ADCC Detection Of Effector Cell Lines To Facilitate Drug Development

Monoclonal antibodies (mAbs) are substances that are produced by B cells and specifically target antigens. In 1975, Köhler and Milstein used hybridoma technology to make it possible to obtain large quantities of pure mAbs. This technology greatly enhanced the basic research and clinical application potential of antibodies, and also greatly promoted the progress of mAbs towards clinical application [1 ] . For this pioneering work, the two and the British-Danish immunologist Niels Kaj Jerne jointly won the 1984 Nobel Prize in Physiology or Medicine.

Antibody drugs used in clinical practice (chimeric monoclonal antibodies, humanized monoclonal antibodies, fully human monoclonal antibodies, ADCs, bispecific antibodies, miniaturized antibodies, and fusion proteins, etc.) have the characteristics of specificity, diversity, and preparation orientation. Specificity is mainly reflected in the ability of antibodies to specifically bind to related antigens and selectively kill tumor target cells, targeted distribution in animals, better efficacy on specific tumors, and definite clinical efficacy; diversity is manifested in the diversity of target antigens, antibody structure and activity, and immunoconjugates and fusion proteins; in addition, antibody drugs can also be prepared with antibodies with different therapeutic effects as needed.

Figure 1: Timeline of the development of therapeutic antibodies and their applications since 1975 [2].

In the process of antibody drug development, it is necessary to detect the obtained antibodies and evaluate the efficacy of antibodies or target cells. The method used is mainly antibody-dependent cell-mediated cytotoxicity (ADCC). Based on the ADCC mechanism of action, it is necessary to detect whether the antibody has ADCC biological activity and further analyze the strength of the antibody ADCC biological activity, which has become a crucial step in the process of antibody drug development and quality control. The ADCC reporter gene bioactivity detection kit uses bioluminescence to measure the activation of the NFAT (Nuclear factor of activated T-cells) signaling pathway and quantitatively detect the Fc effector function of therapeutic antibodies to evaluate the efficacy of antibodies or target cells ( Figure 3 ). When the IgG antibody specifically binds to the surface antigenic determinant cluster of the target cell (virus-infected cells and tumor cells) through the Fab segment, its Fc segment can bind to effector cells such as killer cells (NK cells, monocytes-macrophages, neutrophils) with FcγR, triggering the killing activity of the effector cells and directly killing the target cells (virus-infected cells and tumor cells).

Figure 2: Antibody-dependent cell-mediated cytotoxicity

In the ADCC detection process, effector cells play a key role. They can express Fcγ RIIIa receptors and rely on antibody-mediated killing cells to kill the corresponding target cells. The effector cells provided by YEASEN Biotechnology are modified NFAT-Luc-FcγRIIIa Jurkat stable cell lines, which can be used as target cells to establish ADCC biological activity detection methods using CD20 antibodies and B cell lymphoma cells (Raji, PBMC, WIL2-S cell lines, etc.). The Jurkat effector cell line developed by YEASEN is sterile and free of mycoplasma contamination, and the cell line is a monoclonal cell line.

Cell Line Characteristics
◆ Clear mechanism: Developed based on ADCC mechanism and NFAT signaling pathway;◆ Good stability: monoclonal cell line, low cell variability and high result accuracy;◆ Easy operation: Jurkat effector cells are easy to culture and the experimental procedures are easy to operate;◆ Stable signal: high signal value, good sensitivity, and low background;◆ Good safety: bioluminescence, high safety;◆ Widely used: antibody screening, ADCC mechanism research, Fc functional research of antibody drugs and product quality control, etc.

Cell Line Application
(1) Antibody concentration range detection:
TECAN Spark microplate reader was used for detection, and the luminescence was set to 360-700 nm, the detection time was 10000 m sec; the settlement time was 1 sec; Using a 3 µg/mL stock antibody concentration, dilute the antibody 13 times in 2.5-fold steps; the antibody is: Rituximab, E:T=6:1.
Figure 3: The detection value is relatively obvious (in the range of log -10 to -6)

(2) Optimal efficacy-target ratio detection:
TECAN Spark microplate reader was used for detection, and the luminescence was set to 360-700 nm, the detection time was 10000 m sec; the settlement time was 1 sec;Figure 4 & Figure 5: The starting concentration of the antibody stock solution was 1 µg/mL, and it was diluted 3-fold in 9 gradients; the antibody is: Rituximab , fixed E: 75,000 cells/well. The detection signal value is the highest when the effector-target ratio (E:T is between 2:1 and 1:1). 

Product Information and related products
👉 Jurkat Cell Lines for ADCC Bioassays 
👉 Dual Luciferase Reporter Gene Assay Kit
👉 Luciferase Reporter Gene Assay Kit
👉 Firefly Glo Luciferase Reporter Gene Assay Kit
👉 Dual Glo Luciferase Reporter Gene Assay Kit

References
[1] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495–497. doi: 10.1038/256495a0. [2] Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/s12929-019-0592-z