Idea to IND

Functional Characterization

Nona Biosciences has intensive knowledge and expertise in Immuno-Oncology.We have established many types of in vitro functional assays to characterize the antibodies for Immuno-Oncology targets.

Well-Established Immuno-Oncology Assays

We provide open access to our cell-based assays to accelerate your antibody discovery.

ADCC Assay

ADCC (antibody-dependent cellular cytotoxicity) is dependent on the interaction of the Fc region of the antibody with Fcγ receptors (FcγRs) expressed on immune effector cells such as NK cells. Once NK 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 PBMCs and cancer cells with antibodies, and then monitoring cancer cell death through LDH release (B) or label-free RTCA (Real-time Cell Analysis System) (C).


ADCP Assay

Similar with ADCC, another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). This immune mechanism relies on antibodies’ ability to recognize 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 PBMCs and are differentiated to 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

T cell activation assay is used 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 activates 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 Assay

TDCC (T cell dependent cellular cytotoxicity) assay is used to specifically evaluate the efficacy of candidate antibodies on cytotoxic T cell killing activity. In most of its applications, the TDCC assay 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

Internalization assay is used to evaluate the internalization efficiency of candidate antibodies after binding to target receptors on the cell surface. The assay can be performed with pHrodo or ADC kit format. In pHrodo-based format, Zenon pHrodo iFL IgG Labeling Reagent is incubated with candidate antibodies and then added together with the target-expressing cells. Only after internalization and endocytosis into endosome or lysosome will the pH-sensitive pHrodo dye 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.


Mixed Lymphocyte Reaction (MLR) Assay

The principle of the MLR assay is that T cells from one donor are activated 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-γ are usually measured.