Modified adenoviral fiber and target adenoviruses

• US 6,569,677 B1
• Filed: 10/04/1999
• Issued: 05/27/2003
• Est. Priority Date: 04/02/1997
• Status: Expired due to Fees

• Current Assignee: Transgene S.A. (Compagnie Merieux Alliance SAS)
• Original Assignee: Centre National De La Recherche Scientifique, Transgene S.A. (Compagnie Merieux Alliance SAS)
• Inventors: Boulanger, Pierre, Mehtali, Majid, Legrand, Valérie
• Primary Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US09/402,401
• Time in Patent Office: 1,331 Days
• Field of Search: 424/93.2, 435/320.1, 435/91.4, 514/44
• US Class Current: 435/320.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/10345 Special targeting system for viral vectors
• CPC Class Codes: C12N 2810/851 from growth factors; from growth regulators
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 2830/60 from viruses
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The invention relates to an adenovirus fiber modified by the mutation of one or more residues. The residues are directed towards the natural cell receptor in the three-dimensional structure of said adenovirus. The invention further relates to a DNA fragment, and expression vector, and a cell line expressing said fiber, and also concerns an adenovirus, the process for producing this type of adenovirus, and a infectable host cell, as well as their therapeutic application and a corresponding pharmaceutical composition.

Claims

1. A method for producing an adenovirus whose genome lacks all or part of the sequences encoding a fiber, comprising:

transfecting the genome of said adenovirus into a cell line comprising, either in a form integrated into the genome or in the form of an episome, a DNA fragment or expression vector encoding an adenovirus fiber modified by substitution mutation of one or more residues of a native fiber, wherein said native fiber residues are directed toward the natural cellular receptor recognized by the native adenovirus fiber, and wherein said residues are between the CD loop and the β

sheet I of said fiber, the modification not including deletion of the entire knob region, said DNA fragment or expression vector placed under the control of the elements allowing its expression in said cell line,

culturing said transfected cell line under appropriate conditions in order to allow the production of said adenovirus, and

recovering said adenovirus from the culture of said transfected cell line.

2. The method of claim 1, further comprising substantially purifying said recovered adenovirus.

3. An adenovirus made by the process comprising:

a) identifying one or more residues of a natural adenoviral fiber which are directed toward the natural cellular receptor of a natural adenovirus and which are between the CD loop and the β

sheet I of the adenoviral fiber,

b) modifying said fiber by mutating one or more of said residues so as to eliminate binding to the natural cellular receptor, but not by deleting the entire knob region,

c) either incorporating sequence encoding the modified fiber of step b) into a genome of an adenovirus which lacks a functional native fiber, or providing the modified fiber in trans in a cell line,

d) transfecting the genome of said adenovirus into an appropriate cell line,

e) culturing said transfected cell line under appropriate conditions in order to allow the production of said adenovirus, and

f) recovering said adenovirus from said culture of said transfected cell line.

4. An adenovirus comprising an adenovirus fiber, modified by substitution mutation of one or more residues of a native fiber, wherein said native fiber residues are directed toward the natural cellular receptor recognized by the native adenovirus fiber, and wherein said residues are between the CD loop and the β

sheet I of said fiber and further comprising a ligand inserted at the C-terminal end of the fiber, which is capable of recognizing a cell surface molecule different from the natural cellular receptor of said adenovirus in its natural form.

5. The adenovirus of claim 4, wherein said adenovirus is a replication-defective recombinant adenovirus.

6. The adenovirus of claim 5, wherein said adenovirus is deleted for all or part of the E1 region.

7. The adenovirus of claim 6, wherein said adenovirus is further deleted for all or part of the E2, E4 and/or L1-L5 region.

8. The adenovirus of claim 5, wherein said adenovirus comprises a gene of interest selected from the genes encoding a cytokine, a cellular or nuclear receptor, a ligand, a coagulation factor, the CFTR protein, insulin, dystrophin, a growth hormone, an enzyme, and enzyme inhibitor, a polypeptide with antitumor effect, a polypeptide capable of inhibiting a bacterial infection, a polypeptide capable of inhibiting a parasitic infection, a polypeptide capable of inhibiting a viral infection, a polypeptide capable of inhibiting HIV, an antibody, a toxin, an immunotoxin and a marker.

9. The adenovirus of claim 5, wherein said adenovirus is deleted for all or part of the E3 region.

10. The adenovirus of claim 4, wherein the ligand is a polypeptide.