Cytokine release assay can be used to “de-risk” any potential unwanted effects of novel biotherapeutics in accordance with FDA and EMA recommended guidelines.

Preclinical safety cytokine release assay (CRA)

PBMC were added to wells coated with Alemtuzumab, Muromonab, TGN1412 or with isotype control antibodies, to induce cytokine release, or were stimulated with PHA, LPS, or a cocktail of TLR3/7/8/9 agonists. Cytokine release was measured at 48hr post stimulation. NS = Not stimulated. Data shown is mean of a minimum of five donors. Statistics show one way ANOVA with Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Other readouts: IL-8, IL-13, IL-4, IL-12p70, IL-10, IL-1β

Other formats: Whole Blood CRA

Other immuno-tox assays: include ADCC/ CDC/ ADCP

Enzyme-Linked Immuno Spot (ELISpot) is a technique which quantifies immune cells of low abundance which release biomarkers such as cytokines in response to antigenic stimulation. ELISpot is a highly sensitive method to test immune modulators, novel vaccine candidates or de-risk immunogenicity testing in an antigen-specific CD4 and/or CD8 T cell assay.

ConditionAim
No StimulationNegative control
PMAPositive control
CERI
(CMV, EBV, RSV, Influenza)
MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response
CPI
(CMV, Parainfluenza, Influenza)
Positive protein antigens to evaluate modulation of CD4+ T-cell memory response
CEF
(CMV, EBV, Influenza)
MHC-I restricted peptide pool to evaluate modulation of CD8+ T-cell memory response
Cyclosporin A (CsA)Inhibition of immune response
Figure 1: The ELISpot visual image results showing four donors under four conditions: Unstimulated (negative control), stimulated with PMA (positive control), stimulated with CERI, and stimulated with CPI antigens. Each purple spot represents one IFN-γ producing cell.

Spot Forming Units (SPU) for IFN-γ per 100,000 PBMC from CERI, CPI and CEF antigens for three donors

Figure 2: T cell responses from three donors using stimuli, CERI (I), CPI (II) and CEF (III).  Expressed as spot forming units per 100,000 donor PBMC. Depletion of CD4 or CD8 T cells demonstrates specificity of response to peptide pools. Each value represents the mean of triplicate wells ±SEM.

Evaluation of therapeutic modulation of Th17 differentiation

Figure 1: Polarisation of Th17 cells. Naïve CD4 cells were cultured under Th17 polarising conditions for 12 days in the presence or absence of Ursolic Acid. CD4 T cells were assessed for proliferation by CTV dilution; intracellular cytokine staining (ICS) of IL-17 and IL-10 by flow cytometry. On Day 12, Supernatants were collected and evaluated for IL-17 and IL-10 levels by MSD. Ursolic Acid showed selective inhibition of IL-17 production by ICS and MSD.

Evaluation of therapeutic modulation of antigen-specific memory T cell responses to recall antigens

I. T cell response to Tetanus Toxoid, Influenza and PPD antigens

II. Dose response to Influenza antigen

Antigen specific T cells response to a recall antigens. (I) Healthy donors PBMC were stimulated with PHA-M or triple antigen cocktail (Tetanus Toxoid, Influenza and PPD). Cyclosporin was used as a reference treatment. (II) Dose response to Influenza antigen. CD4 and CD8 T cells proliferation was measured by flow cytometry using CTV dilutions. 

Enhance T cell effector function: benchmark novel therapies against immune checkpoint blocker (anti-PD-1) in a 1-way MLR

Schematic description of 1-way MLR: purified CD14 cells differentiated into DC +/- LPS maturation co-cultured with purified allogenic CTV labelled CD4 T cells.
PD-1 blockade increases IFN-γ production in a 1-way MLR. Levels of IFN-γ were measured by MSD in cell culture supernatants.


PD-1 blockade does not enhance CD4 T cell proliferation in a 1-way MLR as determined by dye dilution of CTV labelled CD4 T cell using flow cytometry.

A THP-1 macrophage cytokine release assay can be used to explore modulators of an inflammatory response or “de-risk” any potential unwanted effects of novel therapeutic delivery systems

THP-1 Macrophage cell line Cytokine Release

THP-1 cells were differentiated with PMA and then stimulated with LPS to induce cytokine release in the presence of different concentrations of the corticosteroid dexamethasone. Cytokine release was measured at 4h and 24h post LPS stimulation.

THP-1 macrophage cell line phagocytosis and efferocytosis

THP-1 macrophage phagocytosis of E.Coli bioparticles and efferocytosis of Heat Shock treated A-375 melanoma tumour cells. THP-1 cells were cultured with E.Coli bioparticles 0-12.5ug/ml; at 1h phagocytosis was evaluated by pHrodo+ cells using flow cytometry. THP-1 cells were cultured with three E:T ratios of A-375 or heat shock treated A-375; at 1h efferocytosis was evaluated by pHrodo+ cells using flow cytometry.