Fusion protein, nanoparticle composed of a plurality of monomers of said fusion protein, and uses thereof

• US 10,640,775 B2
• Filed: 09/29/2015
• Issued: 05/05/2020
• 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
• Primary Examiner(s): Desai, Anand U
• Application Number:
  US15/515,875
• Publication Number:
  US 20170298364A1
• Time in Patent Office: 1,680 Days
• Field of Search: None
• 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 comprising a polypeptide having an amino acid sequence at least 90% identical to the heavy chain of native human ferritin;

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

(c) a third domain comprising a polypeptide consisting essentially of proline, serine and alanine (PAS) residues;

wherein the third domain fusion protein is at least 20 and less than 80 amino acid residues in length; and

wherein the fusion protein is in a linear arrangement of amino-terminus to carboxy-terminus of the first domain, the second domain, and the third domain.

2. The fusion protein according to claim 1, wherein the first domain comprises the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

3. 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.

4. The fusion protein according to claim 3, wherein the amino acid sequence of the matrixmetalloproteinase (MMP) cleavage site is selected from the group consisting of SEQ ID NOs:

3-8.

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

6. 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.

7. 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.

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

9. The fusion protein of claim 1, wherein the first domain comprises a polypeptide having the amino acid sequence of the heavy chain of native human ferritin.

10. The fusion protein according to claim 2, wherein the PAS is between 20 and 80 amino acid residues in length.

11. The fusion protein according to claim 2, wherein the PAS is between 40 and 75 amino acid residues in length.

12. The fusion protein according to claim 7, wherein the first and the second linker sequences are the same.

13. The fusion protein according to claim 7, wherein the first and the second linker amino acid sequences are different from one another.