Mapping the cancer surface proteome in search of target antigens for immunotherapy

Abstract

Immune-based therapeutic interventions recognizing proteins localized on the cell surface of cancer cells are emerging as a promising cancer treatment. Antibody-based therapies and engineered T cells are now approved by the Food and Drug Administration to treat some malignancies. These therapies utilize a few cell surface proteins highly expressed on cancer cells to release the negative regulation of immune activation that limits antitumor responses (e.g., PD-1, PD-L1, CTLA4) or to redirect the T cell specificity toward blood cancer cells (e.g., CD19 and B cell maturation antigen). One limitation preventing broader application of these novel therapeutic strategies to all cancer types is the lack of suitable target antigens for all indications owing in part to the challenges in identifying such targets. Ideal target antigens are cell surface proteins highly expressed on malignant cells and absent in healthy tissues. Technological advances in mass spectrometry, enrichment protocols, and computational tools for cell surface protein isolation and annotation have recently enabled comprehensive analyses of the cancer cell surface proteome, from which novel immunotherapeutic target antigens may emerge. Here, we review the most recent progress in this field.

Keywords: antigen discovery; cell surface proteins; computational tools; immunotherapy; mass spectrometry.

Graphical abstract

Figure 1

Surfaceome chemical enrichment methods Cell surface biotinylation (dark green box): Cells or tissues are treated with reactive biotin esters, which label specific extracellular amino acid residues. Cell surface capture (CSC) (light green box): Cells are exposed to an oxidizing reagent, such as sodium metaperiodate, which oxidizes carbohydrates to produce di-aldehyde groups. These groups are then labeled with biocytin hydrazide. Peroxidase-mediated labeling (orange box): This method uses horseradish peroxidase (HRP) on the cell surface. The reaction is induced by modified biotin molecules and H₂O₂. HRP-linked antibodies or HRP fusion proteins activate reagents to react with cell surface proteins. Metabolic labeling of glycans with azido sugars (yellow box): Azido sugars passively diffuse through the cell membrane. Inside the cell, sugar is modified and exposed on the membrane. Enzymatic processes to examine the surfaceome (blue box): This technique employs a free-ranging biotin ligase, known as BioID, which is merged with a specific protein. When this combined entity is expressed in cells, it can initiate the biotinylation of proteins that are interacting and nearby over several hours, thereby creating a record of protein associations.

Figure 2

Proposed pipeline for target antigen discovery This figure illustrates a stepwise approach for identifying and validating cell surface proteins with therapeutic relevance. The generation of a comprehensive pool of candidates relies on surface proteomics studies and mass spectrometry and integration of RNA-seq data from primary patient samples or cancer cell lines. The purpose is to generate a wide range of potential cell surface protein candidates. The identification of cell surface molecules is based on computational datasets for cell surface molecule annotation based for instance on transmembrane prediction or manually curated repositories. The purpose is to select proteins highly likely localized at the plasma membrane. Normal and malignant tissue annotation serves to select therapeutically relevant candidates by annotating candidate expression in large panels of normal and cancer tissues. It can rely on flow cytometry, immunohistochemistry, or western blot validation analyses in primary tissues and cells. Investigations into the functional relevance of candidate targets can identify biologically and therapeutically relevant antigens and, thus, a therapy targeting such antigens may tackle the critical mechanisms of cancer survival. Targets that are essential for cancer cell survival are advantageous because they limit the opportunity for antigen escape. This workflow provides an approach to discovering and validating cell surface proteins that may have therapeutic significance, particularly in the context of targeted therapies.