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ADCC (antibody-dependent cell-mediated cytotoxicity) is dependent on the interaction of the Fc region of the antibody with Fcγ receptors (FcγRs) expressed on immune effector cells (especially NK cells). Once the effector cells are activated, they will release cytotoxic granules and kill the target cells. The Human CD16a Jurkat reporter cell (Jurkat-CD16-NFAT) was engineered expressing both NFAT-driven luciferase and human CD16a Fcγ receptor, which can be used to evaluate ADCC activity of antibodies in a very simple and cost-effective way (A). ADCC activity can also be evaluated by co-culturing PBMC, cancer cells with candidate antibody molecules, and then monitor cancer cell death through LDH release (B) or label-free RTCA (Real-time Cell Analysis System) (C).

Similar with ADCC, another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). This immune mechanism relies on antibodies’ ability to recognizes target cells and activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification. CD14+ Monocytes are isolated from PBMC and differentiated into macrophages in vitro before co-culturing with target cancer cells. Macrophages and target cancer cells are labeled by different fluorescent dyes, so that phagocytosis rate can be determined after FACS analysis.

T cell activation assay is to evaluate T cell activation through measuring cytokine release. Usually IL-2 or IFN-γ are measured.

In the example below, HBM7008 is a crosslink-dependent bispecific antibody, which only activate T cells in the presence of tumor cells with high expression of the target. On the contrary, Urelumab can activate T cells under all conditions.

TDCC (T cell dependent cell cytotoxicity) is to specifically evaluate the efficacy of antibody inducing cytotoxic T cells’ killing activity. In most of its applications, TDCC is used to measure the cytotoxicity that is specifically induced when a CD3 bispecific antibody molecule engages effector T-cells and redirects cytolysis toward target-positive tumor cells. Cytotoxicity could be measured through LDH (lactate dehydrogenase) release or RTCA (label-free Real-Time Cell Analysis system).

Internalization assay is to evaluate the internalization efficiency of a candidate antibody after binding to it targets on cell surface. The assay can be performed by pHrodo or ADC kit format. In pHrodo-based format, Zenon pHrodo iFL IgG Labeling Reagent is incubated with antibody candidate and then added together with cells. Only after internalization and endocytosed into endosome or lysosome, the pH-sensitive pHrodo dyes undergo a dramatic increase in fluorescence in response to an environmental change from high to low pH. In ADC-kit format, anti-HFc-CL-MMAF forms complexes with the antibody, and then kills cells after internalization.

The principle of a mixed lymphocyte reaction (MLR) is that T cells from one donor will proliferate in the presence of APCs from a different donor. This is caused by the recognition of an HLA mismatch between two unrelated donors, which provokes an immune response from the T cells. MLR is often used to induce generalized stimulation/activation of T cells in culture, in which IL-2 and IFN-g are usually measured.