Therapeutic area

EDA as an immunostimulatory agent.

Pathogens and cancer remain the leading causes of death worldwide. The development of vaccines to prevent diseases for which no vaccine currently exists, such as AIDS or malaria, or to treat chronic infections or cancers, as well as the improvement of efficacy and safety of existing vaccines, remains a high priority. In most cases, the development of such vaccines requires strategies capable of specifically stimulating CD8+ cytotoxic T lymphocytes (CTLs).

CTLs are activated by the presentation to T-cell receptors (TCRs) of short peptides associated with MHC class I molecules. These peptide-MHC class I complexes are present on the surface of antigen-presenting cells (APCs), which are also capable of providing co-stimulatory signals required for optimal CTL activation. Dendritic cells (DC) are the most potent APCs, with a unique capacity to interact with naive T lymphocytes and initiate primary immune responses, activating helper CD4+ and cytotoxic CD8+ T lymphocytes. In peripheral tissues, dendritic cells take up self and non-self antigens. Internalized antigens are then processed into proteolytic peptides, and these peptides are loaded onto MHC class I and II molecules (for CD8+ or CD4+ T lymphocyte activation, respectively). This process of antigen uptake, degradation, and loading is called antigen presentation. However, in the absence of stimulation, peripheral dendritic cells present antigens quite inefficiently. Exogenous signals from pathogens (bacterial or viral products) or endogenous signals (pro-inflammatory cytokines) induce dendritic cells to enter a developmental program, called maturation, which transforms dendritic cells into APCs and T lymphocyte activators. Thus, dendritic cells become the most potent APCs, and the only ones capable of activating naive T lymphocytes and of initiating adaptive immune responses. Concomitant with the modifications of their antigen presenting abilities, maturation also induces massive migration of dendritic cells out of peripheral tissues.

Some of the most efficient maturation stimuli are mediated by toll like receptor TLR (TLR1-9) interaction with their respective ligands. Ligand recognition by TLR provokes rapid activation of innate immunity by inducing production of proinflamatory cytokines and upregulation of costimulatory molecules. Activated innate immunity subsequently leads to effective adaptive immunity. Therefore, a candidate vaccine capable of inducing optimal T cell responses must fulfill several conditions. First, it has to target APC to deliver the antigen-derived T cell epitopes to MHC class I and/or II molecules. Thus, targeting DC could represent the main objective in designing new delivery systems for vaccine development. Furthermore, the vector has to deliver appropriate signals to DC to induce their activation. Antigen delivery to DC without a maturation signal could induce tolerance rather than activation of T helper and cytotoxic cells.  In addition, its efficiency must not be affected by pre-existing immunity against the vector itself.

The present invention relates to fibronectin Extra Domain A  (EDA), a possible natural ligand for TLR4, as a theoretical means for antigen delivery to TLR4 expressing cells that could induce appropriate selection and maturation of APC, and finally lead to an effective specific CTL response. Fibronectin molecules are the products of a single gene, and the resulting protein can exist in multiple forms that arise from alternative splicing of a single pre mRNA. Cellular fibronectins, which contain alternatively spliced EDA, are produced in response to tissue injury. Among other biological functions, EDA has been shown to induce proteoglycan release, and expression of metalloproteinases (MMP 1, 3, and 9) and of pro inflammatory cytokines. We postulated that if EDA could bind to TLR4 on dendritic cells and activate their maturation, EDA could be used as a vehicle to target an antigen to dendritic cells in vivo, favour its uptake and promote, at the same time, dendritic cell maturation to prime a T cell immune response specific for the antigen. We show in figure 1 the mechanism of action of EDA to favour this T cell activation.

 

Figure 1. Targeting antigens to dendritic cells. Mechanism of action for EDA as an adjuvant for vaccine development.

 

We have shown that a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, is able to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4 and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. It was found that fusion of an antigen to EDA protein favours its uptake by dendritic cells, induces their maturation, allows antigen presentation and primes the in vivo activation of strong and specific CTL response (Lasarte et al, J Immunol, 2007). These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.