Drug Evaluation Of An Anti-CD47 Therapeutic Antibody Candidate

Strategies for targeting CD47 are becoming a hot spot of cancer immunotherapy.CD47 is specifically expressed on various kinds of tumor cells,which apply the CD47-SIRPαaxis to escape from immunological surveillance by macrophages.As a potential therapeutic target,CD47 acts more than a promising innate immune checkpoint,but also a motivator to arouse adaptive immune response.The development of the anti-CD47 antibody is still in its early stages without FDA approved drug.However,two CD47-targeting antibodies,Hu5F9-G4 and CC-90002,are currently undergoing clinical development and both apparently have therapeutic potential in cancer.In spite of the rapid development of new drug research,there are still three major questions remained to resolve.Firstly,how to balance the safety and efficacy during CD47 inhibition.Secondly,a more comprehensive study of the antitumor mechanisms of targeting CD47 therapies.Thirdly,how to rich the clinical application by combining the CD47-targeted therapy with other cancer treatment.So,the development of the fully human anti-CD47 therapeutic antibody with potential for drug discovery not only provides new candidate molecules for the research and application of tumor immunotherapy by targeting CD47,but also provides the theoretical results for the research on CD47 and tumor immunology based on comprehensive characterization,pharmacodynamics evaluation,the study of mechanism,the study of combination therapy and the drug evaluation of the antibody candidate.Therefore,in this study,we further developed and assessed a novel fully human anti-CD47 antibody,AMMS4-G4,by using fully synthesized large-capacity phage antibody library and the techniques of antibody affinity maturation.The study finished the research on the characteristics,pharmacodynamics,mechanisms and drug profile especially the safety of AMMS4-G4.The results reveal that AMMS4-G4 exhibited equivalent anticancer effects with Hu5F9-G4,a humanized anti-CD47 antibody in clinical trial,on the potential of inducing significant phagocytosis of tumor cells in vitro and prolonging the survival of leukemia xenografted mice.Additionally,AMMS4-G4significantly inhibited the growth of grafted solid tumors by enhancing macrophage infiltration and modestly enhanced the anti-tumor activity of opsonizing antibody and antiangiogenic therapy.Remarkably,AMMS4-G4 was demonstrated to be a promising candidate with great potential and safe profile for cancer immunotherapy.AMMS4-G4 antibody is a novel molecule with a brand new sequence,which is different from the antibody in research,so it is necessary the possible unique properties.The binding of AMMS4-G4 with the recombinant extracellular domain of human CD47was tested using SPR to assess the antigen-binding activity of AMMS4-G4.The antigen-binding capability of AMMS4-G4 for different tumor cells expressing CD47was detected using ELISA and flow cytometry.In addition,the value of EC₅₀ was calculated to compare with Hu5F9-G4.Moreover,the CD47 specific siRNA was applied to silence the expression of CD47 protein in SKOV3 cells to verify the specificity of binding.Then,the species cross-reactivity of AMMS4-G4 for human,cynomolgus monkey and mouse RBCs was detected using flow cytometry.As a vital property of an efficacious antibody,the blocking activity of AMMS4-G4 was carefully tested using different types of tumor cells.The binding properties of AMMS4-G4 were also analyzed using an epitope analysis.Alanine substitutions were inserted into specific sites that are important for the interaction between SIRP-αand CD47 in the CD47extracellular domain（CD47-ECD）.We analyzed the relative binding activities of AMMS4-G4 to these CD47-ECD mutants by using SPR.The results provide the data support for the consequent in vivo pharmacodynamic evaluation and pharmacological mechanisms of AMMS4-G4.The anti-tumor efficiency of AMMS4-G4 was evaluated the in both U937 and CCRF-SB xenograft leukemia models and subcutaneous xenograft solid-tumor models compared with Hu5F9-G4.Meanwhile,the mechanisms of the anti-tumor activity of AMMS4-G4 were investigated based on the above models.All the mice with continuous administration,and mice morbidity and mortality or the growth of the tumor were observed and recorded in interval observation.At the end of the experiment,the efficacy of the treatment was evaluated by analyzing the observation data.The main mechanism involved in CD47-targeted therapies was investigated by in vitro phagocytosis assay by macrophages.Considering the ADCC function mediated by NK cells maybe an anti-tumor principle exerted by AMMS4-G4,the SKOV3 cell survival assay and anti-tumor efficacy of different subclass of AMMS4 in vivo was respectively applied to investigate whether the Fc-dependent function of AMMS4-G4 was involved in the SKOV3 xenografts.In addition,in our investigation,the wound healing assay using SKOV3 and A549 cells was applied to try to explain for the anticancer effects of anti-CD47 antibodies.The results of the pharmacodynamics and pharmacological mechanisms give the evidence for AMMS4-G4 as an anticancer drug with therapeutic potential for further research and development.The combination therapy for tumor immunotherapy can achieve better therapeutic effects in clinical application.The current research on CD47 targeted therapy also tends to be combined with other tumor therapeutics,such as other immunotherapy,small molecule targeted drugs,chemotherapy,radiotherapy and so on.Therefore,in our further development,the potential of combination therapy of AMMS4-G4 was planned to be investigated.According to the previous research,AMMS4-G4 could significantly inhibit tumor growth combined with an anti-EGFR mAb A2C1,which has been proved to be a cancer-specific opsonizing antibody.So we continuously investigated two kinds of combination immunotherapy:the immunological checkpoint targeted therapy and the antiangiogenic therapy.In order to look into the drug properties of AMMS4-G4 and to clarify its value to develop,the related research,including expression,purification,formulation,in vitro stability,and pharmacokinetics of antibody molecules were investigated,especially for the safety of AMMS4-G4.In terms of safety evaluation,in vitro RBC aggregation assay and acute toxicity on single doses in cynomolgus monkeys were applied.In summary,these studies thus describe a novel,safe and effective fully human anti-CD47 antibody,AMMS4-G4,with good potential as a drug and may expand the clinical applications of immunotherapy.The results further prove that AMMS4-G4 can not only provide new drug selection for clinical tumor immunotherapy,but also provide a new combination drug plan for better tumor treatment efficacy.It is important that the research can provide a theoretical and application basis for tumor immunotherapy to obtain better therapeutic effect clinically.