The Study Of Transgenic Goats About Mammary Gland Specific Expression Of Anti-human CD28/melanoma Bispecific Antibody

Melanoma is a malignant tumor that threatens global health and human health.At present,15 kinds of antibody drugs have been approved by the FDA for the treatment of malignant tumors.However,the incidence and mortality of melanoma gradually increased and the efficacy of chemotherapy drugs such as DTIC was not ideal.The occurrence and development of melanoma is made up of many different links,and the single target antibody drug was limited.The combination of anti-cancer drugs will be more effectively.This is also the future direction of antibody drugs.So far,most bispecific antibodies that mediate the killing of cancer cells harbor a CD3 binding site for activating T cells.Another target site is CD28.As already discovered in the late 80ies the anti-CD28 monoclonal antibody 9.3 provides a requirement signal for bypassing accessory cell in T cell activation.Since then,many bispecific antibodies har-boring a CD28 binding site have been described,that are capable of activating T cells without additional TCR/CD3 engagement.This effect was explained by the formation of a synaptic cleft between the T cells and the engaged cancer cells,generated by the close proximity of these cells.This enables the T cell to release its toxins into that cleft,resulting in a far higher local concentration of toxins in the cleft than by undirected release.The bispecific antibody,r28M,is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells.In order to obtain high expression of this antibody in transgenic goat mammary glands,the genome framework of bovine ?-lactoglobulin was combined with the r28M coding sequence to form a series of new mini gene coding frameworks.Intron insertion sites within r28M affect the correct splicing.In this study,we designed and constructed four bispecific antibody plasmids using pcDNA3.0 as the vector framework GN2017m,GN2017R,GN2017R4,GN2017 for Cell-level expression of cDNA and protein.By RT-PCR and SDS-PAGE detection found that GN2017R4 has a target protein expression at the cellular level.To construct a plasmid pTM-GN2017R4 that can be specifically expressed in goat Mammary Gland Bioreactor,Cre/loxp recombinase system was used to target the pTM-GN2017R4 integrated into human lysozyme transgenic goat cells,and bispecific antibody transgenic goats were finally prepared by somatic cell cloning..The PBS 185 plasmid and pTM-GN2017R4 plasmid were cotransfected into human lysozyme transgenic goat cells which named 3-376?.After screened by puromycin and identified by PCR and sequencing,we got monoclonal cells in which the GN2017R4 was integrated into the predetermined site.This targeted-integrated cell line was used for somatic cell cloning.The 30 donor goats and 20 transplant recipients were induced oestrous synchronization.The results are as follows:The total number of oocytes was 248,and 159 eggs were obtained after fusion.The reconstructed embryos were transplanted into 20 recipients.After the somatic cell nuclear transfer,B-ultrasonography was performed on 20 recipient goat at 35 days.There were 11 recipient goats pregnant.The pregnancy rate was 55%(11/20).One of them was taken out by caesarean section at 35 days.The fetus was pTM-GN2017R4 transgenic rams by PCR detection and sequencing identification.This study laid a solid foundation for the production of GN2017 double specific resistance by transgenic goat mammary gland bioreactor.