Development Of Anti-OX40 Monoclonal Antibody And Anti-CD47 Monoclonal Antibody

Tumor immunotherapy is a major direction of monoclonal antibodies.The clinical trails for immune checkpoint antibodies represented by the PD1-PDL1 pathway had achieved great success,and made the development of antibody drugs which target to immune cells a hot spot.Among those immune checkpoints,OX40 and CD47 had been progressively verified as an effective target.OX40(also known as CD134,TNFRSF4,ACT35)is a costimulatory molecule which express mainly on the activated CD4+ CD8+ T cell surface.The OX40 signal can activate NF-?B,PI3 K and PKB pathways,and the continued activation of these pathways ultimately prolong the survival of T cells and promote the proliferation of T cells.OX40 is widely expressed on T cells locating in the microenvironment of chronic inflammatory and tumor.OX40 L,the ligand of OX40,can promote the activation of B cells and the production of antibodies.Both together can transmit bi-directional signal between activated T cells and B cells,thus the OX40-OX40 L pathway has a strong biological function.Therefore,the development of anti-OX40 antibody on tumor therapy have eseential practical significance.CD47(also known as Integrin,Ovarian cancer marker)is an immune checkpoint molecule which expresses on the surface of a variety of tumor cells and acts on macrophages and DC cells.It can bind SIRP? which is on the surface of the macrophages to pass the "Don't eat me" signal to achieve immune evasion.Blockaed of CD47-SIRP? pathway can not noly promote macrophage phagocytosis of tumor cells,but also improve the immune presentation of tumor-specific antigen to T cells,and activate both innate immune response and adaptive immune response.This study focused on the discovery and evaluation of immune anti-tumor antibodies.On the one hand,we tried to use large fully human single-chain antibody phage library to obtain anti-OX40 agonistic antibodies to provide new candidate molecules for the tumor immune therapy.On the other hand,for an anti-CD47 functional antibody that already obtained in our lab,we explored its application when combined with other targeted antibodies in anti-tumor therapy.The first part of the study was the discovery of anti-tumor antibodies.We choose OX40 as the target antigen and screened for anti-OX40 antibody relying on the large fully human single-chain antibody phage library constructed in our lab.After 3 rounds of screening,we got 8 positive clones and one of them got well enriched.All positive clones were transformed from scFv to the fully human antibody to form recombinant plasmids.Recombinant plasmids were transfected into HEK293-F and fully human antibodies were pured from supernatant based on the AKTA system.Then the properties of the fully human antibodies were verified by the following methods.First,the binding activity of the fully human antibody to the antigen OX40 at the molecular level was verified by ELISA.Second,the binding activity of the fully human antibody to the cell surface antigen was verified by indirect immunofluorescence.Third,the binding activity and specificity of the fully human antibody to the cell surface antigen was verified by flow cytometry.Fourth,using ELISA to determine the specificity of the fully human antibody.Fifth,the serum stability of the fully human antibody was verified by serum placement experiment.Sixth,the affinity of fully human antibodiy was determined by BIAcoreTM3000.The results showed that 8 single-chain antibodies were obtained after 3 rounds of solid screening,among which sc Fv-1 reached 95% enrichment in the screening process.There are 5 scFvs were transformed from scFv to the fully human antibodies successfully and got good expression and purification.After the studies of the characteristics of the antibodies,we found that OX40mab-4,OX40mab-8 had bad binding activities with antigen,and OX40mab-5,OX40mab-6 had bad specifities while OX40mab-1 had the best antigen-binding activity with an EC50 of 0.015 ?M.In addition,OX40mab-1 also had good specificity and serum stability,the affinity of OX40mab-1 was 251 nM.Thus,OX40mab-1 was identified as a candidate antibody molecule for subsequent studies.The second part of the study was the evaluation of immune anti-tumor antibodies.Based on the fully human antibody AMMS4-G4 which already obtained in our lab,we explored the efficacy of AMMS4-G4 in combination with other targeted antibodies on the tumor models.AMMS4-G4 can block the binding of CD47-SIRP?,and can effectively promote macrophage phagocytosis of tumor cells.In vivo experiments confirmed that AMMS4-G4 could prolong the survival of leukemia model mice significantly,and remove the leukemia cells of the model mice when it was administered alone.Therefore based on the above background,we explored the efficacy of AMMS4-G4 in combination with other targeted antibodies in the tumor models,and expected to obtain a new approach to the application of AMMS4-G4 in tumor therapy,laying the foundation for clinical treatment.In this study,another three targeting antibodies were selected: A2C1,Herceptin and Rituximab.A2C1 is another blocking antibody obtained in our lab that targets human EGFR and is undergoing clinical trials for human colorectal cancer.Herceptin is a humanized monoclonal antibody targeting human HER2,it is primarily used for the treatment of HER2-positive metastatic breast cancer clinically.Rituximab is a monoclonal antibody targeting human CD20,it is primarily used for the treatment of CD20-positive Non-Hodgkin's lymphoma clinically.Based on the characteristics of AMMS4-G4 and the other three targeting antibodies,the following tumor models were constructed.The first one was the Lovo tumor in nude mice model which was one of the human colon cancer model and expressed both human CD47 and EGFR.Aiming to investigate the therapeutic effect of AMMS4-G4 and A2C1 in combination.The second was the Skov3 tumor in nude mice model which was one of the human breast cancer model and expressed both human CD47 and HER2.Aiming to investigate the therapeutic effect of AMMS4-G4 and Herceptin in combination.The third was the Raji tumor in NSG mice model which was one of the human Non-Hodgkin's lymphoma model and express both human CD47 and CD20.Aiming to investigate the therapeutic effect of AMMS4-G4 and Rituximab in combination.The evaluation of combination therapies were mainly carried out by the following aspects.First,the binding activity of the antibody with the associated antigen of the tumor cell was detected by flow cytometry.Second,tumor mice were inoculated with tumor cells and grouped to establish tumor-bearing mice models.Third,the drugs were administrated in tumor-bearing mice and the tumor volume was measured and the clinical symptoms were observed.Fourth,according to tumor growth,stopped the administration and continued to observe,then the mice were euthanized,and the tumor weight were analyzed statistically and the efficacy of the combination therapy was evaluated.Fifth,we explored the pharmacological mechanisms of combination therapies.The results showed that in the Lovo tumor model,the combination therapy of A2C1 combined with AMMS4-G4 at the low dose(12.5 mg / kg)have a better efficacy when compared to A2C1 or AMMS4-G4 either alone.At the same time,although the efficacy of combination group was not significantly different from the A2C1 single administration group at high dose(25 mg / kg),but still showed better tumor suppressing tendency.The pharmacokinetic mechanism may be that A2C1 can cooperate with AMMS4-G4 to down-regulate the expression of CD47 on Lovo cells.Besides,AMMS4-G4 may be accumulated in tumor tissue which same to A2C1.In the Skov3 tumor-bearing model,although the efficacy of combination therapy was not significantly improved compared to Herceptin or AMMS4-G4 either alone,but still showed better tumor suppressing tendency.And the efficacy was significantly better than the control antibody hu IgG,and almost equal to the commercial antibody Herceptin when AMMS4-G4 was administrated alone.Similarly,in the Raji tumor-bearing model,AMMS4-G4 had good efficacy when it was administrated alone and can significantly inhibits the transfer of tumor cells to bone marrow.In summary,this study had mainly done two tasks in the discovery and evaluation of anti-tumor monoclonal antibodies.First,monoclonal antibodies targeting the costimulatory molecule OX40 were screened.After 3 rounds of screening,an anti-OX40 monoclonal antibody with good binding activity,specificity,serum stability was obtained,and the affinity was 251 nM.It can provide new candidate for tumor immunotherapy.Second,based on the fully human antibody AMMS4G4 which already obtained in our lab,we explored the efficacy of this antibody in combination with other targeting antibodies in the tumor models.In vivo results showed that in the Lovo tumor model,the combination therapy of A2C1 combined with AMMS4-G4 compared to A2C1,AMMS4-G4 either alone had a better efficacy.The pharmacokinetic mechanism may be that A2C1 can cooperate with AMMS4-G4 to down-regulate the expression of CD47 on Lovo cells.In the Skov3,Raji tumor-bearing models,AMMS4-G4 had good efficacy when it was administrated alone.This provides guidance for AMMS4-G4 in anti-tumor immunotherapy.