Immune Checkpoint Inhibitors Functional Assay

As an immune checkpoint inhibitors CRO, Creative BioMart provides immune checkpoint functional assay service for customers to design immune checkpoint inhibitors roadmap including antibodies, peptides and small molecules. In the functional assay experiments performed, we will firstly activate T-cells in the presence of immune checkpoint inhibitors with several stimulation reagents including PHA, SEB and CD3/CD28, respectively. According to the reagent types, the assays are divided into three sorts, PHA activation assay, SEB activation assay and CD3/CD28 activation assay, respectively.

PHA activation assay

Phytohaemagglutinin (PHA) consisting of PHA-E and PHA-L is a lectin obtained from the red kidney bean which binds to membranes of T cells, and it is well-known as a selective T Cell mitogen to induce T cell activation. To perform PHA activation assay, Creative BioMart will discuss with research teams of customers for designing the best method to help customers testing immune checkpoint inhibitors. Flow cytometry and other related assay tools will be performed to analyze surface receptors located on activated T cells and related cytokines produced by T cells.

SEB activation assay

Staphylococcal enterotoxin B (SEB) also named enterotoxin type B is a kind of enterotoxin produced by gram-positive bacteria Staphylococcus aureus. The function of SEB is to facilitate infection of the host organism. Like PHA, SEB can also stimulate T cell and increase activation markers expression and proliferation. Creative BioMart will also discuss with research teams of customers for designing the best method to help customers testing immune checkpoint inhibitors. Flow cytometry and other related assay tools will be performed to analyze surface receptors located on activated T cells and related cytokines produced by T cells.

CD3/CD28 activation assay

T cells in Creative BioMart will be activated in vitro for their expansion to have enough cells for research studies. There are two signals for T cell activation. The first is activation of TCR complex, and the another is stimulating T cell by CD28. The main principles of anti-CD3 and anti-CD28 antibodies are:

• Anti-CD3 can bind to CD3 and activates TCR complex without antigenic peptides from the antigen presenting cells.
• Anti-CD28 can bind to CD28 and stimulates the T cells without CD80/CD86 from antigen presenting cells.

Although PHA or SEB can induce polyclonal T cell responses, they cannot facilitate T cell expansion, therefore, CD3/CD28 antibodies are vital to stimulate T cell. Usually, two methods are used to activate T cells in CD3/CD28 activation assay including CD3/CD28 stimulation dynabeads and CD3/CD28 antibody complexes. Creative BioMart will also discuss with research teams of customers for designing the best method to help customers testing immune checkpoint inhibitors. Flow cytometry and other related assay tools will be performed to analyze surface receptors located on activated T cells and related cytokines produced by T cells.

Figure 1. Schematic of early T cell activation. (Original author - Mary Cavanagh)

Reference

1. Movafagh A. et al. 2011. The significance application of indigenous Phytohemagglutinin (PHA) mitogen on metaphase and cell culture procedure. Iran J Pharm Res. 10(4):895-903.