Escherichia coli compositions and methods thereof

Patent metadata

Field	Value
Publication number	US-2023000966-A1
Application number	US-202017772610-A
Country	US
Kind code	A1
Filing date	Oct 28, 2020
Priority date	Nov 1, 2019
Publication date	Jan 5, 2023
Grant date	—

Abstract

In one aspect, the invention relates to a polypeptide derived from E. coli and a fragment thereof, including compositions and methods thereof. Also disclosed herein are compositions that include a polypeptide derived from E. coli and a fragment thereof; and modified O-polysaccharide molecules derived from E. coli lipopolysaccharides and conjugates thereof. In a further aspect, disclosed herein are mammalian host cells that include sequence(s) encoding a polypeptide derived from E. coli or fragments thereof.

First claim

1 . A recombinant mammalian cell, comprising a polynucleotide encoding a polypeptide derived from E. coli or a fragment thereof.

2 . The recombinant cell according to claim 1 , wherein the polypeptide is derived from E. coli fimbrial H (FimH).

3 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a phenylalanine residue at the N-terminus of the polypeptide.

4 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a phenylalanine residue within the first 20 residue positions of the N-terminus.

5 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a phenylalanine residue at position 1 of the polypeptide.

6 . The recombinant cell according to claim 5 , wherein the polypeptide does not comprise a glycine residue immediately before the phenylalanine residue at position 1 of the polypeptide.

7 . The recombinant cell according to claim 2 , wherein the polypeptide does not comprise an N-glycosylation site at position 7 of the polypeptide.

8 . The recombinant cell according to claim 6 , wherein the polypeptide does not comprise an Asn residue at position 7 of the polypeptide.

9 . The recombinant cell according to claim 8 , wherein the polypeptide comprises a residue selected from the group consisting of Ser, Asp, Thr, and Gln at position 7.

10 . The recombinant cell according to claim 5 , wherein the polypeptide does not comprise an N-glycosylation site at position 70 of the polypeptide.

11 . The recombinant cell according to claim 10 , wherein the polypeptide does not comprise an Asn residue at position 70 of the polypeptide.

12 . The recombinant cell according to claim 10 , wherein the polypeptide does not comprise a Ser residue at position 70 of the polypeptide.

13 . The recombinant cell according to claim 1 , wherein the polypeptide comprises a residue substitution selected from the group consisting of Ser, Asp, Thr, and Gln at an N-glycosylation site of the polypeptide.

14 . The recombinant cell according to claim 13 , wherein the N-glycosylation site comprises position N235 of the polypeptide.

15 . The recombinant cell according to claim 13 , wherein the N-glycosylation site comprises position N228 of the polypeptide.

16 . The recombinant cell according to claim 13 , wherein the N-glycosylation site comprises position N235 and position N228 of the polypeptide.

17 . The recombinant cell according to claim 2 , wherein the polypeptide comprises SEQ ID NO: 3.

18 . The recombinant cell according to claim 2 , wherein the polypeptide comprises SEQ ID NO: 2.

19 . The recombinant cell according to claim 1 , wherein the polypeptide comprises an aliphatic hydrophobic amino acid residue at position 1 of the polypeptide.

20 . The recombinant cell according to claim 19 , wherein the aliphatic hydrophobic amino acid residue is selected from the group consisting of Ile, Leu, and Val.

21 . The recombinant cell according to claim 1 , wherein the polypeptide comprises a fragment of FimH.

22 . The recombinant cell according to claim 21 , wherein the polypeptide comprises a lectin domain of FimH.

23 . The recombinant cell according to claim 22 , wherein the lectin domain comprises a mass of about 17022 Daltons.

24 . The recombinant cell according to claim 1 , wherein the polypeptide is complexed with a FimC polypeptide or a fragment thereof.

25 . The recombinant cell according to claim 24 , wherein the FimC polypeptide or a fragment thereof comprises a glycine residue at position 37 of the FimC polypeptide or a fragment thereof.

26 . The recombinant cell according to claim 2 , wherein the polypeptide is in the low affinity conformation.

27 . The recombinant cell according to claim 2 , wherein the polypeptide is stabilized by FimG.

28 . The recombinant cell according to claim 2 , wherein the polypeptide is stabilized by a donor-strand peptide of FimG (DsG).

29 . The recombinant cell according to claim 28 , wherein the polynucleotide sequence further encodes a linker sequence.

30 . The recombinant cell according to claim 29 , wherein the linker comprises at least 4 amino acid residues and at most 15 amino acid residues.

31 . The recombinant cell according to claim 29 , wherein the linker comprises at least 5 amino acid residues and at most 10 amino acid residues.

32 . The recombinant cell according to claim 29 , wherein the linker comprises 7 amino acid residues.

33 . The recombinant cell according to claim 1 , wherein the polypeptide does not comprise a signal peptide selected from the group consisting of a native FimH leader peptide, influenza hemagglutinin signal peptide, and a human respiratory syncytial virus A (strain A2) fusion glycoprotein F0 signal peptide.

34 . The recombinant cell according to claim 1 , wherein the polypeptide comprises a murine IgK signal peptide sequence.

35 . The recombinant cell according to claim 1 , wherein the polypeptide comprises any one signal peptide sequence selected from human IgG receptor FcRn large subunit p51 signal peptide and a human IL10 protein signal peptide.

36 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a mutation of arginine to proline at amino acid position 60 (R60P), according to the numbering of SEQ ID NO: 3.

37 . The recombinant cell according to claim 1 , wherein the expression level of the polypeptide is greater than the expression level of the corresponding wild-type polypeptide expressed in the periplasm of a wild-type E. coli cell.

38 . The recombinant cell according to claim 1 , wherein the expression level of the polypeptide is greater than 10 mg/L.

39 . The recombinant cell according to claim 1 , wherein the polynucleotide sequence is integrated into the genomic DNA of said mammalian cell.

40 . The recombinant cell according to claim 1 , wherein the polynucleotide sequence is codon optimized for expression in the cell.

41 . The recombinant cell according to claim 1 , wherein the cell is a human embryonic kidney cell.

42 . The recombinant cell according to claim 40 , wherein the human embryonic kidney cell comprises a HEK293 cell.

43 . The recombinant cell according to claim 42 , wherein the HEK293 cell is selected from any one of HEK293T cells, HEK293TS cells, and HEK293E cells.

44 . The recombinant cell according to claim 1 , wherein the cell is a CHO cell.

45 . The recombinant cell according to claim 44 , wherein said CHO cell is a CHO-K1 cell, CHO-DUXB11, CHO-DG44 cell, or CHO—S cell.

46 . The recombinant cell according to claim 1 , wherein the polypeptide is soluble.

47 . The recombinant cell according to claim 1 , wherein the polypeptide is secreted from the cell.

48 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a N28Q substitution, according to the numbering of SEQ ID NO: 1.

49 . The recombinant cell according to claim 2 , wherein the polypeptide comprises a N28D subst

Assignees

• Pfizer

Inventors

• Anderson Annaliesa Sybil
• Che Ye
• Chen Wei
• Chorro Laurent Oliver
• Chu Ling
• Donald Robert G K
• Griffor Matthew Curtis
• Gu Jianxin
• Guan Zeqiang
• Kim Jin-Hwan
• Kodali Srinivas
• Lomberk Scott Ellis
• Lotvin Jason Arnold
• Merchant Nishith
• Moran Justin Keith
• Pan Rosalind
• Prasad Avvari Krishna
• Ruppen Mark Edward
• Singh Suddham
• Stead David Robert
• Takane Karen Kiyoko

Classifications

• A61K39/0258 Primary

  Escherichia · CPC title

• A61K2039/55577

  Saponins; Quil A; QS21; ISCOMS · CPC title

• A61K2039/55505

  Inorganic adjuvants · CPC title

• A61K2039/627

  characterised by the linker · CPC title

• A61K2039/6037

  Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT] · CPC title