Cancer Immunotherapy: Bispecific T Cell Engagers (BiTEs) and Bispecific Antibodies

ACEA Biosciences xCELLigence

What are Bispecific Antibodies/Bispecific T Cell Engagers (BiTEs)?

The therapeutic efficacy of the antibody-dependent cell-mediated cytotoxicity (ADCC) technique is mitigated by the fact that not all immune cells express the CD16 antibody receptor. In particular, cytotoxic and helper T lymphocytes don’t express CD16 and therefore aren’t recruited to antibody-coated cells.

In order to circumvent this constraint and mobilize the full capacity of the adaptive immune response against tumors, bispecific antibodies have been engineered which simultaneously (1) bind to specific antigens on the surface of tumor cells, and (2) tether and activate cytotoxic and helper T cells by binding the CD3 receptor that is expressed on their surface. This approach has the advantage of bypassing MHC-mediated activation of T cells, and has the potential to target any antigen that is expressed on the surface of tumor cells. Though multiple variations of bispecific antibodies have been studied, one type stands out as being especially promising. Bispecific T cell engagers (BiTEs), which target the CD19 antigen on B cell malignancies, have recently been awarded “Breakthrough Therapy” status by the FDA.

Application Highlight: Analyzing a BiTE Targeting the EpCAM Receptor

To evaluate the utility of xCELLigence RTCA for characterizing BiTEs, killing of adherent PC3 prostate cancer cells by PBMCs was studied in the presence of a BiTE that targets the EpCAM receptor (which is expressed on the surface of most cancer cells of epithelial origin, including PC3 cells). At a PBMC:PC3 ratio of 20, EpCAM/CD3 BiTE increases killing efficacy in a dose dependent manner (left panel). Though PC3 cell killing is still stimulated at the lowest BiTE concentration, complete killing of the PC3 cells is delayed.

As a means of quantifying the time-dependent cytolysis activity of PBMCs towards PC3 cells in the presence of BiTE, the effective time for reduction of Cell Index by 50% (ET50) was plotted (right panel). As expected, ET50 values demonstrate PC3 lysis to be more efficient at higher effector:target cell ratios and at higher BiTE concentrations. The ability of xCELLigence RTCA to assess the effect of BiTEs on the cytolytic activity of effector cells in a continuous manner elucidates killing kinetics that would be impossible to capture with end point assays.

Analyzing the efficacy of a BiTE targeting PC3 prostate cancer cells

Analyzing the efficacy of a BiTE targeting PC3 prostate cancer cells.  Killing of adherent PC3 prostate cancer cells by PBMCs (effector:target ratio = 20) was evaluated in the presence of a BiTE with specificity for both EpCAM (present on PC3 cells) and CD3 (present on PBMCs).  At all BiTE concentrations examined, simultaneous addition of the BiTE and PBMCs causes the destruction of PC3 cells, leading to reduced impedance signal (left panel). Plotting the effective time for reduction of Cell Index by 50% (ET50) enables quantitative evaluation of the differential effects of effector:target ratio and BiTE concentration (right panel). Unpublished data from ACEA Biosciences, Inc.

Key Benefits of Using xCELLigence To Study Bispecific Antibodies/BiTEs:
  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.
Bispecific Antibody/BiTE Supporting information:

  • Adherent target cells tested:
    PC3 prostate cancer cells, Panc89, Colo357, PancTu-I, PDAC, Colo38, MDA-MB435, HBV-transfected HuH7-S
  1. Novel bispecific antibodies increase γδ T-cell cytotoxicity against pancreatic cancer cells. Oberg HH, Peipp M, Kellner C, Sebens S, Krause S, Petrick D, Adam-Klages S, Röcken C, Becker T, Vogel I, Weisner D, Freitag-Wolf S, Gramatzki M, Kabelitz D, Wesch D. Cancer Res. 2014 Mar 1;74(5):1349-60. (Christian-Albrechts-University Kiel, Germany)
  2. Committing cytomegalovirus-specific CD8 T cells to eliminate tumor cells by bifunctional major histocompatibility class I antibody fusion molecules. Schmittnaegel M, Levitsky V, Hoffmann E, Georges G, Mundigl O, Klein C, Knoetgen H. Cancer Immunol Res. 2015 Jul;3(7):764-76. (Roche Pharma Research and Early Development, Germany)