# US 2020/0399649 A1 · Organisms and methods for producing glycomolecules with low sulfation

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- Number: US 2020/0399649 A1
- Document ID: US20200399649A1
- Status: Published application
- Kind: A1
- Application number: 17/012,359
- Filed: 2020-09-04
- Published: 2020-12-24
- Earliest claimed: 2018-03-05
- Assignees: Conagen Inc.
- Applicants: Conagen Inc.
- Inventors: Nicky C. Caiazza; Jun Urano
- Web page: https://patentgrep.com/patent/US20200399649A1

## CPC

- C12N 15/8258: {for the production of oral vaccines (antigens) or immunoglobulins}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N 9/13: {transferring sulfur containing groups (2.8)}
- C12P 21/005: {Glycopeptides, glycoproteins}
- C12Y 208/02: Sulfotransferases (2.8.2)
- C07K 2317/13: isolated from plants
- 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 invention provides recombinant organisms containing a nucleic acid encoding a heterologous glycomolecule that has a low sulfation profile or that is unsulfated. In one embodiment the heterologous glycomolecule is an immunoglobulin molecule. The recombinant organisms have a genetic modification to at least one sulfotransferase gene, such as a deletion, disruption, or other genetic modification. The cells advantageously produce and, optionally secrete, the heterologous glycomolecule. Thus, the invention provides recombinant organisms that provide glycomolecules having a glycosylation profile that is more similar to the glycosylation profile produced in a mammalian cell, and therefore may be safer and more effective for use as a therapeutic in humans or animals. The glycomolecules can be a glycoprotein, glycopeptide, or a glycolipid.

## Claims

### Claim 1

A recombinant cell of the Family Thraustochytriaceae comprising a nucleic acid encoding a heterologous glycomolecule, and further comprising a genetic modification in one or more sulfotransferase gene(s), wherein the one or more sulfotransferase genes comprise a sequence having at least 80% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 28, 31, and 32.

### Claim 2 (depends on 1)

The recombinant cell of claim 1 wherein the genetic modification is selected from the group consisting of: a deletion, a mutation, a disruption, an insertion, an inactivation, an attenuation, and an inversion.

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The recombinant cell of claim 1 wherein the glycomolecule is a glycoprotein or glycopeptide.

### Claim at position 5

5-9. (canceled)

### Claim 10 (depends on 1)

The recombinant cell of claim 1 wherein the cell produces and secretes the heterologous polypeptide molecule or functional portion thereof.

### Claim 11 (depends on 1)

The recombinant cell of claim 1 wherein the glycomolecule is an immunoglobulin.

### Claim 12 (depends on 11)

The recombinant cell of claim 11 wherein the heterologous immunoglobulin comprises fewer sulfated N-glycans relative to a corresponding cell that does not comprise the genetic modification.

### Claim 13 (depends on 12)

The recombinant cell of claim 12 wherein the heterologous immunoglobulin comprises at least 30% unsulfated N-glycans relative to a corresponding cell that does not comprise the genetic modification.

### Claim 14 (depends on 11)

The recombinant cell of claim 11 wherein the heterologous immunoglobulin comprises at least 40% unsulfated N-glycans relative to a corresponding cell that does not comprise the genetic modification.

### Claim 15 (depends on 12)

The recombinant cell of claim 12 wherein the ratio of unsulfated to sulfated N-glycans in the heterologous immunoglobulin is at least 1:2.

### Claim 16 (depends on 12)

The recombinant cell of claim 12 wherein the N-glycans comprise Man3-5GlcNAc2.

### Claim 17 (depends on 1)

The recombinant cell of claim 1 wherein the glycomolecule is an antibody molecule, or portion thereof.

### Claim 18

(canceled)

### Claim 19 (depends on 1)

The recombinant cell of claim 1 wherein the Thraustochytriaceae cell is of a genus selected from the group consisting of: Japanochytrium, Oblongichytrium, Thraustochytrium, Aurantiochytrium, and Schizochytrium.

### Claim 20

(canceled)

### Claim 21 (depends on 1)

The recombinant cell of claim 1 further comprising a genetic modification to a gene encoding a mannosyl transferase.

### Claim 22 (depends on 21)

The recombinant cell of claim 21 wherein the genetic modification is comprised in an alg3 gene.

### Claim 23 (depends on 22)

The recombinant cell of claim 22 wherein the genetic modification is selected from the group consisting of: a deletion, a mutation, a disruption, an insertion, an inactivation, replacement, an attenuation, and an inversion.

### Claim 24 (depends on 1)

The recombinant cell of claim 1 wherein the heterologous glycoprotein or glycopeptide is selected from the group consisting of: trastuzumab, eculizumab, natalizumab, cetuximab, omalizumab, usteinumab, panitummab, and adalimumab, or a functional fragment of any of them.

### Claim at position 21

-26. (canceled)

### Claim 27

A method of producing a glycomolecule having a glycosylation profile with low sulfation, comprising
performing a genetic modification to one or more sulfotransferase genes in an organism of the Family Thraustochytriales, wherein the organism comprises a recombinant nucleic acid encoding a heterologous glycomolecule, or a functional portion thereof and wherein the one or more sulfotransferase gene(s) comprises a sequence having at least 80% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 28, 31, and 32;
cultivating the organism; and
thereby producing the glycomolecule having a glycosylation profile with low sulfation.

### Claim 28 (depends on 27)

The method of claim 27 wherein the genetic modification is selected from the group consisting of: a deletion, a mutation, a disruption, an insertion, an inactivation, an attenuation, and an inversion.

### Claim at position 24

29-31. (canceled)

### Claim 32 (depends on 27)

The method of claim 27 wherein the organism secretes a heterologous glycoprotein or glycopeptide molecule or functional portion thereof.

### Claim 33 (depends on 32)

The method of claim 32 wherein the heterologous glycoprotein or glycopeptide molecule comprises fewer sulfated N-glycans relative to the heterologous glycoprotein or glycopeptide produced in a corresponding cell that does not comprise the genetic modification.

### Claim at position 27

-41. (canceled)