Fibronectin binding protein compositions and methods of use

Details Fibronectin binding protein compositions and methods of use Fibronectin binding protein compositions and methods of use

1998-01-21

2004-02-03

Smith, Lynette R. F. (Department: 1645)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C424S184100, C424S190100, C514S012200, C514S013800, C514S014800, C514S015800

Reexamination Certificate

active

06685943

ABSTRACT:

1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising DNA segments and proteins derived from bacterial species. More particularly, the invention provides fnibA nucleic acid and FnBPA amino acid compositions from
Staphylococcus aureus
. Also disclosed are peptide epitopes and protein sequences comprising site-specifically-modified or truncated fibronectin (Fn) binding site domains, and antibodies derived from immunization of animals with these peptide epitopes and binding site domains. Various methods for making and using these antibodies, peptides and DNA segments, peptides and nucleic acid segments encoding modified ligand binding site domains, and native and synthetic proteins are disclosed, such as, for example, the use of antibodies and/or DNA segments as diagnostic probes and templates for protein production, and the use of antibodies, proteins, fusion protein carriers, peptides and nucleic acid segments in various pharmacological and immunological applications.
1.2 Description of the Related Art
1.2.1 MSCRAMMs
Bacterial adherence to host tissue involves specific microbial surface adhesins of which a subfamily termed MSCRAMMs specifically recognize extracellular matrix (ECM) components. Many pathogenic bacteria have been shown to specifically recognize and bind to various components of the extracellular matrix in an interaction which appears to represent a host tissue colonization mechanism. This adherence involves a group of bacterial proteins termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (Patti et al., 1994; Patti and Höök, 1994).
Several Fn binding MSCRAMMs have been isolated and characterized from different Gram-positive bacteria. Genes encoding Fn binding MSCRAMMs from
Staphylococcus aureus
(Signäs et al., 1989),
Streptococcus pyogenes
(Talay et al., 1994; Hansky et al., 1992) and
Streptococcus dysgalactiae
(Lindgren et al., 1993) have been cloned and sequenced. The deduced amino acid sequences revealed 60-100 kDa proteins with very similar structural organization. The N-terminal signal sequence is followed by a long stretch of unique sequence which in some cases is interrupted by two copies of an approximately 30 amino acid long segment. The ligand binding site is located just N-terminal of a proline-rich domain, which is believed to anchor the proteins in the cell wall. This domain is followed by the sequence LPXTGX (SEQ ID NO: 1) which is a cell wall targeting signal (Schneewind et al., 1995), a stretch of hydrophobic residues representing a trans-membrane unit and a short C-terminal cytoplasmic domain containing a cluster of positively charged residues. The primary Fn-binding sites on these MSCRAMMs consist of 30-42 amino acid long motifs repeated 3-4 times, and most of the repeated units contain a consensus sequence (Lindgren et al., 1993; McGavin et al., 1993). This domain is composed of a unit of 37-40 amino acids, repeated three or four times (FIG.
1
).
Recombinant proteins corresponding to the repeat regions from the different Fn binding MSCRAMMs are all capable of inhibiting the binding of Fn to different Gram-positive bacteria, including
S. aureus, S. dysgalactiae
and
S. pyogenes
(Joh et al., 1994). Furthermore, studies using individual synthetic peptides revealed that a number of the repeat units retain Fn-binding activity, and interfere with binding of Fn by all of the Gram-positive species tested. These data suggest that the binding sites in Fn for the different MSCRAMMs are either overlapping or closely spaced on the matrix protein.
The repeat regions have been overexpressed as recombinant fusion proteins in
Escherichia coli
where the recombinant Fn binding domains (rFnBD) are linked to a stretch of histidine residues which are utilized for affinity purification of the rFnBD proteins. These proteins have been designated as rFnBD-D, rFnBD-A, rFnBD-B, and rFnBD-F, respectively FIG.
1
. The rFnBDs were found to exhibit similar binding kinetics and dissociation constants; for example, the dissociation constants of the four recombinant proteins binding to porcine Fn was determined by biosensor analysis to be in the low nM range with the dominant dissociation rates varying between 1×10
−4
and 6×10
−4
·s
−1
. Additionally, the recombinant proteins have been shown to have cross-species specificity and inhibit binding of Fn to many different bacterial cells (Joh et al., 1994).
The repeated units of the Fn binding domains of the different MSCRAMMs are strikingly similar, and appear to contain a consensus sequence (McGavin et al., 1991; House-Pompeo et al., 1996). The repeat units have a high number of acidic residues, and there are conserved hydrophobic and acidic residues at certain positions. Overall there is a high degree of sequence similarity between repeated units in a specific MSCRAMM as well as between MSCRAMMs from different species. Synthetic peptides, analogous to the repeated units, also bind Fn, and by amino acid substitution in these peptides it has been determined that all conserved residues are not needed for Fn binding (McGavin et al., 1991).
Fn is a disulfide-linked dimeric glycoprotein that is found in a soluble form in body fluids and a fibrillar form in the extracellular matrix. The primary biological function of Fn appears to be related to its ability to serve as a substrate for the adhesion of animal cells. This adhesion is mediated by a family of dimeric receptors which recognize and bind to specific sites in the central part of Fn. The primary binding sites in Fn for MSCRAMMs from Gram-positive bacteria has been localized to the Fn NH
2
-terminal domain (N29) (Mosher and Proctor, 1980; Speziale et al., 1984). This domain is composed of five type I modules which are about 45 amino acids in length. The structure of N29 is a series of anti-parallel &bgr;-sheets stabilized by several disulfide bonds interspersed at regular intervals in the sequences (Potts and Campbell, 1994; Venyaminov et al., 1983). The ability to bind Fn is located exclusively within the C-terminal 20 amino acids of each D-motif (Huff et al., 1994; McGavin et al., 1993; McGavin et al., 1991). These amino acids contain the sequence GG(X3,4)(I/V)DF, which is present in repeated motifs of other Fn-binding adhesins, and within the Fn-binding A2 motif of
S. dysgalactiae
FnBA, changes to either of the GG or IDF sequences resulted in loss of Fn-binding (McGavin et al. 1993).
The
S. aureus
Fn-binding MSCRAMM contains an additional ligand binding site in an approximately 30 amino acid long segment which has been designated Du that encompasses the consensus sequence and is located N-terminal of the repeat region. This segment can also interact with Fn and its N-terminal domain (designated N29) (Jönsson, 1992).
S. aureus
possesses two tandem fnb genes, encoding Fn-binding proteins FnBPA and FnBPB (Jönsson et al., 1991; Signäs et al., 1989), each of which possesses three consecutive 37-or 38 amino acid D-motifs, designated D1, D2, and D3. In tandem, these motifs comprise a high affinity Fn-binding domain, D1-3. Synthetic peptides representing each motif are also individually capable of low affinity Fn-binding, and can competitively inhibit Fn-binding to
S. aureus
(Huff et al., 1994; Signäs et aL, 1989).
1.2.2 Attempts to Generate Antibodies That Block Fn Binding Have Failed
In all of the Fn-binding MSCRAMMs identified so far, the primary ligand binding sites have been located to domains composed of a 37-42 amino acid motif repeated 3-5 times (McGavin et al., 1993). Unfortunately, attempts to generate blocking antibodies employing both synthetic peptides and different forms of the D1-3 immunogen have been largely unsuccessful (Ciborowski et al., 1992; Rozalska et al., 1994; Speziale et al., 1996). Previous attempts to generate high affinity antibodies that could block
S. aureus
binding to Fn have had little success. For example, whe

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