# US 8,540,992 B2 · Methods and vectors for generating asialylated immunoglobulins

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- Number: US 8,540,992 B2
- Document ID: US8540992B2
- Status: Granted
- Kind: B2
- Application number: 12/521,417
- Filed: 2007-12-26
- Published: 2010-07-08
- Granted: 2013-09-24
- Earliest claimed: 2006-12-28
- Applicants: Michael Naso; T. Shantha Raju; Bernard Scallon
- Inventors: Michael Naso; T. Shantha Raju; Bernard Scallon
- Web page: https://patentgrep.com/patent/US8540992B2

## CPC

- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/241: {Tumor Necrosis Factors}
- C12N 15/85: for animal cells
- C12P 21/005: {Glycopeptides, glycoproteins}
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/72: Increased effector function due to an Fc-modification
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C12Y 302/01018: Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)}
- C12Y: ENZYMES

## Abstract

The properties of an Fc-containing protein, for example, an antibody, are controlled by altering the sialylation of the oligosaccharides in the Fc region by transfecting the cell line expressing the Fc-containing protein with a vector sequence encoding a sialidase. The modified Fc-containing proteins have therapeutic utility in diseases or conditions in which it is desirable to control the affinity for one or more of the FcγRI, FcγRIIA, and FcγRIIIA receptors, ADCC activity, macrophage or monocyte activation, serum half-life, and avidity.

## Claims

### Claim 1

A method for providing a sialidase enzymatic activity in a culture comprising a mammalian cell engineered to express an antibody, comprising transfecting the mammalian engineered cell with a vector encoding a catalytic domain of Arthrobacter ureafaciens sialidase enzyme A, wherein the polypeptide is secreted into the culture along with the antibody, whereby the antibody glycans can be acted upon by the polypeptide sialidase enzymatic activity, and wherein one or more of the avidity for multiply localized target proteins, the macrophage or monocyte activation, and serum half-life of the antibody are altered.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mammalian cell line is selected from the group consisting of COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, YB2/0 or Y3, myeloma, or lymphoma cells, or any derivative, immortalized or transformed cell thereof.

### Claim 3 (depends on 2)

The method of claim 2, wherein the antibody is an anti-TNFα antibody and the cell line is C168M.