Specific Binding Members Against Synaptophysin

• US 20080274123A1
• Filed: 03/29/2005
• Published: 11/06/2008
• Est. Priority Date: 03/29/2004
• Status: Active Grant

• Current Assignee: The University Court of the University of Aberdeen
• Original Assignee: The University Court of the University of Aberdeen
• Inventors: Porter, Andy, Wright, Matthew
• Granted Patent: US 7,871,782 B2
• Time in Patent Office: Days
• US Class Current: 424/172.1
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2800/085 Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Abstract

The present invention provides specific binding members that bind synaptophysin and which comprise: an antibody VH domain selected from the group consisting of the C1-3 VH domain (SEQ ID NO. 2) and a VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO. 12 and optionally one or more VH CDR'"'"'s with an amino acid sequence selected from SEQ ID NO. 10 and SEQ ID NO. 11; and/or an antibody VL domain selected front the group consisting of the C1-3 VL domain (SEQ ID NO. 4) and a VL domain comprising one or more VL CDR'"'"'s with an amino acid sequence selected from SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15. The invention further provides related materials such as nucleic acids, kits and compositions, and also methods of use of the binding member, for instance in targeting entities to hepatic stellate cells which are implicated it liver fibrosis.

Claims

1. A specific binding member that binds synaptophysin and which comprises:

an antibody VH domain selected from the group consisting of the C1-3 VH domain (SEQ ID NO.

     2) and a VH domain comprising a VH CDR3 with the amino acid sequence of SEQ ID NO. 12 and optionally one or more VH CDR'"'"'s with an amino acid sequence selected from SEQ ID NO. 10 and SEQ ID NO. 11; and

/or

an antibody VL domain selected from the group consisting of the C1-3 VL domain (SEQ ID NO.

     4) and a VL domain comprising one or more VL CDR'"'"'s with an amino acid sequence selected from SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15.

2. A specific binding member according to claim 1 comprising an antibody VH domain comprising the VH CDR'"'"'s with the amino acid sequences of SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, which specific binding member competes for binding to synaptophysin with an synaptophysin binding domain of an antibody comprising the C1-3 VH domain (SEQ ID NO. 2) and the C1-3 VL domain (SEQ ID NO. 4).

3. A specific binding member according to claim 1 comprising the C1-3 VH domain (SEQ ID NO. 2).

4. A specific binding member according to claim 3 comprising the C1-3 VL domain (SEQ ID NO. 4)

5. A specific binding member according to claim 1 that binds synaptophysin with affinity equal to or better than the affinity of an synaptophysin antigen-binding site formed by the C1-3 VH domain (SEQ ID NO. 2) and the C1-3 VL domain (SEQ ID NO. 4), the affinity of the specific binding member and the affinity of the antigen-binding site being as determined under the same conditions.

6. A specific binding member according to claim 1 which binds an epitope within the amino acid sequence YPFRLHQVYFDAPSC (SEQ ID NO:

9).

7. A specific binding member according to claim 1 that comprises an scFv antibody molecule.

8. A specific binding member according to claim 1 that comprises an antibody constant region.

9. A specific binding member according to claim 8 that comprises a whole antibody.

10. A specific binding member according to claim 1 which comprises additional amino acids providing a further functional characteristic in addition to the ability to bind antigen.

11. A specific binding member according to claim 1 which is conjugated to a detectable label, enzyme, or toxin, optionally via a peptidyl bond or linker.

12. A specific binding member according to claim 11 wherein the toxin is selected from the group comprising tributyl-tin and gliotoxin.

13. A specific binding member according to claim 11 wherein the detectable label is FITC.

14. An isolated nucleic acid which comprises a nucleotide sequence encoding a specific binding member or antibody VH or VL domain of a specific binding member according to claim 1.

15. A host cell transformed with nucleic acid according to claim 14.

16. A method of producing a specific binding member or antibody VH or VL domain, the method comprising culturing host cells according to claim 15 under conditions for production of said specific binding member or antibody VH or VL domain.

17. A method according to claim 16 further comprising isolating and/or purifying said specific binding member or antibody VH or VL variable domain.

18. A method according to claim 16 further comprising formulating the specific binding member or antibody VH or VL variable domain into a composition including at least one additional component.

26. A method according to claim 16, 19 or 20 further comprising binding a specific binding member that binds synaptophysin to synaptophysin or a fragment of synaptophysin.

27. A method comprising binding a specific binding member that binds synaptophysin according to claim 1 to synaptophysin or a fragment of synaptophysin.

28. A method according to claim 26 wherein said binding takes place in vitro.

29. A method according to claim 26 comprising determining the amount of binding of specific binding member to synaptophysin or a fragment of synaptophysin.

30. A method according to claims 16, 19 or 20 further comprising use of the specific binding member in the manufacture of a medicament for treatment of a disease or disorder characterised by liver fibrosis.

31. Use of a specific binding member according to claim 1 in the manufacture of a medicament for treatment of a disease or disorder characterised by liver fibrosis.

32. A method of treatment of a disease or disorder characterised by liver fibrosis, the method comprising administering a specific binding member according to claim 1 to a patient with the disease or disorder or at risk of developing the disease or disorder.

33. A method according to claim 32 wherein the specific binding member directs the delivery of a pharmaceutical composition to target hepatic stellate cells.

34. Use of a specific binding member according to claim 1 and one or more reagents that allow determination of the binding of said member to hepatic stellate cells, in the manufacture of a diagnostic agent for the detection of a disease or disorder characterised by liver fibrosis.

35. A method of diagnosis of a disease or disorder characterised by liver fibrosis, the method comprising administering a specific binding member according to claim 1 and one or more reagents that allow determination of the binding of said member to hepatic stellate cells, to a patient with the disease or disorder or at risk of developing the disease or disorder.

36. A diagnostic kit comprising a specific binding member according to claim 1 and one or more reagents that allow determination of the binding of said member to hepatic stellate cells.

37. A pharmaceutical composition comprising as active principle a specific binding member according to claim 1 in an effective amount, in conjunction with a pharmaceutically acceptable excipient.

19. A method of obtaining a specific binding member that binds synaptophysin, the method comprising

providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the C1-3 VH domain (SEQ ID NO. 2) one or more VH domains each of which is an amino acid sequence variant of the C1-3 VH domain, optionally combining one or more VH domain amino acid sequence variants thus provided with one or more VL domains to provide one or more VH/VL combinations;

and/or

providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the C1-3 VL domain (SEQ ID NO.

     4) a VL domain which is an amino acid sequence variant of the C1-3 VL domain, and combining one or more VL domain amino acid sequence variants thus provided with one or more VH domains to provide one or more VH/VL domain combinations;

and

testing the VH domain amino acid sequence variants or VH/VL combination or combinations for to identify a specific binding member that binds synaptophysin.

21. A method according to claim 19 or claim 20 wherein the specific binding member that binds synaptophysin is an antibody fragment comprising a VH domain and a VL domain.

22. A method according to claim 21 wherein the antibody fragment is an scFv antibody molecule.

23. A method according to claim 21 wherein the antibody fragment is an Fab antibody molecule.

24. A method according to claim 22 further comprising providing the VH domain and/or the VL domain of the antibody fragment in a whole antibody.

25. A method according to claim 19 or 20 further comprising formulating the specific binding member that binds synaptophysin or an antibody VH or VL variable domain of the specific binding member that binds synaptophysin into a composition including at least one additional component.

20. A method of obtaining a specific binding member that binds synaptophysin, which method comprises:

providing starting nucleic acids encoding one or more VH domains which either comprise a CDR3 to be replaced or lack a CDR3 encoding region, and combining said starting nucleic acid with a donor nucleic acid encoding the VH CDR3 amino acid sequence of SEQ ID NO. 12 such that said donor nucleic acid is inserted into the CDR3 region in the starting nucleic acid, so as to provide product nucleic acids encoding VH domains;

or

providing starting nucleic acids encoding one or more VL domains which either comprise a CDR3 to be replaced or lack a CDR3 encoding region, and combining said starting nucleic acid with a donor nucleic acid encoding the VL CDR3 amino acid sequence of SEQ ID NO. 15 such that said donor nucleic acid is inserted into the CDR3 region in the starting nucleic acid, so as to provide product nucleic acids encoding VL domains;

expressing the nucleic acids of said product nucleic acids encoding VH domains and optionally combining the VH domains thus produced with one or more VL domains to provide VH/VL combinations, and/or expressing the nucleic acids of said product nucleic acids encoding VL domains and combining the VL domains thus produced with one or more VH domains to provide VH/VL combinations;

selecting a specific binding member comprising a VH domain or a VH/VL combination that binds synaptophysin; and

recovering said specific binding member that binds synaptophysin and/or nucleic acid encoding the specific binding member that binds synaptophysin.