# US 10,766,946 B2 · Fast-off rate serum albumin binding fibronectin type III domains

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- Number: US 10,766,946 B2
- Document ID: US10766946B2
- Status: Granted
- Kind: B2
- Application number: 15/760,413
- Filed: 2016-09-22
- Published: 2018-09-13
- Granted: 2020-09-08
- Earliest claimed: 2015-09-23
- Assignees: Bristol-Myers Squibb Company
- Applicants: Bristol-Myers Squibb Company
- Inventors: Stanley Richard Krystek, Jr.; Tracy S. Mitchell; Michael L. Gosselin; Dasa Lipovsek; Juhi Juneja
- Web page: https://patentgrep.com/patent/US10766946B2

## CPC

- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C07K 14/765: Serum albumin, e.g. HSA
- C12N 15/85: for animal cells
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C12N 2015/8518: {expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

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

## Claims

### Claim 1

A polypeptide comprising a fibronectin type III tenth (10Fn3) domain, wherein the 10Fn3 domain binds to human serum albumin (HSA) with a KD 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 10Fn3 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.

### Claim 2 (depends on 1)

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).

### Claim 3 (depends on 1)

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).

### Claim 4 (depends on 1)

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).

### Claim 5 (depends on 1)

The polypeptide of claim 1, wherein the FG loop has 1 amino acid substitution relative to the FG loop of the 10Fn3 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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

A method of producing a polypeptide comprising a fibronectin type III tenth (10Fn3) 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.

### Claim 11 (depends on 1)

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