Construction And Study Of The Antitumor Activity Of The Anti-PTK7/MET Bispecific Antibody-drug Conjugate

Objective: A bispecific antibody-drug conjugate(Bs ADC)targeting protein tyrosine kinase 7(PTK7)and cellular-mesenchymal epithelial transition factor(MET),named P91M32-ADC.In vitro affinity,binding endocytosis,killing cells activity,and antibody stability study were subsequently evaluated,and antitumor activity were studied in vivo,which provides valuable research data for the clinical study of bispecific antibody conjugates.Methods: In vitro experiment: P91 and M32 antibodies sequences targeting PTK7 and MET with common light chains were obtained through the Biocytogen’s fully human antibody Ren Lite(?) mice,and then P91M32 bispecific antibody were constructed by knobs-into-holes technology.The Flow cytometry(FCM)was used to detect the binding activity of the P91M32 in a series of tumor cell lines.The internalization efficacy was measured by the FCM and Incucyte.Biacore 8K was used to detect affinity with antigens,antibody stability was detected by hydrophobic interaction chromatography(HIC),capillary isoelectric focusing(c IEF),and size exclusion chromatography(SEC).We conjugated the Bispecific MET×PTK7 antibody to the monomethyl auristatin E(MMAE payload),by using a Val-Cit(VC)linker,to generate the MET×PTK7 Bs ADC with an average drug: antibody ratio of approximately 4.After that,Incucyte was used to compare the in vitro anti-tumor ability of P91M32-ADC to ABBV-647 analog-ADC and ABBV-399 analog-ADC.Furthermore,the tumor inhibitory activity of P91M32-ADC in vivo was explored in the tumor-bearing mice model.Results: In this project,a bispecific antibody P91M32 with a purity of 96.29% was successfully constructed.P91M32 showed superior binding activity with tumor cell line than P91 and M32 and showed stronger endocytosis than ABBV-647 analog and ABBV-399 analog.The affinity of P91M32 with PTK7 and MET human antigen proteins was at the level of 10 n M,and showed good stability in acid treatment and heat treatment experiments.The target antibody-drug conjugate P91M32-ADC with DAR value of 4.20 was constructed by the cysteine coupling method,and the tumor cell killing results showed that P91M32-ADC had stronger cell-killing ability than ABBV-647 analog-ADC and ABBV-399 analog-ADC.In the three CDX models,the efficacy of P91M32-ADC was significantly better than that of the PTK7-positive drug ABBV-647 analog-ADC,and better than or equivalent to the MET-positive drug ABBV-399 analog-ADC in inhibiting tumor growth.Specifically,in the MDA-MB-468 model,P91M32-ADC showed superior antitumor activity to its parent monoclonal antibody ADCs(P91-ADC,M32-ADC),exhibiting a synergistic effect.Conclusion: In this study,we successfully constructed a bispecific antibody-drug conjugate P91M32-ADC targeting PTK7/MET,which improved the endocytosis activity and antitumor activity of PTK7,suggesting that bispecific antibody-drug conjugate are a powerful strategy for future ADC drug development field.