Toll-like receptor 2 binding epitope and binding member thereto
The present invention relates to the identification of a TLR2 binding epitope wherein binding of a binding member to the epitope serves to inhibit TLR2 activation and/or signalling. Polypeptide fragments of TLR2 and three-dimensional structures comprising one or more amino acid residues His318, Pro320, Gln321 or Arg321, Tyr323, Lys347, Phe349, Leu371, Glu375, Tyr376 and His398 of TLR2 which define the identified epitope are provided for use in generating binding members. Also provided are binding members which bind to the identified epitope and methods of using same for the treatment and/or prevention of conditions associated with TLR2 activation and/or signalling.
1. A screening method or assay for identifying a binding member which specifically binds to Toll-like receptor 2 (TLR2) and antagonises TLR2 activation and signalling, the screening method comprising the steps of:
bringing a candidate compound into contact with a polypeptide fragment of Toll-like receptor 2 (TLR2), the polypeptide fragment comprising:
amino acid residues His318, Pro320, Tyr323, Lys347, Phe349, Leu371, Glu375, Tyr376 and His398 of TLR2 as defined in SEQ ID NO:3 or SEQ ID NO:4,
wherein said polypeptide fragment of TLR2 is a polypeptide fragment of TLR2 other than the extracellular domain of TLR2 comprising amino acid residues of SEQ ID NO:7 or SEQ ID NO:8,
wherein the amino acid residues His318, Pro320, Tyr323, Lys347, Phe349, Leu371, Glu375, Tyr376 and His398 form a functional epitope in said polypeptide fragment, wherein binding of the functional epitope by the binding member antagonises TLR2 activation and signalling and wherein the functional epitope is bound by TLR2 antagonistic antibody T2.5 and a humanised version thereof designated OPN-305, and
wherein the polypeptide fragment comprises less than about 200 amino acid residues of TLR2; and
assessing binding between the candidate compound and the polypeptide fragment,
wherein binding between the candidate compound and the polypeptide fragment identifies the candidate compound as an antagonist of TLR2 activation and signalling.
2. The screening method as claimed in claim 1 wherein the polypeptide fragment comprises amino acid residues His318, Pro320, Tyr323, Lys347, Phe349, Leu371, Glu375, Tyr376 and His398 of murine TLR2 as defined in SEQ ID NO:4 and said amino acid residues form the functional epitope.
3. The screening method as claimed in claim 1 wherein the polypeptide fragment comprises amino acid residues His318, Pro320, Tyr323, Lys347, Phe349, Leu371, Glu375, Tyr376 and His398 of human TLR2 as defined in SEQ ID NO:3 and said amino acid residues form the functional epitope.
4. The screening method as claimed in claim 1 wherein the polypeptide fragment consists essentially of SEQ ID NO:5 or SEQ ID NO:6 or a sequence which has at least 85% sequence identity with SEQ ID NO:5 or SEQ ID NO:6.
5. The screening method as claimed in claim 1 wherein the polypeptide fragment comprises less than about 150 amino acid residues of TLR2.
6. The screening method as claimed in claim 5 wherein the polypeptide fragment comprises less than about 102 amino acid residues of TLR2.
7. The screening method as claimed in claim 1 wherein the polypeptide fragment consists essentially of amino acid residues Thr311 to Thr411 of SEQ ID NO:3 or SEQ ID NO:4 or a sequence which has at least 85% sequence identity with the amino acid residues Thr311 to Thr411 of SEQ ID NO:3 or SEQ ID NO:4.
8. The screening method as claimed in claim 1 wherein the polypeptide fragment consists essentially of amino acid residues Leu317 to His398 of SEQ ID NO:3 or SEQ ID NO:4 or a sequence which has at least 85% sequence identity with the amino acid residues Leu317 to His398 of SEQ ID NO:3 or SEQ ID NO:4.
9. The screening method as claimed in claim 1 wherein the polypeptide fragment consists essentially of amino acid residues His318 to His398 of SEQ ID NO:3 or SEQ ID NO:4 or a sequence which has at least 85% sequence identity with the amino acid residues His318 to His398 of SEQ ID NO:3 or SEQ ID NO:4.
10. The screening method as claimed in claim 1 wherein the polypeptide fragment consists essentially of leucine rich repeat regions 11 to 14 of TLR2 or a sequence which has at least 85% sequence identity with the leucine rich repeat regions 11 to 14 of TLR2.
11. The screening method as claimed in claim 1 wherein the polypeptide fragment is a fragment of human TLR2 or murine TLR2.