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

Abstract

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modifiedwith chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferasebased 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.

A novel thermostable beetle luciferase based cytotoxicity assay

“…We wanted to test if the Matador-Glo assay can be combined with the Matador assay to test cytotoxicity against antigen-positive and antigen-negative cell lines in a single experiment. For this purpose, a clone of Raji cells lacking the expression of CD19 was generated using CRISPR/Cas9 technology which was subsequently engineered to stably express Nluc (Raji-CD19KO - Nluc) and served as the antigen-negative target cell line 17 . Nalm6 cells stably expressing Luc146-1H2 (Nalm6-Luc146-1H2) served as the antigen-positive target cell line.…”

“…The pLP/VSVG envelope plasmid was obtained from Invitrogen (Thermo Fisher Scientific). A CD19-specific CAR construct was developed as described in our previous studies 8 , 17 , 28 .…”

“…Cell lines were maintained and cultured as described in our previous studies 8 , 17 , 28 , 29 . K562 (Chronic Myelogenous Leukemia), Raji (Burkitt’s lymphoma), and NK92MI (Natural killer cell) cell lines were obtained from ATCC and maintained as per the instructions provided.…”

“…293FT cells were obtained from Invitrogen (Thermo Fisher Scientific) and cultured as recommended. Peripheral blood mononuclear cells (PBMCs) were obtained and utilized as described in our previous studies 8 , 17 , 28 . PBMCs were isolated by the Ficoll gradient method from platelet depleted donor cells obtained from the Blood Bank at Children Hospital of Los Angeles (CHLA).…”

“…All the cells were cultured at 37 °C in a 5% CO2 humidified incubator. Reagents were made and utilized as described in our previous studies 8 , 17 , 28 . Blinatumomab (BiTE) was obtained from Amgen.…”