Cancer Immunotherapy: Genetically Engineered T Cell-Mediated Cell Killing

ACEA Biosciences xCELLigence

Genetically Engineered T Cells

T cells can be genetically engineered to express a modified T cell receptor (TCR; specific for a tumor antigen) or a chimeric antigen receptor (CAR; composed of an intracellular signaling domain that is linked to an extracellular domain derived from a tumor-specific antibody).

Avoiding the immune tolerance issues associated with non-autologous therapies, and producing T cells that efficiently target tumors without the need for de novo activation in the patient, are primary motivations for genetically modifying T cells. The efficacy of this approach is highlighted by the convincing clinical data that has emerged in recent years (as one example, see: Clin Transl Immunology. 2014;3(5):e16.)

Application Highlight: Measuring the Targeting Efficacy of Engineered TCR vs. CAR

In the example below, multiple assays are used to evaluate the killing efficiency of T cells when they are directed at cancer cells using either an engineered TCR or a CAR. To facilitate this comparison, CD8+ T cells were engineered to express both a TCR (recognizing the OVA257 epitope of ovalbumin) and a CAR (recognizing HER2). At different effector:target cell ratios, these T cells were incubated with adherent MC57 mouse fibrosarcoma cells that expressed the OVA257 epitope, HER2, or no exogenous protein.

After 18 hours of co-incubation, T cell-mediated cytolysis was analyzed using a traditional 51Cr release assay (left panel). Though the engineered T cells are able to kill MC57 cells, killing efficiency increases dramatically when the MC57 cells are expressing either OVA57 or HER2 (as expected). At all effector:target cell ratios analysed, activating these T cells via their TCR or their CAR results in similar killing efficiency (left panel). When the same assay is repeated using xCELLigence real-time impedance monitoring, after 18 hours of co-incubation killing via OVA257 targeting and HER2 targeting are very similar. Importantly, however, when analysed over a longer time period there are substantial differences in the killing efficiencies of these two targeting approaches (right panel).

Genetically Engineered T Cell-Mediated Cell Killing

Targeting T cells using an engineered TCR vs. CAR results in different killing efficiency. T cells engineered to simultaneously express a TCR against the OVA257 epitope of ovalbumin and a CAR against the HER2 protein were co-incubated with adherent mouse fibrosarcoma cells.  Killing efficiency was analyzed by both a traditional 51Cr release assay (left panel) and xCELLigence RTCA impedance monitoring (right panel; here data are plotted in arbitrary units, comparing the cell index value at each time point to the cell index value of the same sample prior to T cell addition).  Both assays show that MC57 cells are killed more efficiently when they express OVA257 or HER2 (as expected).  In the 51Cr release assay (conducted after 18 hours of co-incubation) both targeting approaches produce similar killing efficiencies.  In contrast, real-time monitoring of impedance over a longer time period reveals that in this context the TCR targeting approach results in more robust cell killing.  Figure adopted from Cancer Immunol Res. 2015; 3(5):483-94.

Key Benefits of Using xCELLigence To Study Genetically Engineered T Cell-Mediated Cell Killing:
  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.
Genetically Engineered T Cell-Mediated Cell Killing Supporting Information:

  • Adherent target cells tested:
    A375, SW480, MC57, MC57-HER2, U-251MG, 13-06-MG
  • Genetically Engineered T Cell-Mediated Cell Killing References:
  1. Epigenetic modulation to enable antigen-specific T-cell therapy of colorectal cancer. Chou J, Voong LN, Mortales CL, Towlerton AM, Pollack SM, Chen X, Yee C, Robbins PF, Warren EH. J Immunother. 2012 Feb-Mar;35(2):131-41. (Fred Hutchinson Cancer Research Center, USA)
  2. CAR-T cells inflict sequential killing of multiple tumor target cells. Davenport AJ, Jenkins MR, Cross RS, Yong CS, Prince HM, Ritchie DS, Trapani JA, Kershaw MH, Darcy PK, Neeson PJ. Cancer Immunol Res. 2015 May;3(5):483-94. (Peter MacCallum Cancer Center, Australia)
  3. Serial killers and mass murderers: engineered T cells are up to the task. June CH. Cancer Immunol Res. 2015 May;3(5):470-2. (University of Pennsylvania, USA)
  4. Efficacy of systemic adoptive transfer immunotherapy targeting NY-ESO-1 for glioblastoma. Everson RG, Antonios JP, Lisiero DN, Soto H, Scharnweber R, Garrett MC, Yong WH, Li N, Li G, Kruse CA, Liau LM, Prins RM. Neuro Oncol. 2015 Sep 1. pii: nov153. [Epub ahead of print] (University of California, Los Angeles, USA)
  5. A novel real-time CTL assay to measure designer T-cell function against HIV Env(+) cells. MacLean AG, Walker E, Sahu GK, Skowron G, Marx P, von Laer D, Junghans RP, Braun SE. J Med Primatol. 2014 Oct;43(5):341-8. (Tulane National Primate Research Center, USA)