Anti-Alpha V Immunoliposome Composition, Methods, and Uses

• US 20090175784A1
• Filed: 05/12/2008
• Published: 07/09/2009
• Est. Priority Date: 05/11/2007
• Status: Abandoned Application

• Current Assignee: Centocor Ortho Biotech Incorporated (Johnson & Johnson)
• Original Assignee: Centocor Ortho Biotech Incorporated (Johnson & Johnson)
• Inventors: Sweet, Raymond, Saini, Deepak, Magill, Allen, Goldstein, Joshua, Snyder, Linda A., Weng, Steve P.
• Application Number:
  US12/118,994
• Publication Number:
  US 20090175784A1
• Time in Patent Office: Days
• US Class Current: 424/1.210
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6913 the liposome being modified on its surface by an antibody
• CPC Class Codes: A61K 51/1027 against receptors, cell-surface antigens or cell-surface determinants
• CPC Class Codes: A61K 51/1234 Liposomes
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/2839 against the integrin superfamily
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)

Abstract

An immunoliposome composition targeted to the alphaV-integrin subunit of integrin receptors comprised of ligand-targeted liposomes bearing at least one targeting-ligand derived from an antibody and having binding specificity for at least one integrin receptor comprising an alpha V subunit including αvβ1, αvβ3 αvβ5, αvβ6, or αvβ8 integrin cell receptors is described. The antibody-derived targeting ligand may be a Fab′ fragment, a scFv, or the like. Binding of the immunoliposome to αv-integrin expressing cells, preferably results in internalization of the immunoliposome for cytoplasmic delivery of a liposome-entrapped agent.

Claims

1. An antibody derived targeting-ligand capable of monovalent binding and having specificity for alpha V integrin comprising:

(a) a first polypeptide domain comprising the light chain variable region of a parent monoclonal antibody having specificity for alpha V-integrin or a conservative modification or fragment thereof that retains the binding specificity;

(b) a second polypeptide domain comprising the heavy chain variable region of a parent monoclonal antibody having specificity for alpha V-integrin or a conservative modification or fragment thereof that retains the binding specificity; and

wherein the targeting ligand retains specificity for at least one alphaV integrin.

2. The antibody derived targeting-ligand of claim 1, wherein the light chain variable domain comprises residues 1-108 of SEQ ID NO:

2 and the heavy chain variable domain comprises residues 1-119 of SEQ ID NO;

1.

3. The antibody derived targeting-ligand of claim 1, further comprising at least one peptide linker linking said first and second polypeptides (a) and (b) into a single chain Fv polypeptide having binding affinity specificity for alpha V integrin.

4. The single chain Fv polypeptide of claim 3 wherein the linker has an amino acid sequence of the formula (Gly₄Ser)_n where n is an integer of 1-4.

5. The single chain Fv polypeptide of claim 4 where n is 3.

6. The single chain Fv polypeptide of claim 5 wherein the heavy chain variable region has a leucine substitution at the 18^th amino acid of SEQ ID NO:

1.

7. The single chain Fv polypeptide of claim 3, which competes with the monoclonal antibody CNTO95 for binding to at least one human alpha V integrin subunit.

8. The antibody derived targeting-ligand of claim 1, comprising a Fab fragment wherein the said first and second polypeptides (a) and (b) comprise polypeptides linked by a disulfide bond.

9. The Fab fragment of claim 8, produced by an enzymatic digestion of the monoclonal antibody CNTO95.

10. The Fab fragment of claim 9, wherein the enzymatic digestion step uses pepsin.

11. The Fab fragment of claim 9, wherein the enzymatic digestion step uses papain.

12. The Fab fragment of claim 8, wherein the Fab is secreted from a host cell line.

13. The Fab fragment of claim 12, wherein the host cell line is transfected with a nucleic acid coding for the amino acid sequence of SEQ ID NO:

3.

14. The antibody derived targeting-ligand of any of claims 1-4, wherein the targeting-ligand comprises a predetermined site for chemical conjugation.

15. An isolated nucleic acid molecule encoding the antibody derived targeting-ligand of claim 1.

16. An isolated nucleic acid encoding a single chain Fv polypeptide of claim 1 comprising a nucleic acid sequence according to SEQ ID NO:

5.

17. The isolated nucleic acid molecule of claim 15 wherein the nucleic acid molecule is incorporated into an expression vector.

18. An isolated nucleic acid vector comprising an isolated nucleic acid according to claim 15.

19. A prokaryotic or eukaryotic host cell comprising an isolated nucleic acid according to claims 15, 16, 17, or 18.

20. A host cell according to claim 19, wherein said host cell is at least one selected from E. coli, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma cells, Perc.6, or any derivative, immortalized or transformed cell thereof.

21. A pharmaceutical composition comprising the antibody derived targeting-ligand of claim 1 and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition comprising the antibody derived targeting-ligand of claim 1 covalently linked to, or conjugated to a non-biologic polymer, which polymer optionally is further covalently linked to a lipid, an active, or a reporter molecule.

23. A pharmaceutical composition comprising the antibody derived targeting-ligand of claim 22, in which the biological polymer conjugate is inserted into the surface of a liposome.

24. The composition according to claim 23, wherein the liposome has an anti-neoplastic agent encapsulated within.

25. A composition according to claim 24, wherein the anti-neoplastic agent is selected from a radiopharmaceutical, an estrogen receptor modulator, a retinoid, a topoisomererase inhibitor, a cytotoxin, an alkylating agent, a nitrogen mustard, a nitrosourea, an antimetabolite, a mitotic inhibitor, and a radiosensitizer.

26. A composition according to claim 25, wherein the anti-neoplastic agent is doxorubicin.

27. An isolated nucleic acid molecule encoding the single chain Fv polypeptide of claim 1.

28. A method for producing the single chain Fv polypeptide of claim 1, comprising translating a nucleic acid according to claims 15-18 under conditions in vitro, in vivo or in situ, such that the antibody derived targeting-ligand of claim 1 is expressed in detectable or recoverable amounts.

29. A method of inhibiting growth of a cell expressing alpha v integrin, comprising contacting the cell with an effective amount of a pharmaceutical composition according to claims 21-26 such that the growth of the cell is inhibited.