver functional, precision immune-oncology solutions for patients, providers, and drug development companies. RWJ has received honoraria from Incyte (invited speaker), G1 Therapeutics (advisory board), Bioxcel Therapeutics (invited speaker). RWJ has an ownership interest in U.S. patents US20200399573A9 and US20210363595A1. RWJ's interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. NH holds equity in and advises Danger Bio/Related Sciences, is on the scientific advisory board of Repertoire Immune Medicines and CytoReason, consults for Site Tx, owns equity and has licensed patents to BioNtech, and receives research funding from Bristol Myers Squibb, Moderna, Takeda and Calico Life Sciences. AM has served a consultant/advisory role for Third Rock Ventures, Asher Biotherapeutics, Abata Therapeutics, Clasp Therapeutics, Flare Therapeutics, venBio Partners, BioNTech, Rheos Medicines and Checkmate Pharmaceuticals, was formerly an Entrepreneur-in-Residence at Third Rock Ventures, is currently a Venture Partner for The Column Group, is a co-founder of Monet Lab, an equity holder in Monet Lab, Clasp Therapeutics, Asher Biotherapeutics and Abata Therapeutics, and has received research funding support from Bristol-Myers Squibb. GMB has sponsored research agreements through her institution with Olink Proteomics, Teiko Bio, InterVenn Biosciences, Palleon Pharmaceuticals, Astellas, AstraZeneca. She served on advisory boards for Iovance, Merck, Moderna, Nektar Therapeutics, Novartis, Replimune, and Ankyra Therapeutics. She consults for Merck, InterVenn Biosciences, Iovance, and Ankyra Therapeutics. She holds equity in Ankyra Therapeutics. All other authors have nothing to disclose. Funding Statement We would like to thank funding support from the Prostate Cancer Foundation Young Investigator Award (RJP), Dana Farber Cancer Institute / Harvard CancerCare GI SPORE Career Enhancement Award (AM), Sky Foundation Pancreatic Cancer Research Grant (AM), Doris Duke Charitable Foundation Physician Scientist Fellowship (AM) and DF/HCC K12 (K12CA087723) Paul Calabresi Award for Clinical Oncology (AM). We acknowledge funding provided by the Massachusetts Life Sciences Center Research Infrastructure Program in support of the Mass General Cancer Center Tumor Cartography Center and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committe / IRB of Massachusetts General Hospital gave ethical approval for this work. We have a protocol (11-181) that received approval from IRB. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present work are contained in the manuscript