FUSION PROTEIN, NANOPARTICLE COMPOSED OF A PLURALITY OF MONOMERS OF SAID FUSION PROTEIN, AND USES THEREOF

• US 20170298364A1
• Filed: 09/29/2015
• Published: 10/19/2017
• Est. Priority Date: 09/30/2014
• Status: Active Grant

• Current Assignee: Thena Biotech S.R.L.
• Original Assignee: Thena Biotech S.R.L.
• Inventors: CECI, Pierpaolo, FALVO, Elisabetta
• Granted Patent: US 10,640,775 B2
• Time in Patent Office: Days
• 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: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/001159 Matrix metalloproteinases [MMP]
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

A fusion protein based on the heavy chain of human ferritin is described, which includes at the N terminus of the protein at least one metalloproteinase cleavage sequence and a PAS polypeptide that acts as a masking polymer that increases the protein-drug stability, as well as a nanoparticle composed of multiple monomers of said fusion protein, a nucleic acid encoding for said fusion protein, and diagnostic and therapeutic applications thereof.

Claims

1. A fusion protein comprising at least three domains, wherein:

(a) a first domain comprises the amino acid sequence of the heavy chain of native human ferritin or a variant thereof having at least 90% identity with the amino acid sequence of the heavy chain of native human ferritin;

(b) a second domain comprises the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site; and

(c) a third N-terminal domain consists of the amino acid sequence of a polypeptide of at least 20 amino acid residues and which essentially consists of proline, serine and alanine (PAS).

2. The fusion protein according to claim 1, wherein the PAS has a length which is lower than 80 amino acid residues, preferably comprised between 20 and 80 amino acid residues, more preferably comprised between 40 and 75 amino acid residues.

3. The fusion protein according to claim 1, wherein the first domain comprises the amino acid sequence of the heavy chain of native human ferritin of SEQ ID NO:

1 or the amino acid sequence of the heavy chain variant of human ferritin of SEQ ID NO;

2.

4. The fusion protein according to claim 1, wherein the second domain comprises the amino acid sequence of a matrix metalloproteinase (MMP) cleavage site selected from the group consisting of MMP-2, MMP-3, MMP-7 and MMP-9.

5. The fusion protein according to claim 4, wherein the amino acid sequence of the matrix metalloproteinase (MMP) cleavage site is selected from the group consisting of SEQ ID NO:

3-8.

6. The fusion protein according to claim 1, wherein the proline residues of the aforesaid PAS polypeptide amount to 10-40% of the total amino acid residues thereof.

7. The fusion protein according to claim 1, wherein the amino acid sequence of the PAS polypeptide is selected from SEQ ID NO:

9 and SEQ ID NO;

10.

8. The fusion protein according to claim 1, comprising a first and/or second linker amino acid sequence(s) respectively linking the first domain to the second domain and/or the second domain to the third domain, wherein the first and the second amino acid sequences are the same or different from each other.

9. The fusion protein according to claim 1, which is linked to an active ingredient and/or imaging agent.

10. A nanoparticle comprising a plurality of monomers of a fusion protein according to claim 1.

11. An isolated nucleic acid encoding for a fusion protein according to claim 1 or a nanoparticle comprising a plurality of monomers of the fusion protein.

12. An expression vector comprising the nucleic acid according to claim 11.

13. A host cell comprising the nucleic acid according to claim 11 or a vector comprising the nucleic acid.

14. A pharmaceutical composition comprising:

a fusion protein according to claim 1, a nanoparticle comprising a plurality of monomers of the fusion protein, a nucleic acid encoding for the fusion protein or the nanoparticle, an expression vector comprising the nucleic acid, or a host cell comprising the nucleic acid or the expression vector, in combination with at least one pharmaceutically acceptable excipient, carrier, or diluent.

15. The pharmaceutical composition according to claim 14 further comprising:

a pharmaceutically active ingredient or imaging agent.

16. A method for therapeutic treatment, diagnosis, or both of a hyperproliferative disease, including cancer, the method comprising:

administering a pharmaceutical composition according to claim 14, in an effective amount to a subject, wherein the fusion protein or the nanoparticle acts as a targeted carrier system for a tumour or cancer cell in the subject.

17. The nanoparticle according to claim 10, wherein the plurality of monomers is 24 monomers.