Exploring multivalency and chemical modifications in the development of anti-PD-L1 peptide inhibitors for cancer immunotherapy
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Abstract
The PD-1/PD-L1 immune checkpoint axis is one of the most common pathways exploited by cancers to evade immune surveillance. Many cancers overexpress PD-L1 (programmed cell death ligand 1) on their surface through which they interact with PD-1 (programmed cell death protein 1) on activated immune cells, leading to suppression of the immune response. Significant efforts have been made in oncology research to block the PD-1/PD-L1 interaction. The most common approach is using monoclonal antibodies (mAbs) to competitively antagonize the PD-1/PD-L1 interaction. Among other qualities, the multivalent nature of antibodies (Abs) gives them a high affinity for targets and makes them attractive agents for blocking the PD-1/PD-L1 interaction. However, despite some successes achieved with anti-PD-L1/anti-PD-1 mAbs, there are growing number of reports of suboptimal responses. Additionally, many patients develop resistance and experience immune-related adverse effects due to drawbacks associated with Abs, such as low tumor penetration, high immunogenicity, and prolonged occupancy of target receptors. Over the past decade, peptides have garnered considerable interest as promising alternatives to address the limitations of existing anti-PD-L1 therapies and to overcome some of the drawbacks of mAb-based treatments. Compared to mAbs, peptides have better cell permeability, cost-effectiveness, and the ability for easy formulation for non-parenteral administration. However, the successful translation of anti-PD-L1 peptides is often hindered by challenges related to their limited stability and efficacy. Peptides are typically monovalent and exhibit lower affinities for targets compared to mAbs, which are multivalent. Our group recently discovered a 9-amino acid anti-PD-L1 peptide, TR3. Despite its reasonable potency, it still exhibits poor stability. In this study, we aim to mimic the multivalent nature of antibodies by designing multivalent anti-PD-L1 peptides and exploring how valency influences the blocking efficacy, potency, stability, anti-cancer effect, and tumor penetration. We also aim to optimize the sequence and structure of TR3 to develop more potent and stable TR3 derivatives. This involves modifying sidechains and backbones of the peptide. In the first chapter, we provide an overview of the dissertation research, highlighting checkpoint blockade-based cancer immunotherapy and the significance and challenges of peptides-based therapeutics as potential checkpoint blockade agents. We also present the problems this dissertation aims to address and discusses its objectives. Chapter 2 thoroughly explores the development of peptides as therapeutic agents, focusing on their pivotal role in cancer therapy. We further discuss cancer immunotherapy and the different modalities, especially checkpoint blockade therapy. The chapter concludes with an in-depth examination of the PD-1/PD-L1 pathway and critically reviews the different strategies used to block this interaction. In Chapter 3, we detail our efforts in developing multivalent anti-PD-L1 peptide inhibitors for cancer immunotherapy. We demonstrate that increasing the peptide valency improves its blocking efficiency, with the tetrameric peptide showing a 39-fold improvement over the original peptide. The inhibitory potential of the multivalent peptides was also demonstrated in co-culture experiments involving human peripheral blood mononuclear cells (PBMCs) and DU145 cancer cells. These peptides significantly suppressed cancer cell-mediated T-cell apoptosis, revitalizing T-cells to inhibit the proliferation of cancer cells. Notably, while increasing the valency improved the peptide’s activity, the magnitude of the increase tended to diminish with each subsequent increase in valency. Also, increasing the valency did not adversely affect the peptide’s stability, and the multivalent peptides exhibited similar stability profiles as the monomeric peptide. Peptides achieve good tumor penetration due to their relatively small sizes. A primary concern regarding multimerization of ligands is the corresponding size increase. Using a 3D tumor spheroid model, we showed that the multivalent TR3 peptides had comparable tumor penetration as the monomeric TR3. Additionally, TR3 and TR3-tetramer (TR3-T) exhibited similar tumor distribution profiles in biodistribution studies. However, the multivalent TR3 peptides had significantly higher binding to human serum albumin than the monomeric TR3. While this may be beneficial for achieving a prolonged anti-tumor effect, it could potentially reduce the amount of unbound peptide available for activity. This characteristic necessitates additional consideration when designing multivalent anti-PD-L1 inhibitors. Chapter 4 details our research in developing stable anti-PD-L1 peptides for cancer immunotherapy. In this study, we employed various peptide optimization strategies such as alanine scanning, sidechain optimization, and substitutions with D- and N-methyl amino acids to design peptidomimetic analogs of TR3. These modifications improved the stability of the resulting analogs by about 5-fold. The analogs also exhibited enhanced anti-PD-L1 blocking potency with IC₅₀ values of 355 nM, 359 nM, and 165 nM for TR3-29, TR3-30, and TR3-64, respectively, compared to TR3 with an IC₅₀ of 5,404 nM. The improved activity of the analogs was also evident in an in-vitro coculture experiment involving human PBMCs and DU145 cancer cells. The TR3 analogs showed a significantly higher effect in reducing T-cell apoptosis and inhibiting the proliferation of DU145 cells compared to TR3. Among the analogs, TR3-64 showed the best effect and was further multimerized into dimeric and tetrameric TR3-64 analogs (i.e., TR3-64D and TR3-64T respectively) with IC₅₀ values of 57 nM and 22 nM, respectively. TR3-64 also showed a significant effect in suppressing the growth of murine CT-26 colorectal tumors in a xenograft model. Despite their improved serum stability, the TR3-analogs still exhibited poor gastrointestinal stability. Hence, we used a D-amino acid scan and enzyme-targeted D-amino acid modifications to improve the stability of the peptides. We identified two candidates: TR3-114 and TR3-115, with good serum and gastrointestinal stability and blocking efficiency. TR3-114 and TR3-115 had blocking potency IC₅₀ values of 172 nM and 665 nM, respectively. The peptides also showed good serum stability with half-lives exceeding 24 hours.
Table of Contents
Introduction -- Literature review -- Development of multivalent Anti-PD-L1 peptides for cancer immunotherapy -- Development of stable Anti-PD-L1 peptidomimetics for cancer immunotherapy -- Summary and future directions
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Ph.D. (Doctor of Philosophy)