Antibody dependent Cell Cytotoxicity (ADCC) assessment

Consisting in the targeting of cancer cells for destruction by antibodies to be then killed by effector immune cells, antibody-dependent cell cytotoxicity (ADCC) is thus the basis of several monoclonal antibody therapies which have been proven powerful and highly promising for cancer treatment.
Explicyte offers to perform an ADCC assay based on the full-time course monitoring of both cell proliferation and death of tumor cells, in the presence of immune cells and of candidate antibodies targeting specific tumor antigens. Our kinetic and quantitative cell-based assay allows thus the dynamic assessment and evaluation of the efficacy of test antibodies in eliciting ADCC and directing killing activity towards target tumor cells.

 

Illustrative data

Flow cytometry evaluation of the expression of target tumor antigens and of NK CD16 receptor
Flow cytometry evaluation of the expression of CD16 and CD56 in primary NK cells after their isolation from PBMCs (A), and of HER2 and EGFR target tumor antigens in SKOV3 (B) and A549 (C) tumor cells.  

Flow cytometry evaluation of the expression of CD16 and CD56 in primary NK cells after their ...

Real-time monitoring of NK-mediated SKOV3 cell apoptosis under untreated and antibody-treated conditions
Live cell monitoring of ovarian SKOV3 tumor cell death mediated by activated primary NK cells under untreated, trastuzumab-, or cetuximab-treated conditions. 
SKOV3 tumor cells were cultured under untreated, trastuzumab-, or cetuximab-treated conditions, and activated primary NK cells were then added, at different ratios (0:1 ; 2,5:1 ; 5:1 ; 10:1). Tumor cell death was then monitored during a period of ~72h, by mean of a caspase 3/7 fluorescent probe. Phase-contrast images were acquired on the Incucyte Zoom® imaging platform (Essen Biosciences), with an image acquisition every 2h, and tumor cell apoptosis was quantified and analyzed as a surrogate measure of NK-mediated tumor cytotoxicity. Data were normalized and corrected to the baseline.

Live cell monitoring of ovarian SKOV3 tumor cell death mediated by activated primary NK cells ...

IFNg release by activated NK cells highlights specific anti-HER2 and EGFR antibody-dependent cell cytotoxicity
Combination of NK cells and anti-HER2 or anti-EGFR antibodies enhances NK-mediated cytotoxicity in target tumor cells, in a NK ratio-dependent manner.
SKOV3 ovarian (HER2+/EGFR+, A, B) and A549 lung (HER2-/EGFR+, B, D) tumor cells were cultured under control, trastuzumab-, or cetuximab-treated conditions and, activated primary NK cells were then added at different E:T ratios. 24h later, supernatants were collected and analyzed for IFN𝞬 release as a surrogate of NK-induced cell toxicity.
While IFN𝞬 levels are increased in a NK ratio-dependent manner, thereby reflecting the basal direct NK cytotoxic activity on both SKOV3 and A549 cells (A-D), cetuximab is shown to promote this activity on both SKOV3 and A549 cells (EGFR+, C, D), while trastuzumab induces ADCC only on SKOV3 cells (HER2+, A).

Combination of NK cells and anti-HER2 or anti-EGFR antibodies enhances NK-mediated cytotoxicity ...

To learn more, download our antibody-dependent NK cell-mediated cytotoxicity white paper!


Assay principle

ADCC

  • Co-culture of target tumor cells with effector immune cells (PBMCs, NK cells…).
  • Real-time monitoring of tumor cell count (tumor cells stably expressing a nuclear fluorescent probe) as a surrogate of cell proliferation and of tumor cell death.
  • Automated image analysis & segmentation enables apoptotic death quantitation specifically within tumor cells (caspase 3/7 fluorescent probe within the nuclear probe-expressing cells).
  • IFNgamma release (or other effector cytokines) quantification as a surrogate of functionality of effector immune cells.