Biologically active proteins having increased in vivo and/or in vitro stability

• US 9,938,331 B2
• Filed: 12/16/2015
• Issued: 04/10/2018
• Est. Priority Date: 09/27/2005
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc.
• Inventors: Schellenberger, Volker, Stemmer, Willem P., Wang, Chia-Wei, Scholle, Michael D., Gordon, Nathaniel C., Crameri, Andreas, Popkov, Mikhail
• Primary Examiner(s): Shafer, Shulamith H
• Application Number:
  US14/970,723
• Publication Number:
  US 20160280753A1
• Time in Patent Office: 846 Days
• Field of Search: None
• 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 13/12 of the kidneys
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/06 Antianaemics
• 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 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/53 Colony-stimulating factor [CSF]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/56 IFN-alpha
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 2319/31 : fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/35 : containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 : having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 15/1044 : Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides unstructured recombinant polymers (URPs) and proteins containing one or more of the URPs. The present invention also provides microproteins, toxins and other related proteinaceous entities, as well as genetic packages displaying these entities. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applications.

Claims

1. An unstructured recombinant polymer (URPs), wherein the URP comprising at least about 200 contiguous amino acids, and wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP;

(b) at least 50% of the amino acids of the URP are not present in secondary structure as determined by Chou-Fasman algorithm;

(c) the URP has a Tepitope score equal to or less than −

5;

(d) the URP contains only 3, 4, 5, or 6 different types of amino acids; and

(e) the URP contains greater than 5% glutamic acid and less than 2% lysine or arginine;

wherein incorporation of the URP into a heterologous protein results in a fusion protein having at least a 2-fold increase in apparent molecular weight of the fusion protein as compared to the protein lacking said URP wherein the apparent molecular weight is approximated by size exclusion chromatography, and wherein the fusion protein exhibits an increased half-life as compared to the protein lacking said URP.

2. The URP of claim 1, wherein upon incorporation of the URP into an N-terminus, a C-terminus, into a loop of the heterologous protein, or between domains of the heterologous protein to create a monomeric fusion protein, said heterologous protein exhibits a serum secretion half-life that is at least two-fold longer as compared to the corresponding protein that is deficient in said URP.

3. The URP of claim 1, wherein the URP comprises a non-natural amino acid sequence.

4. The URP of claim 1, wherein the URP comprises at least 400 contiguous amino acids.

5. The URP of claim 1, wherein the URP comprises at least five types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).

6. The URP of claim 1, wherein the URP comprises at least six types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).

7. The URP of claim 1, wherein one type of the amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) constitutes at least 20% of the total amino acids of the URP.

8. The URP of claim 7, wherein the one type of amino acid is glutamate (E).

9. The URP of claim 1, wherein any one type of amino acid is no more than 30% of the total amino acids of the URP.

10. The URP of claim 1, wherein the heterologous protein is a pharmaceutically active protein.

11. The URP of claim 1, wherein the heterologous protein comprises one or more modules selected from the group consisting of binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules.

12. The URP of claim 10, wherein the pharmaceutically active protein is selected from the group consisting of cytokines, growth factors, enzymes, receptors, microproteins, hormones, erythopoetin, adenosine deiminase, asparaginase, arginase, interferon, growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, TNF-receptor, uricase, rasburicase, axokine, RNAse, DNAse, phosphatase, pseudomonas exotoxin, ricin, gelonin, desmoteplase, laronidase, thrombin, blood clotting enzyme, VEGF, protropin, somatropin, alteplase, interleukin, factor VII, factor VIII, factor X, factor IX, dornase, glucocerebrosidase, follitropin, glucagon, thyrotropin, nesiritide, alteplase, teriparatide, agalsidase, laronidase, methioninase.

13. The URP of claim 1, wherein any amino acids of the URP that are not glycine, aspartate, alanine, serine, threonine, glutamate, or proline are not hydrophobic amino acids selected from the group consisting of tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.