PBMC Proliferation Assay

T-lymphocyte mediated drug hypersensitivity reactions mainly target the skin and liver^1,2. These reactions are dependent on antigen presentation to drug-specific T-lymphocytes which result in their activation, expansion, differentiation and the targeting of tissues and organs by activated T-lymphocytes.

The peripheral blood mononuclear cells (PBMC) proliferation assay is an in vitro method which utilises PBMC isolated from consenting healthy donors using Ficoll and density gradient centrifugation. This assay is also suitable for evaluating the priming of naïve T-lymphocytes within the PBMC population and for the assessment of immunogenicity and cross reactivity of small chemical molecules, biologics and peptides. In addition, inter-individual variability in immune responses can be explored by using multiple HLA-typed donor PBMC. Furthermore, follow-up studies involving other endpoints, such as drug-induced cytokine release profiles can be performed using the same cryopreserved donor PBMC.

Cyprotex delivers consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

• Drug hypersensitivity reactions are rare off-target, immune-mediated and mostly delayed-type reactions involving drug-specific T-lymphocytes.
• Common classes of drugs implicated in hypersensitivity include antibiotics, anticonvulsants, antivirals, non-steroidal anti-inflammatory drugs and biologics.
• These reactions are difficult to predict during preclinical drug discovery due to the involvement of specific individual characteristics (genetic and non-genetic) that are present in only susceptible individuals³.
• Current animal models of immunogenicity fail to recapitulate the complexity of the human immune system.
• In vitro drug-induced T-lymphocyte proliferation using PBMC isolated from drug hypersensitive individuals have provided supporting evidence for the diagnosis of clinical cases of drug hypersensitivity reactions^4,5.
• Retrospective genome-wide association studies (GWAS) have identified multiple human leukocyte alleles (HLA) linked to various forms of drug hypersensitivity reactions.
• Unfortunately, these HLA-associated hypersensitivity reactions are currently difficult to prospectively identify using existing immune based in vitro assays during early drug development.
• Hence, this highlights the importance of using relevant human immune cells to screen potentially immunogenic drugs during preclinical drug discovery.
• Our high throughput PBMC proliferation assay evaluates the immunogenic potential of candidate drugs using multiple HLA-typed PBMC donors cryopreserved in our immune cell biobank.
• This assay is non-radioactive and assesses the immunogenic potential of candidate drugs (biologics and small chemicals molecules) based on antigen-induced PBMC proliferation and corresponding increase in cellular ATP.

_{1) Sullivan A et al., (2018). β-Lactam hypersensitivity involves expansion of circulating and skin-resident TH22 cells. J Allergy Clin Immunol 141(1); 235-249
2) Mennicke M et al., (2009). Fulminant liver failure after vancomycin in a sulfasalazine-induced DRESS syndrome: fatal recurrence after liver transplantation. Am J Transplant 9(9); 2197-2202.
3) Gibson A et al., (2018). Genetic and nongenetic factors that may predispose individuals to allergic drug reactions. Curr Opin Allergy Clin Immunol 18(4); 325-332.
4) Pichler WJ and Tilch J, (2004). The lymphocyte transformation test in the diagnosis of drug hypersensitivity. Allergy 59(8); 809-820
5) Nyfeler B and Pichler WJ, (1997). The lymphocyte transformation test for the diagnosis of drug allergy: sensitivity and specificity. Clin Exp Allergy 27(2); 175-181}