Patent Application
20240327502
The present invention relates to antibodies or antigen binding fragments thereof that specifically bind and/or neutralize human metapneumovirus. Another aspect of the invention relates to compositions and kits comprising the antibodies or antigen binding fragments thereof. Another aspect of the invention relates to methods for treating diseases, for example respiratory infections, by administering the antibodies or antigen binding fragments thereof. Another aspect of the invention relates to antigenic human metapneumovirus F proteins. Another aspect of the invention relates to methods for preventing or treating diseases, for example respiratory infections, by administering antigenic human metapneumovirus F proteins.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 27, 2022, is named 25170-WO-PCT_SL.txt and is 495,474 bytes in size.
Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). It is a major pathogen causing acute lower respiratory infection in young children, the elderly and immunocompromised adults. HMPV encodes two major surface glycoproteins, the fusion (F) and glycoprotein (G) proteins. The surface F glycoprotein, which is matured after a proteolytic cleavage process, mediates viral fusion by structure rearrangements and is the primary target of neutralizing antibodies against hMPV. Current understanding of the specificity and functionality of hMPV-F antibody responses induced by natural infection remains elusive. Therapeutic treatments to treat diseases associated with hMPV are needed.
The present disclosure provides antibodies and antigen binding fragments thereof capable of bindings to and/or neutralizing metapneumovirus (MPV), and methods, pharmaceutical compositions, uses and kits of binding to and/or neutralizing an MPV using an antibody or antigen binding fragment thereof. In various embodiments, the MPV is mammalian. For example, the mammalian MPV comprises a human MPV (hMPV). In various embodiments, the hMPV comprises a surface glycoprotein. For example, the glycoprotein comprises a F glycoprotein and/or a G glycoprotein.
The present disclosure provides an antibody or antigen binding fragment thereof which specifically binds to hMPV comprising at least one CDR, three CDRs or six CDRs described in Tables 1-3. The present disclosure provides an antibody or antigen binding fragment thereof which specifically binds to hMPV comprising at least one variable region/domain described in Tables 1-3. For example, the antibody or antigen binding fragment thereof which specifically binds to hMPV comprises a heavy chain variable region, light chain variable region, or heavy chain variable region and light chain variable region.
An aspect of the invention provides an antibody or antigen binding fragment thereof which specifically binds hMPV.
In various embodiments, the antibody or antigen binding fragment thereof comprises a variable region comprising three heavy chain complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR3. In various embodiments, the antibody or antigen binding fragment thereof comprises a variable region comprising any one CDR comprising the amino acid sequences of SEQ ID NOs: 2-340. In various embodiments, the antibody or antigen binding fragment thereof comprises a variable region comprising three light chain CDRs CDR1, CDR2, and CDR3 comprising amino acid sequence selected from the group consisting of: SEQ ID NOs: 341-649.
In various embodiments, the antibody or antigen binding fragment thereof comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, and 338. In various embodiments, the CDR1 is a heavy chain variable region CDR1.
In various embodiments, the antibody or antigen binding fragment thereof comprises a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, and 339. In various embodiments, the CDR2 is a heavy chain variable region CDR2.
In various embodiments, the antibody or antigen binding fragment thereof comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, and 340. In various embodiments, the CDR3 is a heavy chain variable region CDR3.
In various embodiments, the antibody or antigen binding fragment thereof comprises at least one heavy chain variable region and/or at least one light chain variable region.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising three CDRs selected from the group consisting of:
In various embodiments, the antibody or antigen binding fragment thereof comprises a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 341, 344, 347, 350, 353, 356, 359, 362, 365, 368, 371, 374, 377, 380, 383, 386, 389, 392, 395, 398, 401, 404, 407, 410, 413, 416, 419, 422, 425, 428, 431, 434, 437, 440, 443, 446, 449, 452, 455, 458, 461, 464, 467, 470, 473, 476, 479, 482, 485, 488, 491, 494, 497, 500, 503, 506, 509, 512, 515, 518, 521, 524, 527, 530, 533, 536, 539, 542, 545, 548, 551, 554, 557, 560, 563, 566, 569, 572, 575, 578, 581, 584, 587, 590, 593, 596, 599, 602, 605, 608, 611, 614, 617, 620, 623, 626, 629, 632, 635, 638, 641, 644, 647, 650, 653, 656, 659, 662, 665, 668, 671, 674, and 677. In various embodiments, the CDR1 is a light chain variable region CDR1.
In various embodiments, the antibody or antigen binding fragment thereof comprises a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 414, 417, 420, 423, 426, 429, 432, 435, 438, 441, 444, 447, 450, 453, 456, 459, 462, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, 528, 531, 534, 537, 540, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, 579, 582, 585, 588, 591, 594, 597, 600, 603, 606, 609, 612, 615, 618, 621, 624, 627, 630, 633, 636, 639, 642, 645, 648, 651, 654, 657, 660, 663, 666, 669, 672, 675, and 678. In various embodiments, the CDR2 is a light chain variable region CDR2.
In various embodiments, the antibody or antigen binding fragment thereof comprises a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 343, 346, 349, 352, 355, 358, 361, 364, 367, 370, 373, 376, 379, 382, 385, 388, 391, 394, 397, 400, 403, 406, 409, 412, 415, 418, 421, 424, 427, 430, 433, 436, 439, 442, 445, 448, 451, 454, 457, 460, 463, 466, 469, 472, 475, 478, 481, 484, 487, 490, 493, 496, 499, 502, 505, 508, 511, 514, 517, 520, 523, 526, 529, 532, 535, 538, 541, 544, 547, 550, 553, 556, 559, 562, 565, 568, 571, 574, 577, 580, 583, 586, 589, 592, 595, 598, 601, 604, 607, 610, 613, 616, 619, 622, 625, 628, 631, 634, 637, 640, 643, 646, 649, 652, 655, 658, 661, 664, 667, 670, 673, 676, and 679. In various embodiments, the CDR3 is a light chain variable region CDR3.
In various embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising three CDRs selected from the group consisting of:
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, and 904.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence which is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, and 904.
In various embodiments, the antibody or antigen binding fragment thereof comprises a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775,777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, and 905.
In various embodiments, the antibody or antigen binding fragment thereof comprises a light variable region sequence which is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775,777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, and 905.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 680, 682, 684, 686, 688, 690, 692 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, and 904; and comprises a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775,777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, and 905.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 680 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 681.
In various embodiments, the antibody or antigen binding fragment thereof comprises a comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 682 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 683.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 684 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 685.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 686 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 687.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 688 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 689.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 690 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 691.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 692 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 693.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 694 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 695.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 696 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 697.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 698 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 699.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 700 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 701.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 702 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 703.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 704 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 705.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 706 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 707.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 708 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 709.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 710 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 711.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 712 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 713.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 714 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 715.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 716 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 717.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 718 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 719.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 720 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 721.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 722 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 723.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 724 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 725.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 726 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 727.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 728 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 729.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 730 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 731.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 732 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 733.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 734 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 735.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 736 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 737.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 738 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 739.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 740 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 741.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 742 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 743.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 744 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 745.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 746 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 747.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 748 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 749.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 750 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 751.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 752 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 753.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 754 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 755.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 756 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 757.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 758 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 759.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 760 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 761.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 762 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 763.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 764 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 765.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 766 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 767.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 768 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 769.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 770 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 771.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 772 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 773.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 774 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 775.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 776 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 777.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 778 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 779.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 780 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 781.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 782 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 783.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 784 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 785.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 786 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 787.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 788 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 789.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 790 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 791.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 792 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 793.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 794 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 795.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 796 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 797.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 798 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 799.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 800 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 801.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 802 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 803.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 804 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 805.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 806 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 807.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 808 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 809.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 810 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 811.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 812 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 813.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 814 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 815.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 816 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 817.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 818 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 819.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 820 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 821.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 822 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 823.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 824 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 825.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 826 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 827.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 828 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 829.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 830 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 831.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 832 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 833.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 834 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 835.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 836 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 837.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 838 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 839.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 840 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 841.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 842 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 843.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 844 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 845.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 846 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 847.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 848 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 849.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 850 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 851.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 852 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 853.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 854 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 855.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 856 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 857.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 858 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 859.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 860 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 861.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 862 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 863.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 864 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 865.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 866 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 867.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 868 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 869.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 870 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 871.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 872 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 873.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 874 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 875.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 876 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 877.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 878 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 879.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 880 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 881.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 882 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 883.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 884 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 885.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 886 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 887.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 888 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 889.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 890 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 891.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 892 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 893.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 894 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 895.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 896 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 897.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 898 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 899.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 900 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 901.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 902 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 903.
In various embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 904 and comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 905.
In various embodiments, the antibody or antigen binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 906 and/or the amino acid sequence of SEQ ID NO: 907.
In various embodiments, the antibody or antigen binding fragment thereof of any of the preceding claims that neutralizes hMPV A and/or hMPV B. In specific embodiments the antibody or antigen binding fragment thereof neutralizes hMPV A. In specific embodiments the antibody or antigen binding fragment thereof neutralizes hMPV B.
In various embodiments, the antibody or antigen binding fragment thereof inhibits hMPV infection.
In various embodiments, the antibody or antigen binding fragment thereof neutralizes hMPV with a neutralizing potency (IC50) of 10 microgram per milliliter (μg/mL) or less, 9 μg/mL or less, 8 μg/mL or less, 7 μg/mL or less, 6 μg/mL or less, 5 μg/mL or less, 4 μg/mL or less, 3 μg/mL or less, 2 μg/mL or less, or 1 μg/mL or less.
In various embodiments, the antibody or antigen binding fragment thereof neutralizes hMPV with a neutralizing potency (IC50) described herein, e.g., Tables 3A-C.
In various embodiments, the antibody or antigen binding fragment thereof binds and neutralizes hMPV in vitro.
In various embodiments, the antibody or antigen binding fragment thereof neutralizes hMPV in vivo.
In various embodiments, the antibody or antigen binding fragment thereof binds to a hMPV F protein or antigen or a hMPV prefusion F protein or antigen with a KD value of about 1×10−9 M to about 1×10−12 M as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g., KinExa and OCTET). In various embodiments, the antibody or antigen binding fragment thereof binds to a processed F antigen. In various embodiments, the antibody or antigen binding fragment thereof binds to an unprocessed F antigen. In various embodiments, the antibody or antigen binding fragment thereof binds to a processed PreF antigen. In various embodiments, the antibody or antigen binding fragment thereof binds to an unprocessed PreF antigen.
In various embodiments, the antibody or antigen binding fragment thereof binds, cross-reacts and/or neutralizes RSV and/or an RSV antigen.
In various embodiments, the antibody or antigen binding fragment thereof is an antibody.
For example, the antibody is a chimeric, human or humanized antibody.
An aspect of the invention provides an antibody or antigen binding fragment thereof that binds to the same epitope on hMPV as any antibody or antigen binding fragment thereof described herein, e.g., an antibody or antigen fragment thereof as described in Tables 1-4.
In various embodiments, the antibody or antigen binding fragment thereof binds to an epitope of hMPV of SEQ ID NO: 1, SEQ ID NO: 910, SEQ ID NO: 911, SEQ ID NO: 912, and/or SEQ ID NO: 913.
An aspect of the invention provides an antibody or antigen binding fragment thereof which binds to one or more sites: Ø, I, II, III, III′, IV, IV′, V, α, and/or β of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site Ø of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site I of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site II of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site III of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site III′ of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site IV of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site IV′ of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site V of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site α of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site β of hMPV (SEQ ID NO: 1).
An aspect of the invention provides an antibody or antigen binding fragment thereof which neutralizes hMPV and/or binds to one or more sites: Ø, I, II, III, III′, IV, IV′, V, α, and/or β site of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site Ø of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site I of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site II of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site III of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site III′ of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site IV of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site IV′ of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site V of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site α of hMPV (SEQ ID NO: 1). In various embodiments, the antibody or antigen binding fragment thereof binds to site β of hMPV (SEQ ID NO: 1).
In various embodiments, the antibody or antigen binding fragment thereof binds to the particular antigen site described above and have greater than 80% identity (i.e., greater than 80, 85, 90, 95, 97, 98, 99, 100% identity) to the VH and/or VL of the disclosed antibodies or antigen binding fragments thereof described herein, for example, in Table 3B, Table 3C, or Table 3D.
An aspect of the invention provides a multi-specific molecule comprising a first binding region comprising the antibody or antigen binding fragment thereof described herein and a second binding region. In various embodiments, the multi-specific comprises a bispecific molecule. For example, the second binding region binds to a viral-associated antigen. In various embodiments, the second binding region binds to an antigen or ligand associated with a viral condition or viral disease.
An aspect of the invention provides a nucleic acid comprising a nucleotide sequence that encodes any antibody or antigen binding fragment thereof described herein or any bispecific molecule described herein.
An aspect of the invention provides an expression vector comprising any nucleic acid described herein. In one example, the expression vector comprises a nucleic acid encoding at least one of the polypeptides in Table 3D (SEQ ID NOs: 680-905).
An aspect of the invention provides a cell transformed with any expression vector described herein.
An aspect of the invention provides a pharmaceutical composition for preventing, treating or detecting a virus comprising: any antibody or antigen binding fragment described herein, or any bispecific molecule described; and a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition comprises one or more additional prophylactic agent or therapeutic agent. In various embodiments of the pharmaceutical composition, the one or more additional therapeutic agent is selected from the group consisting of: an immunomodulator, a hormone, a cytotoxic agent, an enzyme, a radionuclide, a second antibody conjugated to at least one immunomodulator, an enzyme, a radioactive label, a hormone, an antisense oligonucleotide, or a cytotoxic agent, and a combination thereof.
An aspect of the invention provides a kit for preventing, treating or detecting a virus comprising: any antibody or antigen binding fragment thereof described, any multi-specific molecule described (e.g., bispecific molecule), and/or any pharmaceutical composition described herein; and instructions for use.
An aspect of the invention provides a method of producing an antibody or antigen binding fragment thereof comprising: culturing a host cell comprising a polynucleotide encoding the amino acid sequences of any antibody or antigen binding fragments thereof described herein under conditions favorable to expression of the polynucleotide; and optionally, recovering the antibody or antigen binding fragment thereof from the host cell and/or culture medium.
An aspect of the invention provides a method of selectively detecting hMPV comprising administering or contacting to a sample any antibody or antigen binding fragment thereof described herein and detecting the presence of the antibody or antigen binding fragment thereof. In various embodiments of the method, the sample comprises a bodily fluid. For example, the sample comprises a serum from a subject.
An aspect of the invention provides a method for treating a subject having a viral disease, comprising administering to a subject in need thereof a therapeutically effective amount of any antibody or antigen binding fragment thereof described herein, any multi-specific (e.g., bispecific) molecule described herein, and/or any pharmaceutical composition described herein.
In various embodiments of the method, the viral disease is selected from the group consisting of: an MPV infection (e.g., an hMPV infection), a respiratory virus), clinical symptoms associated with MPV infection, Lower Respiratory tract infections (LTRI) leading to acute respiratory tract infection leading to Bronchiolitis, Pneumonia). In various embodiments of the method, the MPV infection comprises an infection of the lungs and/or respiratory tract. In various embodiments of the method, the respiratory virus is an upper respiratory virus. In various embodiments of the method, the virus disease is associated with hMPV type A. In various embodiments, the virus disease is associated with hMPV type B. In various embodiments, the respiratory virus is a human respiratory syncytial virus (hRSV).
In various embodiments, the method further comprises administering one or more additional therapies.
An aspect of the invention provides any antibody or antigen binding fragment thereof described herein, any multi-specific (e.g., bispecific) molecule described herein, and/or any pharmaceutical composition described herein, for use in the preparation of a medicament to: bind hMPV; neutralize hMPV; and/or treat a disorder or condition associated with hMPV in a subject in need thereof.
An aspect of the invention also provides isolated hMPV F polypeptides, nucleic acids encoding such isolated polypeptides, and immunogenic compositions comprising such isolated hMPV F polypeptides. Such isolated hMPV F polypeptides and immunogenic compositions may be used to immunize a subject and prevent or treat an hMPV infection.
In various embodiments of an isolated hMPV F polypeptide, the invention provides an isolated hMPV F polypeptide comprising residues 1-490 of SEQ ID NO: 911, or a variant thereof, wherein the isolated hMPV F polypeptide does not include residues 491-539 of SEQ ID NO: 1. In various embodiments, the variant comprises an amino acid sequence having 95-99% identity to residues 1-490 of SEQ ID NO: 911. In various embodiments of the isolated hMPV F polypeptide comprising SEQ ID NO: 911, or a variant thereof, the variant amino acid sequence of the isolated hMPV F polypeptide does not vary at residues 491-512 of SEQ ID NO: 911. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 911 by 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 911 by 1-15 amino acids. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 911 by 1-10 amino acids. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 911 by 1-5 amino acids. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 911 by 1-3 amino acids.
In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of SEQ ID NO: 911.
In various embodiments of an isolated hMPV F polypeptide, the isolated hMPV F polypeptide comprises residues 1-490 of SEQ ID NO: 911, or a variant thereof, and a trimerization domain, wherein the isolated hMPV F polypeptide does not include residues 491-539 of SEQ ID NO: 1. In various embodiments, the trimerization domain is a GCN4 domain, a T4 foldon domain, a human type XV collagen domain, or a human type XVIII collagen domain. In various embodiments, the GCN4 domain has the amino acid sequence of SEQ ID NO: 1022, the T4 foldon domain has the amino acid sequence of SEQ ID NO: 1012, the human type XV collagen domain has the amino acid sequence of SEQ ID NO: 1023, and the human type XVIII collagen domain has the amino acid sequence of SEQ ID NO: 1024.
In various embodiments of an isolated hMPV F polypeptide, a protease cleavage sequence at residues 99-102 is replaced with a furin-cleavage sequence. In various embodiments, the furin-cleavage sequence is SEQ ID NO: 1025 or 1026. In various embodiments, the furin-cleavage sequence is SEQ ID NO: 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, or 1066. In various embodiments, the furin-cleavage sequence is SEQ ID NO: 1033.
In various embodiments of an isolated hMPV F polypeptide, the invention provides an isolated hMPV F polypeptide comprising residues 1-490 of SEQ ID NO: 911, or a variant thereof, wherein the isolated hMPV F polypeptide does not include residues 491-539 of SEQ ID NO: 1, wherein the isolated hMPV F polypeptide has a mutation at residue 185. In various embodiments, the mutation is A185P.
In various embodiments of an isolated hMPV F polypeptide, the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910, residues 1-518 of SEQ ID NO: 912, or residues 1-521 of SEQ ID NO: 913, or variants thereof, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517 of SEQ ID NO: 910, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516 of SEQ ID NO: 912, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521 of SEQ ID NO: 913. In various embodiments, the variant comprises an amino acid sequence having 95-99% identity to residues 1-521 of SEQ ID NO: 910, 95-99% identity to residues 1-58 of SEQ ID NO: 912, or 95-99% identity to residues 1-521 of SEQ ID NO: 91, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516, and wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521.
In various embodiments of the isolated hMPV F polypeptide, the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 varies by 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 910 by 1-15 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 910 by 1-10 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 910 by 1-5 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 910 by 1-3 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517.
In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of residues 1-521 of SEQ ID NO: 910.
In various embodiments of the isolated hMPV F polypeptide, the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 varies by 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 912 by 1-15 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 912 by 1-10 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 912 by 1-5 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 912 by 1-3 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516.
In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of residues 1-518 of SEQ ID NO: 912.
In various embodiments of the isolated hMPV F polypeptide, the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 varies by 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 913 by 1-15 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 913 by 1-10 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 913 by 1-5 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence varies from SEQ ID NO: 913 by 1-3 amino acids, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521.
In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of residues 1-521 of SEQ ID NO: 913.
In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of residues 1-521 of SEQ ID NO: 910, residues 1-518 of SEQ ID NO: 912, or residues 1-521 of SEQ ID NO: 913.
In various embodiments of an isolated hMPV F polypeptide, the isolated hMPV F polypeptide comprises a cleavage sequence and at least one affinity tag sequence. In various embodiments, the cleavage sequence is a thrombin cleavage sequence. In various embodiments, the affinity tag sequence is a 6×His sequence (SEQ ID NO: 1014) or a strep tag II sequence.
In various embodiments of an isolated hMPV F polypeptide, the isolated hMPV F polypeptide comprises the amino acid sequence of SEQ ID NO: 910, SEQ ID NO: 912, or SEQ ID NO: 913. In various embodiments, the isolated hMPV F polypeptide consists of the amino acid sequence of SEQ ID NO: 910, SEQ ID NO: 912, or SEQ ID NO: 913.
An aspect of the invention also provides nucleic acids encoding isolated hMPV F polypeptides, isolated hMPV F polypeptides, and immunogenic compositions comprising such isolated hMPV F polypeptides that encode variants of hMPV F protein. Such isolated hMPV F polypeptides and immunogenic compositions may be used to immunize a subject and prevent or treat an hMPV infection
An aspect of the invention also provides an isolated nucleic acid encoding any one of the isolated hMPV F polypeptides encoding an hMPV F protein described above. In some embodiments, the isolated nucleic acid is codon-optimized.
An aspect of the invention provides an isolated nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1006, or a variant thereof, wherein the variant nucleotide sequence of the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO: 911, and wherein the isolated nucleic acid does not include residues 1453-1600 of SEQ ID NO: 1003. In various embodiments, the variant nucleotide sequence of the isolated nucleic acid is SEQ ID NO: 1007.
An aspect of the invention also provides an isolated nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1004 or a variant thereof, SEQ ID NO: 1008 or a variant thereof, or SEQ ID NO: 1010 or a variant thereof, wherein the variant of the isolated nucleic acid comprising SEQ ID NO: 1004 encodes the amino acid sequence of SEQ ID NO: 910, wherein the variant of the isolated nucleic acid comprising SEQ ID NO: 1008 encodes the amino acid sequence of SEQ ID NO: 912, and wherein the variant of the isolated nucleic acid comprising SEQ ID NO: 1010 encodes the amino acid sequence of SEQ ID NO: 913. In various embodiments, the variant of the isolated nucleic acid comprising SEQ ID NO: 1004 is SEQ ID NO: 1005, the variant of the isolated nucleic acid comprising SEQ ID NO: 1008 is SEQ ID NO: 1009, and the variant of the isolated nucleic acid comprising SEQ ID NO: 1010 is SEQ ID NO: 1011. In various embodiments, the variant comprises a nucleic acid sequence having 95-99% identity to the nucleic sequence of SEQ ID NO: 1004, SEQ ID NO: 1008, or SEQ ID NO: 1010.
An aspect of the invention also provides an immunogenic composition comprising an isolated antigenic polypeptide, the isolated antigenic peptide consisting of residues 1-490 of SEQ ID NO: 911, or a variant thereof, and a pharmaceutically acceptable carrier. In various embodiments, the variant comprises an amino acid sequence having 95-99% identity to residues 1-490 of SEQ ID NO: 911.
An aspect of the invention also provides an immunogenic composition comprising an isolated antigenic polypeptide, the isolated antigenic peptide comprising residues 1-521 of SEQ ID NO: 910, residues 1-518 of SEQ ID NO: 912, or residues 1-521 of SEQ ID NO: 913, or a variant thereof, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516, and wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521. In various embodiments, the isolated antigenic polypeptide consists of residues 1-521 of SEQ ID NO: 910, residues 1-518 of SEQ ID NO: 912, or residues 1-521 of SEQ ID NO: 913, or a variant thereof, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516, and wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521. In various embodiments, the variant comprises an amino acid sequence having 95-99% identity to residues 1-521 of SEQ ID NO: 910, residues 1-518 of SEQ ID NO: 912, or residues 1-521 of SEQ ID NO: 913, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 910 does not vary at residues 486-517, wherein the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO: 912 does not vary at residues 102-105 and 490-516, and wherein the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO: 913 does not vary at residues 99-102, 185, and 485-521.
In various embodiments of the immunogenic compositions, the immunogenic composition is multivalent. In various embodiments, the immunogenic composition further comprises an adjuvant. In various embodiments, the immunogenic composition does not comprise an adjuvant.
The invention also provides a method of inducing an immune response in a subject, the method comprising administering to the subject any one of the immunogenic compositions above in an amount effective to produce an antigen-specific immune response in the subject. In various embodiments, the antigen-specific immune response comprises a T cell response or a B cell response. In various embodiments, the subject is administered a single dose of the immunogenic composition. In various embodiments, the subject is administered a booster dose of the immunogenic composition. In various embodiments, the immunogenic composition is administered to the subject by intradermal injection or intramuscular injection. In various embodiments, the subject has been exposed to hMPV, wherein the subject is infected with hMPV, or wherein the subject is at risk of infection from hMPV.
The invention also provides any one of the immunogenic compositions above for use in inducing an antigen-specific immune response in a subject. The invention also provides any one of the immunogenic compositions above for use in preventing or treating hMPV infection in a subject.
The invention provides any one of the isolated hMPV F polypeptides above for use in inducing an antigen-specific immune response in a subject.
The invention provides any one of the isolated hMPV F polypeptides above for use in preventing or treating human metapneumovirus (hMPV) infection in a subject.
The invention provides use of any one of the isolated hMPV F polypeptides or the isolated nucleic acid encoding such polypeptides in the manufacture of a medicament for inducing an antigen-specific immune response in a subject.
The invention provides use of any one of the isolated hMPV F polypeptides above or the isolated nucleic acid encoding such polypeptides or immunogenic compositions comprising such polypeptides in the manufacture of a medicament for preventing or treating human metapneumovirus (hMPV) infection in a subject.
Pneumoviruses including respiratory syncytial virus (RSV) and hMPV are common causes of acute lower respiratory infection (ALRI) [1-6]. Like RSV, the infection of hMPV leads to a variety of symptoms including coughing, wheezing, pneumonia, and bronchiolitis. The high-risk populations, including infants, young children, elderly people, and immunocompromised patients, are more likely to develop severe symptoms that may potentially require hospitalization [7-14]. Co-infection with other respiratory pathogens is common. Recently, cases of co-infection of SARS-CoV-2 and hMPV have also been reported [15-17], and such co-infection with the hMPV appeared to affect monocytes and dampen interferon response in a severe COVID-19 patient [15]. Despite of these medical burdens, currently there is no approved hMPV vaccine or neutralizing antibody available for therapeutic or prophylactic purpose.
The surface glycoprotein F of hMPV mediates the subsequent fusion of viral and cellular membranes [18, 19]. F protein is highly conserved in sequence between different hMPV subtypes, and it appears to be more critical for virus infection than other surface proteins G and SH [20, 21]. It has also been reported that F is the primary viral antigen of hMPV that elicits neutralizing and protective antibodies against hMPV infection, whereas antibodies elicited by G and SH proteins are not protective [22-24]. Therefore, the F protein is an attractive target for neutralizing antibody and vaccine development against hMPV infection.
The hMPV F protein is a class I viral fusion protein that presents as a homo-trimer on viral surface as well as the membrane of host cells. The precursor of F (F0) is synthesized as an intact polypeptide and subsequently subjected to proteolytic processing to become functional [19]. Unlike RSV which has two furin-cleavage sites that can be cleaved twice intracellularly during transport to the cell membrane [25], hMPV only has one protease cleavage site being cleaved at the cell surface or in the virus like particle by transmembrane proteases such as TMPRSS2 [26]. Cleavage of both RSV and hMPV F generates two disulfide-linked chains (F2 and F1). The fusion peptide, which is located at the N-terminus of F1 right after the protease cleavage site, is buried inside a hydrophobic cavity and interact with adjacent monomers, presumably stabilizing the trimer conformation [27, 28]. This globular “prefusion” (PreF) form of proteolytically processed F trimer is metastable and can be triggered into a more stable rod-shaped “postfusion” (PostF) form, through a series of conformational changes [19]. Protein engineering efforts have been made to stabilize hMPV and RSV F in PreF and PostF conformations by introducing additional mutations or modifying protease cleavage sites [27-32]. Despite only about 35% sequence identity, the structures of both stabilized PreF and PostF proteins between RSV F and hMPV F are remarkably conserved [27-31, 33, 34].
Over the past years, a large number of monoclonal antibodies (mAbs) targeting RSV F antigens have been isolated from human B cell repertoire and characterized by various approaches [33, 35-47]. According to these results, six major antigenic sites on RSV F have been defined (Ø, I, II, III, IV, V). PreF-specific antibodies targeting sites Ø and V have been shown to be immunodominant and accountable for a large proportion of neutralizing activity in human sera and antigen-specific memory B cell repertoire of adults [48, 49], suggesting that PreF is a better vaccine candidate of RSV to elicit higher neutralizing antibody (nAb) responses than PostF. In contrast, the immunogenicity potential of stabilized hMPV PreF and PostF appears to be comparable, likely due to the large glycan shield that is only observed at the site Ø of hMPV F protein [28]. Although a limited number of hMPV-F specific mAbs have been discovered from mice immunization [50], phage display [51], and human B cells [52], a comprehensive understanding of human antibody recognition to hMPV F during natural infection remains elusive.
In this study, we reported a large panel of hMPV-F specific mAbs isolated from memory B cells of multiple healthy human donors. Some isolated mAbs exhibited ultra-high neutralization potency in vitro. Investigators have characterized one of the most potent mAbs that recognizes a PreF-specific epitope on site V. Further in-depth characterization of isolated mAbs revealed diverse antigenic sites on hMPV F antigens, including overlapping but distinct epitopes on pre-defined site II and V, and four novel antigenic sites that have not been reported previously. See also Huang et al., 2019 Front. Immunol, pgs. 1-8; Rossey et al., 2018 Trends in Microbiology, vol. 26, issue 3, pgs. 209-219.
Antigenic Sites of hMPV Site Ø—amino acids 54-84; 165-197 of SEQ ID NO: 1
Unlike RSV, the antibody responses to hMPV F appear to be less dominant against the apex of the antigen. Furthermore, a panel of mAbs that bind specifically to the uncleaved PreF have been identified, suggesting the potential immunogenic differences between unprocessed and processed F antigens. This is the first disclosure revealing the comprehensive antigenic epitopes of hMPV F-specific mAbs elicited from natural infection.
As used herein, “Human metapneumovirus” or “hMPV” refers to the single-stranded, negative-sense ribonucleic acid (RNA) virus of the family Pneumoviridae, with a genome of about 13 kb. As used herein, “hMPV F protein,” refers to a fusion glycoprotein F0 that has the amino acid sequence set forth in UniProtKB—G3KCK8 (G3KCK8_9MONO; SEQ ID NO: 1).
As used herein, the term “antibody” refers to any form of immunoglobulin molecule that exhibits the desired biological or binding activity. An antibody that “specifically binds to” hMPV is an antibody that exhibits preferential binding to hMPV as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered “specific” for its intended target if its binding is determinative of the presence of the target protein in a sample, e.g., without producing undesired results such as false positives. Antibodies, or binding fragments thereof, will bind to the target protein with an affinity that is at least two-fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with non-target proteins. Thus, the term “antibody” is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), humanized, fully human antibodies, and chimeric antibodies. “Parental antibodies” are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as humanization of an antibody for use as a human therapeutic.
In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable regions of each light/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are typically joined by a “J” region of about 12 or more amino acids, with the heavy chain also typically including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).
As used herein, “isotype” refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes.
Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (KD) of 10−5 to 10−12 M or less. Any KD greater than about 10−4 M is generally considered to indicate nonspecific binding. As used herein, an antibody that “binds specifically” to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a KD of 10−7 M or less, preferably 10−8 M or less, even more preferably 5×10−9 M or less, and most preferably between 10−8 M and 10−10 M or less, but does not bind with high affinity to unrelated antigens.
As used herein, the term “antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
As used herein, unless otherwise indicated, “antibody fragment” or “antigen binding fragment” or “antigen binding fragment thereof” refers to a fragment of an antibody that retains the ability to bind specifically to the antigen, e.g., fragments that retain one or more CDR regions and the ability to specifically bind to the antigen.
Antigen binding portions include, for example, Fab, Fab′, F(ab′)2, Fd, Fv, fragments including CDRs, and single chain variable fragment antibodies (scFv), and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the antigen (e.g., hMPV).
An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
Antigen binding fragments thereof within the scope of the present invention also include F(ab′)2 fragments which may be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab′)2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain appended to a VH-CH1 chain by a disulfide bridge. A F(ab′)2 fragment is two Fab fragments which, in turn, are appended by two disulfide bridges. The Fab portion of an F(ab′)2 molecule includes a portion of the Fc region between which disulfide bridges are located.
The term “acceptor human framework” refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may have the same amino acid sequence as the naturally-occurring human immunoglobulin framework or human consensus framework, or it may have amino acid sequence changes compared to wild-type naturally-occurring human immunoglobulin framework or human consensus framework. In some embodiments, the number of amino acid changes are 10, 9, 8, 7, 6, 5, 4, 3, or 2, or 1. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence. In some embodiments, the VH acceptor human framework is identical in sequence to the VH human immunoglobulin framework sequence or human consensus framework sequence.
The term “binding protein” as used herein also refers to a non-naturally occurring (or recombinant) protein that specifically binds to at least one target antigen. In various embodiments, the binding protein comprises an anti-hMPV antibody or antigen binding fragment thereof described herein. For example, the binding protein is a multi-specific molecule (e.g., multi-specific antibody) comprising the anti-hMPV antibody or antigen binding fragment thereof, and a second molecule (e.g., a second antibody or antigen binding fragment thereof).
A “multi-specific antibody” is an antibody (e.g., bispecific antibodies, trispecific antibodies) that recognizes two or more different antigens or epitopes. In various embodiments, multi-specific antibodies normally will bind at least two antigens or epitopes (i.e., bispecific antibodies, BsAbs) or more than two antigens. In various embodiments, multi-specific antibodies with additional specificities such as trispecific antibodies are encompassed herein. Examples of BsAbs include those with one arm directed against a viral antigen #1 and the other arm directed against a viral antigen #2. For example, the BsAbs may bind to hMPV strain A and hMPV strain B.
Techniques for making multi-specific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody” technology for making bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, e.g. Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69). Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies”, are also included herein (see, e.g. US 2006/0025576; and Proc Natl Acad Sci USA. 2011 Jul. 5; 108(27): 11187-92).
A “bispecific” or “bifunctional antibody” is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Bifunctional antibodies include, for example, heterodimeric antibody conjugates (e.g., two antibodies or antibody fragments joined together with each having different specificities), antibody/cell surface-binding molecule conjugates (e.g., an antibody conjugated to a non-antibody molecule such as a receptor), and hybrid antibodies (e.g., an antibody having binding sites for two different antigens). Bispecific antibodies include those generated by quadroma technology (Milstein and Cuello (1983) Nature 305(5934): 537-40), by chemical conjugation of two different monoclonal antibodies (Staerz et al. (1985) Nature 314(6012): 628-31), or by knob-into-hole or similar approaches which introduces mutations in the Fc region (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14): 6444-6448), among others.
The term “recombinant antibody,” refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes (e.g., human immunoglobulin genes) or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library (e.g., containing human antibody sequences) using phage display, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences (e.g., human immunoglobulin genes) to other DNA sequences. Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro 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.
“Chimeric antibody” refers to an antibody in which a portion of the heavy and/or light chain contains sequences derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
“Human antibody” refers to an antibody that comprises human immunoglobulin protein sequences or derivatives thereof. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences or derivatives thereof, respectively.
“Humanized antibody” refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The prefix “hum”, “hu” or “h” may be added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.
“Monoclonal antibody” or “mAb” or “Mab”, as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.
Antigen binding fragments (including scFvs) of such immunoglobulins are also encompassed by the term “monoclonal antibody” as used herein. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different epitopes on the antigen, each monoclonal antibody is directed against a single epitope. Monoclonal antibodies can be prepared using any art recognized technique and those described herein such as, for example, a hybridoma method, a transgenic animal, recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), or using phage antibody libraries using the techniques described in, for example, U.S. Pat. No. 7,388,088 and PCT Pub. No. WO 00/31246). Monoclonal antibodies include chimeric antibodies, human antibodies, and humanized antibodies and may occur naturally or be produced recombinantly.
A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.
A “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).
As used herein, the term “single-chain Fv” or “scFv” antibody refers to antibody fragments comprising the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker. For a review of scFv, see Pluckthun (1994) T
The monoclonal antibodies herein also include camelized single domain antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO 94/04678; WO 94/25591; U.S. Pat. No. 6,005,079, which are hereby incorporated by reference in their entireties). In one embodiment, the present invention provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.
As used herein, the term “diabodies” refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.
The antibodies of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571; WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes, such as substitutions, deletions and insertions, glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc may be utilized to alter the half-life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent dosing, with the concomitant increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.
The term “fully human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” refers to an antibody which comprises mouse immunoglobulin sequences only.
“Variable regions” or “V region” or “V chain” as used herein means the segment of IgG chains which is variable in sequence between different antibodies. A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable region of the heavy chain may also be referred to as “heavy chain variable region”, “heavy chain variable domain”, “VH” or “VH” in the instant disclosure. The variable region of the light chain may be referred to as “light chain variable region”, “heavy chain variable domain”, “VL” or “VL” in the instant disclosure. Typically, the variable regions of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.
A “CDR” refers to one of three hypervariable regions (H1, H2, or H3) within the non-framework region of the antibody VH β-sheet framework, or one of three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable domains. CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved b-sheet framework, and thus are able to adapt to different conformation. Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact, and IMGT. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-48; Morea et al., 2000, Methods 20:267-79). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those skilled in the art. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art and shown below in Table 1. In some embodiments, the CDRs are as defined by the Kabat numbering system. In other embodiments, the CDRs are as defined by the IMGT numbering system. In yet other embodiments, the CDRs are as defined by the AbM numbering system. In still other embodiments, the CDRs are as defined by the Chothia numbering system. In yet other embodiments, the CDRs are as defined by the Contact numbering system. See Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and/or those residues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, (1987) J. Mol. Biol. 196: 901-917).
The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Typically, the numbering of the amino acids in the heavy chain constant domain begins with number 118, which is in accordance with the Eu numbering scheme. The Eu numbering scheme is based upon the amino acid sequence of human IgG1 (Eu), which has a constant domain that begins at amino acid position 118 of the amino acid sequence of the IgG1 described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969), and is shown for the IgG1, IgG2, IgG3, and IgG4 constant domains in Béranger, et al., Ibid.
The variable regions of the heavy and light chains contain a binding domain comprising the CDRs that interacts with an antigen. A number of methods are available in the art for defining CDR sequences of antibody variable domains (see Dondelinger et al., Frontiers in Immunol. 9: Article 2278 (2018)). The common numbering schemes include the following.
The following general rules disclosed in www.bioinf.org.uk: Prof Andrew C.R. Martin's Group and reproduced in Table 1 below may be used to define the CDRs in an antibody sequence that includes those amino acids that specifically interact with the amino acids comprising the epitope in the antigen to which the antibody binds. There are rare examples where these generally constant features do not occur; however, the Cys residues are the most conserved feature.
1Some of these numbering schemes (particularly for Chothia loops) vary depending on the individual publication examined.
2Any of the numbering schemes can be used for these CDR definitions, except the Contact numbering scheme uses the Chothia or Martin (Enhanced Chothia) definition.
3The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. (This is because the Kabat numbering scheme places the insertions at H35A and H35B.)
As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues. The residue numbering above relates to the Kabat numbering system and does not necessarily correspond in detail to the sequence numbering in the accompanying Sequence Listing. Amino acid residues in antibodies can also be defined using other numbering systems, such as Chothia, enhanced Chothia, IMGT, Kabat/Chothia composite, Honegger (AHo), Contact, or any other conventional antibody numbering scheme.
As used herein, the term “isolated” used in the context of polypeptides or polynucleotides refers to polypeptides or polynucleotides that are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include other nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. It may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the polypeptides or polynucleotides.
An “isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities.
As used herein, “isotype” refers to the antibody class (e.g., IgG (including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1 and IgA2), IgD, and IgE antibody) that is encoded by the heavy chain constant region genes of the antibody.
An “effector function” refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom. Exemplary “effector functions” include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).
An “Fc region,” “Fc domain,” or “Fc” refers to the C-terminal region of the heavy chain of an antibody. Thus, an Fc region comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL).
The term “epitope” or “antigenic determinant” refers to a site on an antigen (e.g., hMPV) to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids (usually a linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, 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, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in a unique spatial conformation.
The term “epitope mapping” refers to the process of identifying the molecular determinants on the antigen involved in antibody-antigen recognition. Methods for determining what epitopes are bound by a given antibody are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from, e.g., hMPV are tested for reactivity with a given antibody (e.g., anti-hMPV antibody); x-ray crystallography; antigen mutational analysis, two-dimensional nuclear magnetic resonance; yeast display; and hydrogen/deuterium exchange-mass spectrometry (HDX-MS) (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). See also Champe et al. (1995) J. Biol. Chem. 270:1388-1394.
The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same segment or same segments of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the “same epitope on hMPV” with the antibodies described herein include, for example, epitope mapping methods, such as x-ray analyses of crystals of antigen:antibody complexes, which provides atomic resolution of the epitope, and HDX-MS. Other methods monitor the binding of the antibody to antigen fragments thereof (e.g. proteolytic fragments) or to mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component, such as alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081), yeast display of mutant target sequence variants, or analysis of chimeras. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.
Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, may be determined using known binding competition experiments, e.g., BIACORE® surface plasmon resonance (SPR) analysis. In certain embodiments, an antibody competes with, and inhibits binding of another antibody to a target by at least 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may be different depending on which antibody is the “blocking antibody” (i.e., the antibody that when combined with an antigen blocks another immunologic reaction with the antigen). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb. Protoc. 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, an overlapping epitope, or to adjacent epitopes (e.g., as evidenced by steric hindrance). Two antibodies “cross-compete” if antibodies block each other both ways by at least 50%, i.e., regardless of whether one or the other antibody is contacted first with the antigen in the competition experiment.
Competitive binding assays for determining whether two antibodies compete or cross-compete for binding include competition for binding to cells expressing hMPV, e.g., by flow cytometry. Other methods include: SPR (e.g., BIACORE®), solid phase direct or indirect radioimmunoassay (RIA), bio-layer interferometry (BLI) analysis (e.g., OCTET analysis using BLI), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).
As used herein, the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen. Typically, the antibody (i) binds with an equilibrium dissociation constant (KD) of approximately less than 10−7 M, such as approximately less than 10−8 M, 10−9 M or 10−10 M or even lower when determined by, e.g., SPR using a predetermined antigen as the analyte and the antibody as the ligand, or Scatchard analysis of binding of the antibody to antigen positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. Any KD greater than about 10−4 M is generally considered to indicate nonspecific binding.
The term “kassoc” or “ka”, as used herein, refers to the association rate of a particular antibody-antigen interaction, whereas the term “kdis” or “kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “KD”, as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as a molar concentration (M). KD values for antibodies or antigen binding fragments thereof can be determined using methods well established in the art. A preferred method for determining the KD of an antibody or antigen binding fragment thereof is by using SPR, preferably using a biosensor system such as a Biacore® system or flow cytometry and Scatchard analysis, or bio-layer interferometry.
The term “EC50” in the context of an in vitro or in vivo assay using an antibody or antigen binding fragment thereof refers to the concentration of an antibody or antigen binding fragment thereof that induces a response that is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.
The term “cross-reacts,” as used herein, refers to the ability of an antibody or antigen binding fragment thereof described herein to bind to metapneumovirus from a different species. For example, an antibody or antigen binding fragment thereof described herein that binds hMPV may also bind other family members in Pneumoviridae family (e.g., human RSV, bovine RSV, or murine pneumonia virus), or another species of MPV (e.g., avian MPV). Cross-reactivity may be measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA, bio-layer interferometry) or binding to, or otherwise functionally interacting with, cells physiologically expressing the antigen (e.g., HT1080 cells overexpressing hMPV). Methods for determining cross-reactivity include standard binding assays as described herein, for example, by bio-layer interferometry or flow cytometric techniques.
As used herein, the term “linked” refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.
The term “nucleic acid molecule,” as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
The term “isolated nucleic acid molecule,” as used herein in reference to nucleic acids encoding antibodies or antibody binding fragments thereof (e.g., VH, VL, CDR3), is intended to refer to a nucleic acid molecule in which the nucleotide sequences are essentially free of other genomic nucleotide sequences, e.g., those encoding antibodies or antibody binding fragments thereof that bind antigens other than hMPV, which other sequences may naturally flank the nucleic acid in human genomic DNA.
The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
Also provided are “conservative sequence modifications” of the sequences set forth herein, i.e., amino acid sequence modifications which do not abrogate the binding of the antibody or antigen binding fragment thereof encoded by the nucleotide sequence or containing the amino acid sequence, to the antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into a sequence in a table herein (e.g., Table 3B, Table 3C, Table 3D or in Examples 1-6) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an anti-hMPV antibody is preferably replaced with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an anti-hMPV antibody coding sequence or anti-hMPV antigen binding fragment thereof coding sequence, such as by saturation mutagenesis, and the resulting modified anti-hMPV antibodies can be screened for binding activity.
For nucleic acids, the term “substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 80% to 85%, 85% to 90% or 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
For polypeptides, the term “substantial homology” indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80% of the amino acids, usually at least about 80% to 85%, 85% to 90%, 90% to 95%, more preferably at least about 98% to 99.5% of the amino acids, or more preferably at least about 98% to 99.9% of the amino acids.
The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), accounting for the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The nucleic acid and protein sequences described herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.
As used herein, the term “variant” in the context of recombinant hMPV F proteins refers to a molecule that differs in its amino acid sequence or nucleic acid sequence relative to a native sequence or a reference sequence. Sequence variants may possess substitutions, deletions, insertions, or a combination of any two or three of the foregoing, at certain positions within the sequence, as compared to a native sequence or a reference sequence. Ordinarily, variants possess at least 50% identity to a native sequence or a reference sequence. In some embodiments, variants share at least 80% identity or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a native sequence or a reference sequence. In some embodiments, variants share at least between 90%-99%, 91%-99%, 92%-99%, 93%-99%, 94%-99%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% identity with a native sequence or a reference sequence. In some embodiments, a variant differs from the native sequence by 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids or nucleic acids.
As used herein, the term “analog” is meant to include polypeptide variants that differ by one or more amino acid alterations, for example, substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.
The present disclosure provides several types of compositions that are polynucleotide or polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is synonymous with the term “variant” and generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or a starting molecule.
As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences; in particular, the polypeptide sequences disclosed herein are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal residues or N-terminal residues) alternatively may be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence that is soluble or linked to a solid support.
“Substitutional variants” when referring to polypeptides, are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more (e.g., 3, 4 or 5) amino acids have been substituted in the same molecule.
Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild type molecules). The term “identity,” as known in the art, refers to the degree of sequence relatedness between two sequences of polynucleotide or polypeptide molecules as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods.
The term “percent identity” or “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, accounting for the number of gaps and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm in an alignment tool (e.g. the Needleman-Wunsch algorithm in an online tool).
As used herein, the term “global alignment” refers to an alignment of residues between two amino acid or nucleic acid sequences along their entire length, introducing gaps as necessary if the two sequences do not have the same length, to achieve a maximum percent identity. A global alignment can be created using the global alignment tool “Needle” from the online European Molecular Biology Open Software Suite (EMBOSS) (see www.ebi.ac.uk/Tools/psa/emboss_needle/) or the global alignment tool “BLAST®»Global Alignment” from the National Center for Biotechnology Information (NCBI) (see blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&PROG_DEF AULTS=on&BLAST_INIT=GlobalAln&BLAST_SPEC=GlobalAln&BLAST_PROGRAMS=bl astn). Both of these global alignment tools incorporate the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453). In a preferred embodiment, a global alignment of nucleotide sequences using BLAST Global Alignment uses the following default parameters: match score=2; mismatch score=−3; Gap Cost Existence score=5; Gap Cost Extension Score=2. In a preferred embodiment, a global alignment of protein sequences using BLAST Global Alignment uses the following default parameters: Gap Cost Existence=11; Gap Cost Extension=1.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
The term “inhibition” as used herein, refers to any statistically significant decrease in biological activity, including partial and full blocking of the activity. For example, “inhibition” can refer to a statistically significant decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in biological activity (e.g., inhibition of HMPV).
For example, as used herein, the term “inhibits hMPV activity” includes any measurable decrease in hMPV activity, e.g., an inhibition of hMPV activity by at least about 10%, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or about 100%, relative to a control (e.g., a control antibody). The inhibition may be specific to a single mechanism of hMPV activity or may be generalizable to all mechanisms of hMPV activity.
The term “multivalent”, used in the context of an immunogenic composition herein (e.g. “multivalent immunogenic composition”) refers to a pharmaceutical preparation comprising more than one active agent that provides active immunity to a disease or pathological condition caused by more than one strain of pathogen. For example, a multivalent immunogenic composition against hMPV may protect against more than one strain of hMPV.
An “adjuvant,” as used herein, is a substance that serves to enhance the immunogenicity of a composition of the disclosure. An adjuvant may enhance an immune response to an antigen that is weakly immunogenic when administered alone, e.g., inducing no or weak antibody titers or cell-mediated immune response, increase antibody titers to the antigen, and/or lower the dose of the antigen effective to achieve an immune response in the individual. Thus, adjuvants are often given to boost the immune response and are well known to the skilled artisan.
As used herein, the term “prevent” or “preventing” means to administer a prophylactic agent, such as a composition containing any of the polypeptides of the present invention, to a subject or patient at risk of becoming infected by human metapneumovirus (hMPV). Preventing includes reducing the likelihood or severity of a subsequent hMPV infection, ameliorating symptoms associated with a subsequent hMPV infection, and inducing immunity to protect against hMPV infection. Typically, the agent is administered in an amount effective to neutralize hMPV in the body in order to block infection. The amount of a prophylactic agent that is effective to ameliorate any particular disease symptom may vary according to factors such as the age, and weight of the patient, and the ability of the agent to elicit a desired response in the subject. Whether a disease symptom has been ameliorated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom or in certain instances will ameliorate the need for hospitalization.
The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject with a tumor or cancer or a subject who is predisposed to having such a disease or disorder, an anti-hMPV antibody (e.g., anti-human hMPV antibody) or antigen binding fragment thereof described herein, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
“Immunotherapy” refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
“Immunostimulating therapy” or “immunostimulatory therapy” refers to a therapy that results in increasing (inducing or enhancing) an immune response in a subject for, e.g., treating hMPV or an condition associated with hMPV.
As used herein, “immune cell” refers to the subset of blood cells known as white blood cells, which include mononuclear cells such as lymphocytes, monocytes, macrophages, and granulocytes.
As used herein, “administering” refers to the physical introduction of a molecule (e.g., an antibody or antigen binding fragment thereof that binds hMPV) or of a composition comprising a therapeutic agent (e.g., an anti-hMPV antibody or antigen binding fragment thereof) to a subject, using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an antibody described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
The terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or process performed on, or administering an active agent (e.g., an anti-hMPV antibody or antigen binding fragment thereof) to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. Treatment can be of a subject having a disease or a subject who does not have a disease (e.g., for prophylaxis).
As used herein, “adjunctive” or “combined” administration (co-administration) includes simultaneous administration of the agents and/or compounds in the same or different dosage form, or separate administration of the compounds (e.g., sequential administration). For example, at least one agent comprises an anti-hMPV antibody or antigen binding fragment thereof. Thus, a first antibody or antigen binding fragment thereof, e.g., an anti-hMPV antibody or antigen binding fragment thereof, and a second, third, or more antibodies or antigen binding fragments thereof can be simultaneously administered in a single formulation. Alternatively, the first and second (or more) antibodies or antigen binding fragments thereof can be formulated for separate administration and are administered concurrently or sequentially.
“Combination” therapy, as used herein, means administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g. administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. (See, e.g., Kohrt et al. (2011) Blood 117:2423). For example, the anti-hMPV antibody can be administered first followed by (e.g., immediately followed by) the administration of a second antibody (e.g., an anti-viral antibody) or antigen binding fragment thereof, or vice versa. In one embodiment, the anti-hMPV antibody or antigen binding fragment thereof is administered prior to administration of the second antibody or antigen binding fragment thereof. In another embodiment, the anti-hMPV antibody or antigen binding fragment thereof is administered, for example, a few minutes (e.g., within about 30 minutes) or at least one hour of the second antibody or antigen binding fragment thereof. Such concurrent or sequential administration preferably results in both antibodies or antigen binding fragments thereof being simultaneously present in treated patients.
The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve a desired effect, e.g., viral neutralization and viral binding. A “therapeutically effective amount” or “therapeutically effective dosage” of a drug (e.g., anti-hMPV antibody or antigen binding fragment thereof) is any amount of the drug or therapeutic agent that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase or therapeutic agent in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. A therapeutically effective amount or dosage of a drug or therapeutic agent includes a “prophylactically effective amount” or a “prophylactically effective dosage”, which is any amount of the drug or therapeutic agent that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
By way of example, for the treatment of viral infections such as hMPV, a therapeutically effective amount or dosage of the drug or therapeutic agent (e.g., anti-hMPV antibody or antigen binding fragment thereof) inhibits viral activity, viral load, or virus diseases symptoms by at least about 20%, by at least about 30% by at least about 40%, by at least about 50%, by at least about 60%, by at least above 70%, by at least about 80%, or by at least about 90% relative to untreated subjects. In some embodiments, a therapeutically effective amount or dosage of the drug or therapeutic agent completely inhibits viral activity. The ability of a compound or therapeutic agent, including an antibody, to inhibit viral activity can be evaluated using the assays described herein. Alternatively, this property of a composition comprising the compound or therapeutic agent can be evaluated by examining the ability of the composition to inhibit viral activity; such inhibition can be measured in vitro by assays known to the skilled practitioner.
The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject having cancer. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, cats, dogs, cows, chickens, amphibians, reptiles, etc.
The term “sample” refers to tissue, bodily fluid, or a cell (or a fraction of any of the foregoing) taken from a patient or a subject. Normally, the tissue or cell will be removed from the patient, but in vivo diagnosis is also contemplated. Other samples, including urine, tears, serum, plasma, cerebrospinal fluid, feces, sputum, cell extracts etc. can also be useful for particular cancers.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be optionally replaced with either of the other two terms, thus describing alternative aspects of the scope of the subject matter. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. The use of “or” or “and” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration and the like, encompasses variations of up to ±10% from the specified value. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, etc., used herein are to be understood as being modified by the term “about”.
As used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” includes “A and B,” “A or B,” “A” alone, and “B” alone. Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” encompasses each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.
As used herein, the terms “ug” and “microgram” are used interchangeably with “μg” in the following sections. As used herein, the terms “uM” and “micromolar” are used interchangeably with “μM” in the following sections.
Various aspects described herein are described in further detail in the following subsections.
In one aspect, provided herein is an isolated anti-hMPV antibody (i.e., an antibody that binds hMPV) or antigen binding fragment thereof.
In one aspect, provided herein is an isolated anti-hMPV antibody (e.g., recombinant humanized, chimeric, or human antibody) or antigen binding fragment thereof which comprises:
Functional features of the anti-hMPV antibodies or antigen binding fragment thereof provided herein are described below in more detail.
In some embodiments, the anti-hMPV antibody or antigen binding fragment thereof described herein binds to a portion of hMPV (e.g., site α, site β, site II, site III, site IV and site V) with a KD described in the following Examples.
An antibody or antigen binding fragment thereof that exhibits one or more of the functional properties described above (e.g., biochemical, immunochemical, cellular, physiological or other biological activities), as determined using methods known to the art and described herein, will be understood to relate to a statistically significant difference in the particular activity relative to that seen in the absence of the antibody (e.g., or when a control antibody of irrelevant specificity is present). Preferably, the anti-hMPV antibody-induced increases in a measured parameter effects a statistically significant increase by at least 10% of the measured parameter, more preferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% (i.e., 2 fold), 3 fold, 5 fold or 10 fold. Conversely, anti-hMPV antibody-induced decreases in a measured parameter (e.g., hMPV activity) effects a statistically significant decrease by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%.
Also provided herein are anti-hMPV antibodies that bind to the same epitope on hMPV as any of the antibodies described herein. These antibodies have the ability to cross-compete for binding to hMPV with any of the antibodies described herein.
Antibodies disclosed herein include all known forms of antibodies and other protein scaffolds with antibody-like properties. For example, the antibody can be a human antibody, a humanized antibody, a bispecific antibody, an immunoconjugate, a chimeric antibody, or a protein scaffold with antibody-like properties, such as fibronectin or ankyrin repeats.
In some embodiments, the antibody is a bispecific antibody comprising a first and second binding region, wherein the first binding region comprises the binding specificity (e.g., antigen binding region) of an anti-hMPV antibody described herein, and a second binding region that does not bind to hMPV. In some embodiments, the second binding region binds to a protein that is not expressed on platelets.
The antibody also can be a Fab, F(ab′)2, scFv, AFFIBODY, avimer, nanobody, single chain antibody, or a domain antibody. The antibody also can have any isotype, including any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. Full-length antibodies can be prepared from VH and VL sequences using standard recombinant DNA techniques and nucleic acid encoding the desired constant region sequences to be operatively linked to the variable region sequences.
In certain embodiments, the antibodies described herein may have effector function or may have reduced or no effector function. In certain embodiments, the antibodies comprise an effector-less or mostly effector-less Fc, e.g., IgG2 or IgG4. Generally, variable regions described herein may be linked to an Fc comprising one or more modification, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody described herein may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.
In some embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and/or biological activity. For example, modifications can be made in the Fc region in order to generate an Fc variant that (a) has increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement mediated cytotoxicity (CDC), (c) has increased or decreased affinity for C1q and/or (d) has increased or decreased affinity for a Fc receptor relative to the parent Fc. Such Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable. For example, the variant Fc region may include two, three, four, five, etc. substitutions therein, e.g. of the specific Fc region positions identified herein.
A variant Fc region may also comprise a sequence alteration wherein amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even when cysteine residues are removed, single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently. In other embodiments, the Fc region may be modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc region, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. In other embodiments, one or more glycosylation sites within the Fc domain may be removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with non-glycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement, such as the C1q binding site, may be removed from the Fc region. For example, one may delete or substitute the EKK sequence of human IgG1. In certain embodiments, sites that affect binding to Fc receptors may be removed, preferably sites other than salvage receptor binding sites. In other embodiments, an Fc region may be modified to remove an ADCC site. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. Specific examples of variant Fc domains are disclosed for example, in PCT Publication numbers WO 97/34631 and WO 96/32478.
In one embodiment, the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment thereof such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745 by Ward et al.
In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al. In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 by Idusogie et al. In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication number WO 94/29351 by Bodmer et al.
In yet another example, the Fc region may be modified to increase antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity for an Fcγ receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T. Other modifications for enhancing Fc□R and complement interactions include but are not limited to substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.
Fc modifications that increase binding to an Fcγ receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (PCT Patent Publication number WO00/42072).
Other Fc modifications that can be made to Fes are those for reducing or ablating binding to FcγR and/or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index. Exemplary substitutions include but are not limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index. An Fc variant may comprise 236R/328R. Other modifications for reducing FcγR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 3305, 331 S, 2205, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.
Optionally, the Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publication numbers WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217, WO 05/092925 and WO 06/020114).
Fc variants that enhance affinity for an inhibitory receptor FcγRIIb may also be used. Such variants may provide an Fc fusion protein with immunomodulatory activities related to FcγRIIb+ cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcγRIIb relative to one or more activating receptors. Modifications for altering binding to FcγRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index. Exemplary substitutions for enhancing FcγRIIb affinity include but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcγRIIb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.
In certain embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this may be done by increasing the binding affinity of the Fc region for FcRn. For example, one or more of more of following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and/or T256F. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 31 1A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 1 S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/31 IS (Dall Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671. In certain embodiments, hybrid IgG isotypes with particular biological characteristics may be used. For example, an IgG1/IgG3 hybrid variant may be constructed by substituting IgG1 positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. Thus a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other embodiments described herein, an IgG1/IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and/or CH3 region with amino acids from IgG1 at positions where the two isotypes differ. Thus a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to an insertion of a glycine at position 236), and 327A.
Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A, which has been shown to exhibit enhanced FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with S239D/1332E and S239D/1332E/A330L mutations which showed the greatest increase in affinity for FcγRIIIa, a decrease in FcγRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of the triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2/neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, and the S239D/1332E variant showed an enhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgG1 mutants containing L235V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcγRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcγRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that may be used include: S298A/E333A/L334A, S239D/1332E, S239D/1332E/A330L, L235V/F243L/R292P/Y300L/P396L, and M428L/N434S.
When using an IgG4 constant domain, it is usually preferable to include the substitution S228P, which mimics the hinge sequence in IgG1 and thereby stabilizes IgG4 molecules.
In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al. Glycosylation of the constant region on N297 may be prevented by mutating the N297 residue to another residue, e.g., N297A, and/or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.
Additionally, or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies described herein to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication number WO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication number WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).
Another modification of the antibodies described herein is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment thereof. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See for example, European patent number EP 0 154 316 by Nishimura et al. and European patent number EP 0 401 384 by Ishikawa et al.
The affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.
II. Antibodies which Bind to Same Epitope as or Cross-Compete with Anti-hMPV Antibodies
Anti-hMPV antibodies which bind to the same or similar epitopes to the antibodies disclosed herein (and thus also cross-compete with the antibodies disclosed herein) may be raised using immunization protocols. The resulting antibodies can be screened for high affinity binding to hMPV. Selected antibodies can then be studied, e.g., in yeast display assay in which sequence variants of hMPV are presented on the surface of yeast cells, or by hydrogen-deuterium exchange experiments, to determine the precise epitope bound by the antibody.
The epitope to which an antibody binds can be determined using art-recognized methods. An hMPV antibody is considered to bind to the same epitope as a reference anti-hMPV antibody if it, e.g., contacts one or more of the same residues on hMPV as the reference antibody; contacts one or more of the same residues within at least one region of hMPV as the reference antibody; contacts a majority of residues within at least one region of hMPV as the reference antibody; contacts a majority of the same residues within each region of hMPV as the reference antibody; contacts a majority of the same residues along the entire length of hMPV as the reference antibody; contacts all of the same distinct regions of hMPV as the reference antibody; contacts all of the same residues at any one region on hMPV as the reference antibody; or contacts all of the same residues at all of the same regions of hMPV as the reference antibody.
Techniques for determining antibodies that bind to the “same epitope on hMPV” with the hMPV antibodies described herein include x-ray analyses of crystals of antigen:antibody complexes, which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen binding fragments thereof or mutated variations of the antigen where loss of binding due to an amino acid modification within the antigen sequence indicates the epitope component. Methods may also rely on the ability of an antibody of interest to affinity isolate specific short peptides (either in native three-dimensional form or in denatured form) from combinatorial phage display peptide libraries or from a protease digest of the target protein. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that have been shown to map conformational discontinuous epitopes.
The epitope or region comprising the epitope can also be identified by screening for binding to a series of overlapping peptides spanning hMPV. Alternatively, the method of Jespers et al. (1994) Biotechnology 12:899 may be used to guide the selection of antibodies having the same epitope and therefore similar properties to the hMPV antibodies described herein. Using phage display, first, the heavy chain of the anti-hMPV antibody is paired with a repertoire of (e.g., human) light chains to select a hMPV-binding antibody, and then the new light chain is paired with a repertoire of (e.g., human) heavy chains to select a (e.g., human) hMPV-binding antibody having the same epitope or epitope region as an anti-hMPV antibody described herein. Alternatively, variants of an antibody described herein can be obtained by mutagenesis of cDNA sequences encoding the heavy and light chains of the antibody.
Alanine scanning mutagenesis, as described by Cunningham & Wells (1989) Science 244: 1081, or some other form of point mutagenesis of amino acid residues in hMPV may also be used to determine the functional epitope for an anti-hMPV antibody.
The epitope or epitope region (an “epitope region” is a region comprising the epitope or overlapping with the epitope) bound by a specific antibody may also be determined by assessing binding of the antibody to peptides comprising hMPV fragments. A series of overlapping peptides encompassing the hMPV sequence may be synthesized and screened for binding, e.g. in a direct ELISA, a competitive ELISA (where the peptide is assessed for its ability to prevent binding of an antibody to hMPV bound to a well of a microtiter plate), or on a chip. Such peptide screening methods may not be capable of detecting some discontinuous functional epitopes.
An epitope may also be identified by MS-based protein footprinting, such as HDX-MS and Fast Photochemical Oxidation of Proteins (FPOP), structural methods such as X-ray crystal structure determination, molecular modeling, and nuclear magnetic resonance spectroscopy.
Single particle cryo-electron microscopy (SP-Cryo-EM) can also be used to identify the epitope to which an antibody or antigen binding fragment thereof binds. SP-Cryo-EM is a technique for macromolecular structure analysis which uses a high intensity electron beam to image biological specimens in their native environment at cryogenic temperature. In recent years, SP-cryo-EM has emerged as a complementary technique to crystallography and NMR for determining near-atomic level structures suitable for application in drug discovery (Renaud et al. Nat Rev Drug Discov 2018; 17:471-92; Scapin et al. Cell Chem Biol 2018; 25:1318-25; Ceska et al. Biochemical Society Transactions 2019: p. BST20180267). In addition to high resolution information, SP-Cryo-EM has the further advantage of allowing access to larger and more complex biological systems, with the possibility of characterizing multiple conformational or compositional solution states from the same sample, providing insights into more biologically relevant states of the macromolecule. For imaging, a small volume of sample (e.g., 3 microliter, 3 μl, aliquot) is applied onto a grid and flash-frozen in a liquid ethane bath. The frozen grid is then loaded into the microscope and hundreds to thousands of images of different areas of the grids are collected. These images contain two-dimensional projections of the biological macromolecule (particles): using mathematical tools and GPU powered algorithms, the particles are identified, extracted, and classified; in the subsequent step, the different classes are used to compute one or more 3D reconstructions, corresponding to different conformations, oligomerization or binding states if they coexist in the same sample. The individual reconstructions can then be refined to high resolution.
Also provided herein are nucleic acid molecules that encode the anti-hMPV antibodies or antigen binding fragments thereof, as well as the hMPV F proteins and variants thereof described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid described herein can be, for example, DNA or RNA and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule. The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library.
In some embodiments, provided herein are nucleic acid molecules that encode the VH and/or VL sequences, or heavy and/or light chain sequences, of any of the anti-hMPV antibodies or antigen binding fragments thereof described herein. Host cells comprising the nucleotide sequences (e.g., nucleic acid molecules) described herein are encompassed herein. Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (hinge, CH1, CH2 and/or CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.
Also provided herein are nucleic acid molecules with conservative substitutions that do not alter the resulting amino acid sequence upon translation of the nucleic acid molecule.
Monoclonal antibodies that bind hMPV can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
Various methods for making monoclonal antibodies described herein are available in the art. For example, the monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or any later developments thereof, or by recombinant DNA methods (U.S. Pat. No. 4,816,567). For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammer-ling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In another example, antibodies useful in the methods and compositions described herein can also be generated using various phage display methods known in the art, such as isolation from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (e.g., nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
Human antibodies can be made by a variety of methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publication numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, the contents of which are herein incorporated by reference in their entireties. Human antibodies can also be produced using transgenic mice which express human immunoglobulin genes, and upon immunization are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For an overview of this technology for producing human antibodies, see, Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93. Phage display technology (McCafferty et al., Nature 348:552-553 (1990)) also can be used to produce human antibodies and antibody binding fragments thereof in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. Human antibodies can also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275, the contents of which are herein incorporated by reference in their entireties). Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al., 1994, Bio/technology 12:899-903).
Chimeric antibodies can be prepared based on the sequence of a murine monoclonal antibody. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.).
Humanized forms of anti-hMPV antibodies (e.g., humanized forms of mouse anti-hMPV antibodies) are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are typically human immunoglobulins (recipient antibody) in which residues from a CDR or hypervariable region of the recipient are replaced by residues from a CDR or hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework can be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond exactly to either the donor antibody or the consensus framework. As used herein, the term “consensus framework” refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term “consensus immunoglobulin sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see e.g., Winnaker, From Genes to Clones (Veriagsgesellschaft, Weinheim, Germany 1987). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. Where two amino acids occur equally frequently, either can be included in the consensus sequence. As used herein, “Vernier zone” refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and can impact on the structure of CDRs and the affinity of the antibody. Human immunoglobulin (Ig) sequences that can be used as a recipient are well known in the art.
Framework residues in the human framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies can be humanized using a variety of techniques known in the art, including, but not limited to, those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239: 1534 (1988), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publication number WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530, 101, 5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein by reference.
The anti-hMPV antibodies generated using the methods described above can be tested for desired functions, such as particular binding specificities, binding affinities, targeted cell populations, using methods known in the art and described in the Examples, for example, art-recognized protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays. An aspect of the invention provides molecules that may be used to screen for an antibody or antigen binding fragment thereof that binds hMPV. Exemplary assays include, but are not limited to, immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), FACS, enzyme-linked immunoabsorbent assay (ELISA), bio-layer interferometry (e.g., ForteBio assay), and Scatchard analysis.
Further included are embodiments in which the anti-hMPV antibodies or antigen-binding fragments thereof described herein (e.g., antibodies or antigen binding fragments thereof in Examples 1-6) are engineered antibodies to include modifications to framework residues within the variable domains of the parental monoclonal antibody, e.g., to improve the properties of the antibody or antigen binding fragment thereof. Typically, such framework modifications are made to decrease the immunogenicity of the antibody or antigen binding fragment thereof. This is usually accomplished by replacing non-CDR residues in the variable domains (i.e., framework residues) in a parental (e.g., rodent) antibody or antigen binding fragment thereof with analogous residues from the immune repertoire of the species in which the antibody is to be used, e.g., human residues in the case of human therapeutics. Such an antibody or antigen binding fragment thereof is referred to as a “humanized” antibody or antigen binding fragment thereof. In some embodiments, it is desirable to increase the affinity, or alter the specificity of an engineered (e.g., humanized) antibody. One approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody or antigen binding fragment thereof that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody or antigen binding fragment thereof framework sequences to the germline sequences from which the antibody or antigen binding fragment thereof is derived. Another approach is to revert to the original parental (e.g., rodent) residue at one or more positions of the engineered (e.g. humanized) antibody, e.g. to restore binding affinity that may have been lost in the process of replacing the framework residues. (See, e.g., U.S. Pat. Nos. 5,693,762, 5,585,089 and 5,530,101.)
In certain embodiments, the anti-hMPV antibodies and antigen binding fragments thereof are engineered (e.g., modifications in the framework and/or CDRs to improve their properties. Such engineered changes can be based on molecular modeling. A molecular model for the variable region for the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that can interact with the antigen. Conventional CDRs are based on alignment of immunoglobulin sequences and identifying variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and coworkers carefully examined conformations of the loops in crystal structures of antibodies and proposed hypervariable loops. Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There are variations between regions classified as “CDRs” and “hypervariable loops”. Later studies (Raghunathan et al., (2012) J. Mol Recog. 25, 3, 103-113) analyzed several antibody-antigen crystal complexes and observed that the antigen binding regions in antibodies do not necessarily conform strictly to the “CDR” residues or “hypervariable” loops. The molecular model for the variable region of the non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen. In practice, the potential antigen binding regions based on model differ from the conventional “CDR”s or “hyper variable” loops. Commercial scientific software such as MOE (Chemical Computing Group) can be used for molecular modeling. Human frameworks can be selected based on best matches with the non-human sequence both in the frameworks and in the CDRs. For FR4 (framework 4) in VH, VJ regions for the human germlines are compared with the corresponding non-human region. In the case of FR4 (framework 4) in VL, J-kappa and J-Lambda regions of human germline sequences are compared with the corresponding non-human region. Once suitable human frameworks are identified, the CDRs are grafted into the selected human frameworks. In some cases, certain residues in the VL-VH interface can be retained as in the non-human (parental) sequence. Molecular models can also be used for identifying residues that can potentially alter the CDR conformations and hence binding to antigen. In some cases, these residues are retained as in the non-human (parental) sequence. Molecular models can also be used to identify solvent exposed amino acids that can result in unwanted effects such as glycosylation, deamidation and oxidation. Developability filters can be introduced early on in the design stage to eliminate/minimize these potential problems.
Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Pat. No. 7,125,689.
In particular embodiments, it will be desirable to change certain amino acids containing exposed side-chain(s) to another amino acid residue in order to provide for greater chemical stability of the final antibody, so as to avoid deamidation or isomerization. The deamidation of asparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect). Isomerization can occur at DG, DS, DA or DT sequences. In certain embodiments, the antibodies of the present disclosure do not contain deamidation or asparagine isomerism sites. For example, an asparagine (Asn) residue may be changed to Gln or Ala to reduce the potential for formation of isoaspartate at any Asn-Gly sequences, particularly within a CDR.
A similar problem may occur at an Asp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartate formation may debilitate or completely abrogate binding of an antibody to its target antigen. See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In various embodiment, the asparagine is changed to glutamine (Gln). It may also be desirable to alter an amino acid adjacent to an asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation, which occurs at greater rates when small amino acids occur adjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, any methionine residues (typically solvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe or other amino acids in order to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigen binding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Id. Additionally, in order to prevent or minimize potential scissile Asn-Pro peptide bonds, it may be desirable to alter any Asn-Pro combinations found in a CDR to Gln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are subsequently screened to ensure that the substitutions do not decrease the affinity or specificity of the antibody for hMPV, or other desired biological activity to unacceptable levels.
The antibodies (i.e., anti-hMPV antibodies) and antigen binding fragments thereof disclosed herein can also be engineered to include modifications within the Fc region, typically to alter one or more properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity). Furthermore, the antibodies and antigen binding fragments thereof disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more properties of the antibody or antigen binding fragment thereof. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.
The antibodies and antigen binding fragments thereof disclosed herein also include antibodies and antigen binding fragments thereof with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; and PCT Publication numbers WO2003/086310; WO2005/120571; WO2006/0057702. Such modifications can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc regions. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.
In one embodiment, the antibody or antigen binding fragment thereof of the invention is an IgG4 isotype antibody or antigen binding fragment thereof comprising a Serine to Proline mutation at a position corresponding to position 228 (S228P; EU index) in the hinge region of the heavy chain constant region. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al. supra; position 241 is based on the Kabat numbering system).
In one embodiment of the invention, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
In another embodiment, the Fc hinge region of an antibody or antigen binding fragment thereof of the invention is mutated to decrease the biological half-life of the antibody or antigen binding fragment thereof. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody or antigen binding fragment thereof has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745.
In another embodiment, the antibody or antigen binding fragment thereof of the invention is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022.
In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody or antigen binding fragment thereof. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand and retains the antigen binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260.
In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551.
In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication number WO1994/29351.
In yet another example, the Fc region is modified to decrease the ability of the antibody or antigen binding fragment thereof of the invention to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody or antigen binding fragment thereof for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication number WO 00/42072. Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al. (2001) J. Biol. Chem. 276:6591-6604).
In one embodiment of the invention, the Fc region is modified to decrease the ability of the antibody of the invention to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody or antigen binding fragment thereof is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody or antigen binding fragment thereof to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.
In some embodiments, the Fc region of an anti-hMPV antibody is modified to increase or reduce the ability of the antibody or antigen binding fragment thereof to mediate effector function and/or to increase/decrease their binding to the Fc gamma receptors (FcγRs).
The interaction between the constant region of an antigen binding protein and various Fc receptors (FcR) including FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) is believed to mediate the effector functions, such as ADCC and CDC, of the antigen binding protein. The Fc receptor is also important for antibody cross-linking, which can be important for anti-tumor immunity.
Effector function can be measured in a number of ways including for example via binding of the FcγRIII to Natural Killer cells or via FcγRI to monocytes/macrophages to measure for ADCC effector function. For example, an antigen binding protein of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields et al., 2001 J. Biol. Chem., Vol. 276, p 6591-6604; Chappel et al., 1993 J. Biol. Chem., Vol 268, p 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-4010.
Human IgG1 constant regions containing specific mutations or altered glycosylation on residue Asn297 have been shown to reduce binding to Fc receptors. In other cases, mutations have also been shown to enhance ADCC and CDC (Lazar et al. PNAS 2006, 103; 4005-4010; Shields et al. J Biol Chem 2001, 276; 6591-6604; Nechansky et al. Mol Immunol, 2007, 44; 1815-1817).
In one embodiment of the present invention, such mutations are in one or more of positions selected from 239, 332 and 330 (IgG1), or the equivalent positions in other IgG isotypes. Examples of suitable mutations are S239D and 1332E and A330L. In one embodiment, the antigen binding protein of the invention herein described is mutated at positions 239 and 332, for example S239D and 1332E or in a further embodiment it is mutated at three or more positions selected from 239 and 332 and 330, for example S239D and 1332E and A330L. (EU index numbering).
In an alternative embodiment of the present invention, there is provided an antibody comprising a heavy chain constant region with an altered glycosylation profile such that the antigen binding protein has enhanced effector function. For example, wherein the antibody has enhanced ADCC or enhanced CDC or wherein it has both enhanced ADCC and CDC effector function. Examples of suitable methodologies to produce antigen binding proteins with an altered glycosylation profile are described in PCT Publication numbers WO2003011878 and WO2006014679 and European patent number EP1229125.
In a further aspect, the present invention provides “non-fucosylated” or “afucosylated” antibodies. Non-fucosylated antibodies harbor a tri-mannosyl core structure of complex-type N-glycans of Fc without fucose residue. These glycoengineered antibodies that lack core fucose residue from the Fe N-glycans may exhibit stronger ADCC than fucosylated equivalents due to enhancement of FcγRIIIa binding capacity.
The present invention also provides a method for the production of an antibody according to the invention comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising the isolated nucleic acid as described herein, wherein the recombinant host cell does not comprise an alpha-1,6-fucosyltransferase; and b) recovering the antigen binding protein. The recombinant host cell may not normally contain a gene encoding an alpha-1,6-fucosyltransferase (for example yeast host cells such as Pichia sp.) or may have been genetically modified to inactivate an alpha-1,6-fucosyltransferase. Recombinant host cells which have been genetically modified to inactivate the FUT8 gene encoding an alpha-1,6-fucosyltransferase are available. See, e.g., the POTELLIGENT™ technology system available from BioWa, Inc. (Princeton, N.J.) in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies having enhanced antibody dependent cell mediated cytotoxicity (ADCC) activity that is increased relative to an identical monoclonal antibody produced in a cell with a functional FUT8 gene. Aspects ofthe POTELLIGENT™ technology system are described in U.S. Pat. Nos. U.S. Pat. Nos. 7,214,775 and 6,946,292, and PCT Publication numbers WO0061739 and WO0231240. Those of ordinary skill in the art will also recognize other appropriate systems.
It will be apparent to those skilled in the art that such modifications may not only be used alone but may be used in combination with each other in order to further enhance or decrease effector function.
Production of Antibodies with Modified Glycosylation
In still another embodiment, the antibodies or antigen binding fragments thereof of the invention comprise a particular glycosylation pattern. For example, an afucosylated or an aglycosylated antibody or antigen binding fragment thereof can be made (i.e., the antibody lacks fucose or glycosylation, respectively). The glycosylation pattern of an antibody or antigen binding fragment thereof may be altered to, for example, increase the affinity or avidity of the antibody or fragment for hMPV. Such modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody or antigen binding fragment thereof sequence. For example, one or more amino acid substitutions can be made that result in removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity or avidity of the antibody or antigen binding fragment thereof for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.
Antibodies and antigen binding antigen binding fragments thereof disclosed herein may further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian- or human-like glycosylation patterns (See for example, Choi et al., (2003) Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton et al., (2003) Science 301: 1244-1246; Hamilton et al., (2006) Science 313: 1441-1443; Nett et al., Yeast 28(3):237-52 (2011); Hamilton et al., Curr Opin Biotechnol. October; 18(5):387-92 (2007)). A particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Pat. Nos. 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies that have predominantly particular N-glycan structures (See for example, Li et al., (2006) Nat. Biotechnol. 24: 210-215).
In particular embodiments, the antibodies and antigen binding fragments thereof disclosed herein further include those produced in lower eukaryotic host cells and which comprise fucosylated and non-fucosylated hybrid and complex N-glycans, including bisected and multi-antennary species, including but not limited to N-glycans such as GlcNAc(1-4)Man3GlcNAc2; Gal(1-4)GlcNAc(1-4)Man3GlcNAc2; NANA(1-4)Gal(1-4)GlcNAc(1-4)Man3GlcNAc2.
In particular embodiments, the antibodies and antigen binding fragments thereof provided herein may comprise antibodies or antigen binding fragments thereof having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In particular aspects, the hybrid N-glycan is the predominant N-glycan species in the composition.
In particular embodiments, the antibodies and antigen binding fragments thereof provided herein comprise antibodies and antigen binding fragments thereof having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In particular aspects, the complex N-glycan are the predominant N-glycan species in the composition. In further aspects, the complex N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans in the composition. In one embodiment, the antibody and antigen binding fragments thereof provided herein comprise complex N-glycans, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans comprise the structure NANA2Gal2GlcNAc2Man3GlcNAc2, wherein such structure is afucosylated. Such structures can be produced, e.g., in engineered Pichia pastoris host cells.
In particular embodiments, the N-glycan is fucosylated. In general, the fucose is in an α1,3-linkage with the GlcNAc at the reducing end of the N-glycan, an α1,6-linkage with the GlcNAc at the reducing end of the N-glycan, an α1,2-linkage with the Gal at the non-reducing end of the N-glycan, an α1,3-linkage with the GlcNac at the non-reducing end of the N-glycan, or an α1,4-linkage with a GlcNAc at the non-reducing end of the N-glycan. Therefore, in particular aspects of the above the glycoprotein compositions, the glycoform is in an α1,3-linkage or α1,6-linkage fucose to produce a glycoform selected from the group consisting of Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc); in an α1,3-linkage or α1,4-linkage fucose to produce a glycoform selected from the group consisting of GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc1-2)Man3GlcNAc2, GalGlcNAc2(Fuc1-2)Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc1-2)Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2; or in an α1,2-linkage fucose to produce a glycoform selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2, NANAGal2(Fuc1-2)GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2.
In further aspects, the antibodies (e.g., humanized antibodies) or antigen binding fragments thereof comprise high mannose N-glycans, including but not limited to, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or N-glycans that consist of the Man3GlcNAc2 N-glycan structure.
In further aspects of the above, the complex N-glycans further include fucosylated and non-fucosylated bisected and multi-antennary species.
As used herein, the terms “N-glycan” and “glycoform” are used interchangeably and refer to an N-linked oligosaccharide, for example, one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein. The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of the sugar groups occurs co-translationally in the lumen of the ER and continues post-translationally in the Golgi apparatus for N-linked glycoproteins. N-glycans have a common pentasaccharide core of Man3GlcNAc2 (“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). Usually, N-glycan structures are presented with the non-reducing end to the left and the reducing end to the right. The reducing end of the N-glycan is the end that is attached to the Asn residue comprising the glycosylation site on the protein. N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GlcNAc2 (“Man3”) core structure which is also referred to as the “trimannose core”, the “pentasaccharide core” or the “paucimannose core”. N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid). A “high mannose” type N-glycan has five or more mannose residues. A “complex” type N-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a “trimannose” core. Complex N-glycans may also have galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residues that are optionally modified with sialic acid or derivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl). Complex N-glycans may also have intrachain substitutions comprising “bisecting” GlcNAc and core fucose (“Fuc”). Complex N-glycans may also have multiple antennae on the “trimannose core,” often referred to as “multiple antennary glycans.” A “hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core. The various N-glycans are also referred to as “glycoforms”.
With respect to complex N-glycans, the terms “G-2”, “G-1”, “G0”, “G1”, “G2”, “Al”, and “A2” mean the following. “G-2” refers to an N-glycan structure that can be characterized as Man3GlcNAc2; the term “G-1” refers to an N-glycan structure that can be characterized as GlcNAcMan3GlcNAc2; the term “G0” refers to an N-glycan structure that can be characterized as GlcNAc2Man3GlcNAc2; the term “G1” refers to an N-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2; the term “G2” refers to an N-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2; the term “Al” refers to an N-glycan structure that can be characterized as NANAGal2GlcNAc2Man3GlcNAc2; and, the term “A2” refers to an N-glycan structure that can be characterized as NANA2Gal2GlcNAc2Man3GlcNAc2. Unless otherwise indicated, the terms G-2”, “G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycan species that lack fucose attached to the GlcNAc residue at the reducing end of the N-glycan. When the term includes an “F”, the “F” indicates that the N-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2F all indicate that the N-glycan further includes a fucose residue attached to the GlcNAc residue at the reducing end of the N-glycan. Lower eukaryotes such as yeast and filamentous fungi do not normally produce N-glycans that produce fucose.
With respect to multi-antennary N-glycans, the term “multi-antennary N-glycan” refers to N-glycans that further comprise a GlcNAc residue on the mannose residue comprising the non-reducing end of the 1,6 arm or the 1,3 arm of the N-glycan or a GlcNAc residue on each of the mannose residues comprising the non-reducing end of the 1,6 arm and the 1,3 arm of the N-glycan. Thus, multi-antennary N-glycans can be characterized by the formulas GlcNAc(2-4)Man3GlcNAc2, Gal(1-4)GlcNAc(2-4)Man3GlcNAc2, or NANA(1-4)Gal(1-4)GlcNAc(2-4)Man3GlcNAc2. The term “1-4” refers to 1, 2, 3, or 4 residues. With respect to bisected N-glycans, the term “bisected N-glycan” refers to N-glycans in which a GlcNAc residue is linked to the mannose residue at the reducing end of the N-glycan. A bisected N-glycan can be characterized by the formula GlcNAc3Man3GlcNAc2 wherein each mannose residue is linked at its non-reducing end to a GlcNAc residue. In contrast, when a multi-antennary N-glycan is characterized as GlcNAc3Man3GlcNAc2, the formula indicates that two GlcNAc residues are linked to the mannose residue at the non-reducing end of one of the two arms of the N-glycans and one GlcNAc residue is linked to the mannose residue at the non-reducing end of the other arm of the N-glycan.
The antibodies and antigen binding fragments thereof disclosed herein may further contain one or more glycosylation sites in either the light or heavy chain immunoglobulin variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or antigen binding fragment thereof or an alteration of the PK of the antibody due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation has been known to occur at motifs containing an N-X-S/T sequence.
Each antibody or antigen binding fragment thereof will have a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). In general, the TM1 (the temperature of initial unfolding) may be greater than 60° C., greater than 65° C., or greater than 70° C. The melting point of an antibody or antigen binding fragment thereof can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). In a further embodiment, antibodies and antigen binding fragments thereof are selected that do not degrade rapidly. Degradation of an antibody or antigen binding fragment thereof can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).
In a further embodiment, antibodies and antigen binding fragments thereof are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies and antigen binding fragments thereof are acceptable with aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.
Multi-specific antibodies (e.g., bispecific antibodies) provided herein include at least one binding region for a particular epitope on hMPV as described herein, and at least one other binding region (e.g., a viral antigen). Multi-specific antibodies can be prepared as full-length antibodies or antigen binding fragments thereof (e.g. F(ab′)2 antibodies).
Methods for making multi-specific antibodies are well known in the art (see, e.g., PCT Publication numbers WO 05117973 and WO 06091209). For example, production of full length multi-specific antibodies can be based on the co-expression of two paired immunoglobulin heavy chain-light chains, where the two chains have different specificities. Various techniques for making and isolating multi-specific antibody fragments directly from recombinant cell culture have also been described. For example, multi-specific antibodies can be produced using leucine zippers. Another strategy for making multi-specific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported.
Examples of suitable multi-specific molecule platforms include, but are not limited to, Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), Fcab and mAb2 (F-Star), CovX-body (CovX/Pfizer), Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (ImClone/Eli Lilly), Ts2Ab (Medlmmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec), TvAb (Roche), ScFv/Fc Fusions, SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), Dual(ScFv)2-Fab (National Research Center for Antibody Medicine—China), F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), SEED (EMD Serono), mAb2 (F-star), Fab-Fv (UCB-Celltech), Bispecific T Cell Engager (BiTE) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), and Fc-engineered IgG1 (Xencor). Different bispecific formats are described in Spiess et al., 2015 Molecular Immunology, vol. 67, issue 2, Part A, pages 95-106.
In a particular embodiment, the multi-specific antibody comprises a first antibody (or binding portion thereof) which binds to hMPV derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a multi-specific molecule that binds to hMPV and a non-hMPV target molecule. An antibody may be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites and/or target molecules. To create a multi-specific molecule, an antibody disclosed herein can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody or antigen binding fragment thereof, antibody fragment, peptide, receptor, or binding mimetic, such that a multi-specific molecule results.
Accordingly, multi-specific molecules, for example, bispecific antibodies and bifunctional antibodies, comprising at least one first binding specificity for a particular epitope on hMPV and a second binding specificity for a second target are contemplated. In some embodiments, the second target is the second binding region specifically binds to a viral-associated antigen. Viral-associated antigens are well known in the art.
In some embodiments, the antibody is a trispecific antibody comprising a first, second, and third binding region, wherein the first binding region comprises the binding specificity (e.g., antigen binding region) of an anti-hMPV antibody described herein, and the second and third binding regions bind to two different targets (or different epitopes on the same target), for example, the targets described above.
In one embodiment, the multi-specific molecules comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., a Fab, Fab′, F(ab′)2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Pat. No. 4,946,778.
The multi-specific molecules can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-hMPV binding specificities, using methods known in the art. For example, each binding specificity of the multi-specific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).
When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the multi-specific molecule is a mAb×mAb, mAb×Fab, Fab×F(ab′)2 or ligand x Fab fusion protein. A multi-specific molecule can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Multi-specific molecules may comprise at least two single chain molecules. Methods for preparing multi-specific molecules are described for example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.
Binding of the multi-specific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS) analysis, bioassay (e.g., growth inhibition), or western blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA). The radioactive isotope can be detected by such means as the use of a a y-O counter or a scintillation counter or by autoradiography.
In one aspect, provided herein are isolated hMPV F polypeptides and variants thereof. The hMPV F protein polypeptides and variants thereof described herein can be used to induce an active immunogenic response in a subject or to generate antibodies from B cells in vitro.
Cleavable affinity tags may be added to the sequence of the hMPV F polypeptides and variants thereof. As used herein, the term “cleavable affinity tag” refers to a protein sequence on the N or C terminus of the recombinant hMPV F polypeptides described herein for an enzymatic cleavage sequence and a polypeptide sequence which can be used to purify the recombinant hMPV F protein polypeptide by affinity chromatography. Such tags and cleavage sequences are described by Kimple et al., Current Protocols in Protein Science 9.9.1-9.9.23, August 2013. Exemplary affinity tag sequences include, but are not limited to: protein A; lacZ; polyhistidine (HHHHHH, SEQ ID NO: 1014; also called 6×His, His-tag, or His6); Glutathione-S-Transferase (GST); maltose-binding protein (MBP); calmodulin-binding protein (CBP); biotin-based tags (e.g., BCCP); streptavidin-based tags (e.g. strep tag II; SAWSHPQFEK; SEQ ID NO: 1015); mutant thioredoxin (e.g. His-Patch ThioFusion™); FLAG-tag; hemagglutinin (HA); c-myc; T7, Glu-Glu; ALFA-tag; V5-tag; Spot-Tag™; novel epitope tag (NE-tag); β-galactosidase (β-gal); alkaline phosphatase (AP); chloramphenicol acetyl transferase (CAT); horseradish peroxidase (HRP); modified haloalkane dehalogenase (HaloTag™); and modified serine protease subtilisin (Profinity eXact™).
Exemplary cleavage sequences for removing the affinity tag may include, but are not limited to: thrombin protease cleavage sequence (LVPRGS, SEQ ID NO: 1013); factor Xa cleavage sequence (SEQ ID NO: 1016, IEGR; or SEQ ID NO: 1017, IDGR); PreScission™ protease cleavage site (SEQ ID NO: 1018, LEVLFQGP); enterokinase cleavage sequence (SEQ ID NO: 1019, DDDDK); genenase I cleavage sequence (SEQ ID NO: 1020, TQLAYFTDSKNPGAAHYDTFADSLR); and TEV protease cleavage sequence (SEQ ID NO: 1021, ENLYFQ).
In some embodiments, the tag may be recognized directly by a protease and result in cleavage of the affinity tag from the rest of the polypeptide. For example, the tag small ubiquitin-like modifier (SUMO) is recognized by SUMO protease via the SUMO tag's tertiary structure. In another example, a subtilisin prodomain tag (Profinity eXact™; BioRad Laboratories, Hercules CA) is recognized and cleaved by a mutant subtilisin protein. In another example, intein-chitin binding domain tag (intein-CBD; IMPACT™ System, New England Biolabs, Beverley MA) can self-cleave upon activation by a denaturing agent such as dithiothreitol.
Trimerization domain sequences can also be added to the hMPV F polypeptides and variants described herein. Such sequences can allow for trimerization of an antigenic polypeptide. Exemplary trimerization domain sequence may include, but are not limited to: a coiled-coil trimerization domain, such as a GCN4 domain (ARMKQIEDKIEEILSKIYHIENEIARIKKLIGEAGSG; SEQ ID NO: 1022); a foldon domain from T4 fibritin (GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO: 1012); a human type XV collagen domain (SEQ ID NO: 1023); and a human type XVIII collagen domain (SEQ ID NO: 1024).
The protease cleavage site at residues 99-102 of SEQ ID NO: 910 and 911 can be replaced with a furin-cleavage sequence. Furin-cleavage sequences are traditionally described by the consensus sequence RXRR (SEQ ID NO: 1025) or RXKR (SEQ ID NO: 1026), wherein X is any amino acid. In various embodiments, residues 99-102 of SEQ ID NO: 910 are substituted with SEQ ID NO: 1025 or 1026. In various embodiments residues 99-102 of SEQ ID NO: 910 are substituted with SEQ ID NO: 1027 (RGRR), SEQ ID NO: 1028 (RARR), SEQ ID NO: 1029 (RLRR), SEQ ID NO: 1030 (RMRR), SEQ ID NO: 1031 (RFRR), SEQ ID NO: 1032 (RWRR), SEQ ID NO: 1033 (RKRR), SEQ ID NO: 1034 (RQRR), SEQ ID NO: 1035 (RERR), SEQ ID NO: 1036 (RSRR), SEQ ID NO: 1037 (RPRR), SEQ ID NO: 1038 (RVRR), SEQ ID NO: 1039 (RIRR), SEQ ID NO: 1040 (RCRR), SEQ ID NO: 1041 (RYRR), SEQ ID NO: 1042 (RHRR), SEQ ID NO: 1043 (RRRR), SEQ ID NO: 1044 (RNRR), SEQ ID NO: 1045 (RDRR), SEQ ID NO: 1046 (RTRR), SEQ ID NO: 1047 (RGKR), SEQ ID NO: 1048 (RAKR), SEQ ID NO: 1049 (RLKR), SEQ ID NO: 1050 (RMKR), SEQ ID NO: 1051 (RFKR), SEQ ID NO: 1052 (RWKR), SEQ ID NO: 1053 (RKKR), SEQ ID NO: 1054 (RQKR), SEQ ID NO: 1055 (REKR), SEQ ID NO: 1056 (RSKR), SEQ ID NO: 1057 (RPKR), SEQ ID NO: 1058 (RVKR), SEQ ID NO: 1059 (RIKR), SEQ ID NO: 1060 (RCKR), SEQ ID NO: 1061 (RYKR), SEQ ID NO: 1062 (RHKR), SEQ ID NO: 1063 (RRKR), SEQ ID NO: 1064 (RNKR), SEQ ID NO: 1065 (RDKR), or SEQ ID NO: 1066 (RTKR).
The anti-hMPV antibodies or antigen binding fragments thereof disclosed herein can be tested for desired properties, e.g., those described herein, using a variety of assays known in the art.
In one embodiment, the antibodies or antigen binding fragments thereof are tested for specific binding to hMPV. Methods for analyzing binding affinity, cross-reactivity, and binding kinetics of various anti-hMPV antibodies or antigen binding fragments thereof include standard assays known in the art, for example, Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or bio-layer interferometry (e.g., ForteBio assay), as described in the Examples.
In one embodiment, the antibodies or antigen binding fragments thereof are tested for the ability to bind to cells that have been transfected with hMPV.
In one embodiment, the antibodies or antigen binding fragments thereof are screened for the ability to bind to the surface of beads that have been coated with hMPV.
In one embodiment, the antibodies or antigen binding fragments thereof are tested for the ability to bind or affect hMPV. In another embodiment, the antibodies or antigen binding fragments thereof are tested for their effects on hMPV (e.g., inhibition, or no effect).
Also provided herein are compositions (e.g., pharmaceutical compositions) comprising the anti-hMPV antibodies or antigen binding fragments thereof described herein, immunoconjugates comprising the same, or bispecific antibodies comprising the same, and a carrier (e.g., pharmaceutically acceptable carrier). Such compositions are useful for various therapeutic applications.
In some embodiments, pharmaceutical compositions disclosed herein can include other compounds, drugs, and/or agents used for the treatment of various diseases (e.g., respiratory diseases). Such compounds, drugs, and/or agents can include, for example, an anti-viral agent, and/or an anti-inflammatory agent. Exemplary compounds, drugs, and agents that can be formulated together or separately with the anti-hMPV antibodies or antigen binding fragments thereof described herein.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
A pharmaceutical composition described herein may also include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions described herein is contemplated. A pharmaceutical composition may comprise a preservative or may be devoid of a preservative. Supplementary active compounds can be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms described herein are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
For administration of the antibody or antigen binding fragment thereof, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 or 10 mg/kg, of the host body weight. For administration of the antibody or antigen binding fragment thereof, the dosage ranges from about 0.0001 to 100 mg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
An antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions described herein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The therapeutically effective dosage of an anti-hMPV antibody or antigen binding fragment thereof in various embodiments results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. In the context of cancer, a therapeutically effective dose preferably results in increased survival, and/or prevention of further deterioration of physical symptoms associated with cancer. A therapeutically effective dose may prevent or delay onset of cancer, such as may be desired when early or preliminary signs of the disease are present.
A composition described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Alternatively, an antibody or antigen binding fragment thereof described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition described herein can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules for use with anti-hMPV antibodies described herein include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.
In certain embodiments, the anti-hMPV antibodies or antigen binding fragments thereof described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if desired, e.g., for brain cancers), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p 120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
hMPV immunogenic compositions may be formulated or administered alone or in conjunction with one or more other components. For instance, hMPV immunogenic compositions may comprise other components including, but not limited to, adjuvants.
In some embodiments, hMPV immunogenic compositions of the instant disclosure do not include an adjuvant (they are adjuvant-free).
Suitable adjuvants to enhance effectiveness of the immunogenic compositions disclosed herein include, but are not limited to:
In another embodiment, the adjuvant is a mixture of 2, 3, or more of the above adjuvants, e.g., SBAS2 (an oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).
Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1′-2′ dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
In certain embodiments, the adjuvant is an aluminum salt. The aluminum salt adjuvant may be an alum-precipitated immunogenic composition or an alum-adsorbed immunogenic composition. Aluminum-salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in Immunology 143:489-493). The aluminum salt includes, but is not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, Amphogel, aluminum (III) hydroxide, aluminum hydroxyphosphate sulfate, Aluminum Phosphate Adjuvant (APA), amorphous alumina, trihydrated alumina, or trihydroxy aluminum.
APA is an aqueous suspension of aluminum hydroxyphosphate. APA is manufactured by blending aluminum chloride and sodium phosphate in a 1:1 volumetric ratio to precipitate aluminum hydroxyphosphate. After the blending process, the material is size-reduced with a high-shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered against physiological saline and steam sterilized.
In certain embodiments, a commercially available Al(OH)3 (e.g. Alhydrogel or Superfos of Denmark/Accurate Chemical and Scientific Co., Westbury, NY) is used to adsorb proteins in a ratio of 50-200 μg protein/mg aluminum hydroxide. Adsorption of protein is dependent, in another embodiment, on the pI (Isoelectric pH) of the protein and the pH of the medium. A protein with a lower pI adsorbs to the positively charged aluminum ion more strongly than a protein with a higher pI. Aluminum salts may establish a depot of antigen that is released slowly over a period of 2-3 weeks, be involved in nonspecific activation of macrophages and complement activation, and/or stimulate innate immune mechanism (possibly through stimulation of uric acid). See, e.g., Lambrecht et al., 2009, Curr Opin Immunol 21:23.
In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, for example, a CpG-containing oligonucleotide, in particular, a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN 1826, which may be acquired from Coley Pharmaceutical Group.
Methods for use of CpG oligonucleotides are well known in the art and are described, for example, in Sur et al., 1999, J Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst. 23:89-110.
hMPV immunogenic compositions may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, immunogenic compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Immunogenic compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as immunogenic compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). In some embodiments, hMPV immunogenic compositions are administered to humans, human patients, or subjects.
Formulations of the hMPV immunogenic compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., polypeptide or polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
Compositions of this disclosure can be formulated as single dose vials, multi-dose vials or as pre-filled glass or plastic syringes.
In another embodiment, compositions of the present disclosure are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation. Solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
The pharmaceutical composition may be isotonic, hypotonic or hypertonic. However, it is often preferred that a pharmaceutical composition for infusion or injection is essentially isotonic, when it is administered. Hence, for storage the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it may be diluted to become an isotonic solution prior to administration.
The isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate. Examples of ionic isotonic agents include but are not limited to NaCl, CaCl2), KCl and MgCl2. Examples of non-ionic isotonic agents include but are not limited to mannitol, sorbitol and glycerol.
It is also preferred that at least one pharmaceutically-acceptable additive is a buffer. For some purposes, for example, when the pharmaceutical composition is meant for infusion or injection, it is often desirable that the composition comprises a buffer, which is capable of buffering a solution to a pH in the range of 4 to 10, such as 5 to 9, for example 6 to 8.
The buffer may, for example, be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.
The buffer may be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids such as citric and phosphoric; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.
Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, glycols such as propylene glycols or polyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), and Poloxamer 188 (P188) are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
The formulations of the disclosure may also contain a surfactant. Preferred surfactants include, but are not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially PS-20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the TERGITOL NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as BRIJ surfactants), such as triethyleneglycol monolauryl ether (BRIJ 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (SPAN 85) and sorbitan monolaurate.
Mixtures of surfactants can be used, e.g. PS-80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as PS-80) 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
hMPV immunogenic compositions may be administered by any route which results in a therapeutically-effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration. The present disclosure provides methods comprising administering immunogenic compositions to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. hMPV immunogenic compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of immunogenic compositions may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically-effective or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
In some embodiments, hMPV immunogenic compositions may be administered at dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see, e.g., the range of unit doses described in International Publication No WO2013/078199, the contents of which are herein incorporated by reference in their entirety). The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three months, every 6 months, etc. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. In exemplary embodiments, hMPV immunogenic compositions may be administered at dosage levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.
In some embodiments, hMPV immunogenic compositions may be administered once or twice (or more) at dosage levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.
Any of the hMPV immunogenic compositions described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, intranasal and subcutaneous).
hMPV Immunogenic Composition Formulations and Methods of Use
Some aspects of the present disclosure provide formulations of the hMPV immunogenic composition, wherein the immunogenic composition is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to an hMPV antigenic polypeptide). “An effective amount” is a dose of a immunogenic composition effective to produce an antigen-specific immune response. Also provided herein are methods of inducing an antigen-specific immune response in a subject.
In some embodiments, the antigen-specific immune response is characterized by measuring an anti-hMPV antigenic polypeptide antibody titer produced in a subject administered an hMPV immunogenic composition as provided herein. An antibody titer is a measurement of a concentration of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an hMPV antigenic polypeptide) or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.
In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous immunogenic composition was effective, and to identify any recent or prior infections. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by the hMPV immunogenic composition.
The antibodies, antibody compositions, and methods described herein have numerous in vitro and in vivo utilities.
For example, provided herein is a method of treating or preventing a viral disease associated with hMPV comprising administering to a subject in need thereof an anti-hMPV antibody or antigen binding fragment thereof described herein, such that the subject is treated, e.g., such that the viral load and/or viral activity is inhibited or reduced and/or that treatment of the viral disease is achieved.
In one embodiment, provided herein is a method of treating a viral disease associated with hMPV comprising administering to a subject in need thereof an effective amount (e.g., a therapeutically effective amount) of an anti-hMPV antibody described herein (or a bispecific antibody hMPV). In some embodiments, the subject is administered a further therapeutic agent. In some embodiments, the further therapeutic agent is an anti-viral agent, e.g., another anti-viral antibody or anti-viral small molecule
Also encompassed are methods for detecting the presence of hMPV in a sample (e.g., a blood sample), or measuring the amount of hMPV in sample, comprising contacting the sample (e.g., tissue) and a control sample (e.g., corresponding healthy tissue) with an antibody (e.g., monoclonal antibody) or antigen binding fragment thereof which specifically binds to hMPV under conditions that allow for formation of a complex or binding between the antibody or portion thereof and hMPV. The formation of a complex is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative of the presence of hMPV in the sample. The anti-hMPV antibodies or antigen binding fragments thereof described herein can also be used to purify hMPV via immunoaffinity purification.
Diagnostic applications of the anti-hMPV antibodies described herein are also contemplated.
In one embodiment, provided herein is a method of diagnosing a viral infection or viral disease associated with hMPV comprising contacting a biological sample from a patient afflicted with the viral infection or viral disease with an anti-hMPV antibody or antigen binding fragment thereof described herein, wherein positive staining with the antibody indicates the viral infection or viral disease is associated with hMPV.
The anti-hMPV antibodies or antigen binding fragments thereof described herein can be used in combination with various treatments, prophylactic agent, or therapeutic agent (or in the context of a multi-specific antibody or bifunctional partner) known in the art for the prevention or treatment of an infection (e.g., viral infection) and/or a disease (e.g., viral disease), as described herein. In one embodiment, the prophylactic agent or therapeutic agent comprises a second antibody or fragment thereof, an immunomodulator, a hormone, a cytotoxic agent, an enzyme, a radionuclide, a second antibody conjugated to at least one immunomodulator, enzyme, radioactive label, hormone, antisense oligonucleotide, or cytotoxic agent, or a combination thereof. In various embodiments, the therapeutic agent comprises a hormonal therapy, immunotherapy, an antiviral agent, and/or an anti-inflammatory agent.
Suitable anti-viral agents for use in combination therapy with the anti-hMPV antibodies or antigen binding fragments thereof described herein include, but are not limited to, an antiviral agent (e.g., small molecules and antibodies). In various embodiments, the antiviral agent comprises an anti-RSV antibody. In various embodiments, the antiviral agent comprises a nucleotide analogues, which interfere or stop DNA or RNA synthesis. In various embodiments, the antiviral agent comprises an inhibitor of an enzyme involved in DNA or RNA synthesis (e.g., helicase, replicase). In various embodiments, the antiviral agent comprises a compound which inhibits the virus maturation steps during its replication cycle. In various embodiments, the antiviral agent comprises a compound which interferes with cell membrane binding, or virus entry in host cells (e.g., fusion or entry inhibitors). In various embodiments, the antiviral agent comprises an agent which prevents the virus from being expressed within the host cell after its entry. In various embodiments, the antiviral agent comprises a compound that block the virus' disassembly within the cell. In various embodiments, the antiviral agent comprises an agent that restricts virus propagation to other cells.
The compositions and methods as recited herein can include multiple antibodies against the same antigen (i.e., MPV), or against multiple viruses or viral agents/antigens (e.g., RSV and MPV) and/or a bacterial agent/antigen. In various embodiments of the present invention, compositions and methods disclosed herein may include an antibody against MPV (e.g., hMPV), plus an antibody against a bacterial agent/antigen, for example one that infects the respiratory system.
The compositions and methods as recited herein can include an anti-viral antibody that binds to MPV as described herein, and/or a non-immunological anti-viral agent, such as ribavirin, amantadine, rimantadine, or a neuraminidase-inhibitor.
The compositions and methods as recited herein can include anti-infectious agent used in composition with an anti-MPV antibody, including any of the antibodies or antigen binding fragments thereof described herein, may be an anti-bacterial agent, including but not limited to a macrolide, a penicillin, a cephalosporin, or a tetracycline, or may be an antifungal agent, including but not limited to amphotericin b, fluconazole, or ketoconazole, or an anti-parasitic agent, including but not limited to trimethoprim, pentamidine, or a sulfonamide. The anti-infectious agent may be an anti-viral agent such as ribavirin, amantadine, rimantadine, or a neuraminidase-inhibitor. Such additional agents can also include agents useful against other viruses as well as other agents useful against MPV.
Combinations thereof are also specifically contemplated for the methods described herein.
In some embodiments, the combination of therapeutic antibodies discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially.
Also provided are kits comprising the anti-hMPV antibodies or antigen binding fragments thereof, multi-specific molecules, or immunoconjugates disclosed herein, optionally contained in a single vial or container, and include, e.g., instructions for use in treating or diagnosing a disease (e.g., viral respiratory disease). The kits may include a label indicating the intended use of the contents of the kit. The term label includes any writing, marketing materials or recorded material supplied on or with the kit, or which otherwise accompanies the kit. Such kits may comprise the antibody, multi-specific molecule, or immunoconjugate in unit dosage form, such as in a single dose vial or a single dose pre-loaded syringe.
The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.
Commercially available reagents referred to in the Examples below were used according to manufacturer's instructions unless otherwise indicated. Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in the following textbooks: Sambrook et al., supra; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.: N.Y., 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.
The plasmid construction and production of RSV PreF (DS-Cav1) was performed as previously described [31, 55]. The unprocessed hMPV PreF trimeric antigen was derived from a previously published F sequence of strain B2 (nucleotide sequence is SEQ ID NO: 1003, protein sequence is SEQ ID NO: 1) with a C-terminal GCN4 trimerization domain, which adopted a PreF-like structure (see SEQ ID NO: 910 below for entire sequence) [34]. The monomeric hMPV F antigen was derived from the same B2 strain but without the GCN4 domain (see SEQ ID NO: 911 below for entire sequence). The stabilized hMPV PreF construct (1 15BV) adopted the prefusion stabilizing mutations from a previous report [28], as well as a proline mutation A185P and insertion of a furin-cleavage site, on the backbone of wild-type strain B2 hMPV F trimeric antigen with C-terminal GCN4 domain (see
The Unprocessed hMPV PreF Trimeric Antigen Sequence:
The Unprocessed hMPV PreF Monomeric Antigen Sequence (Modified from the Trimeric Construct Above):
hMPV postF Sequence:
The Stabilized hMPV PreF Sequence):
The processed stabilized hMPV PreF construct (115BV) was co-transfected with furin at 1:1 ratio, and other hMPV F constructs were transfected without furin. At day 3 to day 7 post transfection, supernatants were harvested for western blot to confirm expression, for direct ELISA binding assay, and for large-scale purification. The purification of all antigens were performed as previously described [55]. Briefly, harvested supernatants with his-tagged proteins were captured by Ni-Sepharose chromatography (GE Healthcare) and eluted by high imidazole concentration. After an overnight dialysis in the presence of thrombin, the His-tag was cleaved, and concentration of imidazole was reduced. Uncleaved His-tag products as well as initial Ni-Sepharose non-specific binding impurities were removed by negative Ni-Sepharose chromatography (product in flow-through). The protein antigens were further purified by size-exclusion chromatography (Superdex 200, GE Healthcare) and stored in a buffer of 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5 with 300 mM sodium chloride (NaCl).
Blood samples from healthy adults were purchased from a Biological Specialty Company (Colmar PA) after written informed consent. Plasma or serum samples from these donors were screened for activity in an hMPV microneutralization assay. Peripheral blood mononuclear cells (PBMCs) were purified from blood collected in EDTA tubes by density gradient centrifugation in Histopaque™ over Accuspin™ tubes (Sigma Aldrich) according to the manufacturer's instructions. PBMCs were then frozen in 90% heat-inactivated fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide and stored in liquid nitrogen until thawed for use in assays.
Human memory B cells from selected donors were single-sorted with specific antigens as previously described with minor modifications [53, 56]. The purified recombinant unprocessed hMPV PreF trimer and monomer proteins were biotinylated (Thermo) and used as sorting antigens. For a portion of the experiments, B cells were sorted using a mixture of biotinylated unprocessed hMPV PreF and RSV PreF (DS-Cav1) tagged with Alexa-647. Cryopreserved PBMCs were thawed on the day of sorting and the B cell population was enriched using the EasySep™ Human B-cell Enrichment Kit (Stemcell Technologies). Next, B cells were stained with biotinylated or Alexa 647 tagged F antigens and then followed by staining with a panel of monoclonal antibodies including anti-CD3 mAb-PE-Cy™ 7 (BD Biosciences), anti-CD19-FITC (BD Biosciences), anti-human IgG-APC or BV421 (BD Biosciences), and PE-streptavidin. CD3−/CD19+/IgG+/F+ cells were sorted with a BD FACS Jazz or SONY 800S cell sorter in single cell mode into a 96-well plate (
The antibody sequences from the wells with positive ELISA binding to hMPV F were further recovered by regular cloning methods as previously described [56] or by barcode-based next-generation sequencing (NGS). Specifically, for NGS cloning, after RNA extraction and the first step of reverse transcription polymerase chain reaction (RT-PCR) to amplify variable regions, 5′ and 3′ overlap sequences were attached by a second-step of multiplex nested PCR, followed by a third-step of PCR to add 5′ and 3′ barcode and adaptor sequences. The final amplicons, which contain unique 5′ and 3′ barcode combination for each well, were pooled together and sequenced by Illumina MiSeq™. Primer sequences were included and are shown in Table 2.
NGS sequences were checked for quality by FastQC v0.11.2 (bioinformatics.babraham.ac.uk/projects/fastqc) and paired-end reads were assembled using PANDAseq v2.10 [57] with a minimum overlap region of 10 base pair (bp). The assembled B cell receptor (BCR) reads were aligned to V(D)J germline sequences (IMGT v3.1.19) [58] using IgBLAST v1.9.0 [59] and to immunoglobulin heavy-chain constant (IGHC) sequences using blastn (v2.2.29) as a part of the BLAST+suite [60]. The output of IgBLAST was processed using commands from Change-O v0.3.12 [61]. Only functional sequences with E-value no more than 0.001 for the alignment of the V and J genes were retained for downstream analysis. The most abundant sequence obtained from each well were representative of the antibody sequence recovered from the well.
The naturally paired heavy and light chain variable region sequences obtained from single sorted human memory B cell cultures were sub-cloned into pTT5 vector for expression in CHO-3E7, which is a stable Chinese hamster ovary (CHO) clone expressing a truncated but functional form of Epstein-Barr virus nuclear antigen-1 (EBNA1). The CHO-3E7 cells (performed at Genscript). Briefly, CHO-3E7 cells were grown in serum-free FreeStyle™ CHO Expression Medium (Life Technologies). The recombinant plasmids encoding heavy and light chains of each antibody were transiently co-transfected into suspension CHO-3E7 cell cultures. The culture supernatants collected were used for purification with a Protein A Cleaning-in-place (CIP) column (GenScript). The purified antibodies were quality controlled (QC) tested by SDS-PAGE and western blot.
hMPV Plaque Reduction Neutralization Assay
A plaque reduction neutralization assay was developed for hMPV similarly as described previously for RSV [62]. Briefly, antibodies in serial dilutions with OptiMEM™ medium (Gibco) were first added into Poly-D coated 96 well flat bottom plates (Corning Costar) at 50 μl per well. For initial screening, the hMPV A1 and B2 viruses (ZeptoMetrix Corp) at 2000 pfu/ml were mixed with antibody at 50 μl per well and incubated for 1 h at 37° C. with 5% CO2. Rhesus Monkey Kidney Epithelial Cells (LLC-MK2 line) LLC-MK2 is an epithelial line that was established in the 1950s from a pooled suspension prepared from renal tissue excised from six rhesus monkeys (Macaca mulatta). The LLC-MK2 cells (0.8×106 to 1.2×106 cells/ml in OptiMEM™ medium) were then added to the antibody/virus mixtures at 25 μl per well. After 1 hour incubation at 37° C. with 5% CO2, the plates were centrifuged at 1,200 rpm for 10 minutes. A volume (125 μl) of OptiMEM™ medium supplemented with 1% methylcellulose was overlaid in each well. Plates were incubated at 37° C. with 5% CO2 for 4 days. Cells were then fixed with 10% formalin (Fisher Scientific) at 100 μl per well for 30 min at room temperature. The plates were dried for 20 minutes before washing with phosphate-buffered saline with Tween @detergent (PBST). Fixed cells were treated with blocking buffer (Odyssey) for 30 minutes, followed by a 2 hour incubation with a mouse anti-hMPV mAb (EMD Millipore, 1:1000) diluted in blocking buffer at 50 μl per well. Plates were washed again by PBST and then incubated with an anti-mouse IgG Alexa 488 conjugated secondary antibody (Invitrogen, 1:500) in assay diluent at 50 μl per well for 1 hour. After washing off the excessive secondary antibodies using PBST, the plates were analyzed using an EnSight™ plate reader (PerkinElmer). The IC50 values for the antibodies were calculated from a 4 parameter nonlinear fitting algorithm using a Graph Pad Prism software. For the antibodies that showed an IC50<10 μg/ml in initial screening, neutralization assays with the same procedure were repeated in wider range of antibody concentration with hMPV A2 (RL_bx from Baylor) and B2 (Peru6-2003) viruses.
A plaque reduction neutralization assay was utilized by investigators [62]. Briefly, antibodies in serial dilutions with Eagle's Minimum Essential Medium (EMEM) medium supplemented with 2% FBS and 2 mM glutamine were first added into Poly-D coated 96 well flat bottom plates (Corning Costar) at 50 μl per well. The RSV A Long and B Washington strains at 2000 pfu/ml were mixed with antibody at 50 μl per well and incubated for 1 hour at 37° C. with 5% CO2. The HEp-2 cells (ATCC) at 0.8×106 to 1.2×106 cells/ml in EMEM medium supplemented with 2% FBS and 2 mM glutamine were then added to the antibody/virus mixtures at 25 μl per well. After an 1 hour incubation at 37° C. with 5% CO2, the plates were centrifuged at 1,200 rpm for 10 minutes. 125 μl of EMEM medium supplemented with 2% FBS, 2 mM glutamine and 1% methylcellulose were overlaid in each well. Plates were incubated at 37° C. with 5% CO2 for 3 days. Cells were then fixed with ice-cold 80% acetone (Sigma) in phosphate-buffered saline (PBS) at 100 μl per well for 10-20 min at room temperature. The plates were dried for 20 minutes and then washed with PBST. Fixed cells were stained with a mixture of a mouse anti-F monoclonal antibody (1.25 μg/ml) and a mouse anti-N(nucleoprotein) monoclonal antibody (1.25 μg/ml). Plates were washed again by PBST and then incubated with an anti-mouse IgG Alexa 488 conjugated secondary antibody (Invitrogen, 1:500) in assay diluent at 50 μl per well for 1 hour. After washing off the excessive secondary antibodies using PBST, the plates were analyzed using an EnSight™ plate reader (PerkinElmer). The IC50 values were calculated from a four-parameter nonlinear fitting algorithm using a GraphPad Prism software.
Cryo-EM Study of M4B06 and hMPV-F Complex
For sample preparation, the M4B06-hMPV PreF complex was prepared by incubating recombinantly purified M4B06 Fab with a processed hMPV PreF (115BV) trimer (PreF trimer: Fab=1:3) for 1 hour at 4° C., followed by purification using Superdex 200 Increase 10/300 GL column in buffer (25 mM HEPES pH 7.5, 150 mM NaCl). The fraction sample was flashed frozen and pre-checked with negative staining for subsequent cryo-EM study. Three microliters of the purified sample at 0.2 mg/ml were applied onto a glow-discharged holey carbon film grid with Graphene Oxide (Quantifoil, 300 mesh, R2.0/1.0). Samples were blotted for 6 seconds (s) and vitrified by plunging into liquid ethane cooled by liquid nitrogen using a Vitrobot™ Mark IV (Thermo Fischer Scientific) at 4° C. and 100% humidity.
For Cryo-EM data acquisition, a total of −1,800 movie micrographs (36 frames for each movie) were acquired using a FEI Titan Krios™ 300 KV electron microscope. The data acquisition also involved using a Gatan K3™ Summit direct electron detector, operated in super resolution mode applying a total dose of 75 e−/Å2 during a 3 s exposure. During the 3 second period, the total dose is 75 electrons per A2 (angstrom square) of detector (A2 is unit of area size of detector). Each micrograph was acquired (with SerialEM software) at 89,000× nominal magnification (0.5225 Å/pixel at the specimen level) with a nominal defocus range of −1.5 μm to −3.0 μm.
For image processing and 3D reconstruction, all movie frames were aligned and binned by a factor of 2 using MotionCor2 [63] and their contrast transfer function (CTF) parameters were estimated with a Gctf computer program [64]. Approximately 1,000 particles were manually boxed and extracted from ˜30 far-defocus micrographs, and their two dimensional (2D) average classes were used as templates to automatically box particles from all selected micrographs with autopick in a regularized likelihood optimization (RELION) 3.0 computer program [65]. A total of −1,000,000 picked-particle images were extracted (binned by a factor of 8 to give a pixel size of 4.18 Å) and were subjected to reference-free 2D alignment with RELION 3.0. After multiple iterations of 2D classifications, particles (˜218,000) belonging to good classes were three dimensional (3D) classified against the map created from manually-picked particles as the initial model. After 2 iterations of 3D classifications, particles (˜131,000) in good classes were re-centered, re-extracted (binned by a factor of 5 to give a pixel size of 2.6 Å) and refined to yield a map at Nyquist frequency of 5.2 Å resolution. Using this map as a model, particles were further 3D classified into 6 classes (S6 Fig A) without any further origin and orientation determination. Particles (87,876) in those three classes showing better identifiable structural features were re-extracted (binned by a factor of 2 to give a pixel size of 1.045 Å) and further refined to a final map with resolution of 3.75 Å (FSC=0.143) without imposing any symmetry.
For model building, the model of a hMPV PreF trimer of B strain was built using the crystal structure of A strain as the template [28]. The model was docked into CryoEM map using a Chimera visualization program [66]. Structural figures were prepared using the Chimera program.
Transmission electron microscopy and 2D class averaging were performed by NanoImaging Services, Inc. (San Diego, CA). Samples were prepared on continuous carbon films supported by nitrocellulose-coated 400 mesh copper grids (Ted Pella). A 3 μL drop of purified hMPV PostF protein at a concentration of 2-8 μg/mL was applied to a freshly plasma-cleaned grid for 1 minute. The drop was then blotted to a thin film using filter paper, followed by immediate staining with 1% (w/v) uranyl formate. Transmission electron microscopy was performed using a FEI Tecnai T12 electron microscope operating at 120 kilovolt (kV) equipped with an FEI Eagle 4 k×4 k charge-coupled device (CCD) camera. Negative stain grids were transferred into the electron microscope using a room temperature stage. Images of each grid were acquired at multiple scales to assess the overall distribution of the specimen. After identifying potentially, suitable target areas for imaging at lower magnifications, pairs of high magnification images were acquired at nominal magnifications of 110,000× (0.10 nm/pixel) and 67,000× (0.16 nm/pixel) using the automated image acquisition software package Leginon [67]. Images were acquired at a nominal underfocus of −2 μm to −1 μm and electron doses of approximately 30-35e/A2.
Image processing was performed using the Appion software package [68]. Contrast transfer functions of the images were estimated using a CTFFIND4 program [69]. CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Individual particles in the 67,000× or 110,000× high magnification images were selected using automated picking protocols [70], followed by several rounds of reference-free alignment and classification using the X-windows-based microscopy image processing package (Xmipp). See [71]. XMipp contains an algorithm that aligns the selected particles and sorts them into self-similar groups of classes.
Vero kidney epithelial cells were seeded in 6-well plates in OptiPRO™ SFM medium (Gibco), which is a serum-free, animal origin-free culture medium. The medium was supplemented with 1% Pen Strep (Gibco), 2% L-Glutamine (Gibco) and 2% FBS (HyClone) at 37° C. with 5% CO2. In the next day, the cells were infected by a mixture of hMPV A2 virus (RL_bx from Baylor) at 10 plaque forming units per cell (pfu/cell) and M4B06 Fab at 2 μg/mL, 8 μg/mL, and 40 μg/mL in three rounds respectively. In every round, cells were cultured at 37° C. with 5% CO2 and harvested when syncytia appeared (day 6), lysed by three times of freeze/thaw in liquid nitrogen, and centrifuged at 500 g for 10 minutes. One milliliter of supernatant containing virus was used to infect fresh monolayer Vero cells. The remaining supernatant was mixed with 10× sucrose-phosphate-glutamate-albumin (SPGA) buffer (Biological Industries) and frozen for long-term storage. After three rounds of culture and infection, the virus supernatant from the last round was aliquoted into 10-fold serial dilutions and 0.5 mL of supernatant was used to incubate with fresh monolayer the Vero cells at 37° C. with 5% CO2. After a 1 hour incubation, a 3.5 mL volume overlay (OptiMEM™ medium supplemented with 1% methylcellulose) was added, and cells were cultures at 37° C. with 5% CO2 for 1 week.
Twenty-two single plaques were then collected separately and inoculated in fresh monolayer Vero cells prepared in a 24-well plate for 5 days at 37° C. with 5% CO2 before harvesting. The final harvested MARM viruses were lysed in RLT buffer (Qiagen) with 1% 2-mercaptoethonal and viral RNA were extracted using a RNeasy commercial kit (Qiagen). The F genes from individual plaques were amplified by RT-PCR using viral RNAs, followed by PCR analysis with F-specific primers, gel extraction, and sequencing.
BLI-based assays were performed using an Octet Red 96e instrument (ForteBio, Inc.). All experiments were performed at 30° C. with shaking at 1,000 rpm. For the binding assays, the investigators utilized recombinant unprocessed hMPV PreF trimer, processed stabilized hMPV PreF trimer, and hMPV PostF trimer as distinct antigens. The antibodies and antigen proteins were diluted in the kinetics buffer (ForteBio) at concentrations of 0.5 μg/mL and 2 μg/mL, respectively. The antibodies and a reference well with empty buffer were first individually immobilized on anti-human Fc capture (AHC) biosensors (ForteBio) for 180 seconds, followed by the association step with antigen for 600 seconds. Most of antibodies' association curves were within linear range without showing saturation. For data processing, the absolute binding response (nm) of each antibody sample was calculated by substrating the binding response (nm) of the reference well (Table 4). The plots were generated using GraphPad Prism software.
For epitope binning analysis, the 73 isolated mAbs which showed apparent BLI binding response (>0.2 nm) were included in the experiments. Recombinant unprocessed hMPV PreF trimer proteins were used as antigens. All the protein samples were diluted in the kinetics buffer (ForteBio). The antibodies to be characterized or the reference mAbs to be used as internal controls (5 μg/mL) were first individually immobilized on anti-human Fc capture (AHC) biosensors (ForteBio) for 600 seconds. The sensors having the samples and antibodies were then blocked with an irrelevant mAb (5 μg/mL) for 600 seconds. The biosensors were then immersed into wells containing the F antigen (2 μg/mL). Finally, the antigen loaded biosensors were immersed into wells containing the second reference mAbs (5 μg/mL). The normalized secondary mAbs binding was calculated by dividing the binding responses of second reference mAbs binding by the binding responses of antigen loading. The normalized numbers were then plotted as a clustered heatmap by heatmap.2 package in R Studio 1.0.153. MAbs with similar competition profiles as the reference mAbs were clustered into the same categories. The neutralization IC50 values of hMPV A, B and RSV A, B were plotted as top-bars in the heatmap.
For ELISA assays with unpurified antigens in Expi293 supernatants, 96-well Ni-NTA coated plates (Thermo Scientific) were coated with cell culture supernatants for 2 hours at room temperature. Plates were then blocked by adding 2% (v/v) bovine serum albumin (BSA) in PBS. After the blocking step, plates were incubated with serial dilutions of antibodies at room temperature for 90 minutes. Plates were washed by PBST and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (1: 2,000, Southern Biotech) for 45 minutes. Plates were washed again and developed with tetramethylbenzidine (TMB) solution (Virolabs). Any absorbance at 450 nm was detected using a plate reader (Victor III; Perkin-Elmer). The EC50 values were calculated by performing a Hill slope curve fitting using GraphPad Prism software.
To optimize the sequence coverage, several preliminary experiments using various quench conditions, digestion, desalting, liquid chromatography, and mass spectrometry parameters were evaluated. Also, the optimal antigen-antibody ratio was determined in a separate titration experiment. The ratio was optimized by keeping a constant antigen concentration while changing the antibody concentration. Several peptides were identified whose deuteration levels decreased upon addition of the antibody. The optimal Ag:Ab ratio was identified when no additional decrease in deuteration levels were observed which represented equimolar amounts of antigen (Ag) and antibody (Ab) for binding.
The following represents the conditions that were determined to be optimal for the hydrogen-deuterium exchange experiments. Stock solutions of 2.6 μM hMPV prestabilized processed PreF trimer (115BV) with and without an equimolar amount of M4B06 monovalent Fab were prepared in 1×PBS. The Ag:Ab complex was prepared by incubating hMPV F with M4B06 at ambient temperature for 1 hour before cooling to 1° C. The hydrogen-deuterium exchange reactions were initiated by adding 40 μL PBS prepared in D20 to 10 μL of the hMPV F and hMPV F/M4B06 solutions. The labeling reactions were performed at 1° C. for five time points: 15 seconds, 50 seconds, 150 seconds, 500 seconds, and 5000 seconds. After these time points the samples were quenched by mixing 40 μL of the labeled solution with 20 μL of quench solution (2M urea, 1M tris(2-carboxyethyl)phosphine (TCEP), adjusted to pH 3.0 with NaOH) at 1° C. 50 μL of the quenched sample was immediately injected onto an immobilized pepsin column (Waters Enzymate BEH Pepsin column, 2.1×30 mm) held at 10° C. for on-line proteolysis using 0.05% (v/v) trifluoroacetic acid (TFA) in water solvent at a flow rate of 200 μL/minute. The flow, with the digest, continued at the same rate onto a trap column (Waters ultra-performance liquid chromatography (UPLC) ethylene bridged hybrid (BEH) 300 C18, 1.7 μm, 2.1×5 cm) held at 0° C. for desalting. The combined digestion and desalting steps lasted for 90 sec after which the trap column flow was reversed and eluted onto an analytical column (Waters Acquity™ UPLC charged surface hybrid (CSH) carbon 18(C18_, 1.7 μm, 1.0×100 mm) held at 0° C. using a gradient of solvent A: 0.05% (v/v) TFA in water and solvent B: 0.0025% (v/v) TFA in 95% acetonitrile/5% water (v/v). A linear gradient of 13% to 40% B over 9.5 min at a flow rate of 40 μL/min was employed to elute the peptides into a ThermoScientific LTQ-XL Orbitrap mass spectrometer (Thermo Fisher Scientific) operated in positive mode at a resolution of 30,000. The data was acquired over the m/z range of 350-2000 in profile mode using the following MS parameters: heated electrospray ionization (HESI) source voltage 4 kV, capillary voltage 44 V, tube lens 110 V, capillary temperature 250° C., and sheath gas flow 10. The labeling, quench, injection, digestion, desalting, and elution steps were performed using an HTC PAL robot (LEAP Technologies) controlled by HDxDirector software. The isocratic and gradient solvent flows were obtained using Waters nano-Acquity™ UPLC pumps (Waters). Prior to the exchange experiment, the identity of each peptide was confirmed by analyzing a non-deuterated RSV F sample in data-dependent MSMS mode and processed with Proteome Discoverer 1.4 software (Thermo Fisher Scientific). The HDExaminer software (Sierra Analytics) was used to determine the centroid mass of the isotopic envelope of each peptide in the labeling experiment and quantifying deuterium incorporation.
Differential Scanning Fluorimetry for Unprocessed and Processed hMPV F Proteins
Analyses were performed with unprocessed hMPV PreF protein and processed stabilized hMPV PreF protein expressed with and without furin that had been stored at −70° C. Two-fold serial dilutions of hMPV F protein in 50 mM HEPES, 300 mM NaCl at pH 7.5 were prepared from starting concentrations of 1.5 mg/ml, 2.5 mg/ml and 2.7 mg/ml, respectively to obtain samples in a concentration range between (3.125 μM-50 μM). Differential scanning fluorimetry (DSF) measurements were performed using the Prometheus NT.48 instrument (NanoTemper Technologies GmbH, Munich, Germany). In brief, experiments were performed in duplicate using high sensitivity capillaries (PR-C006). Each capillary was filled with 10 μl of protein solution and the fluorescence signal at 350 nm and 330 nm was recorded at temperatures between 20° C. to 95° C. at a temperature increase rate of 1° C./minute. Instrument software was used to calculate the ratio between the 350 nm and 330 nm signal as well as the first derivative. Maxima of the first derivative were used to obtain transition midpoints of protein unfolding events.
Serum samples of four donors were tested in the absorption assay. Each sample (50 μL) is diluted in PBS to a total volume of 970 μL and mixed with 20 μL of 0.5 mg/mL antigen or PBS buffer control for two-hour incubation at room temperature. The samples were then mixed with 10 μl of reconstituted Strep-tag II mAb (GenScript) at 0.5 mg/ml and place on a 3600 rotator at 4° C. for 2 hours. Pre-washed 80 μL of sheep anti-mouse immunoglobulin G Dynabeads™ (2 mg/ml, Life Technologies) were then added to the mixture for 1 hour incubation at 4° C. on a rotator. Dynabeads carrying antigen and antigen-bound antibodies were separated by DynaMag-2 magnet (Life Technologies), leaving absorbed supernatant for further ELISA binding assay with F antigens. ELISA titers were calculated by dilution folds, and averaged ELISA titers from four donors were plotted.
The V gene germline usage, clonal analysis, and somatic hypermutation (SHM) were analyzed by IgBlast [59] based on Kabat delineation system [72]. The clonotype was defined to includes any V(D)J rearrangements that have the same germline V(D)J gene segments, the same productive/non-productive status and the same CDR3 nucleotide as well as amino sequence. Structural visualization was generated by PyMOL 1.7.05 (Schrödinger). The plots were generated by GraphPad Prism software.
To comprehensively profile the human antibody recognition sites to hMPV F antigen, investigators isolated more than 100 mAbs from human memory B cell repertoires of multiple donors by antigen-specific single memory B cell sorting method (
Clonal lineage analysis was performed, and the data showed that the hMPV F-specific B cell repertoires were highly diverse, with 111 out of 113 antibodies being unique. See Table 3D. The frequency of germlines was also diverse (
Among isolated mAbs, clone number M4B06 showed ultra-high hMPV neutralization potency on both tested hMPV A and B subtypes with IC50 at 3.5 ng/mL and 2.2 ng/mL respectively (
To further understand the epitope of M4B06, monoclonal antibody resistant mutants (MARMs) of an hMPV A strain were generated under the selection pressure of M4B06. After three rounds of culture with increasing concentration of M4B06 Fab, it was observed that the hMPV virus was able to replicate under the Fab concentration of 40 μg/mL, which is more than 500 folds of the IC50 of the wild-type virus. Sequencing of single purified plaques of MARMs revealed three types of MARMs: V231A/G264R, G264R/Q434H, and K138Q. The neutralization potency of M4B06 IgG to these three types of MARMs reduced the potency of the immunoglobulin 52-fold, 70-fold, and 49-fold respectively. TheM4B06 Fab failed to neutralize any type of MARM of the hMPV A strain at all, confirming that the mutations generated in these MARMs impaired the neutralizing activity of M4B06 (
Additionally, investigators generated hMPV F variants carrying single and double MARM-derived mutations to determine whether the mutations/variations interrupted M4B06 binding. Data from the ELISA binding assay showed that these mutations did not impact the binding to a control antibody M2D2, which targets a different epitope [53], such that it may be that the mutant proteins were folded similarly as the WT protein (
Moreover, the identified epitope of M4B06 was further confirmed by a CryoEM map of M4B06-hMPV F complex at a resolution of 3.75 Å, which clearly showed that three M4B06 Fabs sit on the epitope area at a nearly perpendicular angle (
To map the targeted antigenic sites of all isolated hMPV F-specific mAbs, investigators designed a panel of hMPV F knock-out mutants and tested ELISA binding with isolated hMPV-F specific mAbs. A panel of mAbs (M1D2, M1C7s, M3D04, M4A10, M3C06, M4B06) were observed to reduce binding to PreF mutants on a variety of residues located at regions equivalent to antigenic site II and V defined on RSV F (
Based on epitope binning, hMPV F-specific mAbs were categorized into eight groups (
To further map its epitope, the investigators analysed the M2A05 antibody binding to a panel of hMPV-B F variants that converted subtype B-specific residues to their counterparts in hMPV-A F. Data show that a single D296K mutation of F antigen significantly reduced binding of M2A05 (
To investigate the binding-specificity of isolated mAbs to different conformations of hMPV F antigens, investigators analysed the binding affinity of isolated mAbs to three forms of hMPV F antigens by BLI. (1) the uncleaved hMPV PreF trimer [34], which was used for B cell sorting and epitope binning (unprocessed PreF, see
Comparing binding of isolated mAbs to unprocessed PreF with PostF showed that there were three groups of mAbs: PreF-specific, PostF-specific, and Pre/Post-dual mAbs (
The PreF/PostF binding-specificity of mAbs was also well-correlated with their antigenic sites: site III′ and site IV mAbs were mostly Pre/Post-dual binders, site II mAbs had similar number of PreF-specific and Pre/Post dual mAbs, and the rest of other sites including site V, site III, site IV′, site α, and site β, were PreF-specific (
The binding of isolated mAbs to unprocessed PreF and as compared to processed PreF was analyzed using assays. Interestingly, there are two apparent groups of mAbs—a group of mAbs showed similar binding affinity to both conformations of PreF; whereas the other group had significantly reduced binding to processed PreF than unprocessed PreF (
To further characterize the differences between unprocessed and processed hMPV PreF antigens, investigators analysed the thermostability of the antigens by differential scanning fluorimetry. The unprocessed hMPV PreF construct, which does not include A185P stabilizing mutation (
Despite the highly similar structural folding and some conserved epitopes between RSV and hMPV F proteins, whether the antigenicity of hMPV F is similar to RSV F remains unclear to date. In this study, investigators isolated and characterized over one hundred mAbs from multiple healthy adult donors, providing a comprehensive antigenic map of hMPV-F specific mAbs elicited from natural infection. Investigators generated 113 hMPV-F specific mAbs isolated from memory B cells of multiple healthy human donors. The investigators analysed the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities to different forms of F antigen: unprocessed (or uncleaved) prefusion (PreF), processed (or cleaved) PreF, and postfusion (PostF). An mAb with ultra-high neutralization potency on both hMPV A and B viruses has been characterized and mapped to antigenic site V. Data showed that unlike RSV-F specific mAbs, antibodies responses to hMPV F are less dominant against the apex of the antigen, possibly due to the additional glycosylation. The majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, novel neutralizing epitopes that differ from previously defined antigenic sites on RSV F were identified, and multiple binding modes of site V and II mAbs have been discovered. Interestingly, mAbs that bind preferentially to the unprocessed PreF showed poor neutralization potency, highlighting the potential differences of immunogenicity between proteolytically unprocessed and processed F antigens. These results elucidate the immune recognition of hMPV infection.
Most of isolated mAbs had significant somatic hypermutations compared with the germline sequences (
Antibodies against two other novel sites, one located in the trimer interface and another one has yet to be characterized, also comprised a significant portion of the total antibodies that were identified. Another mAb MPV458 has been reported to target the 66-87 helix that is located near the trimer interface of MPV F head region [ref. doi.org/10.1371/journal.ppat.1008942]. The epitope of MPV458 does not overlay with the epitope of M8C10, which is completely buried within the MPV F trimer. See
Since nascent hMPV F protein requires proteolytic activation by transmembrane proteases such as TMPRSS2 at the cell surface or in the virus like particle [26], investigators hypothesized that both forms of PreF antigen may be accessible to humoral immunity. By examining the binding specificity of isolated mAbs to both proteolytically unprocessed and processed PreF forms, a large group of mAbs were identified that had apparent binding preference to unprocessed PreF than processed PreF, highlighting the potential differences of immunogenicity between the two PreF form, despite their highly similar structures in monomer forms.
Without wishing to be bound by theory, one possible explanation to this observation is that the proteolytic cleavage generates a hydrophobic end in the N-terminus of F1 segments, forming a hydrophobic core and stabilizing the trimerization of F protein (
Data show that the processed PreF was also able to deplete cross-reactivity to unprocessed PreF in the serum (
In this Example, the detailed structure of M8C10 in complex with MPV-F was analysed and investigators identified a new epitope within the trimer interface. Although antibodies against RSV have been previously seen to partially open the apical end of the F trimer, to our knowledge, a neutralizing antibody capable of disrupting the trimeric fusion protein has only been seen with influenza hemagglutinin.
MPV-F:M8C10 Fab protein complex was screened by sparse matrix using a sitting drop format. The crystals were obtained with a crystallization buffer of 20% PEG 3350 and 200 mM sodium iodide. The crystals were vitrified using a freezing solution containing 20% PEG 3350, 10% PEG 400, and 100 mM sodium iodide. Data were collected at the Advanced Photon Source at Argonne National Labs on beamline, 17-ID.
Data were processed with XDS using an autoprocessing protocol. The phases were determined by molecular replacement using a model of MPV-F (PDB code: 4DAG) and a homology model of the Fab built using the automated antibody homology modeler in MOE. Structures were built iteratively using Coot and refinement with Buster and Phenix. Model geometries were validated using Procheck and Molprobity. Molecular figures were generated using a Pymol molecular visualization system.
Antibody M8C10 was identified from a campaign to isolate MPV-F-reactive antibodies from human donor serum as described in the earlier Examples. Based on a Bio-Layer Interferometry antibody competition assay, investigators were able to identify antibodies that did not compete with previously characterized binding sites of MPV-F, M2B6 for site III, M2D2 for a site IV, and M1C7 for a site V. DS-7 binds a separate site that has been characterized structurally which will be referred to as the DS7-site. In characterizing the antibodies, M8C10 was identified as not competing with these reference antibodies (Table 6) using a bio-layer interferometry competition study.
To further confirm the M8C10 binds to MPV-F, the binding was tested by Surface Plasmon Resonance. MPV-F was captured on the chip and M8C10 Fab was introduced at increasing analyte concentrations. Based on kinetic fitting, the dissociation constant was calculated to be 1.2 nM.
M8C10 was tested for its ability to inhibit MPV infection in a virus neutralization assay. Two strains of MPV were tested from the A1 and B2 subgroups. M8C10 inhibited MPV strain Al with an IC50 of 370.7 nM and stain B2 with an IC50 of 660.5 nM. While many antibodies can bind MPV-F without being about to neutralize the virus, the M8C10 antibody appears to have sub-micromolar activity against the virus.
It was observed that the M8C10 antibody did not compete with one of the reference antibodies, we sought to determine the crystal structure of the complex between MPV-F and a Fab fragment of M8C10. Each component was individually purified, and the antigen-antibody complex was formed by mixing with a molar excess of the Fab. The complex was purified from the unbound Fab by size exclusion chromatography. and concentrated for crystallization. A dataset was collected that diffracted to 3.0 Å (Table 7).
The structure reveals a single copy of the MPV-F:M8C10 Fab complex in the asymmetric unit. The interface of the complex buries a total of 1901 sq. A. All antibody structures against RSV or MPV thus far have demonstrated a binding site that is on the exterior of the surface of the F protein trimer. The structure demonstrates that M8C10 binds to the surface that mediates the trimer interface of F (
Data shows that the M8C10 Fab binds to MPV-F through interactions mediated by four of the six complementarity determining regions (CDRs). The heavy chain CDR1 and CDR2 do not interact with MPV-F in the structure (
Based on the MPV-F epitope residues identified in the structure, mutants were designed to both confirm the structural results and identity the residues that are critical for M8C10 binding. Table 8 lists the mutations generated.
These MPV-F constructs were expressed by transient transfection and the supernatants were tested for M8C10 binding by ELISA. M2D2, an antibody that binds to site IV and should not be affected by the mutations, was used to normalize the signal as a control for expression. (
Other mutations may also affect M8C10 binding, for example, residues A216Y, L219Y, and A249Y appear to have reduced M8C10 binding but are still able to bind. S218Y, D220Y, and D224A appeared to have reduced expression but could also have reduced M8C10 binding. D210Y, N210A, R253A, and R329A did not appreciably interfere with M8C10 binding.
This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The compositions and methods disclosed herein can have other embodiments and can be practiced or carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/015565 | 2/8/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63148920 | Feb 2021 | US | |
| 63276172 | Nov 2021 | US |
