Anti-tumor Effects And Underlying Mechanisms Of A Bispecific Antibody M802 Targeting HER2/CD3 For HER2-positive Cancers

Part ? Construction and characteristics of M802Objective:To construct and purify the bispecific antibody M802 targeting HER2 and CD3,and to measure the characteristics of M802.Methods:Genetic engineering was used to clone the coding sequence of each chain of the bispecific antibody into an eukaryotic expression vector,which was transfected into 293 F cells for expression.The cell supernatant was collected and purified by protein A affinity chromatography and cation exchange chromatography to obtain bispecific antibodies,named M802.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography-size exclusion chromatography were performed to analyze the bispecific antibody.The thermal stability and isoelectric point of the M802 also detected through thermal challenge assay and imaged capillary isoelectric focusing.Liquid phase mass spectrometry were used to to detect the molecular weight of the M802.In addition,the molecular affinity and cell affinity of M802 were evaluated by biomolecular interaction analysis system and flow cytometry.The concentration of M802 in the peripheral blood of mice was measured by enzyme linked immunosorbent assay at different time points to evaluate the pharmacokinetics of M802.Results:M802 had an asymmetric structure,which included a single chain targeting CD3,a light chain and a heavy chain targeting HER2.And the Fc domain of the M802 had been mutated to facilitate the formation of heterodimers.The purification efficiency of M802 reached 98%.The molecular weight of M802 was 127.7KDa.The isoelectric point of M802 was 8.5,which was different from the isoelectric point of other single-chain or dimers.The thermal stability of the M802 antibody was similar to the monoclonal antibody Herceptin(targeting HER2 monoclonal antibody)and L2K(targeting CD3 monoclonal antibody),respectively.The affinity of M802 for the HER2 molecule was 5.78×10-10 M which was similar to the affinity of Herceptin for the HER2 molecule(1.14×10-10 M).While the affinity of M802 for the CD3 molecule(Kd=7.12×10-8 M)was significantly lower than the affinity of L2 K for CD3 molecules(Kd=1.23×10-9 M).The affinity of M802 antibody for HER2 positive cells was similar to that of Herceptin,but the affinity of M802 for CD3 positive cells was significantly lower than that of L2 K.The M802 antibody could simultaneously bind to both HER2 positive cells and CD3 positive cells.The half-life of M802 antibody in mice was 63.665±1.655 h.Conclusions:The asymmetric structure of M802,the different isoelectric points between the polymers and the modification of the Fc domain are all conducive to the correct expression and efficient purification of the bispecific antibody.M802 could recognize and bind to HER2 positive cells and CD3 positive cells simultaneously,which is conducive to the specific killing of the bispecific antibody.Part ? The anti-tumor effects and underlying mechanisms of M802 for HER2-positive tumorsObjective:To evaluate the killing effects of M802 for HER2-positive tumors by in vitro and in vivo studies,and to explore the anti-tumor mechanisms of M802.Methods:The killing effects of M802 for tumor cells and non-tumor cells in an in vitro killing system was evaluated through CFSE/PI staining combined flow cytometry.The effects of the anti-HER2 end of M802 on the proliferation,apoptosis and signaling pathways in HER2-positive cells were verified through CCK-8 assay,Annexin V/PI apoptosis detection assay and western blot.Flow cytometry and enzyme linked immunosorbent assay were performed to evaluate the effects of the anti-CD3 end of M802 on the activation of immune cells(CD25 and CD69)and the secretion of cytokines.NK cells were used to detect the antibody-dependent cell-mediated cytotoxicity(ADCC)mediated by M802 antibody in vitro.In addition,the anti-tumor effects of M802 and M806(an alternative molecule of M802,targeting human HER2 and mouse CD3)for HER2-positive tumors were evaluated by subcutaneous transplantation tumor experiments in mice.The tumor infiltrating lymphocytes were measured by immunohistochemical staining.Results:In the in vitro killing system,M802 showed specific lysis to HER2-positive tumor cells.M802 not only inhibited the proliferation of HER2-positive tumor cells and promotes tumor cell apoptosis,but also had a significant inhibitory effect on the signaling pathways downstream of HER2.In addition,M802 mediated significant ADCC effect.In a mouse model of subcutaneous xenograft tumors,M802 significantly inhibited the growth of NCI-N87 tumors.In the subcutaneous allograft tumor model,M806,showed obvious anti-tumor effects and promoted the infiltration of immune cells into tumor tissues.Conclusions:M802 shows a significant inhibitory effect on HER2-positive tumors in in vivo and in vitro experiments.M802 not only retains the effect of the anti-HER2 end on the proliferation,apoptosis and signal pathway for HER2-positive tumor cells,but also significantly promotes the activation of immune cells and the secretion of cytokines through the anti-CD3 end.Moreover,the Fc structure of the M802 mediates the ADCC effect.Therefore,the M802 had a triple anti-tumor effect.Part ? The synergistic anti-tumor effects of PD-1/PD-L1 inhibitor and M802Objective:To evaluate the synergistic anti-tumor effect of PD-1/PD-L1 inhibitor and M802 by in vivo and in vitro experiments,and to explore the anti-tumor mechanisms of the combination therapy.Methods:In in vitro experiments,flow cytometry was used to detect the basic expression of HER2 and PD-L1 on different tumor cell lines.The in vitro killing experiment was performed to evaluate the anti-tumor effects of M802 in combination with anti-PD-1monoclonal antibody(?PD-1)or anti-PD-L1 monoclonal antibody(?PD-L1)on JIMT-1cells,which express HER2 and PD-L1 at baseline.The expression level of PD-L1 on target cells and PD-1 on efficacy cells in in vitro killing system were measured by flow cytometry.In addition,the supernatant of in vitro killing was collected to stimulate tumor cells and detect the changes of the expression of PD-L1.The luciferase reporter gene experiment was performed to verify the activation of ?PD-1 and ?PD-L1 on CD3 positive cells.Through an allograft tumor model,the anti-tumor effect of M806 combined with ?PD-L1 was evaluated in vivo.Results:We chose JIMT-1 cells for in vitro killing experiments,because JIMT-1 cells express HER2 and PD-L1 at baseline.M802 combined with ?PD-1 or ?PD-L1 had no significant synergistic effect in vitro.In the part II of the study,we found that the level of IFN-? in the supernatant of the killing system was significantly increased.We also found that the PD-L1 and PD-1 expression increased in the killing system.We collected the cell-free killing supernatant after M802 stimulation and applied it to tumor cells,and found that the level of PD-L1 on tumor cells was significantly increased.The results of the luciferase reporter gene experiment demonstrated that the ?PD-1 and ?PD-L1 significantly promoted the activation of CD3 positive cells by M802 in an in vitro killing system.In allograft tumor experiments,M806 combined with ?PD-L1 significantly inhibited the growth of B16-HER2 tumors.In the combination group,75%(6/8)of the mice did not develop tumors.Conclusions:Compared with the single-agent group,the bispecific antibody M806 combined with?PD-L1 shows synergistic anti-tumor effects.In in vitro experiments,M802 significantly promotes the expression of PD-L1 in tumor cells,and ?PD-1 and ?PD-L1 enhances the activation of CD3-positive cells by M802.In summary,the bispecific antibody M802 combined with PD-1/PD-L1 inhibitors could be a promising treatment strategy for HER2-positive tumors.