SERUM ALBUMIN-BINDING FIBRONECTIN TYPE III DOMAINS

• US 20170174748A1
• Filed: 03/19/2015
• Published: 06/22/2017
• Est. Priority Date: 03/20/2014
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: CAMPHAUSEN, Ray, MITCHELL, Tracy S., GOSSELIN, Michael L., PARKER, Rex, FABRIZIO, David, DAVIS, Jonathan, LIPOVSEK, Dasa
• Granted Patent: US 10,442,851 B2
• Time in Patent Office: Days
• 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: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• 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 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction

Abstract

The present invention relates to polypeptides which include tenth fibronectin type III domains (¹⁰Fn3) that binds to serum albumin, with south pole loop substitutions. The invention further relates to fusion molecules comprising a serum albumin-binding ¹⁰Fn3 joined to a heterologous protein for use in diagnostic and therapeutic applications.

Claims

1. A polypeptide comprising a fibronectin type III tenth domain (¹⁰Fn3) wherein the ¹⁰Fn3 domain comprises a) AB, BC, CD, DE, EF, and FG loops, b) a CD loop with an altered amino acid sequence relative to the sequence of the corresponding CD loop of the human ¹⁰Fn3 domain, c) wherein the polypeptide binds to human serum albumin with a K_D of less than 500 nM, and d) wherein the ¹⁰Fn3 domain further binds to one or more of rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin with a K_D of less than 500 nM, for example, less than 100 nM, or less than 10 nM.

2. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain binds to:

a) rhesus serum albumin and cynomolgus serum albumin,

b) mouse serum albumin and rat serum albumin, or

c) rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin.

3. The polypeptide of any one of the preceding claims, wherein the ¹⁰Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4.

4. The polypeptide of any one of the preceding claims, wherein the ¹⁰Fn3 domain binds to domain I-II of HSA.

5. The polypeptide of any one of the preceding claims, wherein the CD loop comprises an amino acid sequence according to the formula G-X₁-X₂-V-X₃-X₄-X₅-S-X₆-X₇-G-X₈-X₉-Y-X₁₀-X₁₁-X₁₂-E, wherein,

(a) X₁ is selected from the group consisting of R or W;

(b) X₂ is selected from the group consisting of H, E, D, Y, or Q;

(c) X₃ is selected from the group consisting of Q or H;

(d) X₄ is selected from the group consisting of I, K, M, Q, L, or V;

(e) X₅ is selected from the group consisting of Y, F, or N;

(f) X₆ is selected from the group consisting of D, V, or E;

(g) X₇ is selected from the group consisting of L, W, or F;

(h) X₈ is selected from the group consisting of P or T;

(i) X₉ is selected from the group consisting of L or M;

(j) X₁₀ is selected from the group consisting of I or V;

(k) X₁₁ is selected from the group consisting of Y or F; and

(l) X₁₂ is selected from the group consisting of T, S, Q, N, or A.

6. The polypeptide of claim 5, wherein the CD loop comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

106, 113, 101-105, 107-112, and 114-125.

7. A polypeptide of any one of the preceding claims, wherein the polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the non-CD loop regions of SEQ ID NOs:

81, 88, 23-80, 82-87, 89-100, 184-209 and 235-260.

8. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs:

;

81, 88, 23-80, 82-87, 89-100, 184-209 and 235-260.

15. A composition comprising a polypeptide of any one of the preceding claims and a carrier.

16. An isolated nucleic acid molecule encoding the polypeptide of any one of claims 1-14.

17. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule has a sequence selected from the group consisting of SEQ ID NOs:

126-151 and 172 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto.

18. An expression vector comprising the nucleotide sequence encoding the polypeptide of any one of claims 1-14.

19. A cell comprising a nucleic acid molecule of claim 16 or 17 or expression vector of claim 18.

20. A method of producing the polypeptides of any one of claims 1-14 comprising culturing the cell of claim 19 under conditions suitable for expressing the polypeptide, and purifying the polypeptide.

9. A fusion polypeptide comprising a fibronectin type III tenth (¹⁰Fn3) domain and a heterologous protein, wherein the ¹⁰Fn3 domain comprises a) AB, BC, CD, DE, EF, and FG loops, b) a CD loop with an altered amino acid sequence relative to the sequence of the corresponding loop of the human ¹⁰Fn3 domain, c) wherein the polypeptide binds to human serum albumin with a K_D of less than 500 nM, and wherein the ¹⁰Fn3 domain further binds to one or more of rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin with a K_D of less than 500 nM, for example, less than 100 nM, or less than 10 nM.

10. The fusion polypeptide of claim 9, wherein the heterologous protein is a therapeutic moiety.

11. The fusion polypeptide of claim 9, wherein the heterologous protein is a polypeptide comprising a ¹⁰Fn3 domain.

12. The fusion polypeptide of claim 11, wherein the ¹⁰Fn3 domain binds to a target protein other than serum albumin, such as PCSK9.

13. The fusion polypeptide of claim 9, wherein the ¹⁰Fn3 domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

81, 88, 23-80, 82-87, 89-100, 184-209 and 235-260.

14. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

81, 88, 23-80, 82-87, 89-100, 168-169, 184-209 and 235-261.