FIBRONECTIN BINDING DOMAINS WITH REDUCED IMMUNOGENICITY

• US 20170137494A1
• Filed: 11/02/2016
• Published: 05/18/2017
• Est. Priority Date: 10/31/2011
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: CAMPHAUSEN, Ray, LIPOVSEK, Dasa, DAVIS, Jonathan
• Granted Patent: US 10,604,556 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: 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

1-224. -224. (canceled)

225. A polypeptide comprising a human fibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain comprises (i) a modification in the amino acid sequence of at least one north pole loop selected from the BC, DE and FG loops relative to the corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO:

1 or

     6), and (ii) a modification in the amino acid sequence of at least one south pole loop selected from the AB, CD and EF loops relative to the corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the at least one modified north pole loop and the at least one modified south pole loop contribute to binding the same target.

226. A library comprising a plurality of polypeptides of claim 225.

229. The polypeptide of claim 225, wherein the ¹⁰Fn3 domain comprises a Front binder, which comprises a modification in the amino acid sequence of the CD loop and the DE loop relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO:

1 or

     6), wherein the modifications contribute to binding the same target.

230. The polypeptide of claim 225, wherein the ¹⁰Fn3 domain comprises a Back binder, which comprises a modification in the amino acid sequence of at least three regions selected from the group consisting of β

-strand A, β

-strand G, the EF loop, the FG loop, the N-terminal region, and the C-terminal region relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target.

239. A nucleic acid encoding a polypeptide of claim 225.

227. A polypeptide comprising a human fibronectin type 3 tenth (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain comprises (i) a modification in the amino acid sequence of at least one of loops AB, BC, CD, DE, EF, or FG relative to the corresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO:

1 or

     6), and (ii) a modification in the amino acid sequence of at least one β

-strand relative to the corresponding β

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

1 or

     6), wherein the at least one modified loop and the at least one modified β

-strand contribute to binding the same target.

228. A library comprising a plurality of polypeptides of claim 227.

231. The polypeptide of claim 227, wherein the ¹⁰Fn3 domain comprises a South Pole binder, which comprises a modification in the amino acid sequence of at least two regions selected from the group consisting of the AB loop, the CD loop, the EF loop, β

-strand A, β

-strand B, β

-strand E, and β

-strand F, relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target.

232. The polypeptide of claim 227, wherein the ¹⁰Fn3 domain comprises a Northwest binder, which comprises a modification in the amino acid sequence of the BC loop, the DE loop, and the FG loop, and a modification in the amino acid sequence of one or more of β

-strand C, β

-strand D, β

-strand F, and β

-strand G, relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target.

233. The polypeptide of claim 227, wherein the ¹⁰Fn3 domain comprises a Northeast binder, which comprises a modification in the amino acid sequence of the BC loop, the DE loop, and the FG loop, and a modification in the amino acid sequence of one or more of the N-terminal region, β

-strand A, β

-strand B, and β

-strand E, relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target.

234. The polypeptide of claim 227, wherein the ¹⁰Fn3 domain comprises a South Front binder, which comprises a modification in the amino acid sequence of one or more of the AB loop, the CD loop, the DE loop, and the EF loop, and a modification in the amino acid sequence of one or more of β

-strand B, β

-strand D, and β

-strand E, relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target.

235. The polypeptide of claim 227, wherein the ¹⁰Fn3 domain comprises a Southwest binder, which comprises a modification in the amino acid sequence of one or more of the CD loop and the EF loop, relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO:

1 or

     6), wherein the modifications contribute to binding the same target.

236. The polypeptide of claim 227, wherein the ¹⁰Fn3 domain comprises a North Pole binder, which comprises a modification in the amino acid sequence of one or more of the BC loop, the DE loop, and the FG loop, and a modification in the amino acid sequence of one or more of the N-terminal region, β

-strand A, β

-strand B, β

-strand C, β

-strand D, β

-strand E, β

-strand F, and β

-strand G, relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target.

240. A nucleic acid encoding a polypeptide of claim 227.

237. A polypeptide comprising a human ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain comprises:

(i) an AG binder, which comprises a modification in the amino acid sequence of β

-strand A and β

-strand G relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target;

(ii) a modification in the amino acid sequence of the residues at positions 77-83 of the FG loop relative to the wild-type human ¹⁰Fn3 domain (SEQ ID NO;

1 or

     6), wherein the modifications contribute to binding the same target;

or

(iii) a modification in the amino acid sequence of the FG loop and a modification in a β

-strand.

238. A library comprising a plurality of polypeptides of claim 237.

241. A nucleic acid encoding a polypeptide of claim 237.