# US 7,610,156 B2 · Methods for rational pegylation of proteins

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- Number: US 7,610,156 B2
- Document ID: US7610156B2
- Status: Granted
- Kind: B2
- Application number: 10/956,352
- Filed: 2004-09-30
- Published: 2005-05-26
- Granted: 2009-10-27
- Earliest claimed: 2003-03-31
- Assignees: Xencor, Inc.
- Inventors: John R. Desjarlais; Jonathan Zalevsky; Gregory L. Moore
- Web page: https://patentgrep.com/patent/US7610156B2

## CPC

- C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- C07K 14/505: Erythropoietin [EPO]
- C07K 14/525: Tumour necrosis factor [TNF]
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- C07K 14/565: IFN-beta
- C07K 14/61: Growth hormone [GH], i.e. somatotropin
- G16B 15/20: Protein or domain folding
- G16B 35/00: ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
- G16B 35/20: Screening of libraries
- G16C 20/60: In silico combinatorial chemistry
- 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)
- C07K 2299/00: Coordinates from 3D structures of peptides, e.g. proteins or enzymes
- G16B 15/00: ICT specially adapted for analysing two-dimensional [2D] or three-dimensional [3D] molecular structures, e.g. structural or functional relations or structure alignment
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16C: COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE

## Abstract

The present invention relates to the use of simulation technology to rationally optimize the locations and sizes of attached polymeric moieties for modification of therapeutic proteins and the proteins generated from this method.

## Claims

### Claim 1

A method of identifying at least one favorable attachment site for a polymeric moiety on a target protein comprising:
a) inputting a set of coordinates into a computer for said target protein;
b) inputting a set of coordinates for said polymeric moiety;
c) selecting a criteria for said favorable attachment site based upon at least one desired characteristic,
d) using a simulation module comprising the steps of:
i) computationally attaching conformers of said polymeric moiety to a plurality of amino acids in said target protein; and
ii) disallowing conformers at each of said amino acids on the basis of a distance cutoff;
e) selecting based upon said criteria one of said amino acids for attachment of said polymeric moiety; and
f) physically making and screening for said at least one desired characteristic at least one target protein with a polymeric moiety attached at said favorable attachment site amino acid position.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said method further comprises identifying a set of polymer sizes for attachment to said favorable attachment site.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the amino acid at said position is a non-naturally occurring amino acid.

### Claim 4 (depends on 3)

The method according to claim 3, wherein said non-naturally occurring amino acid is p-acetyl-L-phenylalanine.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said polymeric moiety for attachment is pharmaceutically acceptable.

### Claim 6 (depends on 5)

The method according to claim 5, wherein said polymeric moiety for attachment is a polyethylene glycol (PEG).

### Claim 7 (depends on 6)

The method according to claim 6, wherein said PEG is a PEG derivative.

### Claim 8 (depends on 1)

The method according to claim 1, wherein said polymeric moiety for attachment has a range of about 1000 daltons to about 100,000 daltons.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said polymeric moiety for attachment is branched.

### Claim 10 (depends on 1)

The method according to claim 1, wherein said polymeric moiety for attachment is unbranched.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said polymeric moiety for attachment is labile.

### Claim 12 (depends on 1)

The method according to claim 1, wherein said protein is a therapeutic protein.

### Claim 13 (depends on 1)

The method according to claim 1, wherein said protein is selected from the group consisting of a human erythropoeitin (EPO), a human tumor necrosis factor (TNF), a human growth hormone (hGH), a human interferon (IFN), a human granulocyte colony stimulating factor (G-CSF) and bone morphogenic protein-7 (BMP7).

### Claim 14 (depends on 1)

The method according to claim 1, wherein said simulation module includes Monte Carlo (MC) simulations, Molecular Dynamics (MD) simulations or combinations thereof.

### Claim 15 (depends on 1)

The method according to claim 1, further comprising substituting at least one amino acid at said favorable attachment site with a cysteine residue.

### Claim 16 (depends on 1)

The method according to claim 1, further comprising substituting at least one lysine or histidine residue of said protein with any other amino acid residue.