Fast-off rate serum albumin binding fibronectin type III domains

Patent metadata

Field	Value
Publication number	US-10766946-B2
Application number	US-201615760413-A
Country	US
Kind code	B2
Filing date	Sep 22, 2016
Priority date	Sep 23, 2015
Publication date	Sep 8, 2020
Grant date	Sep 8, 2020

Abstract

Provided herein are polypeptides which include tenth fibronectin type III domains ( 10 Fn3) that binds to serum albumin. Also provided are fusion molecules comprising a serum albumin binding 10 Fn3 joined to a heterologous protein for use in diagnostic and therapeutic applications.

First claim

We claim:

1. A polypeptide comprising a fibronectin type III tenth ( 10 Fn3) domain, wherein the 10 Fn3 domain binds to human serum albumin (HSA) with a K D of 1 μM or less and comprises AB, BC, CD, DE, EF, and FG loops, wherein the CD loop has 1 amino acid substitution relative to the CD loop of a 10 Fn3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23, and wherein the substitution is selected from the group consisting of:
(i) the arginine (R) at position 2 of the CD loop (SEQ ID NO: 28) is substituted with an amino acid selected from D, E, A, Q, or N;
(ii) the glutamine (Q) at position 5 of the CD loop (SEQ ID NO: 28) is substituted with D;
(iii) the tyrosine (Y) at position 7 of the CD loop (SEQ ID NO: 28) is substituted with an amino acid selected from S or A;
(iv) the leucine (L) at position 10 of the CD loop (SEQ ID NO: 28) is substituted with an amino acid selected from S, T, or A;
(v) the leucine (L) at position 13 of the CD loop (SEQ ID NO: 28) is substituted with an amino acid selected from D, S, or A;
(vi) the tyrosine (Y) at position 14 of the CD loop (SEQ ID NO: 28) is substituted with an amino acid selected from A or T;
(vii) the isoleucine (I) at position 15 of the CD loop (SEQ ID NO: 28) is substituted with an amino acid selected from D, S, or A; and
(viii) the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO: 28) is substituted with an amino acid selected from Q, E, S, or A.

2. The polypeptide of claim 1 , wherein the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO: 28) is substituted with serine (S).

3. The polypeptide of claim 1 , wherein the leucine (L) at position 10 of the CD loop (SEQ ID NO: 28) is substituted with threonine (T).

4. The polypeptide of claim 1 , wherein the tyrosine (Y) at position 16 of the CD loop (SEQ ID NO: 28) is substituted with glutamine (Q).

5. The polypeptide of claim 1 , wherein the FG loop has 1 amino acid substitution relative to the FG loop of the 10 Fn3 domain comprising the amino acid sequence set forth in SEQ ID NO: 23, wherein the lysine (K) at position 12 of the FG loop (SEQ ID NO: 29) is substituted with an amino acid selected from A, T, or S.

6. The polypeptide of claim 1 , wherein the polypeptide comprises an amino acid sequence at least 90% identical to the non-CD loop regions of SEQ ID NOs: 30-34, 36-40, 42-46, 48, 49, 51, 52, 54, 55, 57, 58, 60, 61, 62, 63, 65-68, 70, 71, 73-76, 78, 79, 81-84, 86, 87, 89-98, 100-109, and 111-118.

7. The polypeptide of claim 1 , wherein the 10 Fn3 domain binds to domain I-II of HSA.

8. The polypeptide of claim 1 , wherein the polypeptide comprises a heterologous protein.

9. A composition comprising the polypeptide of claim 1 , and a carrier.

10. A method of producing a polypeptide comprising a fibronectin type III tenth ( 10 Fn3) domain that binds to human serum albumin, the method comprising culturing a cell comprising a nucleic acid which encodes the polypeptide of claim 1 under conditions suitable for expressing the polypeptide, and purifying the polypeptide.

11. A method of enhancing the half-life of a moiety, comprising linking to the moiety a human serum albumin binding polypeptide of claim 1 .

Assignees

• Bristol Myers Squibb Co

Inventors

• Krystek Jr Stanley Richard
• Mitchell Tracy S
• Gosselin Michael L
• Lipovsek Dasa
• Juneja Juhi

Classifications

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C12N2015/8518

  expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title

• C07K14/765

  Serum albumin, e.g. HSA · CPC title

• C12N15/85

  for animal cells · CPC title