T cell mediated cytotoxicity

Redirection of cytotoxic effector T cells is the primary mechanism of action for CD3-engaging bispecific molecules. Using the individuals innate biological systems, these molecules activate and recruit T cells to specifically target disease tissues for destruction through the release of cytotoxic signalling factors.

T cells carry out key roles in immunological function, through the meditation of lasting, antigen-specific, effector and memory responses. Novel therapeutic approaches engage these features to activate, strengthen or engineer a T cell response against aberrant cells in the treatment of cancer and autoimmunity via cytotoxic T-lymphocyte (CTL) responses.

Basic Assay Format

T cell dependent cellular cytotoxicity (TDCC) assays are complex biological assays with many variants reported in the literature however, within each of these assays is a core set of basic conditions necessary for activity. A target cell is selected that expresses the antigen of interest and is incubated with bispecific antibody (bsAb) of interest in the presence of an effector population of T cells. T cell mediated cytotoxicity activity is then visualised by either directly measuring cytotoxicity or other indicator of functional response. Within these core steps there are many important variables to consider when developing an assay for the assessment of TDCC activity of a T-cell engaging therapeutic that should be tailored to the specific objective of the development stage.

Assay Endpoints

Direct cytotoxicity
The most common method of CTL activity for bsAb is the measurement of direct cytotoxicity or the inverse, residual viability.


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Proliferation and Activation Markers
As an orthogonal approach to the direct measurement of cytotoxicity of CTL responses, proliferation and CTL surface protein expression of activation markers can be analysed using flow cytometric techniques.

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Effector cells

Antibody Analytics has established a collection of effector cell formats and biological matrices to support a diverse range of assays, and can offer expert advice on the selection of the most appropriate format to reflect either the need for sensitivity, biological relevance or a combination of both

T cell Format When would this be useful?
PBMC Global measurement of target cell death mediated by T cells, NK cells (if functional Fc domain is present)
Pan T cell (CD3+) T cell mediated cytotoxicity response of CD4+, CD8+ and γδ T cells
CD8+ Specific CD8+ T cell mediated TDCC activity
Expanded T cells Clonal pools of expanded T cells can be created from a primary donor and subsequently used for T cell mediated cytotoxicity assays. This process can also be used to generate antigen-specific Tcells
Healthy and Disease State Donors Healthy and/or disease state tissue can be used to generate data of improved clinical relevance.
Reporter Cells T cell activation measured using a luciferase reporter gene responsive to CD3 engagement

Target Cells

In-house cell line development

Antibody Analytics have developed a proprietary library of common bispecific antigen target-expressing cell lines, offering unparalleled signal to noise response. This allows the development of uniquely sensitive assays for the assessment of CD3-engaging biologics.

With proprietary cell lines expressing common bispecific antigen targets, cell line development capabilities and access to a wide variety of primary T-cells, we can develop industry-leading TDCC assays to support early screening activities through to development of lot release potency methods.

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