The Tumor-promotion Mechanism Of Human Anterior Gradient 2 And The Related Humanized Monoclonal Antibody Development

Tumor microenvironment is one of the most important components of tumor.The tumor microenvironment contains a variety of soluble cytokines that mediate tumor and non-tumor cell-to-cell communication,and is,thereby,involved in the occurrence and development of tumors.Anterior gradient 2(AGR2)is highly expressed in a variety of adenocarcinomas.AGR2 distribution encompasses cytoplasm,cell membrane as well as extracellular space,it is also present in the peripheral blood cells of various cancer patients.The present study of AGR2 mainly focuses on its function to tumor cells and the intracellular mechanism.These studies show that the high AGR2 expression promotes survival,proliferation,migration,malignant transformation and drug resistance development of tumor cells.However,the role of secretory AGR2 on tumor stroma is rarely reported,and its underlying mechanism of action is still missing.This study explored the relationship between secretory AGR2 and tumor angiogenesis.The results showed that tumor interstitial fluid have high AGR2 accumulation(2.67?g / ml).In addition,secreted AGR2 was able to promote the activity of vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(b FGF / FGF2),thus indirectly promote the proliferation and migration of vascular endothelial cells and eventually angiogenesis.Furthermore,AGR2 independently promoted the invasion of fibroblasts.Further mechanism studies have shown that AGR2 binds directly to VEGF and b FGF,increasing the degree of their dimerization,thereby increasing local active-cell factors gradient.This pro-angiogenic activity of AGR2 can be blocked by a specific monoclonal antibody 18A4,thereby inhibiting the growth of tumors expressing AGR2.The maximum tumor inhibition effect was achieved when 18A4 was used in combination with anti-VEGF antibody Avastin.This new mechanism found in this study directly links secreted AGR2 to paracrine signaling network in tumor microenvironment,suggesting that secreted AGR2 is an anti-tumor therapeutic target that can be blocked.In addition,we produced a de-immunized humanized antibody 18A4 Hu I through CDR grafting and de-immunization analysis,and constructed the expression vector using specifically designed humanized antibody fast construction vector system p HAb-FAST.Physicochemical properties and anti-tumor experiments showed that 18A4 Hu I had similar affinity and anti-tumor effect with 18A4.In summary,this study reveals a new mechanism of secretory AGR2 and constructs a humanized antibody 18A4 Hu I that specifically targeting AGR2,laying a good theoretical and physical foundation for antibody therapy targeting AGR2.