Antibody Targeted Fibroblast Activation Protein In Tumor Microenvironment

ObjectiveNumorous studies and clinical application showed that bispecific antibodies can effectively suppress diverse tumors.Fibroblast activation protein(FAP)as a bio-marker of tumors widely exists in tumor microenvironment,which plays an important role in tumor pathogenesis and progress.Therefore,this project intends to establish a bi-specific antibody that targets human FAP and human CD3 simultaneously.This project firstly design double structure specificity of antibody according to base sequence,then pack lentivirus and transfect CHO cells(China pale rat ovarian cells)by lentivirus.To confirm the successful estabilishment of biological engineering double antibody,we would verify the antibody secretion after antibody purification,verify the functionality of the secretion of antibody,including:antibody binding capacity,tumoricidal ability,etc.The aim of the present study is to develop a bi-specific antibody-based drug targeted human FAP/human CD3,that is more effective and safe for cancer treatoment.Methods1.Construction of bispecific antibodies:1.1 Bispecific antibody sequence design(including signal peptide,His Tag,lingker,VH,VL).2.Expression of bispecific antibodies:2.1 double specificity antibodies related sequence lentivirus packaging and contrasts with the empty carrier NC lentivirus packaging(include:1.The cell culture 2.Virus packaging 3.Virus collected 4.Virus drop degree detection,target drop degree is 1 ×109 × 108-1 tu/ml).2.2 CHO cells were transfected with the target gene vector lentivirus or the empty vector NC lentivirus,which lead to the experimental group and NC control group respectively(transfected MOI=1/200,and added Polybrene with transfection auxiliary reagent);2.3 Screening and high expression of seed selection CHO cells(lentivirus carrier options:with GFP green fluorescent protein,and have puro resistant to facilitate cells after transfection screening with monoclonal cell lines selected);2.4 The detection of target antibody in the culture of CHO cells(by Western blot and ELISA to detect the expression of His Tag,and the control test of the supernatant on the NC group);2.5 double raise the volume of specific antibody expression and purification(enrichment choose an intercept relative molecular mass,an ultrafiltration purification choose commercialization kits,at the same time with sds-page,silver stain coomassie brilliant blue staining method contrast ultrafiltration and purification effect,meanwhile with the expected relative molecular mass ratio on stripe location).3.Function verification and research of bispecific antibody:3.1 Bispecific antibody specificity and the two targets binding capability validation(establish the following FAP protein expression/air carrier cell lines:SPC-A-1-FAP/SPC-A-1-NC?K562-FAP/K562-NC,used for the double specificity antibodies and human FAP protein binding capacity CIK and Jurkat cells were used to test the capacity of bispecific antibody binding to human CD3 molecule.Through flow cytometry to detect the specific antibody binding to target cells with different ratio of effected antibodies and target cells.Note:if the streaming antibody FL1 channels of choice,GFP protein should be inactivated in advance);3.2 Tumoricidal ability by in vitro studies:SPC-A-1-FAP/SPC-A-1-NC cells incubated with PBMC/CIK cells plus bispecific antibodies for 24 h,then CCK8 reagent were added to the wells to detect the tumor cell viability by OD450.Results1.The construction and expression of the recombinant lentivirus with bi-specific antibody Hm326.1.1 The genetic sequences needed for antibody design were obtained through NCBI database and reported literature,and the antibody-base sequence was designed.1.2 Tested positive clones,and sequencing showed that the Hm326 antibody expression plasmid vector was successfully constructed.Packaged lentivirus,.lentivirus titer test display compliance with experimental requirements.1.3 CHO cells were transfected with lentivirus and cell lines with high transfection efficiency were selected monoclonally.1.4 Ultrafiltration and purification of the supernatant of cell culture.The expression of HIS-TAG was confirmed by testing.2.Study on the function of Hm326 antibody.2.1 FAP protein was expressed in lung,breast,stomach,pancreas and colon tumor tissue.2.2 Hm326 antibody can bind to the targets human FAP/human CD3.However,the antibody yield is low.Therefore the increase of antibody concentration may enhance its binding ratio with the target cells.2.3 The combination of Hm326 antibody with PBMC/CIK showed the significant cytotoxicity on SPC-A-1-FAP and SPC-A-1-NC cells.And the cytotoxic effects of Hm326 antibody combined with CIK was stronger than the effect of it with PBMC.In addition,the FAP overexpessed cells SPC-A-1-FAP appeared to be more sensitive to the treatment of Hm326 antibody comparing with SPC-A-1-NC,indicating Hm326 antibody possessed a certain degree of specificity to FAP.Conclusion1.To complete the construction of the recombinant lentivirus with dual specific antibody Hm326;2.The construction of CHO engineered cells containing Hm326 gene and the expression of antibodies;3.Complete the basic functional verification of Hm326 antibody:antibody binding capacity,tumoricidal ability.