Cell-Based Assays for Immunogenicity and Immunotoxicity

In vivo animal models are not necessarily predictive for immune responses in humans due to species differences. In vitro immunogenicity and immunotoxicity assays are recommended and can reveal cell-mediated responses.

In vitro immunogenicity and immunotoxicity assays can offer several advantages over the use of animal models , such as:

Accounting for population diversity by using primary cells from a range of human donors
High throughput
Reduced reliance on animal experimentation as well as the associated cost savings
Focused experimentation that targets different stages of the T cell-mediated immune response

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What is immunogenicity and immunotoxicity?

The ability of these therapeutics to evoke and mount an immune response defines the immunogenicity of the treatment. Ideally, treatment with various biological therapeutics will target the specific organ or cell type and is expected to create an efficient and safe intended immune response to address therapeutic needs.

But in reality immunogenic response covers a broad spectrum of secondary immune pharmacologic reactions ranging from the intended on-target response (e.g. desired immunogenicity) to unwanted off-target secondary responses driving undesired immunotoxicity and other secondary immune-related adverse side-effects. These effects range from development of anti-drug antibodies, excessive development of cytokine response and off target toxicity with undesired physiological and cellular responses (Figure 1).

Types of immunogenic reactions

Immunogenic reactions of 2 types:
a) wanted as needed for vaccines or
b) unwanted as for biotherapeutics.

Treatment with therapeutic proteins not meant as a vaccine can also elicit an unwanted immune response, such as the development of anti-drug antibodies (ADAs), produced by the B lymphocytes of the immune system in response to foreign proteins. ADAs can decrease the efficacy of the biologic protein treatments, and can cause high levels of clearance, induce hypersensitivity reactions, or cause severe adverse events. The generation of anti-drug antibodies (humoral immune response) can be T cell dependent or independent which involves the production of antibodies by B cells with and without assistance of T cells.

Assays to investigate innate NK cell cytotoxicity
Natural killer cells (NK cells) are cytotoxic lymphocytes found in peripheral blood that can destroy microbially infected cells or diseased cancerous cells. They are part of the innate immune system and are central to preventing spread of viruses and cancer.
Thus, measurement of NK cell expansion and cytotoxic activity is an important measurement in immune response.
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Assays to investigate innate macrophage activity
Macrophages are a type of white blood cell that is the first line of defense of the immune system that patrols the body seeking any cell types that do not display the proper protein surface markers. Macrophages consume these foreign intruders in a process called phagocytosis. This process is useful for removing a variety of foreign substances, microorganisms, including potentially cancerous cells.
Macrophages differentiate from monocytes into M1 and M2 macrophages. M1 macrophages are mainly involved in pro-inflammatory responses while exhibiting strong microbicidal properties and M2 macrophages are mainly involved in anti-inflammatory responses. Explore Tuning Macrophages for Immunotherapy|to learn more on how macrophages can be leveraged to develop solutions in the field of immune-oncology.

Immune assays to assess off-target toxicity
One of the main cause of drug development failure is the off target binding and reactivity to secreted or surface proteins that results in immunotoxic or sometimes fatal reactions. Thus off target assays are critical cell based assay in preclinical assessment of immunotoxicity. These assays typically consist of a coculture of PBMCs and cells derived from solid organ tissues such as lung, kidney, liver, skin, brain etc.
Human primary cells isolated from tissue of interest are cultured in the presence of PBMCs and the therapeutic of interest. A key characteristic in the development of these assays is the availability of HLA information. Readouts of an immune response typically consist of an assessment of T cell proliferation and activation and or the measurement of cytokines released.

Methods to evaluate immunogenicity/immunotoxicity

Methods for evaluating T cell dependent immune responses include peripheral blood mononuclear cell (PBMC) -based assays and dendritic cell (DC): T cell assays.
Assays can be broadly categorized under the innate category as T cell independent or adaptive category as T cell dependent

Types of assays that investigate innate immunity include uptake, digestion and presentation of biotherapeutic product by antigen presenting cells (APC) like dendritic cells in conjunction with class II HLA molecule.

This step can be tested by following cell based assays:

Biotherapeutic/Antigen internalization by dendritic cells or APCs
Expression of DC activation markers. Download protocol
MAPP (MHC-associated peptide proteomics) assay for protein/biotherapeutic uptake and processing by DC

Types of assays for adaptive immunity include investigation of T cell response to the antigen presentation by proliferation and activation.

This step can be tested by following cell based assays:

DC-T cell assay for T cell proliferation by using CFSE or thymidine incorporation.
Production of cytokines like IFN-Y, IL-2, IL-4 by multiplex ELISA or ELISPOT
T cell phenotyping

Cell-Based Assays for Immunogenicity and Immunotoxicity

What is immunogenicity and immunotoxicity?

Types of immunogenic reactions

Methods to evaluate immunogenicity/immunotoxicity

HLA typed cells

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