In tumor microenvironment with human native immune system, once the tumor-specific targets are recognized by a particular antibody drug, the Fc part of the antibody would bind to the FcγR of native immune cells (such as NK cells and macrophages) and activate them, thereby eliciting effector functions to kill or phagocytose the target cancer cells. The Fc part of the antibody drug may also bind to the complement protein C1q in human serum, activating the complement system and eventually forming membrane attack complex which may act on the membrane of the target cancer cells and lyse them (See the graph below). We may utilize the MoA (Mechanism of Action) of these native immune cells to assess the functionalities of the antibody drugs in vitro.
ADCC, CDC and ADCP assays are relatively complex experiments, and many factors play roles to determine the success of the assay, including:
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There are two formats of ADCC assays at in vitro bioassay platform:
Case 1: Rituximab mediates ADCC effect in a dose-dependent format. Primary human PBMCs and Raji cells are used as the effector and target cells
Case 2: Anti-Claudin 18.2 antibody mediates ADCC effect in a dose-dependent format. ADCC reporter cells and human Claudin 18.2 overexpressing cells are used as the effector and target cells.
1 A large collection of target-overexpressing cell lines is freely available
2 Pooled normal human serum (PNHS) and normal human serum complement protein (NHSC) have comparable effector functions in our CDC assays
Case 1: Adalimumab mediated CDC dose response curves. mTNFα overexpressing cells are used as the target cells, and PNHS (blue) or NHSC(red) are effector molecules.
Case 2: Rituximab mediated CDC dose response curves. Raji cells are used as the target cells, and PNHS (blue) or NHSC (red) are effector molecules.
1 Macrophages can be differentiated into M1 or M2 type
2 GenScript ProBio has a large collection of target-overexpressing cell lines, which are freely available to our clients
3 Early screening refers to the antibody drug discovery stage prior to humanization
*CD20, CD38, CD47/SIRPα and Claudin 18.2, etc
ADCP assay detection:
Two distinct fluorescence dyes are used to label the target cells and the effector cells, respectively. The population of Q2 quadrant which showing double positive is the group of target cells consuming by phagocytosis. The macrophage phagocytosis percentage is calculated as Q2/(Q2+Q3)*100%.