Discovery of Anti-CD47 Peptides for Cancer Immunotherapy and CD47-mimicking Peptides That Inhibit Phagocytic Clearance of Nanoparticles

In the last few decades cancer immunotherapy has emerged as a standard pillar of cancer treatment. Immune checkpoint inhibitors that are currently available target the adaptive immune system, generating T-cell antitumor responses. As critical innate immune cells, macrophages play important roles in homeostasis, preventing pathogen invasion, eliminating tumor cells, and promoting the adaptive immune system responses. Innate immune checkpoint inhibitors targeting innate immune cells can block inhibitory interactions (“don’t eat me” signals) between tumor and both phagocytes and natural killer cells. CD47 is overexpressed in various cancers which act as a potent ‘do not eat me’ signal that contributes to immune evasion of cancer cells. Innate checkpoint inhibitors including CD47/SIRPα antagonists, are now being developed as new strategy for cancer immunotherapy. In addition, CD47 is also known as ‘‘marker of self’’, as it is naturally presented by all cells in the body especially RBCs. This feature was first demonstrated by the rapid clearance of CD47-deficient mouse RBC when they were injected into wild-type mice. There are two major research objectives in this dissertation. The first research objective is to discover anti-CD47 peptides via phage display biopanning that can block CD47/SIRPα interaction to facilitate the macrophage-mediated phagocytosis of tumor cells. These anti-CD47 peptides can be promising innate immune checkpoint inhibitors that can be combined with chemotherapy or other immune checkpoint inhibitors. The second research objective is to discover CD47-mimicking peptides that can bind to SIRPα and provide “don’t eat me signal” to mitigate the impact of immune system on biomaterials conjugated to these peptides. These mini-CD47 peptides can be conjugated to nanoparticles and provide a novel way to inhibit the phagocytic clearance and prolong the circulation time of nanoparticles. Chapter one introduces the background of the dissertation research and presented the statement of problems and objectives. Chapter two reviews immune checkpoint inhibition and the various applications of targeting CD47/SIRPα pathway for cancer immunotherapy and drug delivery. Chapter three discusses the discovery of anti-CD47 peptides using phage display biopanning for cancer immunotherapy. Anti-CD47 peptides that can bind to CD47 and block CD47/SIRPα interaction were identified using novel phage display biopanning strategy. The specificity and blocking efficiency of the peptides to CD47 were examined using various assays. The peptides enhanced macrophage-mediated phagocytosis of human lung carcinoma NCI-H82 tumor cells in macrophage: tumor cell in vitro coculture system. The peptides had no effect on aggregation of RBCs and showed good proteolytic stability in human serum. Peptide CMP-22 demonstrated the ability to significantly increase the antitumor activity of doxorubicin and extends survival of Colon Tumor #26 (CT26) tumor-bearing mice. Our study demonstrated the feasibility of using phage display to discover new innate immune checkpoint inhibitors. Our results also suggested that the anti-CD47 peptides represent a promising innate immune checkpoint inhibitors to promote macrophage-mediated phagocytosis for cancer immunotherapy. Chapter 4 describes the discovery of CD47-mimicking peptides that can bind to SIRPα to provide “don’t eat me signal” that can inhibit the clearance by macrophages. Some studies have shown that coating nanoparticles with the whole CD47 (50 kDa) protein have shown to inhibit the phagocytic clearance of these nanoparticles and prolong the circulation time. It would be more challenging to use the entire CD47 protein due to its large size and the need of a proper protein folding for optimal function. CD47-mimicking peptides might be a good alternative for the stealth functionalization of nanoparticles to prolong their circulation time. Using phage display biopanning, CD47-mimicking peptides were discovered that can bind to SIRPα and inhibit its interaction with CD47. Two peptides (CMPS-4 and CMPS-5) have shown to block CD47/SIRPα with IC₅₀ of 118.4 and 580 nM respectively. These CD47-mimicking peptides are promising strategy to inhibit phagocytic clearance and prolong the circulation time of nanoparticles.

Name thesis:degree_name

Ph.D. (Doctor of Philosophy)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Pharmaceutical Sciences (UMKC)

Grantor

University of Missouri--Kansas City

Year dc:date.issued

2022

Author dc:creator

• Mustafa, Bahaa

Advisor dc:contributor.advisor

• Cheng, Kun (Professor)

Handle dc:identifier.uri

https://hdl.handle.net/10355/90561

OAI identifier oai:identifier

oai:mospace.umsystem.edu:10355/90561