Mitigation of T-cell dependent immunogenicity by reengineering factor VIIa analogue.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog. The reengineered rFVIIa analog variants retained increased intrinsic thrombin generation activity but did not elicit T-cell responses in peripheral blood mononuclear cells isolated from 50 HLA typed subjects representing the human population. Our algorithm, rational immunogenicity determination, offers a broadly applicable deimmunizing strategy for bioengineered proteins.

In vitro cytokine release assays: reducing the risk of adverse events in man.

The induction of cytokine release is a common consequence of the administration of therapeutic antibodies and in most cases is either tolerated by the patient or can be managed clinically by the administration of corticosteroids. However, in 2006, the administration of TGN1412 to six patients in a Phase I trial resulted in a unprecedentedly high level of cytokine release, systemic organ failure and the hospitalization of the subjects. Whilst the path to failure in this incident was multifactorial, at least one contributing factor was the lack of a robust in vitro model that would allow the prediction of the in vivo activity of a therapeutic antibody. In this article we review the current 'state of the art' of in vitro cytokine release assays and explore potential future developments.

An assessment of different DNA delivery systems for protection against respiratory syncytial virus infection in the murine model: gene-gun delivery induces IgG in the lung.

Immunization with plasmid DNA (pDNA) has the potential to overcome the difficulties of neonatal vaccination that may be required for protection against infection with respiratory syncytial virus (RSV); however, little is known about optimal delivery modalities. In this pilot study we compared mucosal delivery of pDNA encoding RSV F protein encapsulated in poly(DL-lactide-co-glycolide) with delivery of pDNA by gene-gun for the induction of immunity in mice. Intra-gastric or intra-nasal immunization with various doses of microparticles induced weak low levels of RSV-specific serum antibodies in a proportion of mice; in contrast, gene-gun vaccination led to protective immunity associated with a humoral response. Interestingly, RSV-specific antibody was detected in lung fragment cultures following intradermal vaccination with the gene-gun.

DNA encoding the attachment (G) or fusion (F) protein of respiratory syncytial virus induces protection in the absence of pulmonary inflammation.

Significant protection against respiratory syncytial virus (RSV) infection was induced in mice vaccinated intramuscularly (i.m.) with DNA encoding the F or G protein of RSV. The amounts of IgG1 of IgG2a antibodies in mice immunized with DNA-G alone were similar. However, the antibody response in mice co-immunized with DNA-G and DNA encoding IL-4 (DNA-IL-4) was strongly biased towards IgG1. In contrast, the antibody response in mice co-immunized with DNA-G and DNA-IL-2, -IL-12 or-IFN-gamma was biased towards IgG2a. Mice vaccinated with DNA-F either alone or in combination with DNA encoding cytokines developed a predominant RSV-specific IgG2a response, which was most pronounced in mice co-immunized with DNA-F and DNA-IL-12 or -IFN-gamma. Vaccinated mice developed only a slightly enhanced pulmonary inflammatory response following RSV challenge. More significantly, and in contrast to mice scarified with recombinant vaccinia virus expressing the G protein, mice vaccinated i.m. with DNA-G did not develop pulmonary eosinophilia, even when the immune response was biased towards a Th2 response by co-administration of DNA-IL-4.