Cancer Immunotherapy: Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

ACEA Biosciences xCELLigence

What is ADCC?

Though the innate and adaptive branches of the immune system are typically described as being distinct and separate from one another, they often work in concert to afford protection and combat disease. Upon encountering a pathogen, cells of the innate immune system typically release cytokines that cross-talk with components of the adaptive immune system, causing them to expand and become activated. Moreover, many cells involved in the innate immune response (including NK cells, neutrophils and eosinophils) also express CD16 (Fc receptor), which is a low affinity receptor for immunoglobulins such as IgG. Immunoglobulin binding by CD16 targets innate immune cells to the immunonglobulin-bound target cell, and triggers target cell destruction. This prophylactic mechanism is known as antibody-dependent cell-mediated cytotoxicity (ADCC) and is the basis of many current monoclonal antibody therapies.

Application Highlight: Erbitux-Mediated NK Cell Killing

Erbitux (Cetuximab) is a therapeutic monoclonal antibody that binds specifically to the human epidermal growth factor receptor (EGFR) that is overexpressed in many tumor types. In the example shown here, real-time impedance monitoring with xCELLigence was used to evaluate the efficacy of Erbitux-mediated NK cell killing. A431 human epidermoid carcinoma cells, which express high levels of human EGFR, were first seeded in the wells of an ACEA electronic microtiter plate (E-Plate). 22 hours post seeding, Erbitux was added at different concentrations. One hour after antibody addition, interleukin 2-activated NK cells were added at an effector:target cell ratio of 20:1. Neither Erbitux nor medium alone have a substantial effect on the real-time impedance trace of the A431 cells (left panel). While NK cell addition alone induces a decrease in the number, size, and/or attachment quality of the adherent A431 cells, the prior addition of Erbitux substantially increases, in a dose-dependent manner, this effect. The fact that MabThera (a monoclonal antibody against the CD20 protein which is not expressed in A431 cells) is ineffective highlights the specific role being played by Erbitux in directing NK cell-mediated killing (left panel). By plotting the Cell Index value (10 hours after NK cell addition) as a function of Erbitux concentration, a dose–response curve was generated and the EC50 of Erbitux calculated (right panel).

Antibody-dependent NK cell-mediated killing of A431 cells. Adherent A431 epidermoid carcinoma cells were incubated with or without the Erbitux monoclonal antibody before being exposed to interleukin 2-activated NK cells. Real-time impedance traces clearly show that the cytolytic activity of the NK cells is accentuated by Erbitux in a dose-dependent manner (left panel). The “Ab” and “NK” arrows denote the times of antibody and NK cell addition, respectively. Plotting the Cell Index, 10 hours after NK cell addition, as a function of antibody concentration yields a dose-response curve and the EC50 of Erbitux (right panel). Figure adapted from Assay Drug Dev Technol. 2006 Oct;4(5):555-63.

Key Benefits of Using xCELLigence to Monitor ADCC:
  1. Label-Free: Allowing for more physiological assay conditions; labeling or secondary assays aren’t required.
  2. Real-Time: Quantitative monitoring of both fast (hours) and slow (days) killing kinetics.
  3. Sensitive: Capable of evaluating low effector cell to target cell ratios that are physiologically relevant.
  4. Simple Workflow: Requires only the addition of effector cells to target cells (in the presence or absence of antibodies); homogeneous assay without additional sample handling.
  5. Automatic Data Plotting: RTCA software enables facile data display and objective analysis, precluding the subjective data vetting that is common to imaging-based assays.
ADCC Supporting Information:

  • Adherent target cells tested:
    MCF-7, A431, BT-474, NCI-N87, SKOV3, PC8, PC9, PC11, PC12, PC13, HD9, HD10, HD11, H322, MCF-7-CD19tm, Colo38, MDA-MB435
  1. Dynamic detection of natural killer cell-mediated cytotoxicity and cell adhesion by electrical impedance measurements. Glamann J, Hansen AJ. Assay Drug Dev Technol. 2006 Oct;4(5):555-63. (Novo Nordisk, Denmark)
  2. Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing. Kute TE, Savage L, Stehle JR Jr, Kim-Shapiro JW, Blanks MJ, Wood J, Vaughn JP. Cancer Immunol Immunother. 2009 Nov;58(11):1887-96. (Wake Forest University School of Medicine, USA)
  3. Pertuzumab in combination with trastuzumab shows significantly enhanced antitumor activity in HER2-positive human gastric cancer xenograft models. Yamashita-Kashima Y, Iijima S, Yorozu K, Furugaki K, Kurasawa M, Ohta M, Fujimoto-Ouchi K. Clin Cancer Res. 2011 Aug 1;17(15):5060-70. (Chugai Pharmaceutical, Japan)
  4. Isolation and characterization of IgG1 with asymmetrical Fc glycosylation. Ha S, Ou Y, Vlasak J, Li Y, Wang S, Vo K, Du Y, Mach A, Fang Y, Zhang N. Glycobiology. 2011 Aug;21(8):1087-96. (Merck Research, USA)
  5. Understanding key assay parameters that affect measurements of trastuzumab-mediated ADCC against Her2 positive breast cancer cells. Kute T, Stehle Jr JR, Ornelles D, Walker N, Delbono O, Vaughn JP. Oncoimmunology. 2012 Sep 1;1(6):810-821. (Wake Forest University School of Medicine, USA)
  6. A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties. Schanzer JM, Wartha K, Croasdale R, Moser S, Künkele KP, Ries C, Scheuer W, Duerr H, Pompiati S, Pollman J, Stracke J, Lau W, Ries S, Brinkmann U, Klein C, Umana P. J Biol Chem. 2014 Jul 4;289(27):18693-706. (Roche Diagnostics, Germany)
  7. γδ T cell-mediated antibody-dependent cellular cytotoxicity with CD19 antibodies assessed by an impedance-based label-free real-time cytotoxicity assay. Seidel UJ, Vogt F, Grosse-Hovest L, Jung G, Handgretinger R, Lang P. Front Immunol. 2014 Dec 2;5:618. (University Children’s Hospital Tübingen, Germany)