1. Scope

This document describes a procedure for purification of human peripheral blood mononuclear cells (PBMC) and their application to determine immunostimulatory activity of Nucleic Acid Nanoparticles (NANPs). PBMCs are purified from freshly donated whole blood of healthy donor volunteers under a protocol approved by the Institutional Review Board (IRB). Safety procedures review and approval by an Institutional Biosafety Committee (IBC), as well as personnel training related to working with whole blood and blood-borne pathogens are required before this protocol can be used. PBMC are purified using Ficoll-Paque density gradient centrifugation. The cells are then cultured in the presence of controls and NANPs, and their culture supernatants are collected and stored at -20 C or -80C before further analysis. The culture supernatants are next analyzed for the presence of cytokines using single-plex or multi-plex Enzyme-Linked Immunosorbent Assay (ELISA). The protocol described here is based on multi-plex chemiluminescent technology. Units - The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Institutional Biosafety Committee (IBC) approval and operators safety training regarding working with whole blood and blood borne pathogens are required prior to the use of this practice.

Keywords

NANPs, nanoparticles, cytokines, cytokine storm

Rationale

Cytokine storm is a condition characterized by high plasma levels of inflammatory cytokines, chemokines and interferons which can be commonly induced by pathogens or their components (endotoxin, lipoproteins, DNA, RNA etc.). Cytokine storm can also be induced in response to certain drugs (e.g., recombinant proteins, therapeutic antibodies, macromolecular nucleic acid-based therapeutics). It is accompanied by fever, hypo- or hypertension and may progress to a more severe life-threatening condition called systemic inflammatory response syndrome (SIRS). For example, cytokine storm was a severe side effect in the phase I clinical trial of the experimental monoclonal antibody therapeutic TGN1412, which resulted in 6 healthy donor volunteers becoming critically ill and requiring intense care. All patients had high serum levels of TNFalpha, IFNgamma and other pro-inflammatory messengers. Cytokine storm to this drug was not observed in preclinical studies involving rats and cynomolgus monkeys, but was easily detectable in vitro using a cytokine release assay in human primary blood cells. Nanoparticles can be used for delivery of therapeutic proteins, antibodies and nucleic acids, or contain biologicals (antibodies, proteins or nucleic acids) as targeting agents. In addition, some nanoparticles can be made of biological molecules (e.g., self-assembling peptides or siRNAs). NANPs are a category of nanomaterials formed by self-assembly of DNA or RNA oligonucleotides. When used without a carrier, these particles are not taken up by the immune cells and do not activate cytokine responses. After the delivery using a carrier, NANPs activate a unique spectrum of cytokines that depends on the type of carrier. These details warrant studying both nanotechnology platforms and their macromolecular payload and targeting agents for the ability to induce inflammatory cytokines. Human PBMC are considered reliable and predictive models for this purpose. The data obtained from such in vitro studies is intended to supplement other preclinical data to create a nanoparticle safety profile towards its clinical development. PBMC derived from healthy donor volunteers are cultured in the presence of controls and nanoparticles in order to identify nanoparticle potential to induce cytokine storm. The culture supernatants prepared according to this protocol can be analyzed by a variety of single and multiplex commercial assays specific to human cytokines, chemokines and interferons, or by a multiplex procedure provided herein

The title and scope are in draft form and are under development within this ASTM Committee.