Stabilized fibronectin domain compositions, methods and uses

• US 9,234,029 B2
• Filed: 09/23/2013
• Issued: 01/12/2016
• Est. Priority Date: 04/30/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Jacobs, Steven
• Primary Examiner(s): Ha, Julie
• Assistant Examiner(s): Hellman, Kristina M
• Application Number:
  US14/033,994
• Publication Number:
  US 20140018519A1
• Time in Patent Office: 841 Days
• Field of Search: None
• US Class Current: 1/1
• 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: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/02 Nasal agents, e.g. decongestants
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/16 Otologicals
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 16/18 : against material from animals or humans
• 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 15/1044 : Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A protein scaffold based on a consensus sequence of fibronectin type III (FN3) proteins, such as the tenth FN3 repeat from human fibronectin (human Tenascin), including isolated nucleic acids that encode a protein scaffold, vectors, host cells, and methods of making and using thereof, exhibit enhanced thermal and chemical stability while presenting six modifiable loop domains which can be engineered to form a binding partner capable of binding to a target for applications in diagnostic and/or therapeutic compositions, methods and devices.

Claims

1. A protein scaffold comprising loop domains topologically similar to loop domains of a fibronectin type III (FN3) domain, the protein scaffold having an amino acid sequence based on the consensus amino acid sequence of SEQ ID NO:

16, wherein one or more specific residues of SEQ ID NO;

16 are replaced to enhance melting temperature (Tm) and chemical stability of the protein scaffold as compared to the Tm and chemical stability of a protein scaffold having the amino acid sequence of SEQ ID NO;

16, and the replacements of SEQ ID NO;

16 are selected from the group consisting of N46V, E14P, L17A, E86I, a combination of N46V and E86I, a combination of N46V and E86I, and a combination of L17A, N46V and E86I.

2. The protein scaffold of claim 1, wherein the Tm is measured by differential scanning calorimetry, the chemical stability is measured as [D] by resistance to guanidinium chloride denaturation, and the Tm is increased as compared to the protein scaffold having the amino acid sequence of SEQ ID NO:

16 from about 1 Kcal to about 12 Kcal.

3. The protein scaffold of claim 1, comprising loop regions for contacting a target protein, wherein the loop regions have been modified to encompass alternate residues to positions in the amino acid sequence of SEQ ID NOS:

16, and 142, 143 and 147-151 at one or more positions selected from the group consisting of residues 13, 15, and 16, 22-28, 38-43, 51-54, 60-64, and 75-81 of an amino acid sequence selected from the group consisting of SEQ ID NOS;

16, and 142, 143 and 147-151.

4. The protein scaffold of claim 3, wherein said protein scaffold binds a target protein with at least one affinity selected from a K_D of at least 10⁻

9M, at least 10<sup>−

10</sup>M, at least 10<sup>−

11</sup>M, at least 10<sup>−

12</sup>M, at least 10<sup>−

13</sup>M, at least 10<sup>−

14</sup>M, and at least 10<sup>−

15</sup>M, as determined by surface plasmon resonance or the Kinexa method.

5. A protein scaffold comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:

142-144 and 147-151.