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Human Antibodies That Bind CXCR4 and Uses Thereof CXCR4-Bindende Humane Antikörper Und Deren Verwendungen Anticorps Humains Liant Le CXCR4 Et Utilisations Associées

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Human Antibodies That Bind CXCR4 and Uses Thereof CXCR4-Bindende Humane Antikörper Und Deren Verwendungen Anticorps Humains Liant Le CXCR4 Et Utilisations Associées (19) TZZ __T (11) EP 2 486 941 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 39/395 (2006.01) C07K 16/28 (2006.01) 15.03.2017 Bulletin 2017/11 (21) Application number: 12155398.6 (22) Date of filing: 01.10.2007 (54) Human antibodies that bind CXCR4 and uses thereof CXCR4-bindende humane Antikörper und deren Verwendungen Anticorps humains liant le CXCR4 et utilisations associées (84) Designated Contracting States: EP-A- 1 316 801 WO-A-2004/059285 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A-2006/089141 US-A1- 2003 206 909 HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR • GHOBRIAL IRENE M ET AL: "The role of CXCR4 Designated Extension States: inhibitors as novel antiangiogenesis agents in RS cancer therapy", BLOOD, W.B.SAUNDERS COMPANY, ORLANDO, FL, vol. 104, no. 11 (30) Priority: 02.10.2006 US 827851 P PART1, 1 November 2004 (2004-11-01), pages 365A-366A, XP002458710, ISSN: 0006-4971 (43) Date of publication of application: • ENDRES M J ET AL: "CD4-INDEPENDENT 15.08.2012 Bulletin 2012/33 INFECTION BY HIV-2 IS MEDIATED BY FUSIN/CXCR4", CELL, CELL PRESS, (62) Document number(s) of the earlier application(s) in CAMBRIDGE, NA, US, vol. 87, 15 November 1996 accordance with Art. 76 EPC: (1996-11-15), pages745-756, XP002920421, ISSN: 07867192.2 / 2 066 351 0092-8674 • BARIBAUD FREDERIC ET AL: "Antigenically (73) Proprietor: E. R. Squibb & Sons, L.L.C. distinct conformations of CXCR4", JOURNAL OF Princeton, NJ 08540 (US) VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 75, no. 19, 1 October (72) Inventors: 2001 (2001-10-01), pages 8957-8967, • Kuhne, Michelle XP002234668, ISSN: 0022-538X San Francisco, CA 94116 (US) • GULENG BAYASI ET AL: "Blockade of the •Brams,Peter stromal cell-derived factor-1/CXCR4 axis Sacramento, CA 95815 (US) attenuates in vivo tumor growth by inhibiting • Tanamachi, Dawn M. angiogenesis in a vascular endothelial growth Milpitas, CA 95035 (US) factor-independent manner", CANCER •Korman,Alan RESEARCH, vol. 65, no. 13, July 2005 (2005-07), Milpitas, CA 95035 (US) pages 5864-5871, XP002491075, ISSN: 0008-5472 • Cardarelli, Josephine M. • VADAY GAYLE G ET AL: "CXCR4 and CXCL12 San Carlos, CA 94070 (US) (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies", CLINICAL (74) Representative: Reitstötter Kinzebach CANCER RESEARCH, THE AMERICAN Patentanwälte ASSOCIATION FOR CANCER RESEARCH, US, Sternwartstrasse 4 vol.10, no. 16, 15 August 2004 (2004-08-15), pages 81679 München (DE) 5630-5639, XP002397863, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-03-0633 (56) References cited: 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 2 486 941 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 486 941 B1 Description Background 5 [0001] Chemokines are a family of about 50 small proteins that modulate cell trafficking and angiogenesis and also play a significant role in the tumor microenvironment (Vicari, A.P. and Caux, C. (2002) Cytokine Growth Factor Rev. 13:143-154). Depending on their structure, chemokines are classified as C-C chemokines (containing a cysteine-cysteine motif) or C-X-C chemokines (containing a cysteine-X-cysteine motif). Receptors that bind such chemokines thus are classified as members of the CCR family or CXCR family, respectively. One member of the CXCR family is CXCR4, a 10 seven transmembrane G-protein coupled receptor that is predominantly expressed on lymphocytes and that activates chemotaxis. CXCR4 binds the chemokine CXCL12 (SDF-1). [0002] CXCR4 plays a role in embryogenesis, homeostasis and inflammation. Studies with mice engineered to be deficient in CXCR4 or SDF-1 implicate the CXCR4/SDF-1 pathway in organ vascularization, as well as in the immune and hematopoietic systems (Tachibana, K. et al. (1998) Nature 393:591-594). Moreover, CXCR4 has been shown to 15 function as a coreceptor for T lymphotrophic HIV-1 isolates (Feng, Y. et al. (1996) Science 272:872-877). CXCR4 also has been shown to be expressed on a wide variety of cancer cell types. Additionally, the CXCR4/SDF-1 pathway has been shown to be involved in stimulating the metastatic process in many different neoplasms (Murphy, P.M. (2001) N. Engl. J. Med. 345:833-835). For example, CXCR4 and SDF-1 have been shown to mediate organ-specific metastasis by creating a chemotactic gradient between the primary tumor site and the metastatic site (Muller, A. et al. (2001) Nature 20 410:50-56; Murakami, T. et al. (2002) Cancer Res. 62:7328-7334; Hanahan, D. et al. (2003) Cancer Res. 63 :3005-3008). [0003] Anti-CXCR4 antibodies are described in WO 2006/089141, US 2003/206909, EP 1 316 801, Ghobrial et al. (Blood 104(11):365A-366A, 2004), Endres et al. (Cell 87:745-756, 1996) and Vaday et al. (Clin Cancer Res 10(16):5630-5639, 2004). 25 Summary [0004] The present disclosure provides a monoclonal antibody or an antigen-binding portion thereof which binds specifically to human CXCR4 expressed on a cell surface and comprises a heavy chain variable region comprising amino acids having a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:49 encoded by a 30 human VH 3-48 germline immunoglobulin gene and a light chain variable region comprising amino acids having a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:50 encoded by a human V K L15 germline immunoglobulin gene, wherein said monoclonal antibody or antigen-binding portion thereof induces apoptosis in cells expressing human CXCR4, and inhibits growth and/or induces apoptosis of CXCR4 + tumor cells in vivo. The monoclonal antibodies of the invention can be isolated monoclonal antibodies, in particular human monoclonal antibodies, that bind 35 to human CXCR4 and that exhibit numerous desirable properties. These properties include the ability to bind to native human CXCR4 expressed on a cell surface, the ability to inhibit SDF-1 binding to human CXCR4, the ability to inhibit SDF-1-induced calcium flux in cells expressing CXCR4, the ability to inhibit SDF-1-induced migration of cells expressing CXCR4, the ability to inhibit capillary tube formation by human umbilical vein endothelial cells (HuVECs), the ability to inhibit tumor cell proliferation in vitro, the ability to inhibit metastases of CXCR4 + tumor cells and/or the ability to increase 40 survival time of a CXCR4+ tumor-bearing subject. [0005] In one embodiment, the antibody also inhibits binding of SDF-1 to human CXCR4, preferably with an EC 50 for inhibition of 50 nM or less, or 30 nM or less, or 15 nM or less, or 10 nM or less, or 5 nM or less, or 3 nM or less (e.g., an EC50 for inhibition of 28.60 nM or less, or 12.51 nM or less, or 2.256 nM or less). In another embodiment, the antibody binds to native human CXCR4 expressed on a cell surface but does not inhibit binding of SDF-1 to human CXCR4. In 45 yet other embodiments, the antibody also inhibits SDF-1-induced calcium flux in cells expressing human CXCR4, pref- erably with an EC50 for inhibition of 3 nM or less, or 2 nM or less, or 1 nM or less, or 0.9 nM or less, or 0.8 nM or less, or 0.7 nM or less, or 0.6 nM or less, or 0.5 nM or less, or 0.4 nM or less e.g( ., 0.9046 nM or less, 0.5684 or less, or 0.3219 nM or less). In yet other embodiments, the antibody also inhibits SDF-1-induced migration of cells expressing human CXCR4, preferably with an EC50 for inhibition of 50 nM or less, or 30 nM or less, or 20 nM or less, or 15 nM or 50 less (e.g., 18.99 nM or less, or 12.44 or less). In still other embodiments, the antibody also inhibits capillary tube formation by HuVECs, induces apoptosis of cells expressing CXCR4, inhibits tumor cell proliferationin vitro, inhibits tumor cell proliferation or induces tumor cell apoptosis in vivo, inhibits metastases of CXCR4 + tumor cells and/or increases survival time of a CXCR4+ tumor-bearing subject. -7 [0006] Preferably, the antibody binds to human CXCR4 with high affinity, such as with a KD of 1 x 10 M or less or 55 -8 with a KD of 5 x 10 M or less. Preferably, the antibodies of this disclosure are full-length antibodies (i.e., comprising variable and constant regions). Furthermore, the antibodies of this disclosure preferably are raised against full-length human CXCR-4 expressed in its native conformation on a host cell or in an artificial membrane. [0007] In a preferred embodiment, the antibody or antigen-binding antibody portion of the invention is an isolated 2 EP 2 486 941 B1 human monoclonal antibody, or an antigen-binding portion thereof, wherein the antibody: (a) binds to native human CXCR4 expressed on a cell surface; (b) inhibits binding of SDF-1 to human CXCR4; 5 (c) inhibits SDF-1-induced calcium flux in cells expressing human CXCR4; (d) inhibits SDF-1-induced migration of cells expressing human CXCR4; and (e) inhibits capillary tube formation by human umbilical vein endothelial cells.
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