(19) TZZ ¥_T (11) EP 2 739 649 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 14/79 (2006.01) A61K 38/40 (2006.01) 27.09.2017 Bulletin 2017/39 A61K 38/00 (2006.01) A61K 39/44 (2006.01) (21) Application number: 12746240.6 (86) International application number: PCT/US2012/049475 (22) Date of filing: 03.08.2012 (87) International publication number: WO 2013/022738 (14.02.2013 Gazette 2013/07) (54) P97 FRAGMENTS WITH TRANSFER ACTIVITY P97-FRAGMENTE MIT TRANSFERAKTIVITÄT FRAGMENTS DE P97 AVEC ACTIVITÉ DE TRANSFERT (84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB WO-A2-02/13873 WO-A2-03/009815 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR • KARKAN DELARA ET AL: "A Unique Carrier for Delivery of Therapeutic Compounds beyond the (30) Priority: 05.08.2011 US 201161515792 P Blood-Brain Barrier", PLOS ONE, vol. 3, no. 6, June 2008 (2008-06), XP002685120, ISSN: (43) Date of publication of application: 1932-6203 11.06.2014 Bulletin 2014/24 • TANG Y ET AL: "Directing adenovirus across the blood-brain barrier via melanotransferrin (P97) (73) Proprietor: Bioasis Technologies Inc. transcytosis pathway in an in vitro model", GENE Richmond, British Columbia V6X 2W9 (CA) THERAPY, MACMILLAN PRESS LTD., BASINGSTOKE, GB, vol. 14, no. 6, 1 March 2007 (72) Inventors: (2007-03-01), pages523-532, XP002517213, ISSN: • JEFFERIES, Wilfred 0969-7128, DOI: 10.1038/SJ.GT.3302888 South Surrey, BC V4A 2V5 (CA) [retrieved on 2006-11-30] • TIAN, Mei Mei • YANG J ET AL: "Deletion of the GPI pre-anchor Coquitlam, BC V3J 7E6 (CA) sequence in human p97-a general approach for • VITALIS, Timothy generating the soluble form of GPI-linked Vancouver, BC V6Z 2N1 (CA) proteins", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, (74) Representative: Gowshall, Jonathan Vallance CA, vol. 34, no. 1, 1 March 2004 (2004-03-01), Forresters IP LLP pages 28-48, XP004487377, ISSN: 1046-5928, Skygarden DOI: 10.1016/J.PEP.2003.09.007 Erika-Mann-Strasse 11 80636 München (DE) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 739 649 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 739 649 B1 Description BACKGROUND 5 Technical Field [0001] The present invention is related to fragments of human melanotransferrin (p97). In particular, this invention relates to treatment of diseases through the introduction of the melanotransferrin fragment conjugated to a therapeutic or diagnostic agent to a subject. 10 Description of the Related Art [0002] Melanotransferrin (MTf) is a bi-lobed protein belonging to the transferrin (Tf) family of iron binding proteins. It has been demonstrated previously that MTf is able to directly bind and transport iron into mammalian cells independent 15 of Tf and Tf receptor (TfR). Unlike other Tf family members, this molecule exists in two forms in humans, a glycosyl- phosphatidylinositol (GPI)-linked cell surface form and a secreted water-soluble form. Additionally, MTf is also found to be expressed on human brain endothelium where it is hypothesized to transport iron across the blood brain barrier (BBB). The role of MTf in the transfer of iron into the brain was assessed by following both radiolabeled soluble MTf and Tf into the mouse brain 20 during a 24-hour period (Moroo et al., 2003, Demeule et al., 2002). It was determined that soluble MTf does have the ability to transcytose across the blood-brain barrier (BBB) and this transport was more efficient than that of Tf. [0003] Subsequently, it has been demonstrated that soluble MTf could be used as a delivery vehicle of therapeutics into the brain (Karkan et al., 2008). Pharmacokinetics studies on soluble MTf demonstrated that the clearance of MTf from serum was much greater than IgG control, and was rapidly distributed to the tissues relative to IgG control. The 25 transport of soluble MTf into the brain as a percentage of injected dose was significantly greater than IgG during the first hour post injection. The accumulation of soluble MTf in the brain was found to be significantly more than that of IgG during the first 6-hours post injection. [0004] Furthermore, it was shown that soluble MTf is able to deliver iron across the BBB (Moroo et al., 2003), as well as paclitaxel covalently linked to MTf (Karkan et al., 2008). In the same study, while both free-adriamycin and MTf- 30 adriamycin conjugates were able to equally inhibit the subcutaneous growth of gliomas outside of the brain, only MTf- adriamycin conjugates significantly prolonged the survival of animals bearing intracranial gliomas when compared to the free-adriamycin control (Karkan et al., 2008). Taken together, these data suggest soluble MTf as a potential drug delivery tool. [0005] However, an even more efficient transfer molecule for delivering a target agent would be useful for therapeutic 35 and diagnostic purposes. The present invention addresses these and other needs. [0006] <Yang et al, Protein Expression And Purification, v34, no 1, pp28-48, 2004 discloses fragments of the p97 protein. WO 03/009815 discloses uptake of p97 in astrocytes and astrocytomas. WO 02/13873 discolses p97 covalently linked to Adriamycin.> 40 BRIEF SUMMARY [0007] The invention is defined in the claims. [0008] The present invention include isolated p97 (melanotransferrin; MTf) polypeptides consisting of the amino acid sequence set forth in SEQ ID NO:1-8 or 9. Also included are compositions comprising a fragment of p97 consisting 45 essentially of SEQ ID NO:1-8 or 9 and a therapeutic or diagnostic agent. [0009] The p97 polypeptide may be labeled with a label selected from the group consisting of fluorescent molecules, luminescent molecules, enzymes, substances having therapeutic activity, toxins, and radionuclides. The p97 polypeptide is conjugated to a therapeutic agent or drug. [0010] Pharmaceutical compositions may comprise a therapeutically effective amount of compound comprising a p97 50 fragment covalently linked to a therapeutic agent and a pharmaceutically acceptable excipient, wherein the p97 fragment consists of the amino acid sequence set forth in SEQ ID NO:1-8 or 9. [0011] Also included are compositions for delivering an agent across the blood brain barrier comprising a p97 fragment conjugated to the agent, a substance which is capable of specifically binding to p97 conjugated to the agent, or a p97 fragment fusion protein containing the p97 fragment fused to the agent, and a pharmaceutically acceptable carrier or 55 diluent, wherein the p97 fragment consists of the amino acid sequence set forth in SEQ ID NO:1-8 or 9. [0012] The invention relates to conjugates, comprising a p97 polypeptide that consists or consists essentially of SEQ ID NO:1, 5 or 6, where the p97 polypeptide is covalently or operatively linked to an agent, to form a p97-agent conjugate. In some embodiments, the agent is a small molecule, a polypeptide, or a label ( i.e., a detectable entity). 2 EP 2 739 649 B1 [0013] In particular embodiments, the small molecule is a cytotoxic or chemotherapeutic or anti-angiogenic agent selected from one or more of alkylating agents, anti-metabolites, anthracyclines, anti-tumor antiobiotics, platinums, type I topoisomerase inhibitors, type II topoisomerase inhibitors, vinca alkaloids, and taxanes. In specific embodiments, the small molecule is selected from one or more of chlorambucil, cyclophosphamide, cilengitide, lomustine (CCNU), mel- 5 phalan, procarbazine, thiotepa, carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin, gefitinib, erlotinib idarubicin, temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecins, irinote- can, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus, everolimus, vincristine, vinblas- tine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasatinib, sorafenib, pazopanib, sunitnib, vatalanib, geftinib, erlotinib, AEE-788, dichoroacetate, tamoxifen, fasudil, SB-681323, semaxanib, donepizil, galantamine, memantine, ri- 10 vastigmine, tacrine, rasigiline, naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatirimer acetate, fingolimod, and mitoxantrone, including pharma- ceutically acceptable salts and acids thereof. [0014] In some embodiments, the polypeptide is an antibody or antigen-binding fragment thereof. In particular em- bodiments, the antibody or antigen-binding fragment thereof specifically binds to one or more of human Her2/neu, 15 Her1/EGFR, CD20, VEGF, CD52, CD33, CTLA-4, tenascin, alpha-4 (α4) integrin, IL-23, amyloid-β, Huntingtin, CD25, nerve growth factor (NGF), TrkA, TNF-α, TNF-β, or α-synuclein, among other targets described herein. [0015] In certain embodiments, the antibody is selected from one or more of trastuzumab, cetuximab, daclizumab, tanezumab, 3F8, abagovomab, adalimumab, adecatumumab, afutuzumab, alemtuzumab, alacizumab (pegol), amatux- imab, apolizumab, bavituximab, bectumomab, belimumab, bevacizumab, bivatuzumab (mertansine), brentuximab ve- 20 dotin, cantuzumab (mertansine), cantuzumab (ravtansine), capromab