Modified host cells and hybrid oligosaccharides for use in bioconjugate production

Patent metadata

Field	Value
Publication number	US-11285200-B2
Application number	US-201916386036-A
Country	US
Kind code	B2
Filing date	Apr 16, 2019
Priority date	Aug 8, 2014
Publication date	Mar 29, 2022
Grant date	Mar 29, 2022

Abstract

Provided herein are host cells capable of producing hybrid oligosaccharides and polysaccharides, wherein said hybrid oligosaccharides and polysaccharides do not comprise a hexose at the reducing end of their first repeat unit. Also provided herein are hybrid oligosaccharides or polysaccharides and bioconjugates which can be produced by the host cells described herein, wherein said bioconjugates comprise a carrier protein linked to a hybrid oligosaccharide or polysaccharide that does not comprise a hexose at the reducing end of its first repeat unit.

First claim

What is claimed is:

1. A E. coli strain W3110 comprising a deletion of the waaL, wherein said E. coli strain comprises an expression vector comprising nucleic acids for expression in the host cell:
a) nucleic acids that encode (i) glycosyltransferases that produce an oligosaccharide or polysaccharide repeat unit, wherein said repeat unit does not comprise a hexose at the reducing end, and wherein said oligosaccharide or polysaccharide repeat unit is derived from a donor oligosaccharide or polysaccharide repeat unit that comprises a hexose at the reducing end, and (ii) heterologous glycosyltransferases sufficient for synthesis of the donor oligosaccharide or polysaccharide repeat unit wherein the donor oligosaccharide or polysaccharide is that of S. pneumoniae ; and
b) a nucleic acid that encodes a carrier protein comprising an N-glycosylation consensus sequence; and
c) a nucleic acid that encodes an oligosaccharyl transferase;
d) a nucleic acid that encodes a wzy polymerase, wherein the polymerase catalyzes the production of a hybrid oligosaccharide or polysaccharide, wherein the hybrid oligosaccharide or polysaccharide (i) comprises a hexose monosaccharide derivative at the reducing end of the first repeat unit and (ii) comprises hexose monosaccharides at the reducing end of all other repeat units;
e) nucleic acid that encodes a heterologous flippase of the donor polysaccharide or oligosaccharide gene cluster;
f) nucleic acid that encodes RcsA; and
g) nucleic acid that encodes an epimerase (in the instant the hybrid oligosaccharide or polysaccharide comprises a hexose monosaccharide derivative at the reducing end of the first repeat unit in addition to the hexose monosaccharide normally present at the reducing end of the first repeat unit of the donor oligosaccharide or polysaccharide).

2. The host cell of claim 1 , wherein said host cell comprises (a) (i) a glycosyltransferase that assembles a hexose monosaccharide derivative onto undecaprenyl pyrophosphate (UND-PP) and (ii) one or more glycosyltransferases capable of adding a monosaccharide to the hexose monosaccharide derivative assembled on UND-PP; or (b) (i) a glycosyltransferase that assembles a hexose monosaccharide derivative onto undecaprenyl pyrophosphate (UND-PP) and (ii) glycosyltransferases that assemble the donor oligosaccharide or polysaccharide repeat unit onto the hexose monosaccharide derivative.

3. The host cell of claim 2 , wherein said hexose monosaccharide derivative is any monosaccharide in which C-2 position is modified with an acetamido group.

4. The host cell of claim 3 , wherein said hexose monosaccharide derivative is N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), 2,4-Diacetamido-2,4,6-trideoxyhexose (DATDH), N-acetylfucosamine (FucNAc), N-acetylquinovosamine (QuiNAc).

5. The host cell of claim 2 , wherein said glycosyltransferase that assembles a hexose monosaccharide derivative onto UND-PP is wecA.

6. The host cell of claim 2 , wherein said one or more glycosyltransferases capable of adding a monosaccharide to the hexose monosaccharide derivative is the wfeD galactosyltransferase from Shigella boyedii; or the wbeY galactofuranosyltransferase from E. coli 028; or the wfdK galactofuranosyltransferase from E. coli 0167; or are the wbeY galactofuranosyltransferase from E. coli 028 and the wfdK galactofuranosyltransferase from E. coli 0167.

7. The host cell of claim 1 , wherein said glycosyltransferases sufficient for synthesis of the repeat units of the donor oligosaccharide or polysaccharide comprise (i) wchL and/or wchM from S. pneumoniae CP14, and/or (ii) wciC, wciD, wciE, and/or wciF from S. pneumoniae CP33F.

8. The host cell of claim 2 , wherein the glycosyltransferases that assemble the donor oligosaccharide or polysaccharide repeat unit onto the hexose monosaccharide derivative comprise a glycosyltransferase that is capable of adding the hexose monosaccharide present at the reducing end of the first repeat unit of the donor oligosaccharide or polysaccharide to the hexose monosaccharide derivative.

9. The host cell of claim 8 , wherein said glycosyltransferase is a wciP galactosyltransferase.

10. The host cell of claim 2 , wherein the glycosyltransferases that assemble the donor oligosaccharide or polysaccharide repeat unit onto the hexose monosaccharide derivative comprise a glycosyltransferase that is capable of adding the monosaccharide that is adjacent to the hexose monosaccharide present at the reducing end of the first repeat unit of the donor oligosaccharide or polysaccharide to the hexose monosaccharide present at the reducing end of the first repeat unit of the donor oligosaccharide or polysaccharide.

11. The host cell of claim 10 , wherein said glycosyltransferase is a wciQ glucosytransferase.

12. The host cell of claim 2 , wherein said host cell further comprises an enzyme capable of modifying a monosaccharide.

13. The host cell of claim 1 , wherein said host cell comprises (a) a nucleic acid that encodes a wzy capsular polysaccharide polymerase or an wzy 0 antigen polymerase and/or (b) a nucleic acid that encodes a wzx flippase.

14. The host cell of claim 13 , wherein said polymerase of (a) and/or (b) is encoded from a gene cluster Streptococcus pneumoniae Capsular Polysaccharide (CP) CP1, CP2, CP3, CP4, CPS, CP6 (A and B), CP7 (A,B, C), CP8, CP9 (A, L,N, V), CP10 (A,B,C,F), CP11 (A, B,C,D,F), CP12(A,B,F), CP13, CP14, CP15(A,B,C,F), CP16(A,F), CP17(A,F), CP18(A,B,C,F), CP19(A,B,C,F), CP20, CP21, CP22(A,F), CP23(A,B,F), CP24(A,B,F), CP25(A,F), CP26, CP27, CP28(A,F), CP29, CP31, CP32(A,F), CP33(A,B,C,D,F), CP34, CP35(A,B,C,D,F), CP36, CP37, CP38, CP39, CP40, CP41(A,F), CP42, CP43, CP44, CP45, CP46, CP47(A,F), or CP48.

15. The host cell of claim 1 , wherein said donor polysaccharide is the Streptococcus pneumoniae capsular polysaccharide (CP) CP2, CP3, CP6 (A, B), CP7 (A, B), CP8, CP9 (A, L, N, V), r[CP1MCP10 (A, F), CP11 JA, B, C, F), CP13, CP14, CP15 (A, B, C, F), CP17 (A, F), CP18 (A, B, C, F), CP19 (A, B, C, F), CP20, CP22 (F), CP23 (F), CP27, CP29, CP31, CP32 (A, F), CP33 (B, F), CP34, CP35 (A, B), or CP37.

16. The host cell claim 1 , wherein said oligosaccharyl transferase is the pglB gene of Campylobacter jejuni.

17. A method of producing a bioconjugate comprising a carrier protein N-linked to an oligosaccharide or polysaccharide that does not comprise a hexose at the reducing end of the first repeat unit, said method comprising (i) culturing the host cell of claim 1 under conditions suitable for the production of proteins and (ii) isolating said bioconjugate.

Assignees

• Glaxosmithkline Biologicals Sa

Inventors

• Faridmoayer Amirreza

Classifications

• C07K14/195

  from bacteria · CPC title

• C12Y204/99

  transferring other glycosyl groups (2.4.99) · CPC title

• C12P19/28

  N-glycosides · CPC title

• A61K39/092 Primary

  Streptococcus · CPC title

• C12N9/1081

  transferring other glycosyl groups (2.4.99) · CPC title