Polypeptides targeting glycosylated MUC2 proteins, methods of synthesis, their nucleic acids and uses thereof

Patent metadata

Field	Value
Publication number	US-10138280-B2
Application number	US-201615178049-A
Country	US
Kind code	B2
Filing date	Jun 9, 2016
Priority date	Sep 9, 2011
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

Abstract

The invention relates to polypeptides, defined through a consensus sequence, having a length from 10 to 80 amino-acid residues, and whose polypeptidic sequence comprises or consists of the consensus sequence P1(Xa)P3(Xb)P5(Xc)P6(Xd)P7 (SEQ ID NO: 1), presenting specific patterns. The polypeptides of the invention target glycosylated Muc2 proteins. The invention also relates to methods of synthesis of such polypeptides, to their nucleic acids and uses thereof. The polypeptidic sequence of the polypeptides of the invention can be part of the N-terminal sequence of a mucus-binding (MUB) domain, especially a mucus-binding (MUB) domain of several species. The invention also relates to chimeric molecule(s) comprising such polypeptides, which are labelled, and vectors, especially plasmids and population of cells or composition comprising polypeptides of the invention. Synthesis methods encompass biotechnological or chemical production. Polypeptides of the invention can be used in staining experiments, as a probe or marker for staining Muc2 protein(s) contained in mucus layer(s), to detect in vitro mucus production or mucus composition in human colon or monitoring any one of the following disease conditions: neoplasic disease(s), including mucinous carcinoma(s), gastric cancer(s) or colorectal cancer(s), especially colon cancer(s), cystic fibrosis, intestine inflammatory disease(s) such as inflammatory bowel disease (IBD) and ulcerative colitis. The invention also relates to a method for manufacturing a medicament. In a particular embodiment, use of polypeptides of the invention can be made for marking neutrophiles.

First claim

The invention claimed is:

1. An isolated nucleic acid molecule that comprises an open reading frame that encodes a polypeptide consisting of:
a) SEQ ID NO: 3 having an additional cysteine residue at the N-terminus,
b) a fragment of SEQ ID NO: 3, wherein the fragment has an additional cysteine residue at the N-terminus, and wherein the fragment has a length of at least 20 contiguous amino acid residues,
c) a variant of SEQ ID NO: 3, wherein the variant has an additional cysteine residue at the N-terminus and has at least 85% identity with SEQ ID NO: 3, or
d) a variant of a fragment of SEQ ID NO: 3, wherein the variant of the fragment has an additional cysteine residue at the N-terminus, has a length of at least 20 contiguous amino acid residues, and has at least 85% identity with the fragment of SEQ ID NO: 3.

2. The isolated nucleic acid molecule according to claim 1 , wherein the polypeptide consists of:
a) SEQ ID NO: 58 having an additional cysteine residue at the N-terminus,
b) SEQ ID NO: 59 having an additional cysteine residue at the N-terminus,
c) SEQ ID NO: 60 having an additional cysteine residue at the N-terminus,
d) SEQ ID NO: 61 having an additional cysteine residue at the N-terminus,
e) a sequence having at least 85% identity with SEQ ID NO: 58 and having an additional cysteine residue at the N-terminus,
f) a sequence having at least 85% identity with SEQ ID NO: 59 and having an additional cysteine residue at the N-terminus,
g) a sequence having at least 85% identity with SEQ ID NO: 60 and having an additional cysteine residue at the N-terminus, or
h) a sequence having at least 85% identity with SEQ ID NO: 61 and having an additional cysteine residue at the N-terminus.

3. The isolated nucleic acid molecule according to claim 1 , wherein the nucleic acid molecule comprises a variant of SEQ ID NO: 66.

4. A recombinant vector comprising a nucleic acid molecule that comprises an open reading frame that encodes a polypeptide consisting of:
a) SEQ ID NO:3,
b) a fragment of SEQ ID NO: 3 having a length of at least 20 contiguous amino acid residues,
c) a variant of SEQ ID NO: 3 having at least 85% identity with SEQ ID NO: 3, or
d) a variant of a fragment of SEQ ID NO: 3 having a length of at least 20 contiguous amino acid residues and having at least 85% identity with the fragment.

5. The vector according to claim 4 , wherein the vector is a plasmid.

6. The vector according to claim 4 , wherein the vector is a 6-methylguanine-DNA-methyltransferase enzyme (MGMT)-based vector.

7. A recombinant vector comprising a nucleic acid molecule encoding in a single open reading frame, from 5′ to 3′:
a) a peptidic secretion signal which is functional in insect cells,
b) a 6-methylguanine-DNA-methyltransferase enzyme or a mutant or a fragment thereof having at least 80% of catalytic activity of native 6-methylguanine-DNA-methyltransferase enzyme, and
c) a polypeptide consisting of:
i) SEQ ID NO:3,
ii) a fragment of SEQ ID NO: 3 having a length of at least 20 contiguous amino acid residues,
iii) a variant of SEQ ID NO: 3 having at least 85% identity with SEQ ID NO: 3, or
iv) a variant of a fragment of SEQ ID NO: 3 having a length of at least 20 contiguous amino acid residues and having at least 85% identity with the fragment.

8. The vector according to claim 7 , wherein the encoded peptidic secretion signal is functional in S2 Drosophilia insect cells.

9. A recombinant cell or a population of recombinant cells comprising a nucleic acid molecule according to claim 1 .

10. A recombinant cell or population of recombinant cells comprising a vector according to claim 4 .

11. The recombinant cell or population of recombinant cells according to claim 10 , wherein the cells are transfected with the vector.

12. The recombinant cell or population of recombinant cells according to claim 11 , wherein the cells are S2 Drosophilia insect cells.

13. A method for producing a polypeptide, comprising:
a. transfecting a vector according to claim 4 in a cell,
b. culturing the cell to form a cell culture containing the polypeptide, and
c. recovering the polypeptide from the cell culture.

14. A method according to claim 13 , wherein the vector is a MGMT-based vector and the cell is a S2 Drosophilia insect cell.

15. The vector according to claim 4 , wherein the polypeptide consists of:
a) a fragment of SEQ ID NO: 3 consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61 or
b) a sequence having at least 85% identity with SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61.

16. The vector according to claim 4 , wherein the polypeptide is a variant of SEQ ID NO: 3 having at least 85% identity with SEQ ID NO: 3, or a variant of a fragment of SEQ ID NO: 3 having a length of at least 20 contiguous amino acid residues and having at least 85% identity with the fragment, and wherein the polypeptide has an additional cysteine residue at the N-terminus.

17. The vector according to claim 4 , wherein the nucleic acid molecule consists of SEQ ID NO: 66.

Assignees

• Pasteur Institut

Inventors

• Marteyn Benoit
• Coic Yves-Marie
• Baleux Francoise
• Sansonetti Philippe

Classifications

• C07K2319/61

  containing an enzyme fusion for detection (lacZ, luciferase) · CPC title

• C07K7/08

  having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) · CPC title

• G01N33/6893

  related to diseases not provided for elsewhere · CPC title

• C07K14/335 Primary

  from Lactobacillus (G) · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title