The Development Of A Novel Anti-VEGF Neutralizing Antibody And Mechanisms Of Its Anti-renal Tumor Effects

Background:Angiogenesis is critical for the growth,maintenance,and metastasis of tumors.The identification of VEGF-A and its role in pathologic angiogenesis has led to the development of anti-VEGF angiogenic therapy for cancer and other diseases.Numerous studies have demonstrated that the overexpression of VEGF-A correlates with advanced tumor stage or tumor invasiveness in various types of human cancers.Renal cell carcinoma(RCC) is often an aggressive tumor,and advanced disease has limited treatment options and bad prognosis,which related to the upregulation of VEGF expression,because the inactivation of the von Hippel-Lindau(VHL) tumour suppressor gene is common in conventional/clear cell RCC.Antibody is a favorable molecule for drug development due to its high binding specificity and affinity to target.To date,the majority of marketed therapeutic monoclonal antibodies are of murine origin.Rabbit antibodies have been widely used in research and diagnostics due to their high specificity,affinity and diversity to various antigens.These properties,which are highly desirable for a good therapeutic antibody,have remained untapped for human disease therapy largely due to the polyclonal nature of rabbit antibodies and the early success of mouse monoclonal antibody.The discovery of a rabbit plasmacytoma cell line as fusion partner for rabbit spleen lymphocytes,makes rabbit hybridomas and monoclonal antibodies feasible. Methods:A panel of anti-VEGF-A rabbit monoclonal antibodies(RabMAbs) were generated by the improved rabbit hybridoma technology.Hybridoma supernatants were screened for binding to human VEGF-A by ELISA and the VEGFR-2 receptor blocking ELISA.The positive hybridomas were cloned and expressed as recombinant RabMAbs for further characterization.The specificity and affinity of antibodies binding to VEGF-A were tested by ELISA and BIAcore method.The inhibition of VEGF-stimulated phosphorylation of VEGFR2 was determined by Western blotting and phospho-ELISA assay.Humanization of one RabMAb was guided by sequence and lineage analysis of the variable regions of these RabMAbs,and non-human residues both in the framework and CDR regions were mutated.The biological potency of humanized antibody was evaluated by the inhibition of HUVEC growth cultured with conditional media from 786-O cells.The expression level of VEGF in 786-O cell was also determined.The efficacy of this antibody was determined in renal cancer xenograft nude mice.The microvessel density(MVD) of tumor biopsies were analyzed by CD34 immunostaining.Results:A total of 15 RabMAbs bound to human VEGF-A can block the VEGF/VEGFR-2 interaction in vitro and inhibit VEGF-stimulated VEGFR-2 phosphorylation in KDR/293 cells.These neutralizing RabMabs were specific to human VEGF-A and did not react to other family members. 12 of them are also crossreactive to mouse VEGF,thus facilitating preclinical study in mouse.The humanized antibody preserved the parental properties and had different epitopes binding to VEGF-A compared to Bevacizumab.In cell based assay,the antibody inhibited the proliferation of HUVECs induced by 786-O cells.We further demonstrated the antibody potently inhibited tumor growth of renal cancer xenograft in mice.It exerted a more prominent inhibition on tumor growth and angiogenesis when administered at the same dosage and schedule as Bevacizumab(p＜0.05,0.042) and in low dosage(0.25mg/kg),it could also effectively reduce MVD(p＜0.01,0.0085 vs.control). Conclusion:We developed a novel humanized anti-VEGF RabMAb for therapeutic application.This antibody could inhibit receptor phosphorylation and endothelial cell proliferation in vitro and reduce microvessel density in renal tumor xenograft model.This study also provides proof of principle on the feasibility of humanized RabMAbs for human therapeutics.