Immunopeptidomics Overview

Introduction to immunopeptidomics

The major histocompatibility complex (MHC) plays a critical role in adaptive immunity through antigen presentation. MHC associated antigens derived from either endogenous or exogenous proteins are recognized by T cells and trigger an immune response. Mass spectrometry-based immunopeptidomics is a rapidly growing proteomics application that enables the identification of MHC associated antigens extracted from biological samples. Targeting MHC antigens isolated using mass spectrometry-based immunopeptidomics provides scientists with novel ways to predict antigens for applications such as cancer treatment, vaccine development, immunotherapy, and drug discovery.

While innate immunity activates a rapid, non-specific inflammatory response, adaptive immunity generates memory T and B cells to allow for a long-lasting, effective response to reinfection over the course of a lifetime. Adaptive immunity can occur after exposure to an antigen from either a pathogen or a vaccination. Antigens are described by where they originate; thus, autoantigens (or self-antigens) are derived from proteins produced within the body’s own cells, while non-self-antigens originate from outside the body and can be derived from external sources such as pathogens, toxins, or transplanted tissues. The ability of the immune system to differentiate between self and non-self-antigens is crucial for maintaining immune homeostasis and preventing autoimmune responses, where the immune system mistakenly targets self-antigens.

Helper T cells, killer T cells, and macrophages are the three main kinds of lymphocytes involved in cell-mediated immunity. When T cells recognize a foreign fragment attached to the MHC molecule, they bind to the MHC-peptide complex and activate an immune response. The maturation and differentiation of naïve T cells into helper or killer T cells are dependent on the binding specificity of MHC proteins to external antigens. The binding of T cell receptors (TCR) to either Class I or Class II MHC molecules directly influences differentiation of the selected cells into either CD4+ (helper) or CD8+ cytotoxic effector and memory cells (killer) T cells, to mediate a direct immune response. The ligand for the TCR is always a peptide bound to an MHC molecule; the CD8 receptor on a cytotoxic T cell can only bind to MHC-I, and the CD4 receptor on a helper T cell can only bind to MHC-II.

Peptides that bind to Class I MHC molecules are usually derived from intracellular proteins (i.e., cytoplasmic) and are generated by the proteasome, transported to the endoplasmic reticulum, loaded onto MHC molecules, and displayed on the cell surface to circulating T lymphocytes. The peptide/MHC-I complex binding to a TCR triggers a cytotoxic response. On Class II MHC molecules, the bound peptides are usually derived from extracellular antigens generated by endosomal proteolysis of proteins after they are endocytosed by specialized antigen presenting cells such as B cells. TCR recognition of such peptide/MHC-II complexes triggers the release of cytokines, creating an inflammatory response among the cells, such as antibody secretion.

While the central role of T cells is to stimulate both humoral and cellular immune responses, it is crucially important that T cells do not react with self-proteins. In healthy cells, MHC molecules present self-peptides, and no reaction occurs from T cells. B cells usually require help from T cells to secrete antibodies. Any given B cell whose receptor mutates to become self-reactive would, under normal circumstances, fail to make antibodies due to lack of self-reactive T cells, which would provide the helper cells to fight the immunity.

Autoimmune disease is caused by autoantibodies and/or autoreactive T cells targeting self-antigens. Polymorphisms, epigenetic modifications, and post translation modifications (PTMs) lead to altered gene expression and function, which in turn can change peptides, leading to abnormal T cell responses that underlie autoimmune disease. Common autoimmune disorders such as celiac disease, type-1 diabetes, inflammatory bowel disease (IBD), multiple sclerosis, autoimmune thyroid disease, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been linked to the immune system mistakenly attacking its own tissues. Mass spectrometry-based immunopeptidomics provides a valuable mechanism for identifying MHC ligands that contain alterations such as PTMs, providing insights into both the pathogenesis and therapeutic options of autoimmune diseases.

The immunopeptidome consists of a complex mixture of peptides at varying concentrations but of similar length, complicating immunopeptidomic analysis. Primary cells and tissue, cell lines, and disease state tissue can be lysed to release MHC-associated peptide complexes into solution for purification. Detecting and identifying MHC-bound peptides on cell surfaces can be challenging due to their low abundance, making it difficult to effectively measure and identify these peptides using traditional working concentrations for mass spectrometry analysis. Thus, immunopeptidomic analysis requires a large amount of peptides from starting tissue material in addition to specialized software to identify the bound peptides that can analyze up to thousands of peptide sequences simultaneously.

To combat these barriers to sample prep, immunopeptidomic samples are generally prepared by isolating desired peptides bound to MHCs using an antibody (allele-specific or pan) or an engineered affinity tag system from lysed cells or tissues to concentrate the sample peptides. In addition to using an immunoaffinity column equipped with MHC or HLA antibodies to capture the molecules and associated peptides or a specific subtype, magnetic and agarose or Sepharose beads with Protein A, G, and A/G can be used to support immunoaffinity purification.

Targeted and non-targeted data acquisition mass spectrometry can be employed in immunopeptidomics. Scientists may require discovery (or non-targeted data acquisition) using label-free quantitation, TMT isobaric labeling or metabolic SILAC labeling techniques. Scientists may also opt to use targeted data validation methods by mass spectrometry to evaluate peptides for applications such as drug discovery. Once MAPs are identified in discovery, targeted quantitation using mass spectrometry for validation of the peptide antigens using products such as AQUA custom peptides or SureQuant assay kits can be performed to complete the analysis.

Immunopeptidomics aids in understanding disease pathogenesis, particularly autoimmune disease and cancers, by using mass spectrometry to quantitatively analyze MHC associated peptides in the immunopeptidome. This approach enables the identification of peptide antigens presented during immune responses or disease states by comparing them against protein sequence databases. Immunopeptidomics has made significant contributions to immunotherapy and vaccine development for cancer by aiding in the development of personalized treatment approaches through the identification of patient tumor specific antigens. Identifying peptide antigens that stimulate a robust immune response also contributes to the generation of effective vaccines against infectious disease. Analysis of the immunopeptidome offers valuable insights for precision medicine, immunotherapy, and advancing the understanding of immune-related diseases.

Characterization for cancer immunotherapy

Identifying and characterizing the immunopeptidome of cancerous tissue enables the discovery of novel tumor specific antigens (TSAs) which are used as targets for cancer immunotherapy. To develop immunotherapies against specific peptides, relevant targets of a tumor are identified using T cell epitopes. Studying the MHC-peptide complexes presented by tumor cells can provide insight into the immune responses against cancer and aid in cancer biomarker identification.

Immunopeptidomics allows for the identification of neoantigens, which are novel peptide antigens derived from mutated proteins that are specifically expressed by cancer cells but not found in normal (control) cells. Since the expression of neoantigens can vary between different cancer types and individuals, their identification and characterization are important for developing personalized immunotherapies, helping lead to improved treatment outcomes. Analysis of the peptide repertoire over time can assess the effectiveness of immunotherapies for tumors, monitor treatment response, discover biomarkers for early cancer detection, and help predict patient outcomes.

Immunopeptidomic studies have been used to identify HLA Class I peptides that are more abundant in triple negative breast cancer samples than normal breast tissue. These peptides are being further validated as targets for cancer vaccines and T cell immunotherapy. Immunopeptidomic analysis is also being used to profile epithelial cells biopsied from ovarian tumors to identify MAPs specifically expressed in cancerous tissue to aid in the development of immunotherapies. Immunopeptidomics brings significant improvements to immunotherapy applications by providing the specificity for minute peptide changes required for detecting de novo mutations and creating personalized medicine that CAR-T therapy affords for better patient outcomes, improving the lives of cancer patients.

Immunopeptidomic analysis aids in vaccine development

Immunopeptidomics has played a significant role in combating infectious disease. The identification of peptides presented by MHC molecules on pathogen infected cells can reveal bacterial and viral antigens as candidates for vaccine development. Immunopeptidomic analysis of pathogen infected cells has identified T cell epitopes for the bacteria Mycobacterium tuberculosis and Chlamydia trachomatis, and the parasite Leishmania major. More recently, this method helped identify viral T cell epitopes and was used to develop potential HIV and SARS-CoV-2 vaccines.

To develop effective cancer vaccines, the identification of epitopes recognized by T cells is crucial. Immunopeptidomics has been a key application in cancer vaccine development, providing an avenue where tumor specific antigens are extracted from biopsied tissues and analyzed. To develop a vaccine targeting cancer cells, several steps are involved.

1. Affinity purification and peptide identification: Immunoprecipitation and tandem mass spectrometry are used to isolate MHC-restricted tumor peptides, then quantify the peptides to help identify cross-reactive T cells for functional characterization of candidate peptides, which should elicit a strong immune response.
2. Validate candidate peptides: Using RNAseq and mass spectrometry analysis, scientists can characterize these key MHC-restricted peptides, then validate and quantify those candidate peptides.
3. Validate candidate peptides for carrier binding (i.e., characterize the binding affinity of the peptides to the oncolytic adenovirus).
4. Assess tumor reactivity to peptides: Determine if tumor tissue growth is impeded.

By analyzing the tumor peptide repertoire, specific peptide signatures or patterns can be identified that correlate with disease progression or treatment response. Immunopeptidomics is used to help improve vaccine development to help cover a wide population of associated peptides with specific antigen expression. Recent research has propelled immunopeptidomics to the forefront for personalized oncolytic cancer vaccines.

Role of PROTACs in MHC peptide presentation

Peptides presented by MHC Class I molecules are derived from endogenous proteins that are degraded through the ubiquitin-proteasome pathway. Targeted protein degradation through proteolysis targeting chimeras (PROTACs®) has been shown to induce changes in the expression of MHC Class I peptides. Some of these peptides originate from the specific protein targeted for degradation, potentially identifying new antigen targets for T cell immunotherapy and increasing the ability of PROTACs to elicit a biological response toward undruggable targets. PROTACs can also be used to generate MHC peptide antigens from mutated proteins that are not easily degraded by the ubiquitin proteasome system, enabling the discovery of additional neoantigens for cancer immunotherapy.

PROTAC® is a registered trademark of Arvinas Operations, Inc.