Method of identifying polypeptide monobodies which bind to target proteins and use thereof

• US 7,598,352 B2
• Filed: 11/19/2001
• Issued: 10/06/2009
• Est. Priority Date: 11/17/2000
• Status: Expired due to Fees

• Current Assignee: University of Rochester
• Original Assignee: University of Rochester
• Inventors: Koide, Shohei
• Primary Examiner(s): Rao; Manjunath N
• Assistant Examiner(s): Shafer; Shulamith H
• Application Number:
  US10/006,760
• Publication Number:
  US 20030186385A1
• Time in Patent Office: 2,878 Days
• Field of Search: 530/380, 530/387.1, 530/381, 530/350
• US Class Current: 530/388.9
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2869 against hormone receptors for antibodies against neuromediator receptors C07K16/286
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

A method of identifying a polypeptide monobody having target protein binding activity, said method comprising: providing a host cell comprising (i) a reporter gene under control of a 5′ regulatory region operable in the host cell, (ii) a first chimeric gene which encodes a first fusion polypeptide comprising a target protein, or fragment thereof, fused to a C-terminus of a DNA-binding domain which binds to the 5′ regulatory region of the reporter gene, and (iii) a second chimeric gene which encodes a second fusion polypeptide comprising a polypeptide monobody fused to a transcriptional activation domain; and detecting expression of the reporter gene, which indicates binding of the polypeptide monobody of the second fusion polypeptide to the target protein such that the transcriptional activation domain of the second fusion polypeptide is in sufficient proximity to the DNA-binding domain of the first fusion polypeptide to allow expression of the reporter gene.

Claims

1. A fibronectin type III (Fn3) polypeptide monobody derived from the amino acid sequence of SEQ ID NO:

2 or SEQ ID NO;

3, the polypeptide monobody comprising;

at least two adjacent Fn3 β

-strand domain sequences selected from the group of A (residues 9-14 of SEQ ID NO;

2 and SEQ ID NO;

     3), B (residues 17-21 of SEQ ID NO;

2 and SEQ ID NO;

     3), C (residues 31-38 of SEQ ID NO;

2 and SEQ ID NO;

     3), D (residues 46-50 of SEQ ID NO;

2 and SEQ ID NO;

     3), E (residues 55-59 of SEQ ID NO;

2 and SEQ ID NO;

     3), F (residues 67-75 of SEQ ID NO;

2 and SEQ ID NO;

     3), and G (residues 88-94 of SEQ ID NO;

2 and SEQ ID NO;

     3), with an associated loop region sequence linked between each pair of adjacent β

-strand domain sequences, each of the loop region sequences being selected from the group of loops AB (residues 15-16 of SEQ ID NO;

2 and SEQ ID NO;

     3), BC (residues 22-30 of SEQ ID NO;

2 and SEQ ID NO;

     3), CD (residues 39-45 of SEQ ID NO;

2 and SEQ ID NO;

     3), DE (residues 51-54 of SEQ ID NO;

2 and SEQ ID NO;

     3), EF (residues 60-66 of SEQ ID NO;

2 and SEQ ID NO;

     3), and FG residues 76-87 of SEQ ID NO;

2 and SEQ ID NO;

     3); and

optionally, an N-terminal tail of at least about 2 to about 33 amino acids, a C-terminal tail of at least about 2 to about 25 amino acids, or both;

wherein at least one loop region sequence comprises a modified amino acid sequence which varies from a corresponding loop region present in SEQ ID NO;

2 or SEQ ID NO;

3 by deletion of up to all but one amino acid residue, insertion of two to about 25 amino acid residues, or replacement of two to all amino acid residues, and

wherein the polypeptide monobody exhibits estrogen receptor binding activity via interaction of the at least one loop region sequence with the estrogen receptor.

2. The polypeptide monobody according to claim 1, wherein the polypeptide monobody exhibits estrogen receptor binding activity in the presence of an estrogen receptor agonist or an extrogen receptor antagonist.

3. The polypeptide monobody according to claim 2, wherein the estrogen receptor agonist is estradiol, estriol, diethylstilbestrol, or genistein.

4. The polypeptide monobody according to claim 2, wherein the estrogen receptor antagonist is hydroxy tamoxifen, ICI182780, or raloxifene.

5. The polypeptide monobody according to claim 1, wherein said at least two Fn3 β

-strand domain sequences comprises each of β

-strand domain sequences A through G of SEQ ID NO;

2 or SEQ ID NO;

3, wherein the loop region sequences comprise the AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop.

6. The polypeptide monobody according to claim 5, wherein the loop region sequence that comprises the modified amino acid sequence is selected from the group consisting of the AB loop region sequence, the BC loop region sequence, the DE loop region sequence, and the FG loop region sequence.

7. The polypeptide monobody according to claim 5, wherein the BC loop region sequence and the FG loop region sequence both comprise one of said modified amino acid sequences.

8. A fusion protein comprising:

a first portion comprising a polypeptide monobody according to claim 1 and

a second portion fused to the first portion.

9. The fusion protein according to claim 8, wherein the second portion comprises a label.

10. The fusion protein according to claim 9, wherein the label is an alkaline phosphatase tag or a His₍₆₎ tag.

11. The fusion protein according to claim 8, wherein the second portion comprises a transcriptional activation domain.

12. The polypeptide monobody according to claim 1, wherein the FG loop region sequence comprises the amino acid sequence selected from the group of SEQ ID NO:

20 and SEQ ID NO;

32.

13. The polypeptide monobody according to claim 1, wherein the BC loop region sequence comprises the amino acid sequence of SEQ ID NO:

23.

14. The polypeptide monobody according to claim 1, wherein the FG loop region sequence comprises the amino acid sequence of SEQ ID NO:

67.

15. The polypeptide monobody according to claim 1, wherein the AB loop region sequence comprises the amino acid sequence of SEQ ID NO:

34.

17. The polypeptide monobody according to claim 1, wherein the BC loop region sequence comprises the amino acid sequence selected from the group of SEQ ID NO:

22 and SEQ ID NO;

24.

18. The polypeptide monobody according to claim 1, wherein the FG loop region sequence comprises the amino acid sequence selected from the group of SEQ ID NO:

48 and SEQ ID NO;

49.

19. The polypeptide monobody according to claim 1, wherein the AB loop region sequence comprises the amino acid sequence of SEQ ID NO:

35.

16. A fibronectin type III (Fn3) polypeptide monobody comprising the formula β

_A-L_AB-β

_B-L_BC-β

_C-L_CD-β

_D-L_DE-β

_E-L_EF-β

_F-L_FG-β

_G, wherein;

β

_A, β

_B, β

_C, β

_D, β

_E, β

_F, and β

_G are, respectively, β

-strand domain sequences A through G of a tenth Fn3 domain of fibronectin; and

L_AB, L_BC, L_CD, L_DE, L_EF, and L_FG are, respectively, loop region sequences AB, BC, CD, DE, EF, and FG, wherein at least one loop region sequence selected from the group of AB, BC, and FG varies from a corresponding loop region present in the tenth Fn3 domain of fibronectin by deletion of up to all but one amino acid residue, insertion of two to about 25 amino acid residues, or replacement of two to all amino acid residues; and

wherein the polypeptide monobody exhibits estrogen receptor binding activity via interaction of the at least one loop region sequence with the estrogen receptor.

20. A fibronectin type III (Fn3) polypeptide monobody derived from the amino acid sequence of SEQ ID NO:

2 or SEQ ID NO;

3, the polypeptide monobody comprising;

at least two adjacent Fn3 β

-strand domain sequences selected from the group of A (residues 9-14 of SEQ ID NO;

2 and SEQ ID NO;

     3), B (residues 17-21 of SEQ ID NO;

2 and SEQ ID NO;

     3), C (residues 31-38 of SEQ ID NO;

2 and SEQ ID NO;

     3), D (residues 46-50 of SEQ ID NO;

2 and SEQ ID NO;

     3), E (residues 55-59 of SEQ ID NO;

2 and SEQ ID NO;

     3), F (residues 67-75 of SEQ ID NO;

2 and SEQ ID NO;

     3), and G (residues 88-94 of SEQ ID NO;

2 and SEQ ID NO;

     3), with an associated loop region sequence linked between each pair of adjacent β

-strand domain sequences, each of the loop region sequences being selected from the group of loops AB, BC, CD, DE, EF, and FG; and

optionally, an N-terminal tail of at least about 2 to about 33 amino acids, a C-terminal tail of at least about 2 to about 25 amino acids, or both;

wherein at least one loop region sequence is modified from the corresponding loop region of SEQ ID NO;

2 or SEQ ID NO;

3, the at least one loop region sequence being selected from the group of (i) the BC loop region sequence comprising the amino acid sequence of SEQ ID NO;

23, (ii) the FG loop region sequence comprising the amino acid sequence of SEQ ID NO;

67, and (iii) the AB loop region sequence comprising the amino acid sequence of SEQ ID NO;

34; and

wherein the polypeptide monobody exhibits estrogen receptor binding activity via interaction of the at least one loop region sequence with the estrogen receptor.