A Fast and Sensitive Luciferase-based Assay for Antibody Engineering and Design of Chimeric Antigen Receptors

• Published in: Scientific reports Vol. 10; no. 1; p. 2318
• Main Authors: Natarajan, Venkatesh, Gopalakrishnan, Ramakrishnan, Matta, Hittu, Choi, Sunju, Gong, Songjie, Jeronimo, Alberto, Keerthipati, Pooja Smruthi, Morales, Anthony, Venkatesh, Harishwar, Chaudhary, Preet M
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 11.02.2020; Nature Publishing Group UK
• Subjects: Animals; Antigen (tumor-associated); Antigens; Aquatic Organisms - enzymology; Cell surface; Cell therapy; Chimeric antigen receptors; Genetic engineering; Genetic Engineering - methods; Humans; Immunoglobulins; Luciferases - genetics; Luciferases - metabolism; Lymphocytes T; Mammalian cells; Protein purification; Receptors, Antigen, T-Cell - genetics; Receptors, Antigen, T-Cell - immunology; Receptors, Chimeric Antigen - genetics; Receptors, Chimeric Antigen - immunology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; Surface antigens; Malibu California; United States > US
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-59099-9

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modified with chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferase-based assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.