title: Successful construction and massive expression of a novel Anti-CD19 human-mouse chimeric antibody Hm2E8b.
abstract: CD19 antigen is a major target for human B cell malignancies. Many studies have shown that the antibodies recognizing this antigen hold clinical therapeutic potential, while CD19 antibody of mouse origin requires genetic engineering to reduce the potential side effects of the antibody for their clinical use. There are many clones of CD19 antibodies available with different subclasses of immunoglobulin. IgM type antibody holds a high affinity and high complement activating capacities facilitating the targeting efficacy when it is used in targeting therapy. However, engineering the murine IgM antibody into a functional humanized antibody remains a challenge. The aim of this study was to construct a chimeric antibody composed of a CD19 specific murine IgM antibody 2E8 single-chain antibody fragment (scFv) and human IgG1 Fc region, which was named 2E8scFv-Fc or Hm2E8b. The function and the biological activities of this engineered antibody were characterized using a variety of approaches including cellular, immunological, flow cytometric, and molecular biological approaches. After switching from IgM- to IgG-like type antibody, Hm2E8b retained full antigen-binding activity to membrane CD19 antigen as its parental antibody 2E8, and the immune effector function analysis revealed that it could mediate complement-dependent cytotoxicity (CDC) to kill the target cells via IgG1 Fc domain. The yield of the engineered antibody Hm2E8b in the supernatant was 13.3 μg/mL expressed and secreted in the CHO cell system, which reached the secretory quantity of a regular mouse hybridoma cells. Our conclusion is that the IgM type of CD19 mouse antibody can be successfully engineered into an IgG1 type human-mouse chimeric antibody with similar affinity and biological activity. The yield of the Hm2E8b expression and secretion in CHO cell system was adequate to facilitate further development for therapeutic purpose.
authors.affiliation: Division of Hematology and Oncology, The Children's Hospital of Zhejiang University School of Medicine , Hangzhou, China .
authors.forename: Diying
authors.initials: D
authors.lastname: Shen
authors.forename: Yongmin
authors.initials: Y
authors.lastname: Tang
authors.forename: Sisi
authors.initials: S
authors.lastname: Li
authors.forename: Weiqun
authors.initials: W
authors.lastname: Xu
authors.forename: Lingyan
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D018941:Antigens, CD19 / Q000276:immunology*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D018941:Antigens, CD19; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies