Universal carrier for targeting molecules to GB3 receptor expressing cells

Patent metadata

Field	Value
Publication number	US-9603923-B2
Application number	US-201414478687-A
Country	US
Kind code	B2
Filing date	Sep 5, 2014
Priority date	Feb 1, 2001
Publication date	Mar 28, 2017
Grant date	Mar 28, 2017

Abstract

The present invention concerns an universal polypeptidic carrier for targeting directly or indirectly a molecule to Gb3 receptor expressing cells and having the following formula STxB-Z(n)-Cys, wherein:
STxB is the Shiga Toxin B subunit or a functional equivalent thereof,
Z is an amino-acid devoided of sulfydryl group, n being 0, 1 or a polypeptide, Cys is the amino-acid Cysteine,

and the use thereof for MHC class I and MHC class II presentation of antigens.

First claim

The invention claimed is:

1. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising the nucleotide sequence STxB encoding the Shiga Toxin B subunit or a functional equivalent thereof bearing at its 3′ end the codon TGT, or the codon TGC encoding cysteine, wherein the functional equivalent thereof binds to the Gb3 receptor and triggers the internalization of an antigen and its presentation in an MHC class I and MHC class II restricted pathway on the same antigen presenting cell;
b) a polynucleotide comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence encoding the Shiga Toxin B subunit or a functional equivalent thereof, bearing at its 3′ end the codon TGT or TGC, wherein the functional equivalent thereof binds to the Gb3 receptor and triggers the internalization of an antigen and its presentation in an MHC class I and MHC class II restricted pathway on the same antigen presenting cell and
c) a nucleotide sequence complementary to the sequence in a) or b).

2. The polynucleotide according to claim 1 , comprising SEQ ID No. 2.

3. A recombinant vector or plasmid, comprising a polynucleotide sequence according to claim 1 .

4. A recombinant cell line obtained by transformation with the recombinant vector according to claim 3 .

5. The recombinant cell line according to claim 4 , which is a prokaryotic cell line.

6. The recombinant cell line according to claim 5 , which prokaryotic cell line is E. coli.

7. The recombinant cell line according to claim 6 , deposited at the CNCM on Dec. 19, 2000 under accession number I-2604.

8. A method for constructing a recombinant vector according to claim 3 comprising:
a) providing a plasmid comprising a STxB sequence;
b) applying two PCR amplification steps using two couples of primers AA′ and BB′ wherein A and B are complementary to each other and comprise the Cys codon and A′ and B′ are outside the STxB sequence;
c) isolating the amplified fragments;
d) hybridizing the amplified fragments;
e) applying a PCR amplification on the hybridized fragments; and
f) inserting the amplified fragments into a plasmid.

9. The method according to claim 8 , wherein in step f) the fragments are inserted into a SphI and SalI restriction site of the plasmid pSU108.

10. A process for producing an isolated polypeptide comprising:
a). culturing a recombinant cell line of claim 4 ;
b) obtaining a periplasmic extract of said cells; and
c) purifying said polypeptide.

11. The process according to claim 10 wherein in step a) the cell line is E. coli and in step c) the purification is by anion exchange column chromatography followed by a gel filtration column chromatography.

Assignees

• Inst Curie
• Centre Nat Rech Scient
• Inst Nat Sante Rech Med
• Univ Pierre Et Marie Curie (Paris Vi)

Inventors

• Johannes Ludger
• Tartour Eric
• Goud Bruno
• Fridman Wolf Herve

Classifications

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• C07K14/25

  Shigella (G) · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• A61K2039/6037

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

• A61K2039/627

  characterised by the linker · CPC title