title: A humanized monoclonal antibody targeting secreted anterior gradient 2 effectively inhibits the xenograft tumor growth.
abstract: Anterior Gradient 2 (AGR2) is a potential anti-tumor target and we previously reported a murine antibody 18A4 with specific binding to AGR2. However, humanization is a must to overcome immunogenicity before considering for clinical use and optimized vectors for mammalian expression are also necessary for following industrialized manufacture. Here, we describe an anti-tumor humanized antibody blocking secreted AGR2 activity. We employed the CDR grafting technique and deimmunization analysis to construct humanized antibody variants of 18A4, and 18A4Hu I was selected as the best humanization candidate, characterized by physical and chemical property comparison. Mouse xenograft study showed that 18A4Hu I could effectively inhibit the xenograft tumor growth, antibody blocking epitope analysis using AGR2 mutants indicated that the inhibition activity of 18A4Hu I is exerted probably through blocking the AGR2 functions which rely on the amino acid sites of E60-H76 and A86-E153. What's more, in this report, we also describe a pHAb-FAST vector system which is specifically designed for humanized antibody mammalian expression vector fast construction. With pHAb-FAST system, expression vector of 18A4Hu I could be quickly constructed only through twice overlapping PCR reactions. To our knowledge, AGR2-targeted 18A4Hu I is a promising humanized anti-tumor drug candidate, and pHAb-FAST system is a useful optimized mammalian expression vector construction tool. Our findings are supposed to accelerate the development of antibody-based cancer therapy.
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; Engineering Research Center of Cell and Therapeutic Antibody of Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Guo
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; Engineering Research Center of Cell and Therapeutic Antibody of Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Yu
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Rong
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Siva B
authors.initials: SB
authors.lastname: Merugu
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Hitesh B
authors.initials: HB
authors.lastname: Mangukiya
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China; Engineering Research Center of Cell and Therapeutic Antibody of Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. Electronic address: daweili@sjtu.edu.cn.
authors.forename: Dawei
authors.initials: D
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007114:Immunization; D051379:Mice; D008819:Mice, Nude; D009088:Mucoproteins; D015513:Oncogene Proteins; D010051:Ovarian Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D010053:Ovary / Q000276:immunology / Q000473:pathology; D011506:Proteins / Q000276:immunology*
chemical_list: C502296:AGR2 protein, human; D061067:Antibodies, Monoclonal, Humanized; D018984:Epitopes, T-Lymphocyte; D009088:Mucoproteins; D015513:Oncogene Proteins; D011506:Proteins
keywords: Anterior gradient 2; Anti-tumor humanized antibody; Antibody humanization; Vector construction