Construction And Anti-tumor Effects Of A Novel Bispecific Fusion Protein Targeting PD-L1 And CD47

Tumor immune escape was one of the crucial hallmarks of cancer.The immune system recognizes and is poised to eliminate cancer but is held in check by inhibitory receptors and ligands.Cancer immunotherapies could enlist natural immune system functions to fight tumor cells.As monoclonal antibodies are homogeneous in structure and binding specificity,more than 30 mAbs targeting tumor antigens(EGFR?Her2?VEGF,CD19,CD20)were approved for cancer treatedment.In recent years,preliminary clinical findings with mAbs targeting immune-checkpoint proteins,such as CTLA-4(Iplimumab)? PD-1(Nivolumab ? Pembrolizumab),PD-L1(Avelumab ? Atezolizumab,Durvalumab),CD47(CV1,Velcro-CD47),indicate broad and diverse opportunities to enhance antitumour immunity with the potential to produce durable clinical responses.Therefore,Science magazine ranked checkpoint protein antagonists,representative of cancer immunotherpy,as the number one breakthrough in all scientific disciplines for 2013.PD-1(Program Death-1)expressed on effctor T cells participate in peripheral tolerance and play a key role in immune suppression in cancer and chronic infectious diseases.When binding to its ligand PD-L1 which is highly expressed on the surface of tumor cells,PD-1 could send to the immune systema “Don't find me” signal,which inhibits T cell-mediated immue response and contributes to tumor immune evasion.Thus far,anti-PD-1/L1 therapy has generated significant clinical benefits by inducing regression of advanced and metastatic tumors and improving survival in patients.However,each treatment modality has limitations,treatment with immune checkpoint blockade achieved about only a 30% objective response rate(ORR)in multiple clinical trials.Therefor,the PD-1/PD-L1 antibodies are being combined with other anticancer agents such as chemotherapy,targeted therapy,radiotherapy,and other immunotherapy.CD47 belongs to the immunoglobulin superfamily,and is expressed as a 50 kDa cell surface antigen in wide variety of tissues,which serves as both a receptor for thrombospondin(TSP)and a ligand for the transmembrane signal regulatory protein SIRP?.CD47 has a number of different functions such as platelet activation,cell motility,leukocyte adhesion and migration.Moreover,CD47 transmits an anti-phagocyticsignal,known as the “Don't eat me” signal,to macrophages upon engaging its receptor signal regulatory protein(SIRP?).Recently studies revealed that CD47 is overexpressed on cancer cells,and CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer.As all human solid and blood tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination,CD47 is therefore a validated target for cancer therapies.However,these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47.These factors reduce bioavailability of anti-CD47 mAbs and increase the risk of blood toxicity in vivo.In our preliminary studies,it was found that CD47 and PD-L1 were often co-overexpressed on tumors in vivo,they may suppress both innate and adaptive immune response and thereby evade immune surveillance.Thus,we proposed that targeting both innate and adaptive immune checkpoints would likely maximize anti-tumor therapeutic effectand elicit more durable responses.Atezolizumab,an engineered IgG1 m Ab that targets PD-L1 and blocks the interaction between PD-1 and PD-L1,was approved by FDA for cancer patients with locally advanced or metastatic malignancies;CV1 was engineered high-affinity SIRP? variants monomers with about a 50,000-fold increased affinity for human CD47 relative to wild-type SIRP?,which exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosisin vitro and enhancing antitumor responses in vivo.Based on the Fab region of Atezolizumab and CV1 monomer,we constructed a bispecific fusion protein targeting both CD47 and PD-L1 using “Knobs-into-Holes” technology,denoted as IAB.It was effective in inducing the phagocytosis of tumor cells and stimulating T cell activation in vitro.Meanwhile,IAB exhibited potent antitumor activity in immune-competent mouse model.Moreover,the anti-tumor effect of IAB was impaired by anti-CD8 antibody or clodronate liposomes,which implied that both CD8+ T cells and macrophages were required for the anti-tumor efficacy of IAB,and IAB play an essential and sufficient role to engage innate and adaptive immune responses.Colletively,the strategies used in this study open up avenues for synergistic innate and adaptive immune responseagainst tumors,which may have great impact in antitumor drug discovery,and the IAB targeting PD-L1 and CD47 was also a potential drug candidate for cancer treatment.