Purification and top-down proteomic analysis of PD-1 (programmed cell death 1 protein) from CD8+ T lymphocytes of patients with head and neck cancer

• Grant number: 23/08471-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2023
• End date: October 13, 2024
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Adriana Franco Paes Leme
• Grantee: Reynaldo Magalhães Melo
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 18/18496-6 - The role of alcohol treated-extracellular vesicles in oral cells transformation, AP.TEM

Abstract

Head and neck cancer (HNC) is the seventh most common type of cancer, with a survival rate of approximately 50% after 5 years. The checkpoint inhibitors nivolumab and pembrolizumab are used as immunotherapies in the treatment of HNC; however, this procedure has a suboptimal durable response rate (~15%). These inhibitors target the PD-1 protein (programmed cell death 1 protein), which is highly expressed in T lymphocytes and is capable of inhibiting the activation of these cells, and also affecting anti-tumor immunity. PD-1 has refined mechanisms of interaction, degradation, and activity regulations through post-translational modifications (PTMs), as well as being susceptible to alternative splicing. Top-down (TD) proteomics has an exceptional capacity for discovering PTMs and is considered the prime technique for characterizing proteoforms. Therefore, this project was designed to analyze the PD-1 proteoforms present on the membranes of CD8+ T lymphocytes from HNC patients, with or without lymph node metastasis, using TD proteomics. The analysis was conducted as follows: 1) global analysis of membrane proteoforms; 2) characterization of PD-1 proteoforms purified through membrane enrichment and liquid chromatography; 3) identification and quantification of immunopurified PD-1 proteoforms using different primary antibodies. These analyses will reveal the complexity of PD-1 proteoforms. This new perspective has the potential to discover proteoforms that impact the recognition of antibodies used in HNC immunotherapy, as well as identify regions in the protein with potential for the development of immunotherapies based on PD-1 inhibition. (AU)