Discovery and delivery of peptide-based immune checkpoint inhibitors targeting Pd-L1 for cancer immunotherapy
No Thumbnail Available
Authors
Meeting name
Sponsors
Date
Journal Title
Format
Subject
Abstract
The objective of this dissertation is to develop peptide-based immune checkpoint inhibitors that block the interaction between programmed cell death ligand-1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) for cancer immunotherapy. We recently discovered a low-molecular-weight linear anti-PD-L1 peptide using phage display. The peptide specifically blocks the PD-L1/PD-1 interaction and shows promising anti-tumor activity in a mouse model of colon cancer. However, like most peptides, a major impediment to the advancement of this native peptide is its short serum half-life and relatively low affinity compared to monoclonal antibodies. Therefore, we hypothesize that structural modification of the linear anti-PD-L1 peptide leads to derivatives with improved bioactivity and stability. Strategies like peptide truncation, D-amino acid substitution, cyclization, N-methylation, and unnatural amino acid incorporation were applied to optimize the activity and stability of the linear peptide. In the first Chapter, we briefly introduced the background of the dissertation research, cancer immunotherapy, peptide drug discovery, and strategies to optimize the pharmacokinetic properties of peptide drugs. We also presented the Statement of the Problems and Objectives. In the second chapter, we reviewed cancer immunotherapy, peptide drug discovery, and strategies to optimize the pharmacokinetic properties of peptide drugs. We also discussed a few peptides that have been successfully employed as checkpoint inhibitors for cancer immunotherapy. In the third chapter, we presented a detailed account on the discovery of cyclic peptide inhibitors of PD-L1 for cancer immunotherapy. Here, we applied the innovative strategy of macrocyclization scanning to improve the stability and activity of a linear anti-PD-L1 peptide. The cyclic peptides demonstrated up to a 34-fold improvement in the PD-1/PD-L1 blocking activity and a 4-fold improvement in serum stability. These cyclic peptides prevent apoptosis of human CD3+ T cells co-cultured with cancer cells and inhibits the proliferation of cancer cells co-cultured with human peripheral blood mononuclear cells (PBMCs). More importantly, the cyclic peptides significantly inhibit tumor growth in vivo even at a low dose of 0.5 mg/kg. Our results demonstrate that macrocyclization scanning is an effective way to improve the serum stability and bioactivity of the anti-PD-L1 linear peptide. This strategy can be employed in the optimization of other bioactive peptides, particularly those for blocking protein-protein interactions. The fourth chapter discussed the discovery of proteolytically stable and orally bioavailable peptidomimetic inhibitors of PD-L1 for cancer immunotherapy. Progress in the development of small molecule immune checkpoint inhibitors is not at pace with monoclonal antibodies due to inherent challenges associated with the application of small molecules as protein-protein interaction modulators. Most protein-protein interactions involve a relatively large surface area, typically from 1500 to 3000 Å2. On the other hand, small molecules form a much smaller surface (300 - 1000 Å2), thus making such modulation nearly impossible. By combining strategies like peptide truncation, alanine scanning, and unnatural amino incorporation, we developed small molecular weight peptidomimetics that exhibited biological activity like large biomolecules while maintaining the physicochemical properties of small molecules. These modified peptides effectively block the PD-1/PD-L1 interaction at sub-nanomolar concentrations. The modified peptide CJP117 showed the best blocking activity in the competitive surface plasmon resonance (SPR) assay with an IC50 of 90 nM and a blocking efficiency of 99.06 % at 10 µM. From a cell specificity assay using flow cytometry, these peptides demonstrated high specificity to PD-L1 overexpressing cells compared with PD-L1 deficient cells. The anti-PD-L1 peptidomimetics also suppressed cancer cell proliferation but reversed apoptosis of PBMCs cocultured with tumor cells. In a biodistribution study, we showed that Cy5-labeled anti-PD-L1 modified peptides accumulate in EMT-6 tumors orthotopically implanted in Balb/c mice. This data together with the in vitro specificity data suggest that the anti-PD-L1 peptidomimetics have very high target specificity, thus having a minimal tendency for off-target effects. The in vivo anti-tumor activity study demonstrates the high efficacy of the modified peptides as they significantly suppressed tumor growth at a low dose of 0.5 mg/kg when compared with saline and the negative control peptide. Pharmacokinetics studies of CJP117 revealed that CJP117 has good oral bioavailability. The reduced size, optimum lipophilicity, and improved proteolytic stability culminated in a modified peptide with high oral bioavailability. We have also demonstrated that oral administration of CJP117 at 5 mg/kg can suppress tumor growth in a CT-26 mouse colorectal cancer model. In the fifth chapter, we described the development of chitosan-coated liposomes for the oral delivery of CJP117 modified peptide. We assessed the surface coating of the liposome using dynamic light scattering (DLS) and SPR. DLS was used to examine the surface charge, while SPR was used to evaluate mucin binding. Our results showed that the surface charge of the liposomes changed from negative to positive after coating with chitosan. Also, mucin from porcine stomach was coated onto a CM-dextran SPR sensor chip using the EDC/NHS chemistry, and different dilutions of the liposomes were injected into the chip. The chitosan-coated liposomes showed higher binding to mucin when compared with the uncoated liposomes. This suggests that our liposomal formulation will have high binding to mucus in the gastrointestinal tract thus increasing the gastric residence time of the formulation and ultimately improving the bioavailability of the peptide. Transmission electron microscopy (TEM) and DLS data suggest that the liposomes have particle sizes of 130-200 nm.
Table of Contents
Introduction -- Literature review -- Discovery of cyclic peptide inhibitors targeting PD-L1 for cancer immunotherapy -- Discovery of orally bioavailable peptidomimetic inhibitors of PDL1 for cancer immunotherapy -- Development of chitosan-coated liposomes to orally deliver ANTIPD-L1 peptidomimetics for cancer immunotherapy -- Summary and future directions
DOI
PubMed ID
Degree
Ph.D. (Doctor of Philosophy)