Fibronectin binding domains with reduced immunogenicity

• US 9,522,951 B2
• Filed: 10/31/2012
• Issued: 12/20/2016
• Est. Priority Date: 10/31/2011
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Davis, Jonathan, Lipovsek, Dasa, Camphausen, Ray
• Primary Examiner(s): Boesen, Christian
• Application Number:
  US14/355,155
• Publication Number:
  US 20150051149A1
• Time in Patent Office: 1,511 Days
• Field of Search: None
• US Class Current: 1/1
• 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 14/47 from mammals
• 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 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2833 against MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 16/2857 against nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, orphan receptor
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2318/20 Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue

Abstract

Fibronectin type III (10Fn3) binding domains having novel designs that are associated with reduced immunogenicity are provided. The application describes alternative 10Fn3 binding domains in which certain immunogenic regions are not modified when producing a binder in order to maintain recognition as a self antigen by the host organism. The application also describes 10Fn3 binding domains in which HLA anchor regions have been destroyed thereby reducing the immunogenic contribution of the adjoining region. Also provided are 10Fn3 domains having novel combinations of modified regions that can bind to a desired target with high affinity.

Claims

1. A polypeptide comprising a modified human ¹⁰Fn3 domain, which comprises AB, BC, CD, DE, EF and FG loops and β

-strands A, B, C, D, E, F and G, wherein the modified ¹⁰Fn3 domain comprises modifications only in;

(i) the amino acid sequence of the BC loop relative to the BC loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1);

(ii) the amino acid sequence of the FG loop relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1), and

(iii) the amino acid sequence of a β

-strand selected from the group consisting of β

-strand B, β

-strand C, and both β

-strand B and β

-strand C, relative to the β

-strand B and the β

-strand C of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1),

wherein the modifications in the BC and FG loops contribute to binding the same target, and the modified ¹⁰Fn3 domain has reduced immunogenicity relative to a modified ¹⁰Fn3 domain comprising the β

-strand B and β

-strand C of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1).

2. A library comprising a plurality of polypeptides of claim 1.

3. A nucleic acid encoding a polypeptide of claim 1.

4. A method of treating or diagnosing a disease or disorder in a subject in need thereof, comprising administering to the subject the polypeptide of claim 1.

5. The polypeptide of claim 1, wherein the modification in the FG loop is in the amino acid residues corresponding to positions 77-83 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO:

1).

6. The polypeptide of claim 1, wherein the modification in the BC loop is in the amino acid residues corresponding to positions 28-29, 27-29, 26-29, 25-29, or 24-29 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO:

1).

7. The polypeptide of claim 5, wherein the modification in the BC loop is in the amino acid residues corresponding to positions 28-29, 27-29, 26-29, 25-29, or 24-29 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO:

1).

8. The polypeptide of claim 1, comprising a modification of one or more amino acid residues corresponding to positions 18-21 of the β

-strand B of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1).

9. The polypeptide of claim 8, wherein the modification the β

-strand B is at a position corresponding to amino acid residues 19 and/or 21 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1).

10. The polypeptide of claim 1, comprising a modification of one or more amino acid residues relative to the corresponding β

-strand C of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1).

11. The polypeptide of claim 10, wherein the modification the β

-strand C is at a position corresponding to amino acid residues 33 and/or 35 of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

     1).

12. The polypeptide of claim 1, wherein the modifications are insertions, substitutions, or deletions.

13. The polypeptide of claim 5, wherein the modifications are substitutions.

14. The polypeptide of claim 6, wherein the modifications are substitutions.

15. The polypeptide of claim 1, wherein the modified ¹⁰Fn3 domain further comprises a pharmacokinetic moiety.

16. The polypeptide of claim 15, wherein the pharmacokinetic moiety is selected from the group consisting of a polyoxyalkylene moiety, a human serum albumin binding protein, a sialic acid, a human serum albumin, a transferring, an IgG, an IgG binding protein, and an Fc fragment.

17. The polypeptide of claim 1, comprising a second modified human ¹⁰Fn3 domain.

18. The polypeptide of claim 17, wherein each of the modified human ¹⁰Fn3 domains are connected by a polypeptide linker selected from any one of SEQ ID NOs:

32-43.

19. The polypeptide of claim 17, wherein each of the modified human ¹⁰Fn3 domains binds to the same target molecule.

20. The polypeptide of claim 17, wherein each of the modified human ¹⁰Fn3 domains binds to a different target molecule.