# Patent

## Title
Human antibodies to human RANKL, encoding nucleic acids and methods of treatment

## Abstract

Isolated human antibodies or antigen-binding fragments thereof, which specifically bind to human RANKL and block hRANKL binding to a RANK receptor, are provided. The antibodies are useful in preventing or treating disorders and/or diseases, which are associated with RANKL/RANK interactions, including bone disorders or cancer.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase Application based on the International Application No. PCT/US2009/054262 filed Aug. 19, 2009, which claims priority to U.S. Provisional Application Nos. 61/089,918 filed Aug. 19, 2008 and 61/160,588 filed Mar. 16, 2009, all of which are herein specifically incorporated by reference in their entirety.


FIELD OF THE INVENTION
The present invention is related to human antibodies and antigen-binding fragments of human antibodies that specifically bind human RANKL, and therapeutic methods of using those antibodies and antibody fragments.
SEQUENCE LISTING
An ASCII compliant text file of the sequence listing is filed concurrently herewith pursuant to 37 CFR §1.52(e) and 37 CFR §1.821. The text file containing the sequence listing is named “6080A-WO_SeqList”, was created on Aug. 14, 2009, and contains approximately 427 kilobytes. The contents of the text file are herein incorporated by reference.
STATEMENT OF RELATED ART
Receptor activator of NF-κB (RANK), found on the cell surface of osteoclasts and osteoclast precursors, and its ligand (RANKL) expressed by T cells and bone marrow stromal cells, are both members of the TNF superfamily. RANKL, which may be a membrane-bound or secreted ligand, interacts with RANK and regulates the differentiation, activation, and survival of osteoclasts (human RANKL nucleic acid and protein sequences are shown in SEQ ID NO:850-851, respectively). It has been shown that both RANKL-knockout mice and RANK-knockout mice die with severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and with smaller limbs, skull and vertebrae, due to a lack of osteoclasts.
Osteoprotegerin (OPG), produced by osteoblasts, bone marrow stromal cells and other cells, acts as a natural antagonist of RANKL by binding to RANKL and blocking RANKL-RANK interactions. OPG significantly affects the development of osteoclasts and inhibits bone resorption. OPG-knockout mice exhibited severe osteoporosis due to enhanced osteoclast formation and function (Mizuno et al. 1998, Biochem Biophys Res Commun 237:610-615; and Bucay et al. 1998, Genes Dev 12:1260-1268). Thus, antagonists to RANKL, such as anti-RANKL antibodies that inhibit the binding of RANKL to RANK, can modulate the differentiation and the function of osteoclasts and interfere with pathological conditions, such as osteoporosis and osteopenia. Antibodies that bind to human RANKL polypeptides are disclosed in, for example, U.S. Pat. Nos. 6,740,522; 7,411,050; EP0951551.
SUMMARY OF THE INVENTION
In the first aspect, the invention provides fully human monoclonal antibodies and antigen-binding fragments of antibodies that specifically bind to human RANKL (SEQ ID NO: 851) with high affinities, neutralize the biological activity of RANKL, and blocks the binding of RANKL to its RANK receptor. The antibodies can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, CDR, Fab, F(ab′)2 or scFv fragment), and may be modified to effect functionality, e.g., to eliminate residual effector functions (Glu which eliminates residual effector functions (Reddy et al. 2000, J. Immunol. 164:1925-1933).
In one embodiment, the antibody or antigen-binding fragment of the invention comprises a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO:2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334, 338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434, 450, 454, 458, 474, 478, 482, 498, 502, 506, 522, 526, 530, 546, 550, 554, 570, 574, 578, 594, 598, 602, 618, 622, 626, 642, 646, 650, 666, 670, 674, 690, 694, 698, 714, 718, 722, 738, 742, 746, 762, 766, 770, 786, 790, 794, 810, 814, 818, 834, 838, and 857, or a substantially identical sequence thereof having at least 90%, 95%, 98% or 99% sequence identity. In one embodiment, the HCVR is SEQ ID NO:694 or 738.
In one embodiment, the antibody of the invention comprises a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO:10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260, 264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370, 380, 384, 394, 404, 408, 418, 428, 432, 442, 452, 456, 466, 476, 480, 490, 500, 504, 514, 524, 528, 538, 548, 552, 562, 572, 576, 586, 596, 600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682, 692, 696, 706, 716, 720, 730, 740, 744, 754, 764, 768, 778, 788, 792, 802, 812, 816, 826, 836, 840, 859, and 861, or a substantially identical sequence thereof having at least 90%, 95%, 98% or 99% sequence identity. In one embodiment, the LCVR is SEQ ID NO:696 or 740.
In one embodiment, the antibody of the invention comprises a HCVR and LCVR (HCVR/LCVR) selected from the group consisting of SEQ ID NO:2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740, 742/744, 746/754, 762/764, 766/768, 770/778, 786/788, 790/792, 794/802, 810/812, 814/816, 818/826, 834/836, 838/840, 857/859, and 746/861. In specific embodiments, the HCVR/LCVR is SEQ ID NO:694/696 or 738/740.
In a second aspect, the invention features a human antibody or antigen-binding fragment of an antibody which specifically binds human RANKL and blocks RANKL-RANK interaction, comprising a heavy chain complementarity determining region 3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 is selected from the group consisting of SEQ ID NO:8, 32, 56, 80, 104, 128, 152, 176, 200, 224, 248, 272, 296, 320, 344, 368, 392, 416, 440, 464, 488, 512, 536, 560, 584, 608, 632, 656, 680, 704, 728, 752, 776, 800 and 824, or a substantially identical sequence thereof having at least 90%, 95%, 97%, 98% or 99% sequence identity, and the LCDR3 is selected from the group consisting of SEQ ID NO:16, 40, 64, 88, 112, 136, 160, 184, 208, 232, 256, 280, 304, 328, 352, 376, 400, 424, 448, 472, 496, 520, 544, 568, 592, 616, 640, 664, 688, 712, 736, 760, 808 and 832, or a substantially identical sequence thereof having at least 90%, 95%, 97%, 98% or 99% sequence identity.
In one embodiment, the human antibody or antigen-binding fragment of an antibody further comprises a heavy chain CDR1 (HCDR1) and CDR2 (HCDR2), and light chain CDR1 (LCDR1) and CDR2 (LCDR2), wherein the HCDR1 is selected from the group consisting of SEQ ID NO:4, 28, 52, 76, 100, 124, 148, 172, 196, 220, 244, 268, 292, 316, 340, 364, 388, 412, 436, 460, 484, 508, 532, 556, 580, 604, 628, 652, 676, 700, 724, 748, 772, 796 and 820, or a substantially identical sequence thereof having at least 90%, 95%, 97%, 98% or 99% sequence identity; the HCDR2 is selected from the group consisting of SEQ ID NO:6, 30, 54, 78, 102, 126, 150, 174, 198, 222, 246, 270, 294, 318, 342, 366, 390, 414, 438, 462, 486, 510, 534, 558, 582, 606, 630, 654, 678, 702, 726, 750, 774, 798, and 822, or a substantially identical sequence thereof having at least 90%, 95%, 97%, 98% or 99% sequence identity; the LCDR1 is selected from the group consisting of SEQ ID NO:12, 36, 60, 84, 108, 132, 156, 180, 204, 228, 252, 276, 300, 324, 348, 372, 396, 420, 444, 468, 492, 516, 540, 564, 588, 612, 636, 660, 684, 708, 732, 756, 780, 804 and 828, or a substantially identical sequence thereof having at least 90%, 95%, 97%, 98% or 99% sequence identity; and the LCDR2 is selected from the group consisting of SEQ ID NO:14, 38, 62, 86, 110, 134, 158, 182, 206, 230, 254, 278, 302, 326, 350, 374, 398, 422, 446, 470, 494, 518, 542, 566, 590, 614, 638, 662, 686, 710, 734, 758, 782, 806 and 830, or a substantially identical sequence thereof having at least 90%, 95%, 97%, 98% or 99% sequence identity.
In specific embodiments, the invention provides a human antibody or antigen-binding fragment of an antibody comprising HCDR3 and LCDR3 domains shown in SEQ ID NO:680 and 688 or 728 and 736. In a more specific embodiment, the human antibody or antigen-binding fragment of an antibody comprise heavy and light chain CDR sequences (HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 of SEQ ID NO:676/678/680 and 684/686/688; or 724/726/728 and 732/734/736.
In a specific embodiment, the invention encompasses a human antibody or antigen-binding fragment of an antibody that specifically binds human RANKL (SEQ ID NO:851) with KD of about 30 pM or less, as measured by surface plasmon resonance, wherein the antibody or antibody fragment comprises HCDR3 and LCDR3, wherein the HCDR3 and the LCDR3 comprise amino acid sequences that have at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 680 and 688, respectively. In a further embodiment, the antibody or antibody fragment further comprise HCDR1, HCDR2, LCDR1 and LCDR2, wherein the HCDR1 is SEQ ID NO:676, HCDR2 is SEQ ID NO:678, LCDR1 is SEQ ID NO:684 and LCDR2 is SEQ ID NO:686.
In a specific embodiment, the invention encompasses a human antibody or antigen-binding fragment of an antibody that specifically binds human RANKL with KD of about 30 pM or less, as measured by surface plasmon resonance, wherein the antibody or antibody fragment comprises HCDR3 and LCDR3, wherein the HCDR3 and the LCDR3 comprise amino acid sequences that have at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 728 and 736, respectively. In a further embodiment, the antibody or antibody fragment further comprises HCDR1, HCDR2, LCDR1 and LCDR2, wherein the HCDR1 is SEQ ID NO:724, HCDR2 is SEQ ID NO:726, LCDR1 is SEQ ID NO:732 and LCDR2 is SEQ ID NO:734. In further embodiments, CDR sequences are those within HCVR and LCVR of SEQ ID NO:694/696 or 738/740.
In a specific embodiment, the invention encompasses an antibody or antigen-binding fragment of an antibody that specifically binds human RANKL with KD of about 30 pM or less, as measured by surface plasmon resonance, wherein the antibody or antibody fragment comprises HCVR and LCVR, wherein the HCVR and the LCVR are amino acid sequences comprising at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 694 and 696, or SEQ ID NO: 738 and 740.
In a third aspect, the invention provides nucleic acid molecules encoding the antibodies, or antigen-binding portions, of the invention. Recombinant expression vectors carrying the antibody-encoding nucleic acids of the invention, and host cells into which such vectors have been introduced, are also encompassed by the invention, as are methods of producing the antibodies by culturing the host cells under conditions permitting producing of the antibodies, and recovering the antibodies produced.
In one embodiment, the antibody of the invention comprises a HCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:1, 17, 21, 25, 41, 45, 49, 65, 69, 73, 89, 93, 97, 113, 117, 121, 137, 141, 145, 161, 165, 169, 185, 189, 193, 209, 213, 217, 233, 237, 241, 257, 261, 265, 281, 285, 289, 305, 309, 313, 329, 333, 337, 353, 357, 361, 377, 381, 385, 401, 405, 409, 425, 429 433, 449, 453, 457, 473, 477, 481, 497, 501, 505, 521, 525, 529, 545, 549, 553, 569, 573, 577, 593, 597, 601, 617, 621, 625, 641, 645, 649, 665, 669, 673, 689, 693, 697, 713, 717, 721, 737, 741, 745, 761, 765, 769, 785, 789, 793, 809, 813, 817, 833, 837, and 856, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or 99% homology thereof. In one embodiment, the antibody or fragment thereof comprises a HCVR encoded by a nucleic acid sequence shown in SEQ ID NO:693 or 737.
In one embodiment, the antibody or antigen-binding fragment of an antibody further comprises a LCVR encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:9, 19, 23, 33, 43, 47, 57, 67, 71, 81, 91, 95, 105, 115, 119, 129, 139, 143, 153, 163, 167, 177, 187, 191, 201, 211, 215, 225, 235, 239, 249, 259, 263, 273, 283, 287, 297, 307, 311, 321, 331, 335, 345, 355, 359, 369, 379, 383, 393, 403, 407, 417, 427, 431, 441, 451, 455, 465, 475, 479, 489, 499, 503, 513, 423, 527, 537, 547, 551, 561, 571, 575, 585, 595, 599, 609, 619, 623, 633, 643, 647, 657, 667, 671, 681, 691, 695, 705, 715, 719, 729, 739, 743, 753, 763, 767, 777, 787, 791, 801, 811, 815, 825, 835, 839, 858, and 860, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or 99% homology thereof. In one embodiment, the antibody or fragment thereof comprises a LCVR encoded by a nucleic acid sequence shown in SEQ ID NO:695 or 739. In a more specific embodiment, the antibody or fragment thereof comprises HCVR and LCVR encoded by the nucleic acid sequences shown in SEQ ID NO:693 and 695; or 737 and 739. Also encompassed by the invention are nucleic acid molecules encoding the CDR domains of heavy and light chains shown in amino acid sequences SEQ ID NO:694/696 or 738/740.
In a fourth aspect, the invention features a human antibody or antigen-binding fragment of an antibody comprising a HCDR3 encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NO:7, 31, 55, 79, 103, 127, 151, 175, 199, 223, 247, 271, 295, 319, 343, 367, 391, 415, 439, 463, 487, 511, 535, 559, 583, 607, 631, 655, 679, 703, 727, 751, 775, 799 and 823, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or at least 99% homology thereof; and a LCDR3 encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NO:15, 39, 63, 87, 111, 135, 159, 183, 207, 231, 255, 279, 303, 327, 351, 375, 399, 423, 447, 471, 495, 519, 543, 567, 591, 615, 639, 663, 687, 711, 735, 759, 783, 807 and 831, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or at least 99% homology thereof.
In a further embodiment, the antibody or antigen-binding fragment thereof further comprises a HCDR1, HCDR2, LCDR1 and LCDR2 encoded by a nucleotide sequence, wherein HCDR1 is encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, 27, 51, 75, 99, 123, 147, 171, 195, 219, 243, 267, 291, 315, 339, 363, 387, 411, 435, 459, 483, 507, 531, 555, 579, 603, 627, 651, 675, 699, 723, 747, 771, 795 and 819, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or at least 99% homology thereof; HCDR2 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 29, 53, 77, 101, 125, 149, 173, 197, 221, 245, 269, 293, 317, 341, 365, 389, 413, 437, 461, 485, 509, 533, 557, 581, 605, 629, 653, 677, 701, 725, 749, 773, 797 and 821, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or at least 99% homology thereof; LCDR1 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:11, 35, 59, 83, 107, 131, 155, 179, 203, 227, 251, 275, 299, 323, 347, 371, 395, 419, 443, 467, 491, 515, 539, 563, 587, 611, 635, 659, 683, 707, 731, 755, 779, 803 and 827, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or at least 99% homology thereof; and LCDR2 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:13, 37, 61, 85, 109, 133, 157, 181, 205, 229, 253, 277, 301, 325, 349, 373, 397, 421, 445, 469, 493, 517, 541, 565, 589, 613, 637, 661, 685, 709, 733, 757, 781, 805 and 829, or a substantially similar sequence having at least 90%, 95%, 97%, 98% or at least 99% homology thereof.
In a fifth aspect, the invention features an isolated antibody or antibody fragment that specifically binds and neutralizes human RANKL, comprising (a) HCDR1 comprising an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8 (SEQ ID NO:841), wherein X1 is Gly; X2 is Phe; X3 is Thr; X4 is Phe; X6 is Ser; X6 is Gly; X7 is Tyr or Phe; and X8 is Gly; (b) HCDR2 comprising an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8 (SEQ ID NO:842), wherein X1 is Ile; X2 is Ser; X3 is Tyr; X4 is Asp; X5 is Gly; X6 is Arg, Thr or Ser; X7 is Asn; and X8 is Lys; (c) HCDR3 comprising an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8—X9—X10—X11—X12—X13—X14—X15 (SEQ ID NO:843), wherein X1 is Ala; X2 is Arg; X3 is Cys or Ser; X4 is Leu; X5 is Ser; X6 is Gly; X7 is Thr; X8 is Tyr; X9 is Trp; X10 is Gly; X11 is Tyr; X12 is Gly; X13 is Met; X14 is Asp; and X15 is Val.
In a further embodiment, the isolated antibody or antibody fragment further comprises (d) LCDR1 comprising an amino acid sequence of the formula X1—X2—X3—X4—X5—X6 (SEQ ID NO:844), wherein X1 is Gln; X2 is Ser or Asn; X3 is Ile; X4 is Arg, Gly or Ser; X5 is Ser or Asn; and X6 is Trp; (e) LCDR2 comprising an amino acid sequence of the formula X1—X2—X3 (SEQ ID NO:845), wherein X1 is Lys; X2 is Ala; and X3 is Ser; and (f) LCDR3 comprising an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8—X9 (SEQ ID NO:846), wherein X1 is Gln; X2 is Gln or Arg; X3 is Tyr; X4 is Asn; X5 is Ser or Arg; X6 is Tyr; X7 is Leu or Ser; X8 is Trp; and X9 is Thr.
In a specific embodiment, the invention encompasses a human antibody or antigen-binding fragment of an antibody that specifically binds human RANKL with KD of about 30 pM or less, comprising HCDR3 and LCDR3, wherein HCDR3 comprises an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8—X9—X10—X11—X12—X13—X14—X15 (SEQ ID NO:843), wherein X1 is Ala; X2 is Arg; X3 is Ser; X4 is Leu; X5 is Ser; X6 is Gly; X7 is Thr; X8 is Tyr; X9 is Trp; X10 is Gly; X11 is Tyr; X12 is Gly; X13 is Met; X14 is Asp; and X15 is Val; and LCDR3 comprises an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8—X9 (SEQ ID NO:846), wherein X1 is Gln; X2 is Gln or Arg; X3 is Tyr; X4 is Asn; X5 is Ser; X6 is Tyr; X7 is Leu or Ser; X8 is Trp; and X9 is Thr.
In a more specific embodiment, the antibody or antibody fragment further comprising HCDR1, HCDR2, LCDR1 and LCDR2, wherein HCDR1 comprises an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8 (SEQ ID NO:841), wherein X1 is Gly; X2 is Phe; X3 is Thr; X4 is Phe; X5 is Ser; X6 is Gly; X7 is Tyr or Phe; and Xe is Gly; HCDR2 comprises an amino acid sequence of the formula X1—X2—X3—X4—X5—X6—X7—X8 (SEQ ID NO:842), wherein X1 is Ile; X2 is Ser; X3 is Tyr; X4 is Asp; X5 is Gly; X6 is Arg or Thr; X7 is Asn; and X8 is Lys; LCDR1 comprises an amino acid sequence of the formula X1—X2—X3—X4—X5—X6 (SEQ ID NO:844), wherein X1 is Gln; X2 is Ser or Asn; X3 is Ile; X4 is Arg or Gly; X5 is Ser or Asn; and X6 is Trp; and LCDR2 comprising an amino acid sequence of the formula X1—X2—X3 (SEQ ID NO:845), wherein X1 is Lys; X2 is Ala; and X3 is Ser.
In a sixth aspect, the invention features a fully human antibody or antibody fragment which binds hRANKL with an IC50 of less than about 300 pM, as measured in an in vitro assay or ELISA-based hRANKL blocking assay. In a further embodiment, the antibody of the invention exhibits an IC50 of about 250 pM or less. In more specific embodiments, the antibody of the invention exhibits an IC50 of about 100 pM or less or about 50 pM or less.
In one embodiment, the invention provides an isolated human antibody, or an antigen-binding portion thereof, that binds hRANKL with a KD of less than about 100 pM, preferably less than about 60 pM, even more preferably less than about 50 pM, less than about 30 pM, less than about 20 pM, or less than about 10 pM or less, as determined by surface plasmon resonance (BIACORETM), for example, using hRANKL.
The invention encompasses an expression vector comprising a nucleic acid molecule as defined above. Further, the invention encompasses a method of producing an anti-human RANKL antibody or antigen-binding fragment of an antibody comprising the steps of introducing the expression vector of the invention into an isolated host cell, growing the cell under conditions permitting production of the antibody or fragment thereof, and recovering the antibody or fragment so produced. In specific embodiments, the host cell is an E. coli cell, a CHO cell, or a COS cell.
The invention encompasses anti-hRANKL antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of a galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
The invention includes anti-hRANKL antibodies, which bind specific epitopes of hRANKL and are capable of blocking the biological activity of hRANKL, in particular, its RANK-binding activity. The RANK-binding region of RANKL is located between amino acid residues 125 and 317 of the amino acid sequence represented by SEQ ID NO:851. Thus, the anti-hRANKL antibodies of the invention bind the epitopes located between the residues 125 and 317 of SEQ ID NO:851, thereby blocking the binding of RANKL to RANK.
In a seventh aspect, the invention features a composition comprising a recombinant human anti-RANKL antibody and a pharmaceutically acceptable carrier. Further included in the invention are vectors and host cells comprising vectors which contain nucleic acid molecules encoding the human anti-hRANKL antibody of the invention, as well as methods of producing these novel antibodies, comprising growing a host cell comprising the nucleic acid molecules encoding the anti-RANKL antibody of the invention or an antibody fragment, under conditions permitting production of the protein and recovering the protein so produced.
In an eighth aspect, the invention features methods for inhibiting RANKL activity using an antibody, or antigen-binding portion thereof, of the invention. In one embodiment, the method comprises contacting hRANKL with the instant antibody or an antigen-binding fragment thereof, such that hRANKL is inhibited from binding to RANK. In another embodiment, the method comprises administering the antibody or antibody fragment of the invention, to a human subject suffering from a disorder that is ameliorated by inhibition of RANKL activity. The disorder treated may be a bone disease, such as, osteoporosis, hypercalcemia, bone loss due to arthritis or metastasis, Paget's disease, and osteopetrosis, or cancer, such as a cancer of the breast, colon, liver, prostrate, lung, pancreas, bone, etc.
The invention encompasses an antibody or antigen-binding fragment of an antibody as described above for use to attenuate or inhibit a RANKL-mediated disease or condition in a human. The RANKL-mediated disease or condition is a bone disorder or cancer.
The invention encompasses use of an antibody or antigen-binding fragment of an antibody as described above in the manufacture of a medicament for use to attenuate or inhibit a RANKL-mediated disease or condition in a human. The RANKL-mediated disease or condition is osteoporosis, hypercalcemia, bone loss due to arthritis or metastasis, Paget's disease, osteopetrosis, breast cancer, colorectal cancer, lung cancer, prostrate cancer, liver cancer, pancreatic cancer, or bone cancer.
Further encompassed by the invention is a method of treating a disease, disorder or condition which is ameliorated or inhibited by inhibition of RANKL, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment of an antibody as described above to a human subject in need thereof, wherein the disease, disorder or condition is ameliorated or inhibited. In specific embodiments, the disease is a bone disease or cancer.
Other objects and advantages will become apparent from a review of the ensuing detailed description.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Sequence comparison table of heavy chain variable regions and CDRs of antibodies H4H008G, H4H009P and H4H010P.
FIG. 2. Sequence comparison table of light chain variable regions and CDRs of antibodies H4H008G, H4H009P and H4H010P.



DETAILED DESCRIPTION
It is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Definitions
“Receptor activator of NF-κB ligand”, “human RANKL” and “hRANKL” are used interchangeably to refer to the human protein encoded by the nucleic acid sequence of SEQ ID NO:850 and the protein having the amino acid sequence of SEQ ID NO:851.
The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The term “high affinity” antibody refers to those antibodies having a binding affinity to hRANKL of at least 10−9 M; preferably 10−19M; even more preferably 10−11 M, as measured by surface plasmon resonance, e.g., BIACORETM or solution-affinity ELISA.
By the term “slow off rate” or “Koff” is meant an antibody that dissociates from hRANKL with a rate constant of 1×10−3 s−1 or less, preferably 1×104s−1 or less, as determined by surface plasmon resonance, e.g., BIACORETM.
The term “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hRANKL). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” or “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. 1989, Nature 241:544-546), which consists of a VH domain; and (vi) an isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988, Science 242:423-426; and Huston et al., 1988, PNAS 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” or “antigen-binding fragment” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (e.g., Holliger et al. 1993, PNAS 90:6444-6448; Poljak et al. 1994, Structure 2:1121-1123).
Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al. 1995, Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al., 1994, Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al., 1992, Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hRANKL is substantially free of antibodies that specifically bind antigens other than hRANKL). An isolated antibody that specifically binds hRANKL may, however, have cross-reactivity to other antigens, such as hRANKL molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
A “neutralizing antibody”, as used herein (or an “antibody that neutralizes hRANKL activity”), is intended to refer to an antibody whose binding to hRANKL results in inhibition of the biological activity of hRANKL. This inhibition of the biological activity of hRANKL can be assessed by measuring one or more indicators of hRANKL biological activity by one or more of several standard in vitro or in vivo assays known in the art (see examples below).
The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORETM system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
The term “KD”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
The term “epitope” includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by a specific antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In preferred embodiments, an antibody is said to specifically bind an antigen when the equilibrium dissociation constant is less than or equal to 10−8 M, more preferably when the equilibrium dissociation constant is less than or equal to 10−9 M, and most preferably when the dissociation constant is less than or equal to 10−19 M.
The term “substantial similarity”, or “substantial sequence similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, preferably at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
As applied to polypeptides, the term “substantial identity” or “substantially identical” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80% sequence identity, preferably at least 90% or 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., 1992, Science 256: 1443-45. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
Sequence similarity or sequence identity for polypeptides, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences can be also compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 2000, supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al., 1990, J Mol. Bio. 215: 403-410 and Altschul et al., 1997, Nucleic Acids Res 25:3389-402.
Preparation of Human Antibodies
Methods for generating human antibodies are known. Antibodies may be therapeutically useful in blocking a ligand-receptor interaction or inhibiting receptor component interaction, rather than by killing cells through fixation of complement and participation in complement-dependent cytotoxicity (CDC), or killing cells through antibody-dependent cell-mediated cytotoxicity (ADCC). The constant region of an antibody is thus important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.
Human immunoglobulins can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.
The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al., 1993 Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.
Antibodies of the invention are preferably prepared with the use of VELOCIMMUNETM technology. In general, the antibodies of the instant invention possess very high affinities, typically possessing KD of from about 10−9 through about 10−12 M or higher, for example, at least about 10−9M, at least 10−10 M, at least 10−11 M or at least 10−12 M, when measured by binding to antigen either immobilized on solid phase or in solution phase.
Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As described below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4 (for example, SEQ ID NO: 847, 848 or 849). While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
Epitope Mapping and Related Technologies
To screen for antibodies which bind to a particular epitope (e.g., those which block binding of IgE to its high affinity receptor), a routine cross-blocking assay such as that described in Harlow and Lane 1990, supra can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke 2004, Methods Mol Biol 248:443-63), or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer 2000 Protein Science 9: 487-496).
The term “epitope” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, at least 6, or 8-10 amino acids in a unique spatial conformation.
Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (U.S. patent Publication No. 2004/0101920). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the hRANKL antibodies of the invention into groups of antibodies binding different epitopes.
Immunoconjugates
The invention encompasses a human anti-hRANKL monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxin agents include any agent that is detrimental to cells. Examples of suitable cytotoxin agents and chemotherapeutic agents for forming immunoconjugates are known in the art, see for example, WO 05/103081.
Bispecifics and Multispecifics
The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al. 1991 J. Immunol. 147:60-69. The human anti-hRANKL antibodies of the invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity.
Bioequivalents
The anti-RANKL antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind human RANKL. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the anti-RANKL antibody-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an anti-RANKL antibody or antibody fragment that is essentially bioequivalent to an anti-RANKL antibody or antibody fragment of the invention. Examples of such variant amino acid and DNA sequences are discussed above.
Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.
Bioequivalent variants of anti-RANKL antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
Therapeutic Administration and Formulations
The invention provides therapeutic compositions comprising the anti-hRANKL antibodies or antigen-binding fragments thereof of the present invention. The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA 1998 J Pharm Sci Technol 52:238-311.
The dose may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When the antibody of the present invention is used for treating various conditions and diseases associated with hRANKL, including bone diseases such as osteoporosis, hypercalcemia, bone loss due to arthritis or metastasis, Paget's disease, osteropetrosis, and the like, in an adult patient, it is advantageous to intravenously administer the antibody of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted.
Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer 1990 Science 249:1527-1533; Treat et al. 1989 in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.
In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton 1987 CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984). Other controlled release systems are discussed in the review by Langer 1990 Science 249:1527-1533.
The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to 100 mg and in about 10 to 250 mg for the other dosage forms.
A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
Numerous reusable pen delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENTTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly).
Single and combination therapies. The invention provides therapeutic methods in which the antibody or antibody fragment of the invention is useful to treat a variety of conditions involving hRANKL. The anti-hRANKL antibodies or antibody fragments of the invention are particularly useful for the treatment of bone diseases such as osteoporosis, hypercalcemia, bone loss due to arthritis or metastasis, Paget's disease, osteropetrosis, and the like. The anti-hRANKL antibodies and fragments of the invention are useful for treating a variety of cancers, such as metastatic, breast, bone, colorectal, pancreatic, liver and prostrate cancers. Combination therapies may include the anti-RANKL antibody of the invention with, for example, one or more of a bis-phosphonate or a RANK antagonist, or a chemotherapeutic agent.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1
Generation of Human Antibodies to Human RANKL
Mice may be immunized by any method known in the art (see, for example, Harlow and Lane, supra). The antibody immune response is monitored by standard antigen-specific immunoassay. When a desired immune response is achieved, antibody-expressing B cells were harvested and fused with mouse myeloma cells to preserve their viability, forming hybridoma cell lines. Such hybridoma cell lines are screened and selected to identify cell lines that produce antigen-specific antibodies using assays as described below.
Alternatively, antigen-specific hybridoma cells may be isolated by flow cytometry. Briefly, after fusion to myeloma cells, pooled hybridoma cells are grown for 10 days in HAT medium. The cells are harvested and stained with biotin-labeled hRANKL at 2 μg/ml for one hour, followed by addition of phycoerythrin-streptavidin. The fluorescence-labeled cells are sorted by flow cytometry (one cell per well into 96 well plates containing hybridoma growth medium), cultured for 8-10 days, and conditioned media is screened for the presence of functionally desirable monoclonal antibodies, as described below.
Anti-hRANKL mAbs may be derived via direct isolation of splenocytes. Antigen-specific antibodies may also be isolated directly from antigen-immunized B cells without fusion to myeloma cells, as described in U.S. Patent Publication 2007/0280945A1. Stable recombinant antibody-expressing CHO cell lines are established from isolated selected recombinants.
Example 2
Gene Utilization Analysis
To analyze the structure of antibodies produced in accordance with the invention, the nucleic acids encoding antibody variable regions were cloned and sequenced. The predicted amino acid sequences of the antibodies were confirmed by N-terminal amino acid sequencing. From the nucleic acid sequence and predicted amino acid sequence of the antibodies, gene usage was identified for each antibody chain. For example, antibody 2H10 (HCVR/LCVR SEQ ID NO:722/730) and antibody 2G10 (SEQ ID NO: 674/682): HCVR: 3-30 (VH), 1-26 (D), 6 (JH); LCVR: 1-5 (VK), 1 (JK).
Example 3
Antigen Binding Affinity Determination
Equilibrium dissociation constants (KD values) for antigen binding to the selected antibodies generated by hybridoma cell lines described above were determined by surface kinetics in a real-time biosensor surface plasmon resonance assay (BIACORETM 2000). Each selected antibody was captured on a goat anti-mouse IgG polyclonal antibody surface created through direct chemical coupling to a BIACORETM chip to form a captured antibody surface. Human RANKL at 25 nM was injected over the captured antibody surfaces, and antigen-antibody binding and dissociation was monitored in real time for 1.5 hours at room temperature.










TABLE 1







Antibody
KD (pM)



















7B10-1
455



1F2-6
2030



3B11-7
88.5



5A6-1
519



1G5-8
699



2A12-1
140



6A10-1
491



2D3-3
383



1G9-9
92.4



5C11-6
100



5E3-3
236



6B6-4
27.7



7G12-7
373



6A8-2
107



3G10-3
275



4D2-3
346



7G11-4
239



2E3-29
76.4



2F8-9
65










Equilibrium dissociation constants (KD values) for antigen binding to the selected anti-hRANKL mAbs generated via direct isolation of splenocytes described above were also determined by surface kinetics in a real-time biosensor surface plasmon resonance assay (BIACORETM 2000) described above. Each selected antibody was captured on a goat anti-hFc polyclonal antibody (Jackson Immuno Research Lab) surface created through direct chemical coupling to a BIACORETM chip to form a captured antibody surface (Table 2).










TABLE 2







Antibody
KD (pM)



















1G8
8.42



2H10
15.8



3D4
26



2E11
16



2H12
33.1



1E12
36.9



1F8
7.85



2A3
94.3



2E12
95.7



2G10
28



3A6
200



1F12
40.7



2G8
96.8



1B5
59.8



2E10
17.2



1G12
24.6










Antigen binding affinities of selected purified antibodies for hRANKL at either 25° C. or 37° C. were also determined by surface kinetics employing a real-time biosensor surface plasmon resonance assay (BIACORETM). Antibodies 1E12, 1F8, 1G8, 2A3, 2E10, 2E11, 2E12, 2G10, 2H10, and 2H12 were redesignated as H4H001N, H4H002N, H4H003N, H4H004N, H4H005N, H4H006N, H4H007N, H4H008N, H4H009N, and H4H010N, respectively (Table 3: Control: Human anti-hRANKL mAb, SEQ ID NO: 2 and 4 of U.S. Pat. No. 7,364,736). Sequence identifier numbers correspond to HCVR/LCVR.








TABLE 3







Antibody
Purified Antibody














Clone ID
SEQ ID NO
Name
SEQ ID NO
Name
SEQ ID NO
Name
SEQ ID NO





1E12
482/490
H4H001N
482/490
H4H001P
498/500
H4H001G
502/504


1F8
530/538
H4H002N
530/538
H4H002P
546/548
H4H002G
550/552


1G8
578/586
H4H003N
578/586
H4H003P
594/596
H4H003G
598/600


2A3
602/610
H4H004N
602/610
H4H004P
618/620
H4H004G
622/624


2E10
818/826
H4H005N
818/826
H4H005P
834/836
H4H005G
838/840


2E11
626/634
H4H006N
626/634
H4H006P
642/644
H4H006G
646/648


2E12
650/658
H4H007N
650/658
H4H007P
666/668
H4H007G
670/672


2G10
674/682
H4H008N
674/682
H4H008P
690/692
H4H008G
694/696


2H10
722/730
H4H009N
722/730
H4H009P
738/740
H4H009G
742/744


2H12
746/754
H4H010N
746/754
H4H010P
762/764
H4H010G
766/768




















TABLE 4







Antibody
KD (pM) 25° C.
KD (pM) 37° C.




















Control
12.2
49.2



H4H001P
186
41



H4H001G
228
49.5



H4H005P
601
45.3



H4H005G
76.2
24.1



H4H006P
172
44



H4H006G
265
41.5



H4H008P
162
63.1



H4H008G
7.0
163



H4H009P
173
24.8



H4H009G
198
42.4



H4H010P
69.7
77.9



H4H010G
146
7.45



H4H002P
262
53.1



H4H003P
251
17



H4H004P
199
14.9



H4H007P
335
57.8










In addition, antigen binding affinities of selected purified antibodies for hRANKL at either 25° C. or 37° C. were also determined by surface kinetics employing an alternative real-time biosensor surface plasmon resonance assay (BIACORETM). Briefly, hRANKL was captured at 10 μl/min for 5 min on an anti-penta-his antibody surface created through direct chemical coupling to a BIACORETM chip to form a captured hRANKL surface. Three different concentrations (25 nM, 50 nM, and 100 nM) of each selected anti-hRANKL mAb were injected over the captured hRANKL surface at 10 μl/min for 2.5 min at either 25° C. or 37° C. Binding of antigen to antibody and dissociation of the bound complex were monitored in real time for 1.5 hour (Table 5, KD expressed in pM, T12 expressed in hrs).











TABLE 5





Antibody
KD @ 25° C.
T1/2 @ 25° C.
KD @ 37° C.
T1/2 @ 37° C.



















Control
59.5
11
118
4


H4H008G
11.9
6
39.9
1


H4H009P
376
1
881
0.13









Example 4
Inhibition of hRANKL or mfRANKL and hRANK Interaction
Selected antibodies were tested for the ability to block human RANK (hRANK) binding to human RANKL (hRANKL, SEQ ID NO:852) or monkey (Macaca fascicularis) RANKL (mfRANKL, SEQ ID NO:854). Briefly, hRANK-hFc protein was coated on a 96-well plate in PBS buffer overnight at 4° C. at 2 μg/ml, and nonspecific binding was blocked with BSA, to form a hRANK-hFc surface. The hRANK-hFc surface was used to measure binding of either penta-his-tagged hRANKL (his-hRANKL) or penta-his-tagged mfRANKL (his-mfRANKL) pre-equilibrated with varying concentrations of anti-hRANKL mAbs. Briefly, a constant amount of either his-hRANKL (150 pM) or his-mfRANKL (150 pM) was pre-mixed with varied amounts of a selected antibody, ranging from 0 to about 100 nM in serial dilutions, followed by a 1 hr incubation at room temperature to allow antibody-antigen binding to reach equilibrium. The equilibrated sample solutions were then transferred to the RANK-hFc coated plates for measurement of free his-hRANKL. After 1 hr binding, the plate was washed, bound his-hRANKL or his-mfRANKL was detected using a mouse anti-penta-his antibody and subsequently detected by HRP-conjugated anti-mFc antibodies. IC50 values (pM) were determined (Table 6).











TABLE 6







Antibody
150 pM his-hRANKL
150 pM his-mfRANKL




















Control
95
165



H4H001G
140
825



H4H005G
3135
9585



H4H008G
20
160



H4H009G
65
875



H4H010G
55
175



H4H001P
105
710



H4H002P
165
1360



H4H003P
85
900



H4H004P
155
1270



H4H005P
1160
4320



H4H006P
85
420



H4H007P
335
2760



H4H008P
115
6510



H4H009P
60
345



H4H010P
45
150



H4H006G
195
1235










Example 5
Sequential hRANKL Binding Competition
The ability of selected anti-hRANKL mAbs to bind control antibody-bound hRANKL was measured on a BIACORETM 1000. Briefly, human RANKL-mFc was first captured on a goat-anti-mouse IgG surface at about 350 RU. Control antibody (first antibody) at 50 μg/ml was then allowed to bind to the hRANKL-mFc surface, and the extent of binding of the control antibody was measured. Each control antibody-bound surface was then incubated with one of the selected anti-hRANKL mAbs (second antibody) at 50 μg/ml for 4 min at 25° C. The amount of second antibody bound was measured as a change in RU. Among the antibodies tested, only 7B10-1 displayed significant binding to the control antibody-bound surface.
Example 6
Neutralization of Biological Effect of hRANKL In Vitro
The ability of selected anti-hRANKL mAbs to neutralize hRANKL-mediated cellular function was measured in vitro using an engineered HK293 cell line that contains RANK/NFκB-luc-eGFP. Inhibition of hRANKL-inducible luciferase activity by selected anti-hRANKL mAbs was measured in the engineered HK293 cells. Briefly, cells were seeded onto 96-well plates at 2×104 cells/well in media and incubated overnight at 37° C., 5% CO2. Antibody protein (in serial dilutions starting from 25 nM in cell media) was added to the wells containing HK293/RANK/NFκB-luc-eGFP cells, in triplicate, followed by addition of hRANKL (final concentration of 33.3 pM hRANKL native trimer) to each well. The plates were incubated at 37° C., 5% CO2 for 6 hours. Luciferase activity was determined by adding BRIGHT-GLOTM Substrate (Promega). Data are expressed as IC50 values (Table 7; NB: no blocking was observed in the presence of 25 nM of antibody).










TABLE 7







Antibody
IC50 (pM)









1F2
NB



1G5
1000



1G9
420



2A12
440



2D3
300



2D3-1
433



2F8
160



2F8-1
223



3B11
2100



3G10
1100



4D2
350



5A6
1200



5C11
330



5C11-1
1067



5E3
870



6B6
400



6B6-1
952



7B10
NB



7G12
0.570



Control
34



8D2
516



8D2-1
1321



6F6
885



10C1
245



10C1-1
905



10F4
332










The ability of selected purified antibodies to neutralize hRANKL-mediated cellular function in vitro was also determined as described above (Table 8).











TABLE 8







Antibody
IC50 (pM)
N




















Control
34
11



Control
23
1



H4H001P
106
9



H4H002P
214
5



H4H003P
181
8



H4H004P
143
6



H4H005P
1168
5



H4H006P
80
5



H4H007P
452
5



H4H008P
118
6



H4H009P
45
10



H4H009P
84
1



H4H010P
51
5



H4H001G
111
5



H4H005G
1058
4



H4H006G
171
3



H4H008G
20
5



H4H008G-1
4
1



H4H009G
102
4



H4H010G
53
4










Example 7
Cross-Reactivity of Anti-hRANKL mAbs with Mouse RANKL
Cross-reactivity of the selected antibodies with mouse RANKL (mRANKL) protein (SEQ ID NO:855) was determined. Each antibody (1-20 μg/ml) was first captured on either a goat anti-mouse IgG polyclonal antibody surface (for antibodies generated from hybridomas) or goat-anti-human IgG polyclonal antibody surface (for antibodies derived via direct isolation of splenocytes) created through direct chemical coupling to a BIACORETM chip. Mouse RANKL at 50-100 nM was injected over the antibody surfaces. Only antibody 1F2-6 bound to mRANKL weakly. None of the remaining antibodies showed any cross-reactivity with mRANKL.
Example 8
Neutralization of Biological Effect of hRANKL In Vivo
To assess the biological effect of anti-hRANKL mAbs, mice expressing hRANKL but not mRANKL (RANKLhu/hu mice) were generated. The efficacy of selected anti-hRANKL mAbs for inhibiting hRANKL was determined in ovariectomized (OVX) female RANKLhu/hu mice. Briefly, 28 OVX female RANKLhu/hu mice, approximately 5 to 6.5 months old, were divided evenly into 4 groups. In addition, a group of 7 sham-operated RANKLhu/hu mice, approximately 5 to 6.5 months old, was also used as a sham-vehicle control. Ovariectomies were performed on day—14. On day 0, a selected mAb (Control, H4H008G, or H4H009P) was injected at 10 mg/kg/dose subcutaneously (sc) weekly for 3 weeks. Dual energy X-ray absorptiometry (DEXA) was performed pre-OVX, at day 0, and weekly after the treatment. Bone mineral densities (BMDs) of whole body (Table 9), lumbar (Table 10), and femur (Table 11) were determined and normalized to pre-OVX baseline. In addition, bone mineral content (BMC) of whole body (Table 12), lumbar (Table 13), and femur (Table 14) were also determined (percent over baseline±SEM).












TABLE 9





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
100.00 ± 0.00
100.00 ± 0.00 
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00


8
100.64 ± 0.83
98.99 ± 0.93
104.40 ± 1.31
102.07 ± 1.53
104.47 ± 0.56


15
100.27 ± 0.81
97.13 ± 1.13
106.93 ± 1.52
105.51 ± 1.14
105.51 ± 1.05


22
 98.41 ± 0.68
95.43 ± 1.08
107.24 ± 1.22
106.37 ± 1.96
106.46 ± 0.99





















TABLE 10





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
100.00 ± 0.00 
100.00 ± 0.00 
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00


8
98.56 ± 2.66
92.30 ± 1.61
107.91 ± 1.85
104.67 ± 1.56
107.69 ± 1.93


15
98.80 ± 2.34
89.94 ± 1.70
106.27 ± 2.91
108.33 ± 2.45
109.50 ± 2.16


22
94.00 ± 0.90
88.76 ± 2.03
115.53 ± 2.20
109.33 ± 2.18
110.41 ± 3.18





















TABLE 11





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
100.00 ± 0.00
100.00 ± 0.00 
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00


8
104.77 ± 1.79
101.34 ± 2.52 
104.10 ± 2.04
103.70 ± 1.40
103.61 ± 1.25


15
100.54 ± 1.39
98.41 ± 2.85
103.10 ± 2.57
105.96 ± 2.56
103.91 ± 1.19


22
100.31 ± 1.78
96.74 ± 2.80
105.64 ± 2.18
105.64 ± 2.23
106.99 ± 1.45





















TABLE 12





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
100.00 ± 0.00 
100.00 ± 0.00 
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00


8
98.86 ± 2.25
94.46 ± 1.01
104.76 ± 1.16
104.47 ± 1.36
104.51 ± 2.51


15
96.99 ± 1.31
94.14 ± 1.01
104.10 ± 1.39
104.84 ± 1.44
107.31 ± 1.82


22
98.80 ± 1.22
91.23 ± 0.82
109.10 ± 1.87
108.76 ± 2.34
107.77 ± 2.60





















TABLE 13





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
100.00 ± 0.00 
100.00 ± 0.00 
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00


8
98.81 ± 2.81
88.19 ± 1.97
108.63 ± 1.91
105.80 ± 2.58
109.07 ± 3.28


15
103.16 ± 3.07 
91.41 ± 2.61
103.93 ± 3.80
107.64 ± 2.89
109.10 ± 3.45


22
96.56 ± 0.95
85.76 ± 2.26
115.16 ± 3.29
112.54 ± 3.91
112.50 ± 4.16





















TABLE 14





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
100.00 ± 0.00
100.00 ± 0.00 
100.00 ± 0.00
100.00 ± 0.00
100.00 ± 0.00


8
104.80 ± 4.07
99.23 ± 1.37
106.43 ± 2.63
105.27 ± 1.39
103.69 ± 2.21


15
104.13 ± 1.69
101.77 ± 2.42 
111.94 ± 2.30
111.00 ± 1.56
108.54 ± 2.94


22
104.31 ± 2.51
98.97 ± 2.49
111.43 ± 2.26
112.56 ± 1.79
112.66 ± 1.70









Serum samples were obtained through retro-orbital sinus bleeding pre-OVX, and on days 0, 1, 8, 15, and 22. Serum osteocalcin, a bone formation marker, was measured using Mouse Osteocalcin EIA Kit (Cat# BT-470, BTI, Stoughton, Mass.) (Table 15). Serum tartrate-resistant acid phosphatase 5b (TRAP 5b), a bone resorption marker, was measured using a MouseTRAPTM Assay kit (TRACP 5b mouse) (Cat# SB-TR103, IDS Inc, AZ) (Table 16: percent change from baseline±SEM).












TABLE 15





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
0.00 ± 0.00
  0.00 ± 0.00
 0.00 ± 0.00
 0.00 ± 0.00
  0.00 ± 0.00


1
−4.67 ± 3.66  
−0.20 ± 4.75
 1.18 ± 2.85
 −8.26 ± 6.17
−8.98 ± 3.91


8
−7.92 ± 3.65  
0.172 ± 5.29
−16.92 ± 3.01
−25.24 ± 5.36
−29.24 ± 4.64 


15
−7.72 ± 4.09  
−7.77 ± 3.74
−26.14 ± 4.17
−33.45 ± 5.79
−20.95 ± 11.94


22
0.03 ± 9.57
−9.73 ± 4.61
−24.43 ± 5.21
−31.91 ± 7.32
−26.53 ± 14.45





















TABLE 16





Days
Sham-Veh
OVX-Veh
Control
H4H008G
H4H009P




















0
 0.00 ± 0.00
 0.00 ± 0.00
 0.00 ± 0.00
 0.00 ± 0.00
  0.00 ± 0.00


1
−19.84 ± 9.21
 0.50 ± 6.85
−52.05 ± 3.22
−63.92 ± 5.06
−52.98 ± 12.37


8
 −6.16 ± 8.02
−10.08 ± 4.82
−96.23 ± 1.18
−95.82 ± 1.09
−88.87 ± 7.40 


15
−15.40 ± 5.46
−26.84 ± 2.10
−96.49 ± 1.17
−99.21 ± 0.36
−62.51 ± 19.67


22
 1.49 ± 9.89
 −5.73 ± 8.99
−88.10 ± 8.42
−78.23 ± 6.37
 −4.86 ± 24.48









The efficacy of selected anti-hRANKL mAbs for inhibiting hRANKL was also determined in intact male RANKLhu/hu mice. Effects of anti-mAbs on BMD, BMC, serum osteocalcin, and TRAP 5b are comparable to those observed in OVX female RANKLhu/hu mice.
Example 9
Pharmacokinetics of Anti-hRANKL mAbs in Wild-Type and RANKLhu/hu Mice
A total of 36 mice, approximately 16 to 21 weeks old, were divided equally into 6 groups (n=6). Three of the groups were wild-type (WT) mice and the other three groups were RANKLhu/hu mice. A single dose of anti-hRANKL mAb (Control, H4H008G, H4H009P) was given subcutaneously at 10 mg/kg. Blood samples were taken at 0 hr, 6 hr, day 1, day 3, day 6, day 10, day 14, day 21, and day 28. Anti-hRANKL mAb was detected using HRP-conjugated anti-hFc antibody. Concentrations of anti-hRANKL mAbs in WT and RANKLhu/hu mice were determined (Table 17). Results are expressed as mean concentration (μg/ml)±SD.










TABLE 17









Wild-Type (WT)
RANKLhu/hu













Days
Control
H4H008G
H4H009P
Control
H4H008G
H4H009P
















0
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00


0.25
19.27 ± 9.70 
18.42 ± 10.12
31.49 ± 17.86
24.78 ± 4.06 
22.81 ± 5.62 
24.84 ± 7.90 


1
63.85 ± 8.87 
55.47 ± 13.03
85.52 ± 22.76
79.40 ± 7.34 
58.15 ± 9.71 
84.17 ± 12.62


3
69.05 ± 11.89
50.05 ± 10.05
74.06 ± 20.21
72.37 ± 10.18
46.67 ± 8.42 
70.00 ± 7.27 


6
73.71 ± 10.61
60.06 ± 2.47 
81.15 ± 26.55
72.32 ± 7.23 
51.20 ± 8.73 
76.50 ± 9.29 


10
64.79 ± 12.11
38.35 ± 13.83
54.08 ± 21.06
63.23 ± 11.61
15.86 ± 12.51
27.96 ± 23.68


14
49.38 ± 11.13
18.50 ± 10.22
33.12 ± 20.84
42.37 ± 12.80
4.23 ± 6.72
4.90 ± 8.10


21
36.77 ± 5.35 
5.11 ± 7.63
21.56 ± 23.35
25.61 ± 10.08
0.30 ± 0.65
0.05 ± 0.08


28
33.74 ± 7.76 
3.54 ± 6.53
18.55 ± 20.36
17.81 ± 10.59
0.05 ± 0.08
0.02 ± 0.00









Example 10
Anti-hRANKL Antibody Variants
Anti-hRANKL antibody variants were made that exhibit either increased production of the antibody or that exhibit reduced glycosylation. H4H008P2 (SEQ ID NO:857 and 859 for HCVR and LCVR, respectively) is an H4H008G variant in which the HCVR and LCVR frameworks of H4H008G were replaced with an HCVR and a mutated LCVR (A50V and S59Y) frameworks of a high-expressing, non-hRANKL-specific fully human antibody, respectively. H4H010P2 (SEQ ID NO:746 and 861 for HCVR and LCVR, respectively) is an H4H010P variant containing a single point mutation of N28S on the LCVR.
Antigen binding affinities of the antibody variants for hRANKL at either 25° C. or 37° C. were also determined by surface kinetics employing a real-time biosensor surface plasmon resonance assay (Table 19). Briefly, human trRANKL-his (R&D) was first captured over an anti-his surface. Anti-hRANKL antibodies at various concentrations ranging from 12.5 nM to 50 nM was injected over the captured RANKL surfaces at either 25° C. or 37° C. The antigen and antibody binding and dissociation were monitored for 1 hr. KD is expressed in pM, T1/2 in hrs.











TABLE 19






KD





Antibody
@ 25° C.
T1/2 @ 25° C.
KD @ 37° C.
T1/2 @ 37° C.



















Control
57.2
8
227
2


H4H008G
11.9
6
39.5
1


H4H008P2
25.4
2
245
0.2


H4H010P2
134
1
848
0.15









Antibody variants were also tested for their ability to inhibit the interaction between hRANKL or mfRANKL to hRANK as described above (IC50 (pM), Table 20).











TABLE 20








150 pM




Antibody
his-hRANKL
150 pM his-mfRANKL




















H4H008G
90.0
290



H4H008P2
56.9
1800



H4H010P2
51.9
353










The ability of antibody variants to neutralize hRANKL-mediated cellular function was measured in vitro using an engineered HK293 cell line that contains RANK/NFκB-luc-eGFP as described above (Table 20).











TABLE 21







Antibody
IC50 (pM)
n




















H4H008G
10.8
3



H4H008P2
15.0
3



H4H010P2
18.7
3

## Claims

1. A human antibody or antigen-binding fragment of a human antibody that binds to human receptor activator of NF—KB ligand (RANKL), and comprises a heavy chain variable domain and light chain variable domain combination selected from the group consisting of SEQ ID NOs: 594/596, 618/620, 838/840, 694/696, 738/740, and 766/768.

2. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to claim 1, and a pharmaceutically acceptable carrier.

3. A nucleic acid molecule encoding the human antibody or antigen-binding fragment according to claim 1.

4. An expression vector comprising the nucleic acid molecule according to claim 3.

5. A method of producing an anti-human RANKL antibody or antigen-binding fragment thereof, comprising the steps of introducing the expression vector of claim 4 into an isolated host cell, growing the cell under conditions permitting production of the antibody or fragment thereof, and recovering the antibody or fragment so produced.

6. The method according to claim 5, wherein the host cell is an E. coli cell, a CHO cell, or a COS cell.

7. A method of treating a disease, disorder or condition which is ameliorated or inhibited by inhibition of osteoclast differentiation, survival, or activity, by inhibition of RANKL, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment according to claim 1 to a human subject in need thereof, wherein the disease, disorder or condition is ameliorated or inhibited.

8. The method of claim 7, wherein the disease is a bone disease or cancer.

9. A human antibody or antigen-binding fragment of a human antibody that binds to human receptor activator of NF—KB ligand (RANKL), and comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 724, SEQ ID NO: 726, SEQ ID NO: 728, SEQ ID NO: 732, SEQ ID NO: 734, and SEQ ID NO: 736, respectively.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to claim 9, and a pharmaceutically acceptable carrier.

11. A nucleic acid molecule encoding the human antibody or antigen-binding fragment according to claim 9.

12. An expression vector comprising the nucleic acid molecule according to claim 11.

13. A method of producing an anti-human RANKL antibody or antigen-binding fragment thereof, comprising the steps of introducing the expression vector of claim 12 into an isolated host cell, growing the cell under conditions permitting production of the antibody or fragment thereof, and recovering the antibody or fragment so produced.

14. The method according to claim 13, wherein the host cell is an E. coli cell, a CHO cell, or a COS cell.

15. A method of treating a disease, disorder or condition which is ameliorated or inhibited by inhibition of osteoclast differentiation, survival, or activity, by inhibition of RANKL, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment according to claim 9, to a human subject in need thereof, wherein the disease, disorder or condition is ameliorated or inhibited.

16. The method of claim 15, wherein the disease is a bone disease or cancer.