Fibronectin binding domains with reduced immunogenicity

Patent metadata

Field	Value
Publication number	US-11279751-B2
Application number	US-201916581877-A
Country	US
Kind code	B2
Filing date	Sep 25, 2019
Priority date	Oct 31, 2011
Publication date	Mar 22, 2022
Grant date	Mar 22, 2022

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.

First claim

What is claimed:

1. A polypeptide comprising a human fibronectin type 3 tenth ( 10 Fn3) domain, which comprises AB, BC, CD, DE, EF, and FG loops and β-strands A, B, C, D, E, F, and G, wherein: (i) the BC loop has the amino acid sequence of the corresponding loop of a wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), (ii) the amino acid sequences of the CD and FG loops are modified relative to the corresponding loops of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), and (iii) the amino acid sequence of at least one of the β-strands is modified relative to the corresponding β-strand of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

2. The polypeptide of claim 1 , wherein the at least one modified β-strand is adjacent to the modified CD loop or the modified FG loop in a linear sequence of the 10 Fn3 domain.

3. The polypeptide of claim 1 , wherein the amino acid sequences of at least two of the β-strands have been modified relative to the corresponding β-strands of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

4. The polypeptide of claim 3 , wherein each of the at least two modified β-strands is adjacent to each side of the modified CD loop or the modified FG loop in the linear sequence of the 10 Fn3 domain.

5. The polypeptide of claim 3 , wherein the modified CD loop, the modified FG loop, and each of the at least two modified β-strands contribute to binding to a target.

6. The polypeptide of claim 3 , wherein the amino acid sequences of the β-strand C, the β-strand D, the β-strand F, and the β-strand G are modified relative to the corresponding β-strands of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

7. The polypeptide of claim 1 , wherein the AB loop, the DE loop, the EF loop, or a combination thereof has the amino acid sequence of the corresponding loop of the wild-type human 10 Fn3 domain (SEQ ID NO: l or 6).

8. The polypeptide of claim 1 , which has reduced immunogenicity relative to a corresponding polypeptide comprising the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

9. The polypeptide of claim 1 , wherein the amino acid sequence of the modified CD loop, the modified FG loop, or both have been extended in length relative to the amino acid sequence of the corresponding loop of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

10. The polypeptide of claim 1 , wherein the amino acid sequence of the modified CD loop, the modified FG loop, or both have been reduced in length relative to the amino acid sequence of the corresponding loop of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

11. The polypeptide of claim 1 , which has at least 50% sequence identity to the amino acid sequence of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

12. The polypeptide of claim 1 , wherein the polypeptide which has at least 65% sequence identity to the amino acid sequence of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

13. A library comprising a plurality of polypeptides comprising a human fibronectin type 3 tenth ( 10 Fn3) domain, which comprises AB, BC, CD, DE, EF, and FG loops and β strands A, B, C, D, E, F, and G, wherein: (i) the BC loop has the amino acid sequence of the corresponding loop of a wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), (ii) the amino acid sequences of the CD and FG loops are modified relative to the corresponding loops of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), and (iii) the amino acid sequence of at least one of the β-strands is modified relative to the corresponding β-strand of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

14. The library of claim 13 , which comprises at least 10 5 polypeptides each comprising a different 10 Fn3 domain sequence.

15. The polypeptide of claim 1 , wherein at least four of the amino acid residues of the CD loop are modified relative to the corresponding amino acid residues of the CD loop of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

16. The polypeptide of claim 1 , wherein at least one amino acid residue of the CD loop is the same as the wild type amino acids at those corresponding amino acid residue of the CD loop of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

17. The polypeptide of claim 16 , wherein the at least one amino acid residue of the CD loop that is the same as the wild-type amino acid residue corresponds to amino acid residue 46 of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

18. The polypeptide of claim 16 , wherein the at least one amino acid residue of the CD loop that is the same as the wild-type amino acid residue corresponds to amino acid residue 47 of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

19. The polypeptide of claim 6 , wherein each of modified β-strands C, D, F, and G comprises a hydrophobic core amino acid residue, and wherein one or more of the hydrophobic core amino acid residues are not modified relative to the corresponding amino acid residues of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6).

20. The polypeptide of claim 6 , wherein: (i) two of the amino acid residues of the β-strand C are modified relative to the corresponding amino acid residues of β-strand C of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), (ii) one of the amino acid residues of the β-strand D is modified relative to the corresponding amino acid residue of β-strand D of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), (iii) three of the amino acid residues of the β-strand F are modified relative to the corresponding amino acid residues of the β-strand F of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), (iv) two of the amino acid residues of the β-strand G are modified relative to the corresponding amino acid residues of the β-strand G of the wild-type human 10 Fn3 domain (SEQ ID NO: 1 or 6), or (v) any combination thereof.

Assignees

• Bristol Myers Squibb Co

Inventors

• Davis Jonathan
• Lipovsek Dasa
• Camphausen Ray

Classifications

• C12N15/1062 Primary

  mRNA-Display, e.g. polypeptide and encoding template are connected covalently · CPC title

• C07K16/2857

  against nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, orphan receptor · CPC title

• C07K2318/20

  Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics · CPC title

• C07K16/244

  Interleukins [IL] · CPC title

• C07K16/241 Primary

  Tumor Necrosis Factors · CPC title