# US 5,707,826 A · Enzymatic method for modification of recombinant polypeptides

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- Number: US 5,707,826 A
- Document ID: US5707826A
- Status: Granted
- Kind: A
- Application number: 08/470,220
- Filed: 1995-06-06
- Granted: 1998-01-13
- Earliest claimed: 1993-07-20
- Assignees: BioNebraska, Inc.
- Inventors: Fred W. Wagner; Jay Stout; Dennis Henriksen; Bruce Partridge; Shane Manning
- Web page: https://patentgrep.com/patent/US5707826A

## CPC

- C07K 14/60: Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
- A61P 3/08: for glucose homeostasis (pancreatic hormones A61P5/48)
- A61P 13/02: of urine or of the urinary tract, e.g. urine acidifiers
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/605: Glucagons
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- 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 2319/00: Fusion polypeptide
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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)

## Abstract

The method of the invention provides for the formation of a recombinant polypeptide which has been modified at the C-terminal end through the use of a transpeptidation process. The method is suitable for modifying recombinant polypeptides of any source including those which may be commercially available, those derived from recombinant single copy or multicopy polypeptide constructs, or those derived from single or multicopy recombinant fusion protein constructs. The transpeptidation reaction involves contacting an endopeptidase enzyme with a recombinant polypeptide to substitute an addition unit, of one or more amino acids, for a leaving unit, linked to a core polypeptide through a cleavage site recognized by the endopeptidase enzyme. Recombinant polypeptides derived from multicopy polypeptide constructs may be cleaved from the multicopy polypeptide at the N-terminal and C-terminal ends and simultaneously under go substitution of the leaving unit by the desired addition unit. The invention utilizes known and newly discovered cleavage recognition sites to effectuate the desired modification products.

## Claims

### Claim 1

A process for modifying a polypeptide by transpeptidation comprising: contacting together an addition unit, trypsin, and a multicopy polypeptide which includes at least two cores, adjacent cores being linked by an enzyme cleavage site, and a leaving unit linked to a terminal core by the enzyme cleavage site, such that transpeptidation occurs simultaneously with cleavage of the multicopy polypeptide to produce modified polypeptide products having the addition unit connoted to the core: and
wherein the core includes a GLP1(7-34) amino acid sequence (SEQ ID NO:4); and the enzyme cleavage site is a -Lys-Xxx- peptide bond, wherein the Xxx residue is not an amino acid residue having a side chain carboxylic acid group.

### Claim 2 (depends on 1)

The process of claim 1, wherein the core corresponds to GLP1 (7-34) (SEQ ID NO:4) or GLP1 (1-34) (SEQ ID NO:6); and adjacent cores are linked directly to each other.

### Claim 3

A process for modifying a recombinant polypeptide by transpeptidation comprising:
(a) forming a recombinant polypeptide which includes a core and a leaving unit, the leaving unit being linked to the core by an enzyme cleavage site; and
(b) contacting an addition unit and tHe recombinant polypeptide with trypsin to produce a modified recombinant polypeptide having the addition unit attached to the core and substituted for the leaving unit; and
wherein the core includes a GLP1(7-34) amino acid sequence (SEQ ID NO:4); and the enzyme cleavage site is a -Lys-Xxx- peptide bond, wherein the Xxx residue is not an amino acid residue having a side chain carboxylic acid group.

### Claim 4

A process for modifying a polypeptide comprising:
(a) cleaving the polypeptide, which includes a leaving unit linked to a core by an enzyme cleavage site, with trypsin to form a hydrolyzed product; and
(b) contacting the hydrolyzed product and an addition unit with the trypsin to produce a modified polypeptide having the addition unit attached to the core; and
wherein the core includes a GLP1(7-34) amino acid sequence (SEQ ID NO:4); and the enzyme cleavage site is a -Lys-Xxx- peptide bond,
wherein the Xxx residue is not an amino acid residue having a side chain carboxylic acid group.

### Claim 5

A process for modifying a polypeptide comprising:
contacting (a) a first polypeptide including a leaving unit linked to a GLP1(7-34) amino acid sequence (SEQ ID NO:4) by a C-terminal -Lys-Xxx- peptide bond and (13) an addition unit with (c) trypsin to cleave the leaving unit from the GLP1(7-34) amino acid sequence and produce a second polypeptide having the addition unit linked to a C-terminus of the GLP1(7-34) amino acid sequence;
wherein the --Xxx- residue is not an amino acid residue having a side chain carboxylic acid group.

### Claim 6 (depends on 5)

The process of claim 5 wherein the -Lys-Xxx- peptide bond is a -Lys-Gly-, -Lys-Ala- or -Lys-His- peptide bond.

### Claim 7 (depends on 5)

The process of claim 5 wherein the first polypeptide comprises the leaving unit linked to a GLP1(1-34) amino acid sequence (SEQ ID NO:6) by the C-terminal Lys-Xxx peptide bond.

### Claim 8 (depends on 5)

The process of claim 5 comprising contacting the first polypeptide and the addition unit with the trypsin at a trypsin:polypeptide molar ratio of about 1:10 to 1:500,000.

### Claim 9 (depends on 5)

The process of claim 5 wherein the contacting step occurs in a solution at a pH of about 5 to about 11.

### Claim 10 (depends on 5)

The process of claim 5 comprising contacting (a) the first polypeptide and (b) the addition unit with (c) the trypsin in a solution which includes an organic solvent.

### Claim 11 (depends on 10)

The process of claim 10 wherein the organic solvent includes N,N-dimethylacetamide, dimethylsulfoxide, or a mixture thereof.

### Claim 12 (depends on 5)

The process of claim 5 wherein the leaving unit comprises an amino acid residue.

### Claim 13 (depends on 5)

The process of claim 5 wherein the addition unit comprises an amino acid residue.

### Claim 14 (depends on 13)

The process of claim 13 wherein the addition unit is Gly-Arg-NH.sub.2.

### Claim 15 (depends on 13)

The process of claim 13 wherein the addition unit is Gly-Arg-Gly.

### Claim 16 (depends on 5)

The process of claim 5 wherein the first polypeptide comprises a GLP1 (7-34)-Ala-Phe-Ala amino acid sequence (SEQ ID NO:2).

### Claim 17

A method of producing GLP1(7-36)-NH.sub.2 comprising contacting (a) a first polypeptide including a leaving unit linked to a GLP1(7-34) amino acid sequence (SEQ ID NO:4) by a -Lys-Xxx- peptide bond and (b) a Gly-Arg-NH.sub.2 addition unit with (c) trypsin to cleave the leaving unit from the GLP 1(7-34) amino acid sequence and produce a second polypeptide having a C-terminal GLP1(7-36)-NH.sub.2 sequence (SEQ ID NO:4); wherein the -Lys-Xxx- peptide bond is not a -Lys-Glu- or -Lys-Asp- peptide bond.

### Claim 18 (depends on 17)

The method of claim 17 wherein the -Lys-Xxx- peptide bond is a -Lys-Ala- , -Lys-Gly- or -Lys-Ris- peptide bond.

### Claim 19 (depends on 17)

The method of claim 17 wherein the first polypeptide comprises at least two copies of the GLP1(7-34) amino acid sequence (SEQ ID NO:4).

### Claim 20 (depends on 17)

The method of claim 17 wherein the first polypeptide comprises a GLP1(7-34)-Ala-Phe-Ala ammino acid sequence (SEQ ID NO:2).