Preparation And Identification Of Monoclonal Antibody Targeting Axl

Objective: Receptor tyrosine kinases(RTK) are cell-surface transmembrane receptors which play an important role in cell survival, cell proliferation and cell migration in malignant cells. Axl is a member of TAM subfamily of receptor tyrosine kinase. Axl and its ligand Gas6 are aberrantly over-expressed in numerous human cancers. The binding of Gas6 and Axl activate multiple signaling pathways, which involve in the development and progression of cancer, including cell survival, migration, invasion, and angiogenesis. In recent years, Axl and Gas6 have been considered as a promising novel target for cancer therapeutics. The monoclonal antibody drug therapeutics have the advantage of strong specificity, high safety, low toxicity,and good clinic effect, which play an important role in tumor treatment.The development of antibody drugs specifically targeting Axl could provides more options for clinical treatment of tumors.Methods: In this study, the anti-Axl monoclonal antibody was screened by hybridoma technique. The sequence and the structure of Axl extracellular segment were analyzed by bioinformatics and structural biology. The gene of Axl functional domain which binds to Gas6 was cloned into the p GEX-4T-1expression vector. Then transformed into E. coli BL21 the recombinant plasmids and induced the target protein expression by IPTG. The target protein were obtained by affinity purification. BALB/c mice were immunized with the target protein, and after cell fusion and cloning culture of hybridomacells, the monoclonal antibody was screened out by ELISA, FACS and Western blotting. Furthermore, the biological activities of the anti-Axl monoclonal antibody were evaluated the in vitro by competitive ELISA,wound healing assay, transwell assay,and proliferation inhibition assay. The gene of variable region of the anti-Axl monoclonal antibody was cloned,blasted and analyzed.Results:1.Preparation of AntigenThe result of human Axl extracellular sequence and structure analysis suggested that the Ig-like-C2-type-1(27-128aa) of Axl extracellular domain was the Axl functional domain which could bind to Gas6.The fragment from27 to 128 aa of Axl was cloned into p GEX-4T-1expression vector. The recombinant plasmid was transformed into E. coli BL21 and induced to express fusion protein GST-F1 by IPTG.Then obtain the high purity of the target protein through the optimization of purification conditions. The target protein GST-F1 was purified by affinity chromatography and identified by SDS-PAGE and Western Blotting. The excepted fusion protein GST-F1 was obtained successfully, which laid the foundation for immunization for the production of monoclonal antibodies.2. Preparation, screening and identification of anti Axl m AbThe anti-Axl monoclonal antibodies(m Abs) were prepared by hybridoma technique. BALB/c mice were immunized with GST-F1 fusion protein as antigen, and after cell fusion the anti-Axl monoclonal antibody was screened by ELISA. Twohybridoma cell lines secrete anti-Axl monoclonal antibody steadily were screened by FACS.The isotypes of two anti-Axl monoclonal antibodies were Ig G3. The results of ELISA showed that the two anti-Axl monoclonal antibodies specifically recognized not only GST-F1 but also Fc-Axl-ECD. Moreover, the results of Western blot and FACS indicate that the two anti-AXLmabs could recognized AXL specifically.The gene of variable region of the anti-Axl monoclonal antibody was cloned and sequenced. The sequences of anti-Axl monoclonal antibody was compared with known sequences listed in the Gen Bank. As a result, the variable gene of antibodies was reliably obtained. The framework region and complementary determining region were divided, as the analysis of variable region of the anti-Axl monoclonal antibody.3.The biological activity of anti-Axlm AbThe biological activity of the anti-Axlm Ab was evaluated in vitro by competitive ELISA, wound healing assay, transwell assay,and proliferation inhibition assay. The results of competitive ELISA showed that the anti-Axl m Ab could effectively block the binding of Gas6 to Axl. Two selected anti-Axl m Abs induced Axl downregulation and inhibited its phosphorylationin MDA-MB-231. The results of proliferation inhibition assay revealed that the anti-Axlm Abs could not obvious inhibit cell proliferation of MDA-MB-231 cells. The results of anti-Axl m Abs wound healing assay and transwell assay indicated that the anti-Axlm Abs can significantly inhibit the migration of MDA-MB-231 cells.Conclusions:1.The target protein GST-F1 was obtained by prokaryotic expression and purification after determining the domains of Axl which could bind to Gas6 with the computer aided theoretical analysis.2.Two anti-Axl m Abs(8-11,16-7) were obtained by hybridoma technique.3.The anti-Axl m Abs could specifically block Gas6 binds to Axl,downregulation the expression of Axl and inhibit its phosphorylation; The anti-Axlm Abs inhibited the migration of MDA-MB-231 cells, but had no significant effect on the inhibition of the proliferation of MDA-MB-231 cells.