Preparation and characterization of a new monoclonal antibody against CXCR4 using lentivirus vector
Xinyi Li1, Yu Kuang2, Xiaojun Huang3
1Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China; West China School of Stomatology, Sichuan University, Chengdu 610041, China.
Abstract:
CXCR4 is a member of chemokine receptors and plays a vital role in numerous diseases and cancer processes, which makes the CXCR4/CXCL12 chemotactic axis a potential therapeutic target. In this study, we used lentiviral vectors as a novel technology to produce a monoclonal antibody against CXCR4. Lentivirus vector pLV-CXCR4-Puro was successfully constructed and a hybridoma cell line 1A4 was generated. The CXCR4 monoclonal antibody (MAb) 1A4 had high titer and affinity, and the isotype was identified as IgG1a. The recombinant lentivirus vector could effectively stimulate the production of 39kDa CXCR4 antibody in vivo after immunization. Western blot analysis showed that the MAb could recognize the CXCR4 antigen expressed on transfected 293T cells as well as various human cancer cell lines. Immunofluorescence assays showed that MAb 1A4 mainly localized and strongly stained on the membrane of transfected 293T cells. Immunohistochemistry assays demonstrated that 1A4 could recognize strong expression of CXCR4 on the hepatocellular carcinoma (HCC). Thus, the method using lentiviral vectors may have application on effective and large-scale production of the CXCR4 monoclonal antibody, which will be a potential tool for the diagnosis and treatment of human cancers.
Insights
Researchers developed a novel lentiviral vector method to produce a CXCR4 monoclonal antibody (MAb). This antibody shows promise for diagnosing and treating cancers by targeting the CXCR4/CXCL12 axis.
Area of Science:
- Immunology
- Oncology
- Molecular Biology
Background:
- CXCR4 chemokine receptor is crucial in disease and cancer progression.
- The CXCR4/CXCL12 axis presents a viable therapeutic target.
Purpose of the Study:
- To develop a novel lentiviral vector technology for producing a monoclonal antibody against CXCR4.
- To characterize the efficacy of this antibody in recognizing CXCR4 in cancer cells.
Main Methods:
- Construction of lentivirus vector pLV-CXCR4-Puro.
- Generation of hybridoma cell line 1A4 producing CXCR4 monoclonal antibody (MAb) 1A4.
- Validation using Western blot, immunofluorescence, and immunohistochemistry assays.
Main Results:
- Successful construction of the lentivirus vector and generation of high-titer, high-affinity IgG1a MAb 1A4.
- The MAb 1A4 effectively recognized CXCR4 on transfected cells and various human cancer cell lines.
- Immunohistochemistry confirmed 1A4's recognition of CXCR4 in hepatocellular carcinoma (HCC).
Conclusions:
- Lentiviral vector technology offers an effective method for large-scale production of CXCR4 monoclonal antibodies.
- The developed MAb 1A4 is a potential tool for cancer diagnosis and therapy targeting the CXCR4 pathway.
