In nearly 5 years we have developed over 30 types of CAR-T/NK cells and target cells with several patented innovations in the CAR construct design. We can construct a CAR based on your antibody sequence or we can generate the entire construct from a mouse monoclonal antibody or fully human single-chain variable antibody fragment (scFv) from our existing library.
ProMab Biotechnologies Custom Chimeric Antigen Receptor Cell Engineering: A complete service, from antigen to clinically relevant solution.
At ProMab Biotechnologies, we perform real-time cytotoxicity assays (RTCA), both with hematological cancers and solid tumor cell lines. The RTCA system we use incorporates a microelectronic biosensor system which continuously measures the impedance caused by adherent cells between the electrodes. An increase in the number of cells leads to an increase in impedance. In contrast, killing of the adherent target cells by CAR-T cells causes a decrease in impedance.
We also measure target cell-induced cytokine production by the CAR-T cells, either by ELISA or high-throughput cytokine screening. Cytokine release is a critical metric when validating CAR-T cells since a number of adverse reactions in patients is directly related to an extreme level of cytokines secreted from the CAR-T cells.
Below is an overview of the RTCA equipment and assay.
Figures A - C:
(B) Equipment for real-time cytotoxicity assay (RTCA).
(C) RTCA with CD19 CAR-T effector cells and HeLa-CD19 target cells. CD19 CAR-T cells killed Hela-CD19 target cells (C2) but not parental Hela cells (C1). The effector: target ratio was 10:1, and each line shows the average of three wells.
Flow Cytometry as a means of performing cytotoxicity assays.
Figure D. CAR-T cells exhibit cytotoxic activity as quantified by flow cytometry
We also measures cell cytotoxicity by flow cytometry, using multiple ratios of CAR-T effector (E) and target (T) cells (E:T = 5:1, 10:1, 20:1) Green fluorescent probes such as CFSE are used to label the target cells, and red fluorescent probes such as 7-AAD are used to identify dead cells. The extent of cytotoxicity is calculated as the percentage of green fluorescent cells that are also red fluorescent.
ELISA as a means of performing cytotoxicity assays.
Figure E. CD19-CAR-T cells produce IFN-γ and IL-2 when cultured in the presence of CD19+ target cells
We quantify cytokine secretion using enzyme linked immuno-sorbent assays (ELISA)s to measure IFN-γ and IL-2 with a high degree of sensitivity. However, we are not limited to these important cytotoxic markers and can provide our clients with a complete cytokine profile and other panels of interest for preclinical studies.