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Methods and Compositions for the Selective Activation of Protoxins Through Combinatorial Targeting

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Methods and Compositions for the Selective Activation of Protoxins Through Combinatorial Targeting (19) TZZ Z¥_T (11) EP 2 046 375 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 16/28 (2006.01) C07K 14/195 (2006.01) 05.04.2017 Bulletin 2017/14 (86) International application number: (21) Application number: 07810652.3 PCT/US2007/016475 (22) Date of filing: 20.07.2007 (87) International publication number: WO 2008/011157 (24.01.2008 Gazette 2008/04) (54) METHODS AND COMPOSITIONS FOR THE SELECTIVE ACTIVATION OF PROTOXINS THROUGH COMBINATORIAL TARGETING VERFAHREN UND ZUSAMMENSETZUNGEN ZUR SELEKTIVEN AKTIVIERUNG VON PROTOXINEN DURCH KOMBINATORISCHES TARGETING PROCÉDÉS ET COMPOSITIONS PERMETTANT UNE ACTIVATION SÉLECTIVE DE PROTOXINES PAR UN CIBLAGE COMBINATOIRE (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A1-01/14570 WO-A2-98/20135 HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE WO-A2-2004/094478 US-A1- 2003 054 000 SI SK TR US-A1- 2004 048 784 (30) Priority: 20.07.2006 US 832022 P • TAIT J F ET AL: "PROUROKINASE-ANNEXIN V CHIMERAS", JOURNAL OF BIOLOGICAL (43) Date of publication of application: CHEMISTRY, THE AMERICAN SOCIETY OF 15.04.2009 Bulletin 2009/16 BIOLOGICAL CHEMISTS, INC, US, vol. 270, no. 37, 15 September 1995 (1995-09-15), pages (73) Proprietor: The General Hospital Corporation 21594-21599, XP002920655, ISSN: 0021-9258, Boston, MA 02114 (US) DOI: 10.1074/JBC.270.37.21594 • WELS W ET AL: "CONSTRUCTION, BACTERIAL (72) Inventors: EXPRESSION AND CHARACTERIZATION OF A • SEED, Brian BIFUNCTIONAL SINGLE-CHAIN Derry, NH 03038 (US) ANTIBODY-PHOSPHATASE FUSION PROTEIN • WOLFE, Jia, Liu TARGETED TO THE HUMAN ERBB-2 Winchester, MA 01890 (US) RECEPTOR", BIO/TECHNOLOGY, NATURE • CHO, Glen, S. PUBLISHING CO. NEW YORK, US, vol. 10, no. 10, Newton, MA 02458 (US) 1 October 1992 (1992-10-01), pages 1128-1132, • TSAI, Chai-Iun XP000647729, ISSN: 0733-222X, DOI: Winchester, MA 01890 (US) 10.1038/NBT1092-1128 • CHIRON ET AL.: ’Furin-mediated cleavage of (74) Representative: Tomkins & Co Pseudomonas exotoxin-derived chimeric toxins’ 5 Dartmouth Road J.BIOL. CHEM. vol. 272, no. 50,1997, pages 31707 Dublin 6 (IE) - 31711, XP008102903 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 2 046 375 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 046 375 B1 Description Field of the Invention 5 [0001] In general, the present invention relates to a therapeutic strategy for targeting cyotoxic or cytostatic agents to particular cell types while reducing systemic adverse effects. More specifically, the present invention involves the use of a therapeutic modality comprising two or more individually inactive components with independent targeting principles, which are activated through their specific interaction at the targeted cells. The invention also provides related methods and compositions. 10 Background of the Invention [0002] Selective killing of particular types of cells is desirable in a variety of clinical settings, including the treatment of cancer, which is usually manifested through growth and accumulation of malignant cells. An established treatment 15 for cancer is chemotherapy, which kills tumor cells by inhibiting DNA synthesis or damaging DNA (C habner and Roberts, Nat. Rev. Cancer 5:65 (2005)). However, such treatments often cause severe systemic toxicity due to nondiscriminatory killing of normal cells. Because many cancer chemotherapeutics exert their efficacy through selective destruction of proliferatingcells, increased toxicities to normal tissues with high proliferationrates, such as bone marrow, gastrointestina l tract, and hair follicles, have usually prevented their use in optimal doses. Such treatments often fail, resulting in drug 20 resistance, disease relapse, and/or metastasis. To reduce systemic toxicity, different strategies have been explored to selectively target a particular cell population. Antibodies and other ligands that recognize tumor-associated antigens have been coupled with small molecule drugs or protein toxins, generating conjugates and fusion proteins that are often referred to as immunoconjugates and immunotoxins, respectively (Allen, Nat. Rev. Cancer 2:750 (2002)). [0003] In addition to dose-limiting toxicities, another limitation for chemotherapy is its ineffectiveness for treatment of 25 cancers that do not involve accelerated proliferation, but rather prolonged survival of malignant cells due to defective apoptosis (Kitada et al., Oncogene 21:3459 (2002)). For example, B cell chronic lymphocytic leukemia (B-CLL) is a disease characterized by slowly accumulating apoptosis-resistant neoplastic B cells, for which currently there is no cure (Munk and Reed, Leuk. Lymphoma 45:2365 (2004)). [0004] Cancer stem cells (CSCs) are a small fraction of tumor cells that have a capacity for self-renewal and unlimited 30 growth, and therefore are distinct from their progeny in their capacity to initiate cancers (Schulenburg et al., Cancer 107:2512 (2006)). Current cancer therapies do not target these cancer stem cells specifically, and it is hypothesized that the persistence of CSCs results in an ineradicable subset of cells that can give rise to progeny cells exhibiting drug resistance and/or contributing to the formation of metastases. In those tumors which harbor CSCs it is highly attractive to be able to eliminate these cells. CSCs have been thought to possess many properties similar to that of normal stems 35 cells, e.g., long life span, relative mitotic quiescence, and active DNA repair capacity, as well as resistance to apoptosis and to drug/toxins through high level expression of ATP-binding cassette drug transporters such as P-glycoprotein. Consequently, CSCs are thought to be difficult to target and destroy by conventional cancer therapies (Dean et al., Nat. Rev. Cancer 5:275 (2005)). Conversely, it is critically important to distinguish CSCs from normal stem cells because of the essential roles that normal stem cells play in the renewal of normal tissues. 40 [0005] To increase the selectivity of highly toxic anti-tumor agents, various attempts have been made to take advantage of specific features of the tumor microenvironment, such as the low pH, low oxygen tension, or increased density of tumor specific enzymes, that are not found in the vicinity of normal cells in well-perfused tissues. Environmentally sensitive anti-tumor agents have been developed that are hypothesized to exhibit increased toxicity in the solid tumor. For example "bioreductive prodrugs" are agents that can be activated to cytotoxic agents in the hypoxic environment of a solid tumor 45 (Ahn and Brown, Front Biosci. 2007 May 1;12:3483-501.) Similarly Kohchi et al. describe the synthesis of chemothera- peutic prodrugs that can be activated by membrane dipeptidases found in tumors (Bioorg Med Chem Lett. 2007 Apr 15;17(8):2241-5.) The use of selective antibody conjugated enzymes to alter the tumor microenvironment has also been explored by many groups. In the strategy known as antibody-directed enzyme prodrug therapy (ADEPT), enzymes conjugated to tumor-specific antibodies are intended to be delivered to the patient, followed by a chemotherapeutic 50 agent that is inactive until subject to the action of the conjugated enzyme (see for example Bagshawe, Expert Rev Anticancer Ther. 2006 Oct;6(10):1421-31 or Rooseboome et al. Pharmacol Rev. 2004 Mar;56(1):53-102) To date the clinical advantages of these strategies remain undocumented and there remains a high interest in developing more selective and more potent agents that can show therapeutic utility. 55 Summary of the Invention [0006] In one aspect the invention provides a composition according to claim 1. In another aspect the invention provides proteins for use according to claim 2. In a further aspect the present invention provides a method of destroying or inhibiting 2 EP 2 046 375 B1 a target cell according to claim 13. [0007] In one aspect, the invention features a protoxin activator fusion protein including one or more cell-targeting moieties and a modification domain. The protoxin activator fusion protein comprises a natively activatable domain. The modification domain is inactive prior to activation of the natively activatable domain. Desirably, the protoxin activator 5 fusion protein is non-toxic to a target cell (e.g., the protoxin activator fusion protein has less than 10% of the cytotoxic or cytostatic activity of the combination of the protoxin activator fusion protein and the protoxin upon which the protoxin activator fusion protein acts). [0008] In the above aspects, the modification domain can be a protease containing the catalytic domain of a human protease (desirably an exogenous human protease), or a non-human protease, including a viral protease (e.g., retroviral 10 protease, a potyviral protease, a picornaviral protease, or a coronaviral protease). In a related aspect, the modification domain can be a phosphatase. [0009] In another aspect, a protoxin fusion protein including one or more non-native cell-targeting moieties, a selectively modifiable activation domain, and a toxin domain (e.g., an activatable toxin domain) is disclosed. [0010] In this aspect, the modifiable activation domain may include a substrate for an 15 exogenous enzyme. [0011] In this aspect, the exogenous enzyme can be, for example, a protease or phosphatase. Examples of proteases include an exogenous human protease or a non-human (or non-mammalian) protease, including a viral protease (e.g., a retroviral protease, a potyviral protease, a picornaviral protease, or a coronaviral protease). [0012] Also in this aspect, the activatable toxin domain can include an activatable pore forming toxin or an activatable 20 enzymatic toxin. Examples of such domains include an AB toxin, a cyotoxic necrotizing factor toxin, a dermonecrotic toxin, and an activatable ADP-ribosylating toxin.
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