Synthesis Of PD-L1 And PD-1 Targeting Peptide-Polymer Conjugates And Their Anti-Tumor Applications

Immune checkpoint blockade based on targeting programmed death 1（PD-1）/programmed death-ligand 1（PD-L1）has been used in the treatment of a variety of cancers.However,the monoclonal antibodies used in the clinical practice place a double burden on patients physically and economically,due to problems including complicated preparation process,low response rate,and systemic side effects.Compared with macromolecular antibodies,peptides have the advantages of low synthesis cost,high biological safety,low immunogenicity,and high tissue and tumor penetration.However,most peptide drugs have short circulation time in vivo,less tumor accumulation,poor inhibition of the targeting protein in vivo,and other problems that affect the interaction with the targeting protein and weaken the antitumor immune effects.On the one hand,peptides are attached to polymers to construct different types of peptide-polymer conjugated drugs,endowing peptides with longer circulation time,inhibition of target protein with higher efficiency,and some new functions（such as adhesion and bridging cells）,which is a feasible strategy to address the bottleneck problem of peptide-based immune checkpoint inhibitors.On the other hand,in order to further improve the anti-tumor efficacy of patients,chemotherapy and immunotherapy are usually combined in clinical practice.However,the type of chemotherapy drugs has a great impact on the effect of combined therapy.The combination of ill-suited chemotherapeutics with immune checkpoint inhibitors may cause serious adverse reactions in patients.As a result,it is another key issue to choose the appropriate combination therapy of chemotherapeutics and peptide drugs for safe and efficient synergistic anti-tumor immunotherapy.Given the problems of short circulation time in vivo,low tumor accumulation,and poor immunotherapeutic effect of peptides,a tumor microenvironment-associated reactive oxygen species（ROS）responsive polymer with PDL1-bound peptide（PDLlpep）conjugated（PDLlpep-PAP）was prepared to form a synergistic drug delivery system（PDL1pep-PAPM@PTX）for combined cancer immunotherapy and chemotherapy.On the one hand,PDL1pep-PAPM can bind to PD-L1 protein on the cell surface in the form of polyvalent to drive lysosomal degradation of PD-L1.On the other hand,in response to high ROS levels in the tumor environment,the nanomicelle structure of PDLlpep-PAPM@PTX dissociates and rapidly releases PTX,thereby effectively killing tumor cells.The synergy between PTX-based chemotherapy and PD-L1-blockade-based immunotherapy significantly increased the infiltration of cytotoxic T cells in tumors and increased cytokines that activate antitumor immunity,achieving safe and efficient anti-tumor effects in triple-negative breast cancer models.Secondly,to solve the problem that peptide-polymer conjugates based on a single peptide cannot shorten the distance between effector T cells and tumor cells in space to help effector T cells in killing tumor cells more efficiently,the idea of bispecific peptide-polymer conjugate was proposed that utilize two targeting peptides for dualtargeting two types of receptors in target-effector cells.In the second part,a bispecific peptide-polymer conjugate was prepared with PD-1-targeted peptide（PD1pep）and PD-L1-targeted peptide（PDL1pep）conjugated to the eight-armed PEG polymer for dual targeting PD-1 and PD-L1 on tumor cells and lymphocytes,respectively.The bispecific peptide polymer ^octaPEG-PD1-PDL1 targeting PD-1/PD-L1 has been shown to have an affinity for PD-1 and PD-L1 proteins.^octaPEG-PD1-PDL1 can bind to tumor cells and lymphocytes through PD-1 and PD-L1 proteins,shortening the distance between T cells and tumor cells in time and space by increasing their bridging,thus enhancing the killing of T cells on tumor cells.In vivo experiments confirmed it has excellent tumor accumulation with biological safety.^octaPEG-PD1PDL1 can effectively reverse the tumor immunosuppressive microenvironment and exert efficient anti-tumor immune efficacy against CT26 tumor models.Finally,to explore the effects of PEG linkers and peptides with different grafting rates on bispecific peptide-polymer for binding cell surface receptors,bridging targeteffector cells,and the effects of effector T cells,two types of peptide-polymers using PAMAM as the carrier linked PEG or not,and with different proportions of PD1pep and PDL1pep peptide grafting were designed and synthesized.One type is the bispecific peptide-polymer of PD1pep and PDL1pep directly bounded to the amineterminated PAMAM in different proportions（named PAMAM-PD1H-PDL1H,PAMAM-PD1M-PDL1M,PAMAM-PD1L-PDL1L according to the grafting rate of PD1pep and PDL1pep from high to low）.The other type is PD1pep and PDL1pep bounded to PEG-terminated PAMAM in different proportions(named PAMAMPD1₂₀-PDL1₂₀,PAMAM-PD1₁₀-PDL1₁₀,PAMAM-PD1₅-PDL1₅ according to the grafting rate of PD1pep and PDL1pep from high to low).Their effects on cell adhesion,bridging between tumor cells and lymphocytes,and inducing lymphocytekilling tumor cells were studied in detail.Meanwhile,the anti-tumor effect of bispecific peptide-polymer and the combination with chemotherapy were also explored by CT26 subcutaneous tumor models.In summary,peptide-polymer conjugates of different systems were constructed from the conjugation of a single type of peptide to degrading PD-L1 to the conjugation of two types of peptides for bi-targeting tumor cells and lymphocytes.In addition,the effect of peptide-polymer-based immunotherapy was enhanced by the combination of chemotherapy.