Study On The Preparation,Characterization And Antitumor Activity Of A Novel Anti-CD30 Antibody-drug Conjugate

Part ?.The preparation of a novel enediyne-integrated antibody-drug conjugate and its antitumor efficacy against CD30+ lymphomasPurpose:The treatment paradigms of hematological malignancies have been changed with the advent of monoclonal antibody-drug conjugates(ADCs).CD30 is a 120-kDa type I trans-membrane glycoprotein belonging to the tumor necrosis factor receptor superfamily(TNFRS).Overexpression of CD30 has been reported in Hodgkin lymphomas(HLs)and anaplastic large cell lymphomas(ALCLs).CD30-targeted treatment with ADCs can lead to promising clinical benefit.Lidamycin(LDM),consisting of an apoprotein LDP and an active enediyne chromaphore AE,is a member of the enediyne antibiotic family and one of the most potent antitumor agents.Notably,AE and LDP can be dissociated and reconstituted under certain conditions in vitro.LDM is considered as an ideal cytotoxic payload for the preparation of ADCs.In this study,we show the generation,production and antitumor activity of anti-CD30-LDM,a novel ADC which consists of the intact anti-CD30 antibody and lidamycin.Methods:LDP was fused to the N-terminal of the light chains of a CD30-targeting antibody to constitute the fusion protein anti-CD30-LDP.And then,the fusion protein was expressed in CHO cells.Its antigen-binding activity to recombinant CD30 protein and tumor cells was assayed by ELISA,Biacore and flow cytometry.Immunofluorescence confocal microscopy was used to detect whether it could be delivered into cancer cells by receptor-mediated endocytosis.The in vivo tumor-targeting ability of anti-CD30-LDP was observed by living images.Next,anti-CD30-LDM was prepared by assembling the enediyne molecule AE to anti-CD30-LDP.Induction of cell death in a panel of tumor cell lines was analyzed using flow cytometry,cell viability assays and immunoblotting.The in vivo anti-tumor effect was determined in a lymphoma Karpas299 xenograft model.Results:In the study,after transfection,we selected and identified the CHO single cell clone which expressed high level of recombinant fusion protein anti-CD30-LDP.After monoclonal expansion and protein purification,we obtained the anti-CD30-LDP fusion protein with purity of more than 98%.Anti-CD30-LDP showed specific and high affinity binding to recombinant CD30 and CD30+ cell lines.The antigen-antibody interaction demonstrated that anti-CD30-LDP had potent binding affinity to recombinant CD30 with the equilibrium dissociation constant of 2.08 nmol/L.Besides,it could be internalized into target cells through the antigen-mediated endocytosis and trafficked to lysosomes.Anti-CD30-LDP also exhibited excellent tumor-targeting capability in vivo and it significantly prolong the retention time of LDP in the tumor.After the assembling of AE to anti-CD30-LDP,anti-CD30-LDM was highly cytotoxic to HL and ALCL cell lines,with IC50 values of 0.005?0.05 nmol/L.It can also induce G2/M cell cycle arrest and cell apoptosis.In the Karpas299 xenograft model,the tumor growth was inhibited by 87.76%in mice treated with anti-CD30-LDM with no discernible adverse effects.Conclusions:In this study,we generated the new ADC anti-CD30-LDM by a novel two-step method.Anti-CD30-LDM shows attractive antigen-binding activity,tumor-targeting capability and anti-tumor efficacy both in vitro and in vivo and could be a promising candidate for the treatment of CD30+ lymphomas.Part ? The antitumor efficacy of anti-CD30-LDM combined with crizotinib in anaplastic large cell lymphomasPurpose:The treatment of relapsed and refractory anaplastic large cell lymphomas(ALCLs)is a tough challenge in clinical research,and finding effective therapeutic targets and therapeutic modes are still important goals for the treatment of such cancers.We have previously described a novel antibody-drug conjugate(ADC),anti-CD30-LDM,which displayed specific affinity and extremely potent cytotoxicity to CD30 overexpressed ALCL cells and highly therapeutic efficacy against CD30-positive xenografts in NOD/SCID mice.Combination therapy has gained momentum in oncology in recent years,and studies have shown that ADCs in combination with targeted small-molecule inhibitor can enhance the antitumor effects more significantly than monotherapy.Studies have shown that NPM-ALK fusion protein was excessively expressed in over 60%of cases in ALCLs.NPM-ALK activates downstream signaling events of MEK-ERK,JAK-STAT3 and PI3K/Akt pathways which promote proliferation,prevent apoptosis,and enhance tumor progression and migration in ALK+ ALCLs.Crizotinib is an ALK tyrosine kinase inhibitor that has been approved for the treatment of patients with EML4-ALK-positive non-small cell lung cancers(NSCLCs).The clinical studies have shown that crizotinib also is an effective and safety treatment in NPM-ALK-positive ALCLs.Based on these above,this research attempts to study the anti-tumor effect of anti-CD30-LDM combined with crizotinib on CD30+ALK+ ALCL cells,and explores the molecular mechanisms.It aimed to provide a potential therapeutic strategy for the treatment of ALCLs.Methods:The combined therapeutic effects of anti-CD30-LDM and crizotinib were investigated in ALCL cell lines Karpas299 and SU-DHL-1 with the expression of CD30 and ALK.The CCK-8 assay was used to analysis the effects of anti-CD30-LDM both alone and in combination with crizotinib on cell proliferation.Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry analysis.Caspase-3/7 activity was detected by the Caspase-3/7 Activity Assay Kit.Western Blot was used to detect the changes of related proteins after single or combined action of the two drugs.The in vivo antitumor effects of combination were evaluated in Karpas299 and SU-DHL-1 xenograft models.Results:The results of CCK-8 assay showed that the combination of anti-CD30-LDM and crizotinib exhibited significant synergistic inhibition(CDI<1)in ALK-positive Karpas299 and SU-DHL-1 cells.Annexin V-FITC/PI staining and flow cytometry analysis indicated that the combination of anti-CD30-LDM and crizotinib induced a higher apoptotic rate when compared with the single drug treated groups.The activity of caspas-3/7 levels in cells treated with anti-CD30-LDM and crizotinib were 6-8 times higher than that of the control group and 2.5 times more than that of the single drug treated groups.Combination of anti-CD30-LDM with crizotinib induced apoptosis of Karpas299 and SU-DHL-1 cells by activating the caspas3/7 and inactivating the PARP.Besides,low-dose of anti-CD30-LDM can make DNA damage,phosphorylate p53 protein,regulate DNA repair and induce apoptosis,but it activates the ERK1/2 signaling pathway that promotes cell proliferation,simultaneously.However,crizotinib can block the EKR1/2 signaling through the inhibition of NPM-ALK phosphorylation,and then enhance the anti-CD30-LDM-induced cell apoptosis.Excitingly,combination of anti-CD30-LDM and crizotinib has significant antitumor efficacy on both Karpas299 and SU-DHL-1 xenograft models in vivo.Conclusions:Crizotinib enhances anti-CD30-LDM-induced antiproliferative and apoptotic effects on ALCL cells by inhibiting the activation of ERK1/2 signaling.This research for the first time proved that the combination of anti-CD30-LDM and crizotinib shows synergistic inhibition effect in tumor cells.The findings present here will profit the clinical applications of anti-CD30-LDM and other DNA-damaging agents for the treatment of lymphoma as well as other hematologic malignancies in the future.