Introduction to in vitro Applications of Cynomolgus Monkey samples (PBMC, plasma, serum, etc.)

What is a cynomolgus monkey?

Cynomolgus monkeys, also known as long-tailed macaques, are non-human primates (NHPs) commonly used in biomedical research. The body length of an adult cynomolgus monkey is about 40–47 cm, and the tail length is 50–60 cm. Male cynomolgus monkeys weigh 5–7 kg, and the females generally weigh about 3–4 kg. They are also known as crab-eating macaques because of their habit of foraging for crabs and shellfish at the beach after low tide. Typically the life span of cynomolgus ranges between 25 and 30 years. Cynomolgus aged 19 to 31 months are termed juveniles, whereas those aged 32 to 44 months are called adolescents (not sexually mature). Cynomolgus monkeys reach sexual maturity at the ages of 4 for females and 6 for males.

Causes of using cynomolgus monkeys in studies

Due to their 90–93% genetic similarity to humans, cynomolgus monkeys can exhibit common age-related diseases in humans, such as cardiovascular disease, Alzheimer’s disease, bone loss, knee osteoarthritis, obesity, diabetes, and diabetic complications. Cynomolgus monkeys are also widely used as animal models for allogeneic or xenografts. Researchers therefore believe that cynomolgus monkeys are an ideal model for basic research such as disease pathology, vaccine development, immunology, and cardiology. According to guidelines, cynomolgus monkeys are also used in the pharmaceutical industry for toxicology and efficacy studies in drug development.

However, to conduct experiments on cynomolgus monkeys, the mechanism of the drug needs to be comprehensively considered. For example, HIV modeling failed due to a lack of AIDS progression in cynomolgus monkeys. For example, when developing anti-CD28 agonist antibodies, cynomolgus monkeys are a poor model because CD4+ T cells do not express CD28. Therefore, the high doses of antibodies used in toxicology studies have no effect on cynomolgus monkeys. While, when the antibodies are injected into the body, they can cause cytokine release syndrome, often with fatal side effects.

But by conducting in vitro studies on cynomolgus monkey samples, such as cynomolgus monkey peripheral mononuclear cells (PBMCs) or cynomolgus monkey whole blood, researchers can gain some insight into whether the subsequent in vivo studies in humans would be beneficial.

In vitro applications of cynomolgus monkey samples (PBMC, serum, plasma, etc.)

Cynomolgus monkey PBMC can be applied to the study of the Fc-mediated functions of therapeutic antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) assays. Studies on the efficacy of different human antibody subtypes in different cell types showed similar effector cells in human and cynomolgus monkey ADCC. At the same time, PBMC from cynomolgus monkeys can also be used as target cells for the ADCC assay. Rituximab is capable of performing ADCC on CD20-expressing cynomolgus monkey B cells, so in vivo studies in cynomolgus monkeys showed B cell depletion when using rituximab. Appropriate reagents, such as control antibodies, must be used when performing these assays to ensure that the observed cytotoxicity is driven by the antibody/targeted antigen and not by some molecule unrelated to human effector cells.

Although the functions of B cell proliferation and B cell antibody secretion in cynomolgus monkeys are relatively similar to those in humans, there are obvious differences in T cell proliferation and cytokine secretion between humans and cynomolgus monkeys. The immune response function of T cells in humans is stronger than that in cynomolgus monkeys. Therefore, the extrapolation of T cell toxicity evaluation from animal data to human data needs to be carefully considered. Studies have verified the comparison of T cell and B cell functions between humans and cynomolgus monkeys in three ways.

(1) Monoclonal antibody-induced cytokine release test: Human and cynomolgus monkey PBMC cells were incubated with ANC28.1 (Anti-CD28 Mouse Mab) for 2h, 24h, and 48h, and then ELSIA detection of TNF-α and IFN-γ was performed.

(2) T/B cell proliferation test: PBMC cells from the peripheral blood of humans and cynomolgus monkeys were collected and given different concentrations of phytohemagglutinin (PHA) and lipopolysaccharide (LPS). After incubation for 3 days, cck-8 was used to detect the cell proliferation rate.

(3) Collect human and cynomolgus monkey peripheral blood PBMC cells and administer influenza vaccine, inhibitor (anti-CD20 monoclonal antibody) or cell culture medium. After 6 days of incubation, ELISPOT was used to detect antibody secretion.

Result

(1) Monoclonal antibody-induced cytokine release test: As the action time of ANC28.1 prolongs, the TNF-α and INF-γ released by human PBMC cells gradually increase. However, cynomolgus monkey PBMC cells did not release TNF-α and INF-γ (because cynomolgus monkey CD4+ does not express CD28).

(2) T/B cell proliferation test: After the peripheral blood PBMC cells of human and cynomolgus monkeys were given PHA, the number of cells increased significantly. However, the proliferation rate of human PBMC cells was significantly higher than that of cynomolgus monkeys. However, under the stimulation of different concentrations of LPS, the proliferation trends of PBMC in the peripheral blood of humans and cynomolgus monkeys were very similar.

(3) B cell antibody formation function test: After human and cynomolgus monkey peripheral blood PBMC cells were stimulated by the influenza vaccine, the number of cells secreting corresponding antibodies was significantly higher than that in the negative control group. After administration of the anti-CD20 monoclonal antibody, the number of antibody-secreting cells in the inhibition group of cynomolgus monkeys was significantly reduced. Under the same treatment conditions, the number of antibody-secreting cells in humans and cynomolgus monkeys increased at a similar rate.

Serum from cynomolgus monkeys can be used in complement-dependent cytotoxicity (CDC) assays. The component proteins required for CDC are present in cynomolgus monkey serum and can be used for CDC detection of human target cells expressing target antigens. Thus, human therapeutic antibodies recruit cynomolgus complement proteins to promote the lysis of target cells.

Cynomolgus monkey serum and plasma are also commonly seen in the protocol to set baselines and generate concentration reference ranges for various assays (e.g., cytokine release measurements). They also act as critical reagents in various tests. In immunoassays like ELISA, ELISPOT, and WB that require initial blocking to prevent non-specific binding, cynomolgus monkey serum works to achieve this function.

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Reference: Lin Zhi., et al. ” Comparison of T cell and B cell functions between humans and cynomolgus monkeys.” 2013 (3rd) Chinese Annual Conference on Pharmaceutical Toxicology And drug non-clinical safety evaluation research forum.