The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine.

Ziad Julier,Laura Jeanbart,Alexandre De Titta,Mikaël M Martino,Jeffrey A Hubbell

doi:10.1038/srep08569

Abstract

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8+ T cell responses in two murine cancer models.

Highlights

The Toll-like receptor 4 (TLR4) Agonist Fibronectin Extra Domain A is Cryptic, Exposed by Elastase-2; use in a fibrin matrix cancer vaccine
We sought to explore the importance of the local molecular context of fibronectin type III (FNIII) EDA in its immunological activities, as the domain resides in the vicinity of other active FNIII repeats in the natural sequence FNIII 9-10-11EDA-12-13-14 (Fig. 1a)
The FNIII EDA domain, present in a splice variant of FN, which is found in sites of transient inflammation as in tissue damage[4,5,6] and of chronic inflammation such as psoriatic lesions[10,11,12] and scleroderma lesions[15], has been shown to agonize

Summary

Introduction

The TLR4 Agonist Fibronectin Extra Domain A is Cryptic, Exposed by Elastase-2; use in a fibrin matrix cancer vaccine. Certain functions of FN depend on the presence of the alternatively spliced type III repeats extra domains A (FNIII EDA) and B (FNIII EDB) These extra domains are expressed in the vicinity of developing vessels during embryogenesis[2,3] and later in particular cases such as after tissue injury[4,5,6], within tumors[7,8,9], and at sites of chronic inflammation such as psoriatic lesions[10,11,12]. Based on these recent findings, we were motivated to investigate the potential importance of the molecular context of FNIII EDA’s placement within the FN chain upon its activity in inducing immune responses

Methods

Results

Conclusion