Abstract
The immune system possesses a remarkable ability to distinguish between “self” and “non-self” antigens to help maintain homeostasis. A key mechanism underlying this capability is the presentation of peptides on the major histocompatibility complex (MHC). This process is essential for mounting immune responses against infectious disease and cancer. Notably, many FDA-approved cancer immunotherapies, including checkpoint inhibitors and cancer vaccines rely on the recognition of MHC-displayed antigens by effector immune cells. However, the continued success of these therapies depends on the identification of conserved cancer antigens and a deeper understanding of the biological mechanisms governing antigen presentation.
Cancers can escape immune detection by eliminating cancer-specific antigens. One class of these antigens includes neoantigens, which are modified peptides presented on MHC that arise from tumor-specific alterations. Cancer cells can avoid immune recognition of their neoantigens by either (1) losing neoantigen expression or (2) downregulating MHC molecules, thereby reducing antigen presentation. Given the central role of antigen presentation in immune surveillance, understanding the mechanisms that drive neoantigen loss and MHC downregulation is critical for developing more effective immunotherapies. Additionally, understanding the biological pathways behind the generation of neoantigens also aids in identifying promising neoantigens to target. By elucidating these pathways, we can refine existing immunotherapies and identify new approaches to improve immune targeting of cancer cells.
Chapter 3 details the selective delivery of a neoantigen to cancer cells using a pH(low) insertion peptide (pHLIP). pHLIP selectively integrates into the membranes of cells in acidic environments and facilitates the transport of large molecules, including peptides, across the cell membrane. The acidic tumor microenvironment is a well-conserved feature across cancer, driven by the increased energy demand and the resulting production of lactic acid through glycolysis. Here, we demonstrate that pHLIP can efficiently deliver a model neoantigen across the cell membrane and into the antigen presentation pathway in a pH-dependent manner. Furthermore, brief treatment with the conjugate enables neoantigen-specific T cells to recognize the peptide-MHC complex, highlighting the potential of this strategy for enhancing immune targeting of tumors.
Next, we addressed the challenge of MHC-I downregulation, a common immune evasion strategy, by developing a screening platform to identify small molecules that enhance MHC-I surface expression. Loss of MHC-I is observed in various tumor types, including colorectal cancer, and is associated with resistance to checkpoint inhibition therapy. In Chapter 4, we identify promising chemical scaffolds that increase MHC-I display on colorectal cancer cells. Through screening diverse classes of compounds reported to upregulated MHC-I, we found that a purine-based scaffold derived from heat shock protein 90 inhibitors exhibited the most robust activity. To further optimize this scaffold, we employed a click-chemistry-based approach to rapidly generate and evaluate hundreds of derivatives, selecting those that enhance MHC-I expression while minimizing toxicity relative to the parent compound.
In Chapter 5, we investigated how enzymatic post-translational modifications (PTMs) influence the antigen presentation pathway. Peptides modified with PTMs represent a significant class of cancer-specific neoantigens. However, the molecular basis of their immunomodulatory effects remains poorly understood for most PTMs. We hypothesized that PTMs play a critical role in modulating peptide affinity for MHC-I as well as recognition by T cells through their T cell receptors (TCRs). To test this, we integrated experimental data with computational modeling to assess the impact of various PTMs on peptide-MHC-I binding affinity. Additionally, we observed substantial changes in TCR recognition of modified peptides, suggesting that TCR specificity is a key determinant in the immune response to modified self-antigens.
While most modified self-antigens presented on MHC-I arise from nonenzymatic modifications, their impact on the immune response remains less understood. In Chapter 6, we investigated how nonenzymatic modifications influence the antigen presentation pathway. Specifically, we assessed their effects on MHC-I binding affinity and TCR recognition. Additionally, we also identify key changes in peptide presentation between unmodified and modified cancer-associated MHC peptides. Finally, we are developing an enrichment strategy to map sites of nonenzymatic modifications displayed on MHC to provide insights onto the role of the modification on immune responses. This is particularly significant given that many inflammatory conditions involve the release of electrophilic molecules capable of nonenzymatically modifying proteins, potentially shaping immune recognition and response.
Finally, in Chapter 7, we examine how intracellular pathogens evade both immune defenses and antibiotic treatment. Bacteria have evolved strategies that allow them to survive within host cells, where they are shielded from immune effectors and antimicrobial agents. A key challenge in treating intracellular infections is determining whether reduced antibiotic efficacy stems from bacterial phenotypic changes or limited antibiotic accumulation within intracellular compartments. To address this, we developed a method to incorporate a bioorthogonal alkyne handle into the peptidoglycan of Staphylococcus aureus, enabling the quantification of azide-modified antibiotic accumulation. By comparing the kinetics of azide-modified antibiotic accumulation with alkyne-labeled peptidoglycan in S. aureus residing within macrophages, we aimed to elucidate the extent to which antibiotics reach intracellular pathogens.
