Metapneumovirus, antibodies that bind to antigenic metapneumovirus proteins, and uses thereof

Abstract

The present invention relates to anti-human metapneumovirus (hMPV) antibodies and the use of such antibodies in the treatment of viral infections. The invention also includes antigenic hMPV proteins and immunogenic compositions comprising such antigenic hMPV proteins, as well as the use of such antigenic hMPV proteins and related compositions to prevent or treat viral infections in subjects.

Description

[Technical Field]

[0001] The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to and / or neutralizes human metapneumovirus. Another aspect of the present invention relates to compositions and kits comprising the antibody or antigen-binding fragment thereof. Another aspect of the present invention relates to methods for treating diseases, such as respiratory infections, by administering the antibody or antigen-binding fragment thereof. Another aspect of the present invention relates to an antigenic human metapneumovirus F protein. Another aspect of the present invention relates to methods for preventing or treating diseases, such as respiratory infections, by administering an antigenic human metapneumovirus F protein.

[0002] Reference to an electronically submitted sequence listing

[0003] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on January 27, 2022 is titled 25170-WO-PCT_SL.txt and is 495,474 bytes in size. [Background technology]

[0004] 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 tract infections in children, the elderly, and immunocompromised adults. HMPV encodes two major surface glycoproteins: the fusion (F) protein and the glycoprotein (G) protein. The surface F glycoprotein, which matures after proteolytic cleavage, mediates viral fusion by structural rearrangement and is the primary target of neutralizing antibodies against hMPV. Current understanding of the specificity and functionality of the hMPV-F antibody response induced by natural infection remains unclear. Therapeutic interventions to treat hMPV-associated diseases are needed. Summary of the Invention

[0005] The present disclosure provides antibodies and antigen-binding fragments thereof capable of binding to and / or neutralizing metapneumovirus (MPV), as well as methods, pharmaceutical compositions, uses, and kits for using the antibodies or antigen-binding fragments thereof to bind to and / or neutralize MPV. In various embodiments, the MPV is mammalian. For example, the mammalian MPV includes human MPV (hMPV). In various embodiments, the hMPV includes a surface glycoprotein. For example, the glycoprotein includes an F glycoprotein and / or a G glycoprotein.

[0006] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to hMPV, comprising at least one CDR, three CDRs, or six CDRs listed in Tables 1 to 3. The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to hMPV, comprising at least one variable region / domain listed in Tables 1 to 3. For example, antibodies or antigen-binding fragments thereof that specifically bind to hMPV comprise a heavy chain variable region, a light chain variable region, or a heavy chain variable region and a light chain variable region.

[0007] One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to hMPV.

[0008] 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 sequence 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 the amino acid sequence selected from the group consisting of SEQ ID NOs: 341-649.

[0009] In various embodiments, the antibody or antigen-binding fragment thereof is 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, and a CDR1 comprising an amino acid sequence selected from the group consisting of: 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.

[0010] In various embodiments, the antibody or antigen-binding fragment thereof is 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, and a CDR2 comprising an amino acid sequence selected from the group consisting of: 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.

[0011] In various embodiments, the antibody or antigen-binding fragment thereof is 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.

[0012] 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.

[0013] 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:

[0014] (a) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively;

[0015] (b) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively;

[0016] (c) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively;

[0017] (d) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively;

[0018] (e) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively;

[0019] (f) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 17, 18, and 19, respectively;

[0020] (g) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 20, 21, and 22, respectively;

[0021] (h) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively;

[0022] (i) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0023] (j) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively;

[0024] (k) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively;

[0025] (l) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 35, 36, and 37, respectively;

[0026] (m) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 38, 39, and 40, respectively;

[0027] (n) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 41, 42, and 43, respectively;

[0028] (o) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 44, 45, and 46, respectively;

[0029] (p) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively;

[0030] (q) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52, respectively;

[0031] (r) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 53, 54, and 55, respectively;

[0032] (s) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 56, 57, and 58, respectively;

[0033] (t) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 59, 60, and 61, respectively;

[0034] (u) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 62, 63, and 64, respectively;

[0035] (v) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 65, 66, and 67, respectively;

[0036] (w) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 68, 69, and 70, respectively;

[0037] (x) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 71, 72, and 73, respectively;

[0038] (y) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 74, 75, and 76, respectively;

[0039] (z) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 77, 78, and 79, respectively;

[0040] (aa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 80, 81, and 82, respectively;

[0041] (bb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 83, 84, and 85, respectively;

[0042] (cc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 86, 87, and 88, respectively;

[0043] (dd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 89, 90, and 91, respectively;

[0044] (ee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 92, 93, and 94, respectively;

[0045] (ff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 95, 96, and 97, respectively;

[0046] (gg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 98, 99, and 100, respectively;

[0047] (hh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 101, 102, and 103, respectively;

[0048] (ii) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 104, 105, and 106, respectively;

[0049] (jj) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 107, 108, and 109, respectively;

[0050] (kk) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 110, 111, and 112, respectively;

[0051] (ll) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 113, 114, and 115, respectively;

[0052] (mm) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 116, 117, and 118, respectively;

[0053] (nn) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively;

[0054] (oo) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 122, 123, and 124, respectively;

[0055] (pp) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 125, 126, and 127, respectively;

[0056] (qq) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 128, 129, and 130, respectively;

[0057] (rr) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 131, 132, and 133, respectively;

[0058] (ss) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 134, 135, and 136, respectively;

[0059] (tt) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 137, 138, and 139, respectively;

[0060] (uu) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 140, 141, and 142, respectively;

[0061] (vv) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 143, 144, and 145, respectively;

[0062] (ww) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 146, 147, and 148, respectively;

[0063] (xx) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 149, 150, and 151, respectively;

[0064] (yy) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 152, 153, and 154, respectively;

[0065] (zz) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 155, 156, and 157, respectively;

[0066] (aaa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 158, 159, and 160, respectively;

[0067] (bbb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 161, 162, and 163, respectively;

[0068] (ccc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 164, 165, and 166, respectively;

[0069] (ddd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 167, 168, and 169, respectively;

[0070] (eee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 170, 171, and 172, respectively;

[0071] (fff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 173, 174, and 175, respectively;

[0072] (ggg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 176, 177, and 178, respectively;

[0073] (hhh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 179, 180, and 181, respectively;

[0074] (iii) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 182, 183, and 184, respectively;

[0075] (jjj) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 185, 186, and 187, respectively;

[0076] (kkk) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 188, 189, and 190, respectively;

[0077] (lll) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 191, 192, and 193, respectively;

[0078] (mmm) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 194, 195, and 196, respectively;

[0079] (nnn) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 197, 198, and 199, respectively;

[0080] (ooo) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 200, 201, and 202, respectively;

[0081] (ppp) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 203, 204, and 205, respectively;

[0082] (qqq) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 206, 207, and 208, respectively;

[0083] (rrr) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 209, 210, and 211, respectively;

[0084] (sss) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 212, 213, and 214, respectively;

[0085] (ttt) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 215, 216, and 217, respectively;

[0086] (uuu) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 218, 219, and 220, respectively;

[0087] (vvv) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 221, 222, and 223, respectively;

[0088] (www) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 224, 225, and 226, respectively;

[0089] (xxx) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 227, 228, and 229, respectively;

[0090] (yyy) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 230, 231, and 232, respectively;

[0091] (zzz) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 233, 234, and 235, respectively;

[0092] (aaaa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 236, 237, and 238, respectively;

[0093] (bbbb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 239, 240, and 241, respectively;

[0094] (cccc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 242, 243, and 244, respectively;

[0095] (dddd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 245, 246, and 247, respectively;

[0096] (eeee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 248, 249, and 250, respectively;

[0097] (ffff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 251, 252, and 253, respectively;

[0098] (gggg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 254, 255, and 256, respectively;

[0099] (hhhh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 257, 258, and 259, respectively;

[0100] (iiii) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 260, 261, and 262, respectively;

[0101] (jjjj) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 263, 264, and 265, respectively;

[0102] (kkkk) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 266, 267, and 268, respectively;

[0103] (llll) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 269, 270, and 271, respectively;

[0104] (mmmm) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 272, 273, and 274, respectively;

[0105] (nnnn) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 275, 276, and 277, respectively;

[0106] (oooo) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 278, 279, and 280, respectively;

[0107] (pppp) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 281, 282, and 283, respectively;

[0108] (qqqq) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 284, 285, and 286, respectively;

[0109] (rrrr) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 287, 288, and 289, respectively;

[0110] (ssss) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 290, 291, and 292, respectively;

[0111] (tttt) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 293, 294, and 295, respectively;

[0112] (uuuu) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 296, 297, and 298, respectively;

[0113] (vvvv) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 299, 300, and 301, respectively;

[0114] (www) a variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 302, 303 and 304, respectively;

[0115] (xxxx) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 305, 306, and 307, respectively;

[0116] (yyyy) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 308, 309, and 310, respectively;

[0117] (zzzz) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 311, 312, and 313, respectively;

[0118] (aaaaa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 314, 315, and 316, respectively;

[0119] (bbbbb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 317, 318, and 319, respectively;

[0120] (ccccc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 322, respectively;

[0121] (ddddd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 323, 324, and 325, respectively;

[0122] (eeeee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 326, 327, and 328, respectively;

[0123] (fffff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 329, 330, and 331, respectively;

[0124] (ggggg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 332, 333, and 334, respectively;

[0125] (hhhhh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 335, 336, and 337, respectively; and

[0126] (iiiii) A variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 338, 339, and 340, respectively.

[0127] In various embodiments, the antibody or antigen-binding fragment thereof is 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.

[0128] In various embodiments, the antibody or antigen-binding fragment thereof is 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.

[0129] In various embodiments, the antibody or antigen-binding fragment thereof is 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, 51 514, 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.

[0130] 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:

[0131] (a) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 341, 342, and 343, respectively;

[0132] (b) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 344, 345, and 346, respectively;

[0133] (c) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 347, 348, and 349, respectively;

[0134] (d) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 350, 351, and 352, respectively;

[0135] (e) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 353, 354, and 355, respectively;

[0136] (f) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 356, 357, and 358, respectively;

[0137] (g) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 359, 360, and 361, respectively;

[0138] (h) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 362, 363, and 364, respectively;

[0139] (i) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 365, 366, and 367, respectively;

[0140] (j) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 368, 369, and 370, respectively;

[0141] (k) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 371, 372, and 373, respectively;

[0142] (l) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 374, 375, and 376, respectively;

[0143] (m) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 377, 378, and 379, respectively;

[0144] (n) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 380, 381, and 382, ​​respectively;

[0145] (o) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 383, 384, and 385, respectively;

[0146] (p) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 386, 387, and 388, respectively;

[0147] (q) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 389, 390, and 391, respectively;

[0148] (r) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 392, 393, and 394, respectively;

[0149] (s) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 395, 396, and 397, respectively;

[0150] (t) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 398, 399, and 400, respectively;

[0151] (u) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 401, 402, and 403, respectively;

[0152] (v) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 404, 405, and 406, respectively;

[0153] (w) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 407, 408, and 409, respectively;

[0154] (x) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 410, 411, and 412, respectively;

[0155] (y) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 413, 414, and 415, respectively;

[0156] (z) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 416, 417, and 418, respectively;

[0157] (aa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 419, 420, and 421, respectively;

[0158] (bb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 422, 423, and 424, respectively;

[0159] (cc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 425, 426, and 427, respectively;

[0160] (dd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 428, 429, and 430, respectively;

[0161] (ee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 431, 432, and 433, respectively;

[0162] (ff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 434, 435, and 436, respectively;

[0163] (gg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 437, 438, and 439, respectively;

[0164] (hh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 440, 441, and 442, respectively;

[0165] (ii) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 443, 444, and 445, respectively;

[0166] (jj) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 446, 447, and 448, respectively;

[0167] (kk) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 449, 450, and 451, respectively;

[0168] (ll) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 452, 453, and 454, respectively;

[0169] (mm) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 455, 456, and 457, respectively;

[0170] (nn) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 458, 459, and 460, respectively;

[0171] (oo) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 461, 462, and 463, respectively;

[0172] (pp) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 464, 465, and 466, respectively;

[0173] (qq) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 467, 468, and 469, respectively;

[0174] (rr) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 470, 471, and 472, respectively;

[0175] (ss) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 473, 474, and 475, respectively;

[0176] (tt) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 476, 477, and 478, respectively;

[0177] (uu) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 479, 480, and 481, respectively;

[0178] (vv) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 482, 483, and 484, respectively;

[0179] (ww) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 485, 486, and 487, respectively;

[0180] (xx) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 488, 489, and 490, respectively;

[0181] (yy) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 491, 492, and 493, respectively;

[0182] (zz) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 494, 495, and 496, respectively;

[0183] (aaa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 497, 498, and 499, respectively;

[0184] (bbb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 500, 501, and 502, respectively;

[0185] (ccc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 503, 504, and 505, respectively;

[0186] (ddd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 506, 507, and 508, respectively;

[0187] (eee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 509, 510, and 511, respectively;

[0188] (fff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 512, 513, and 514, respectively;

[0189] (ggg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 515, 516, and 517, respectively;

[0190] (hhh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 518, 519, and 520, respectively;

[0191] (iii) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 521, 522, and 523, respectively;

[0192] (jjj) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 524, 525, and 526, respectively;

[0193] (kkk) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 527, 528, and 529, respectively;

[0194] (lll) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 530, 531, and 532, respectively;

[0195] (mmm) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 533, 534, and 535, respectively;

[0196] (nnn) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 536, 537, and 538, respectively;

[0197] (ooo) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 539, 540, and 541, respectively;

[0198] (ppp) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 542, 543, and 544, respectively;

[0199] (qqq) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 545, 546, and 547, respectively;

[0200] (rrr) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 548, 549, and 550, respectively;

[0201] (sss) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 551, 552, and 553, respectively;

[0202] (ttt) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 554, 555, and 556, respectively;

[0203] (uuu) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 557, 558, and 559, respectively;

[0204] (vvv) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 560, 561, and 562, respectively;

[0205] (www) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 563, 564, and 565, respectively;

[0206] (xxx) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 566, 567, and 568, respectively;

[0207] (yyy) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 569, 570, and 571, respectively;

[0208] (zzz) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 572, 573, and 574, respectively;

[0209] (aaaa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 575, 576, and 577, respectively;

[0210] (bbbb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 578, 579, and 580, respectively;

[0211] (cccc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 581, 582, and 583, respectively;

[0212] (dddd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 584, 585, and 586, respectively;

[0213] (eeee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 587, 588, and 589, respectively;

[0214] (ffff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 590, 591, and 592, respectively;

[0215] (gggg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 593, 594, and 595, respectively;

[0216] (hhhh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 596, 597, and 598, respectively;

[0217] (iiii) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 599, 600, and 601, respectively;

[0218] (jjjj) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 602, 603, and 604, respectively;

[0219] (kkkk) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 605, 606, and 607, respectively;

[0220] (llll) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 608, 609, and 610, respectively;

[0221] (mmmm) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 611, 612, and 613, respectively;

[0222] (nnnn) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 614, 615, and 616, respectively;

[0223] (oooo) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 617, 618, and 619, respectively;

[0224] (pppp) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 620, 621, and 622, respectively;

[0225] (qqqq) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 623, 624, and 625, respectively;

[0226] (rrrr) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 626, 627, and 628, respectively;

[0227] (ssss) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 629, 630, and 631, respectively;

[0228] (tttt) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 632, 633, and 634, respectively;

[0229] (uuuu) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 635, 636, and 637, respectively;

[0230] (vvvv) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 638, 639, and 640, respectively;

[0231] (www) a variable region comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 641, 642 and 643, respectively;

[0232] (xxxx) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 644, 645, and 646, respectively;

[0233] (yyyy) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 647, 648, and 649, respectively;

[0234] (zzzz) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 650, 651, and 652, respectively;

[0235] (aaaaa) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 653, 654, and 655, respectively;

[0236] (bbbbb) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 656, 657, and 658, respectively;

[0237] (ccccc) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 659, 660, and 661, respectively;

[0238] (ddddd) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 662, 663, and 664, respectively;

[0239] (eeeee) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 665, 666, and 667, respectively;

[0240] (fffff) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 668, 669, and 670, respectively;

[0241] (ggggg) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 671, 672, and 673, respectively;

[0242] (hhhhh) a variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 674, 675, and 676, respectively; and

[0243] (iiiii) A variable region comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 677, 678, and 679, respectively.

[0244] In various embodiments, the antibody or antigen-binding fragment thereof is 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, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, and 904.

[0245] In various embodiments, the antibody or antigen-binding fragment thereof is 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.

[0246] In various embodiments, the antibody or antigen-binding fragment thereof is 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, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, and 905.

[0247] In various embodiments, the antibody or antigen-binding fragment thereof is 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, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to an amino acid sequence selected from the group consisting of: 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903 and 905.

[0248] In various embodiments, the antibody or antigen-binding fragment thereof is 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, 800, 802, 804, 806, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 840, 842, 844, 846, 848, 8, 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 heavy chain variable region sequences, 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, 794, 795, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, and 905.

[0249] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:681.

[0250] 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:682 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:683.

[0251] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:685.

[0252] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:687.

[0253] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:689.

[0254] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:691.

[0255] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:693.

[0256] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:695.

[0257] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:697.

[0258] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:699.

[0259] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:701.

[0260] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:703.

[0261] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:705.

[0262] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:707.

[0263] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:709.

[0264] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:711.

[0265] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:713.

[0266] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:715.

[0267] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:717.

[0268] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:719.

[0269] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:721.

[0270] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:723.

[0271] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:725.

[0272] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:727.

[0273] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:729.

[0274] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:731.

[0275] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:733.

[0276] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:735.

[0277] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:737.

[0278] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:739.

[0279] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:741.

[0280] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:743.

[0281] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:745.

[0282] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:747.

[0283] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:749.

[0284] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:751.

[0285] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:753.

[0286] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:755.

[0287] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:757.

[0288] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:759.

[0289] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:761.

[0290] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:763.

[0291] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:765.

[0292] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:767.

[0293] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:769.

[0294] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:771.

[0295] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:773.

[0296] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:775.

[0297] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:777.

[0298] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:779.

[0299] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:781.

[0300] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:783.

[0301] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:785.

[0302] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:787.

[0303] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:789.

[0304] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:791.

[0305] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:793.

[0306] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:795.

[0307] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:797.

[0308] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:799.

[0309] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:801.

[0310] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:803.

[0311] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:805.

[0312] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:807.

[0313] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:809.

[0314] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:811.

[0315] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:813.

[0316] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:815.

[0317] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:817.

[0318] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:819.

[0319] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:821.

[0320] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:823.

[0321] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:825.

[0322] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:827.

[0323] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:829.

[0324] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:831.

[0325] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:833.

[0326] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:835.

[0327] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:837.

[0328] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:839.

[0329] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:841.

[0330] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:843.

[0331] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:845.

[0332] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:847.

[0333] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:849.

[0334] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:851.

[0335] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:853.

[0336] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:855.

[0337] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:857.

[0338] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:859.

[0339] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:861.

[0340] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:863.

[0341] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:865.

[0342] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:867.

[0343] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:869.

[0344] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:871.

[0345] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:873.

[0346] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:875.

[0347] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:877.

[0348] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:879.

[0349] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:881.

[0350] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:883.

[0351] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:885.

[0352] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:887.

[0353] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:889.

[0354] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:891.

[0355] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:893.

[0356] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:895.

[0357] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:897.

[0358] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:899.

[0359] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:901.

[0360] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:903.

[0361] 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 a light chain variable region comprising the amino acid sequence of SEQ ID NO:905.

[0362] 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.

[0363] In various embodiments, the antibody or antigen-binding fragment thereof of any one of the preceding claims neutralizes hMPV A and / or hMPV B. In certain embodiments, the antibody or antigen-binding fragment thereof neutralizes hMPV A. In certain embodiments, the antibody or antigen-binding fragment thereof neutralizes hMPV B.

[0364] In various embodiments, the antibody or antigen-binding fragment thereof inhibits hMPV infection.

[0365] In various embodiments, the antibody or antigen-binding fragment thereof neutralizes hMPV with a neutralizing potency (IC50) of 10 micrograms 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.

[0366] In various embodiments, the antibodies or antigen-binding fragments thereof neutralize hMPV with a neutralizing potency (IC50) described herein, eg, Tables 3A-C.

[0367] In various embodiments, the antibody or antigen-binding fragment thereof binds to and neutralizes hMPV in vitro.

[0368] In various embodiments, the antibody or antigen-binding fragment thereof neutralizes hMPV in vivo.

[0369] In various embodiments, the antibody or antigen-binding fragment thereof has a cytoplasmic affinity of about 1×10 as determined by surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., KinExa and OCTET). -9 M ~ approx. 1×10 -12 The antibody or antigen-binding fragment thereof binds to an hMPV F protein or antigen or an hMPV prefusion F protein or antigen with a KD value of M. 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 a non-processed 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 a non-processed PreF antigen.

[0370] In various embodiments, the antibody or antigen-binding fragment thereof binds to, cross-reacts with, and / or neutralizes RSV and / or a RSV antigen.

[0371] In various embodiments, the antibody or antigen-binding fragment thereof is an antibody, e.g., the antibody is a chimeric, human, or humanized antibody.

[0372] One aspect of the present invention provides any of the antibodies or antigen-binding fragments thereof described herein, for example, antibodies or antigen-binding fragments thereof that bind to the same epitope on hMPV as the antibodies or antigen-binding fragments thereof described in Tables 1 to 4.

[0373] 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.

[0374] One aspect of the invention provides antibodies or antigen-binding fragments thereof that bind to one or more of the following sites of hMPV (SEQ ID NO:1): ?, I, II, III, III', IV, IV', V, alpha, and / or beta. In various embodiments, the antibody or antigen-binding fragment thereof binds to site ? 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).

[0375] One aspect of the invention provides antibodies or antigen-binding fragments thereof that neutralize hMPV and / or bind to one or more of the following sites of hMPV (SEQ ID NO:1): ?, I, II, III, III', IV, IV', V, alpha, and / or beta. In various embodiments, the antibody or antigen-binding fragment thereof binds to site ? 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).

[0376] In various embodiments, the antibody or antigen-binding fragment thereof binds to a specific antigenic site as described above and has greater than 80% identity (i.e., greater than 80, 85, 90, 95, 97, 98, 99, 100% identity) with the VH and / or VL of an antibody or antigen-binding fragment thereof disclosed herein, e.g., in Table 3B, Table 3C, or Table 3D.

[0377] One aspect of the present invention provides multispecific molecules comprising a first binding region comprising an antibody or antigen-binding fragment thereof described herein and a second binding region. In various embodiments, the multispecific comprises a bispecific molecule. For example, the second binding region binds to a virus-associated antigen. In various embodiments, the second binding region binds to an antigen or ligand associated with a viral condition or disease.

[0378] One aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding any of the antibodies or antigen-binding fragments thereof described herein, or any of the bispecific molecules described herein.

[0379] One aspect of the present invention provides an expression vector comprising any of the nucleic acids described herein. In one example, the expression vector comprises a nucleic acid encoding at least one of the polypeptides of Table 3D (SEQ ID NOS: 680-905).

[0380] One aspect of the invention provides a cell transformed with any of the expression vectors described herein.

[0381] One aspect of the present 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 herein, and a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition comprises one or more additional prophylactic or therapeutic agents. In various embodiments of the pharmaceutical composition, the one or more additional therapeutic agents are selected from the group consisting of an immunomodulatory agent, a hormone, a cytotoxic agent, an enzyme, a radionuclide, a second antibody conjugated to at least one immunomodulatory agent, an enzyme, a radiolabel, a hormone, an antisense oligonucleotide, or a cytotoxic agent, and combinations thereof.

[0382] One aspect of the invention provides a kit for preventing, treating, or detecting a virus, the kit comprising any of the described antibodies or antigen-binding fragments thereof, any of the described multispecific molecules (e.g., bispecific molecules), and / or any of the pharmaceutical compositions described herein; and instructions for use.

[0383] One aspect of the invention provides a method for producing an antibody or antigen-binding fragment thereof, the method comprising culturing a host cell containing a polynucleotide encoding the amino acid sequence of any antibody or antigen-binding fragment thereof described herein under conditions favoring expression of the polynucleotide, and optionally recovering the antibody or antigen-binding fragment thereof from the host cell and / or culture medium.

[0384] One aspect of the invention provides a method for selectively detecting hMPV, comprising administering or contacting a sample with any of the antibodies or antigen-binding fragments 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 serum from a subject.

[0385] One aspect of the present invention provides a method of treating a subject having a viral disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of any antibody or antigen-binding fragment thereof described herein, any multispecific (e.g., bispecific) molecule described herein, and / or any pharmaceutical composition described herein.

[0386] In various embodiments of the method, the viral disease is selected from the group consisting of an MPV infection (e.g., hMPV infection), a respiratory virus), clinical symptoms associated with an MPV infection, a lower respiratory tract infection (LTRI) leading to acute respiratory tract infection leading to bronchiolitis, and 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 tract virus. In various embodiments of the method, the viral disease is associated with hMPV type A. In various embodiments, the viral disease is associated with hMPV type B. In various embodiments, the respiratory virus is human respiratory syncytial virus (hRSV).

[0387] In various embodiments, the method further comprises administering one or more additional therapies.

[0388] One aspect of the present invention provides any antibody or antigen-binding fragment thereof described herein, any multispecific (e.g., bispecific) molecule described herein, and / or any pharmaceutical composition described herein for use in the preparation of a medicament for binding to hMPV, neutralizing hMPV, and / or treating a disorder or condition associated with hMPV in a subject in need thereof.

[0389] One aspect of the present 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 can be used to immunize subjects and prevent or treat hMPV infection.

[0390] In various embodiments of the 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 comprise 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 is unchanged at residues 491-512 of SEQ ID NO:911. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs 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 acid. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs by 1 to 15 amino acids from SEQ ID NO: 911. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs by 1 to 10 amino acids from SEQ ID NO: 911. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs by 1 to 5 amino acids from SEQ ID NO: 911. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs by 1 to 3 amino acids from SEQ ID NO: 911.

[0391] In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of SEQ ID NO:911.

[0392] In various embodiments of the 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 comprise 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.

[0393] In various embodiments of the isolated hMPV F polypeptide, the 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.

[0394] In various embodiments of the 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 comprise residues 491-539 of SEQ ID NO:1, and the isolated hMPV F polypeptide has a mutation at residue 185. In various embodiments, the mutation is A185P.

[0395] In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide comprises 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 isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517 of SEQ ID NO:910; the isolated hMPV F polypeptide variant comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516 of SEQ ID NO:912; and the isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521 of SEQ ID NO:913. In various embodiments, the variants comprise 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 an isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517, an isolated hMPV F polypeptide variant comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516, and an isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521.

[0396] In various embodiments of the isolated hMPV F polypeptide, a variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:910 differs 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 acid, and a variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs by 1-15 amino acids from SEQ ID NO:910, and a variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:910 by 1-10 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:910 by 1-5 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:910 by 1-3 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517.

[0397] In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of residues 1-521 of SEQ ID NO:910.

[0398] In various embodiments of the isolated hMPV F polypeptide, a variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO:912 differs 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 acid, and a variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs by 1-15 amino acids from SEQ ID NO:912, and a variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:912 by 1-10 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:912 by 1-5 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:912 by 1-3 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516.

[0399] In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of residues 1-518 of SEQ ID NO:912.

[0400] In various embodiments of the isolated hMPV F polypeptide, a variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:913 differs 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 acid, and a variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs by 1-15 amino acids from SEQ ID NO:913, and a variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:913 by 1-10 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:913 by 1-5 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521. In various embodiments of the isolated hMPV F polypeptide, the variant amino acid sequence differs from SEQ ID NO:913 by 1-3 amino acids, and the variant of the isolated hMPV F polypeptide comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521.

[0401] In various embodiments of the isolated hMPV F polypeptide, the isolated hMPV F polypeptide consists of residues 1-521 of SEQ ID NO:913.

[0402] 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.

[0403] In various embodiments of the 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 6xHis sequence (SEQ ID NO: 1014) or a strep tag II sequence.

[0404] In various embodiments of the 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.

[0405] One aspect of the present invention also provides nucleic acids encoding isolated hMPV F polypeptides, isolated hMPV F polypeptides, and immunogenic compositions comprising such isolated hMPV F polypeptides encoding variants of the hMPV F protein. Such isolated hMPV F polypeptides and immunogenic compositions can be used to immunize subjects and prevent or treat hMPV infection.

[0406] One aspect of the invention also provides an isolated nucleic acid encoding any one of the isolated hMPV F polypeptides encoding the hMPV F protein described above. In some embodiments, the isolated nucleic acid is codon-optimized.

[0407] One aspect of the invention provides an isolated nucleic acid comprising the 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 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.

[0408] One aspect of the invention also provides an isolated nucleic acid comprising the 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 an isolated nucleic acid variant comprising SEQ ID NO:1004 encodes the amino acid sequence of SEQ ID NO:910, an isolated nucleic acid variant comprising SEQ ID NO:1008 encodes the amino acid sequence of SEQ ID NO:912, and an isolated nucleic acid variant comprising SEQ ID NO:1010 encodes the amino acid sequence of SEQ ID NO:913. In various embodiments, an isolated nucleic acid variant comprising SEQ ID NO:1004 is SEQ ID NO:1005, an isolated nucleic acid variant comprising SEQ ID NO:1008 is SEQ ID NO:1009, and an isolated nucleic acid variant comprising SEQ ID NO:1010 is SEQ ID NO:1011. In various embodiments, a variant comprises a nucleic acid sequence having 95-99% identity to the nucleic acid sequence of SEQ ID NO:1004, SEQ ID NO:1008, or SEQ ID NO:1010.

[0409] One aspect of the invention also provides an immunogenic composition comprising an isolated antigenic polypeptide, an 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.

[0410] One aspect of the present invention also provides an immunogenic composition comprising an isolated antigenic polypeptide, wherein the isolated antigenic peptide comprises 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 isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:910 has unchanged residues 486-517; the isolated hMPV F polypeptide variant comprising residues 1-518 of SEQ ID NO:912 has unchanged residues 102-105 and 490-516; and the isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:913 has unchanged 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 variants thereof, wherein the isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517, the isolated hMPV F polypeptide variant comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516, and the isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521. In various embodiments, the variants comprise 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 an isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:910 is unchanged at residues 486-517, an isolated hMPV F polypeptide variant comprising residues 1-518 of SEQ ID NO:912 is unchanged at residues 102-105 and 490-516, and an isolated hMPV F polypeptide variant comprising residues 1-521 of SEQ ID NO:913 is unchanged at residues 99-102, 185, and 485-521.

[0411] In various embodiments of the immunogenic composition, 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.

[0412] The present invention also provides methods of inducing an immune response in a subject, comprising administering to the subject any one of the above immunogenic compositions in an amount effective to generate 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 or intramuscular injection. In various embodiments, the subject has been exposed to hMPV, the subject is infected with hMPV, or the subject is at risk of infection from hMPV.

[0413] The invention also provides any one of the above immunogenic compositions for use in inducing an antigen-specific immune response in a subject.The invention also provides any one of the above immunogenic compositions for use in preventing or treating hMPV infection in a subject.

[0414] The invention provides any one of the above-described isolated hMPV F polypeptides for use in inducing an antigen-specific immune response in a subject.

[0415] The invention provides any one of the above isolated hMPV F polypeptides for use in the prevention or treatment of human metapneumovirus (hMPV) infection in a subject.

[0416] The invention provides the use of any one of an isolated hMPV F polypeptide or an isolated nucleic acid encoding such a polypeptide in the manufacture of a medicament for inducing an antigen-specific immune response in a subject.

[0417] The invention provides the use of any one of the above-described isolated hMPV F polypeptides, or isolated nucleic acids 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. [Brief explanation of the drawings]

[0418] [Figure 1A] FIG. 1A shows the workflow for antibody discovery by selection, culture, and cloning of single memory B cells.

[0419] [Figure 1B] Figure 1B shows a representative set of images demonstrating the B cell sorting gating strategy. Enriched B cells were gated using light backscatter (BSC) and forward scatter (FSC), followed by forward scatter height per area (FSC-H) to exclude debris and doublet cells. Cells were then gated on CD3 followed by CD19+IgG+ cells. These cells were then used to gate antigen-specific cells (hMPV F+).

[0420] [Figure 2A] Figure 2A is a schematic diagram of hMPV unprocessed wild-type PreF, unprocessed stabilized PreF, and processed stabilized PreF. Figure 2A discloses the amino acid sequences of SEQ ID NO: 1000 (ENPRQSRFVL) and SEQ ID NO: 1001 (ENPRRRRFVL), respectively, in order of appearance. SEQ ID NO: 1000 (ENPRQSRFVL) is included in SEQ ID NO: 910. SEQ ID NO: 1001 (ENPRRRRFVL) is included in SEQ ID NO: 913. [Figure 2B] FIG. 2B is a photograph of a denaturing SDS-PAGE gel of the constructs described in FIG. 2A.

[0421] [Figure 3A]Figures 3A, 3B, and 3C show CDRH3 length and somatic hypermutation (SHM) data for hMPV F-specific mAbs. Figure 3A shows the germline frequencies of VH and VK / VL listed in Tables 3A, 3B, 3C, and 3D. [Figure 3B] FIG. 3B is a graph showing CDRH3 length in VH. [Figure 3C] Figure 3C is a graph showing nucleotide substitutions in VH (excluding CDRH3) and VK / VL. Horizontal bars (identified by arrows) indicate median values. Analysis was based on the Kabat delineation system [1].

[0422] [Figure 4A] Figure 4A is a graph showing antibody M4B06 IgG neutralization assay data for hMPV strains A and B. Error bars indicate the standard deviation of three replicates. [Figure 4B] Figure 4B is a graph showing biolayer interferometry (BLI) binding of M4B06 IgG to untreated PreF trimer, treated PreF trimer, and PostF trimer. [Figure 4C] Figure 4C is a set of graphs and IC50 tables showing binding of M4B06 IgG to untreated hMPV PreF WT and PreF-bearing monoclonal antibody-resistant mutant (MARM) mutants, as determined by ELISA using Expi293 cell culture supernatants containing expressed antigen. Error bars indicate the standard deviation of duplicate replicates. [Figure 4D] Figure 4D is a graph and EC50 table showing neutralization assay data for M4B06 IgG and Fab, and an irrelevant control antibody (DD1L), against hMPV PreF and three MARM viruses. Error bars indicate standard deviation of triplicates. [Figure 4E] FIG. 4E shows the M4B06 epitope identified by MARM and hydrogen / deuterium exchange mass spectrometry (HDX-MS) mapped onto the hMPV PreF structure, with epitope residues identified by shading. [Figure 4F]Figure 4F shows a cryoEM map of three M4B06 Fabs complexed with the PreF trimer processed at 3.75 angstroms (Å). A hMPV PreF model with the same shading as in Figure 4E was fitted to the density.

[0423] [Figure 5] Figure 5 shows an epitope map of the hMPV PreF trimer protein and M4B06 by HDX-MS. Figure 5 discloses SEQ ID NO: 1002, which corresponds to amino acids 19 to 542 of SEQ ID NO: 913.

[0424] [Figure 6A] Figure 6A is a diagram showing the various epitopes in antigenic sites II and V, and Figure 6B is a set of graphs. Figure 6A is an epitope map showing contact residues of different site II and V mAbs mapped onto the hMPV PreF trimer structure [2]. [Figure 6B] Figure 6B is a graph showing binding of selected site II and V mAbs to unprocessed hMPV PreF and epitope knockout mutants as determined by ELISA using Expi293 cell culture supernatants containing expressed antigen. Error bars indicate standard deviation of duplicate replicates.

[0425] [Figure 7A] Figure 7A is a graph showing binding of antibody M2D2 (positive control) IgG to untreated hMPV PreF and PreF bearing MARM mutants. Error bars indicate standard deviation of two replicates. [Figure 7B] FIG. 7B is a graph showing binding of M1A04 IgG to unprocessed hMPV PreF and epitope knockout mutants. [Figure 7C] Figure 7C is a graph showing binding of M2A05 IgG to unprocessed hMPV PreF and epitope knockout mutants. Binding was determined by ELISA using Expi293 cell culture supernatants containing expressed antigen.

[0426] [Figure 8A] Figures 8A, 8B, and 8C are a set of figures and graphs showing epitope mapping of hMPV F-specific mAbs. Figure 8A is a heat map showing epitope binning of selected mAbs using untreated hMPV PreF antigen. Seventy-three isolated mAbs that showed apparent BLI binding responses (>0.2 nM) to the tested antigen were included. The heat map shows epitope binning from a sandwich-based BLI binding assay, with markers / shading indicating greater competition between the first and second antibodies. The top sidebar shows the neutralization potency (IC50) of the mAbs against hMPV A, hMPV B, RSV A, and RSV B virus strains. [Figure 8B] Figure 8B shows the major antigenic sites II, III, III', IV, IV', and V (internal antigenic site α is not visible, and antigenic site β has not yet been clearly characterized) mapped onto the hMPV PreF trimeric protein structure. The DS7 site is also labeled for reference. [Figure 8C] Figure 8C is a graph showing the proportion of antibodies targeting each antigenic site, grouped by neutralizing potency. For each mAb, the stronger neutralizing potency of hMPV A and B was selected for grouping.

[0427] [Figure 9] Figure 9 is a series of photographs showing negative stain EM and 2D averages of engineered hMPV postF trimeric proteins. Most class averages show particles with distinct heads (top regions) and elongated tails (middle portions). In some averages (bottom / tail), the tail appears longer, with a small, distinct section at the end of the tail. In some averages, a depression in the "head" portion of the particle was visible (identified by an arrow). All figures are to scale.

[0428] [Figure 10A]Figures 10A, 10B, 10C, 10D, and 10E are a set of graphs showing the correlation between neutralization potency, antigenic site, and binding specificity for different hMPV F conformations. Figures 10A and 10B are graphs showing the BLI binding response of hMPV-F-specific mAbs to unprocessed hMPV PreF trimers and PostF trimers. Each dot represents an isolated hMPV F-specific antibody: neutralization potency (Figure 10A) or [Figure 10B] Mapped antigenic sites are distinguished by: V, II, III, III', IV, IV', site α, site β, or uncharacterized (Figure 10B). [Figure 10C] Figures 10C and 10D are graphs showing the BLI binding responses of hMPV-F-specific mAbs to unprocessed hMPV PreF and processed stabilized hMPV PreF (115BV) antigens. Each dot represents the neutralization potency (Figure 10C) or [Figure 10D] Isolated hMPV F-specific antibodies are depicted shaded by mapped antigenic sites: V, II, III, III', IV, IV', site α, site β, or uncharacterized (Fig. 10D). [Figure 10E] Figure 10E is a graph showing the correlation between neutralization potency (after Log10 transformation) and the ratio of treated PreF / untreated PreF BLI binding. The dashed line and R-squared indicate linear regression. Each dot represents an isolated hMPV F-specific antibody, which is shaded and identifies the mapped antigenic site to which it binds (V, II, III, III', IV, IV', site α, site β, or uncharacterized).

[0429] [Figure 11A] FIG. 11A shows the format of the magnetic bead-based serum absorption assay used to analyze untreated hMPV PreF, treated hMPV PreF, hMPV PostF, and RSV PreF trimers. [Figure 11B] Figure 11B is a graph showing the mean ELISA titers from four donors after antigen depletion, normalized by the titer of undepleted serum. Error bars indicate SEM.

[0430] [Figure 12A] Figures 12A, 12B, and 12C are a set of graphs showing differential scanning fluorimetry (DSF) spectra: of unprocessed hMPV PreF protein (1.5 mg / ml; Figure 12A); [Figure 12B] Fig. 12B is of processed stabilized hMPV PreF protein (2.5 mg / ml) expressed without furin; [Figure 12C] Figure 12C shows unprocessed, stabilized hMPV PreF protein (2.7 mg / ml) expressed without furin cleavage. Spectra were recorded in duplicate at a scan rate of 1 °C / min. The graph shows the ratio of fluorescence between 350 nm and 330 nm (top) and the first derivative of this ratio (bottom).

[0431] [Figure 13] Figure 13 is a graph showing the distribution of isolated hMPV-F-specific mAbs and their mapped antigenic sites (II, V, III, III', IV, IV', site α, or uncharacterized), grouped by donor. The left Y-axis indicates the number of mAbs discovered from each donor. The right Y-axis indicates the serum neutralization titer (fold dilution) of each donor. Markers / dots indicate the neutralization titer of donor serum at fold dilution. One donor was not included because only one mAb was isolated from this donor.

[0432] [Figure 14A] FIG. 14A shows unprocessed hMPV PreF monomers. [Figure 14B] Figure 14B shows side and top views of the processed stabilized hMPV PreF trimer, identifying the N-terminal segment of F1 near the cleavage site.

[0433] [Figure 15A]Figures 15A-E are a set of figures and tables showing the interaction of antibody M8C10 with MPV. Figure 15A is a stereogram / drawing of the MPV-F:M8C10 complex (top) with the MPV-F trimer (PDB: code 5wb0) shown from the same perspective (bottom) for reference. [Figure 15B] Figure 15B is a set of individual views / drawings of the MPV-F:M8C10 complex (top panel) from the Fab perspective (left), side (center), and top (right). The same views of the MPV-F trimer are shown below for each panel. [Figure 15C] Figure 15C lists the sequences of the M8C10 heavy chain (HC) and light chain (LC), with the CDR regions highlighted and shaded as follows: HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Figure 15C also shows a diagram of the surface representation of the complex on the right, with the CDRs shaded as sequences and identifying the F protein, heavy chain, and light chain. [Figure 15D] FIG. 15D is a diagram showing the interactions mediated by the four interacting CDR regions. [Figure 15E] FIG. 15E lists the residues involved in the interaction between MPV-F and M8C10, with residues in the CDRs shaded.

[0434] [Figure 16A] Figure 16A is a series of graphs showing the effect of epitope residue mutations on M8C10 binding, and Figure 16B is a series of figures. Figure 16A is a set of graphs showing ELISA binding analysis of MPV-F mutations. Binding to M8C10 is shown in blue across a range of concentrations. As a control, the site IV binder, M2D2, is shown in red. [Figure 16B] FIG. 16B is a set of structure-based diagrams showing the interactions mediated by R198, N202, and A216.

[0435] [Figure 17]Figure 17 is a set of figures showing a comparison of the M8C10-binding epitopes and MPV458-binding epitopes on the hMPV F monomer. The crystal structures M8C10 (bottom) and MPV458 (top) complexed with the hMPV F monomer were superimposed with hMPV F, revealing their distinct epitopes. The indicated regions indicate direct contact residues between hMPV F and M8C10. The left portal shows the hMPV F trimer structure with one protomer (left) and the other two protomers shaded (right). DETAILED DESCRIPTION OF THE INVENTION

[0436] Pneumoviruses, including respiratory syncytial virus (RSV) and hMPV, are common causes of acute lower respiratory tract infections (ALRI) [1-6]. Similar to RSV, hMPV infection results in a variety of symptoms, including cough, wheezing, pneumonia, and bronchiolitis. High-risk populations, including infants, young children, the elderly, and immunocompromised patients, are more likely to develop severe symptoms that may require hospitalization [7-14]. Co-infection with other respiratory pathogens is common. Recently, cases of coinfection with SARS-CoV-2 and hMPV have also been reported [15-17], and such coinfection with hMPV appeared to affect monocytes and attenuate interferon responses in patients with severe COVID-19

[15] . Despite these medical burdens, there are currently no approved hMPV vaccines or neutralizing antibodies available for therapeutic or prophylactic purposes.

[0437] The surface glycoprotein F of hMPV mediates the subsequent fusion of the viral membrane with the cellular membrane [18,19]. The F protein is highly conserved in sequence among different hMPV subtypes and appears to be more important for viral infection than the other surface proteins G and SH [20,21]. F is the primary viral antigen of hMPV that elicits neutralizing and protective antibodies against hMPV infection, but it has also been reported that antibodies elicited by the G and SH proteins are not protective [22-24]. Therefore, the F protein is an attractive target for the development of neutralizing antibodies and vaccines against hMPV infection.

[0438] The hMPV F protein is a class I viral fusion protein that is displayed as a homotrimer on the viral surface and on the membrane of host cells. The precursor of F (F0) is synthesized as an intact polypeptide and subsequently undergoes proteolytic processing to become functional

[19] . Unlike RSV, which has two furin cleavage sites that can be cleaved twice intracellularly during transport to the cellular membrane

[25] , hMPV has only one protease cleavage site that is cleaved on the cell surface or within virus-like particles by a transmembrane protease such as TMPRSS2

[26] . Cleavage of both RSV and hMPV F generates two disulfide-linked chains (F2 and F1). The fusion peptide, located at the N-terminus of F1 immediately after the protease cleavage site, is buried inside a hydrophobic cavity and interacts with adjacent monomers, likely stabilizing the trimeric conformation [27, 28]. This proteolytically processed F trimer in the spherical "prefusion" (PreF) form is metastable and can be induced to 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 the PreF and PostF conformations by introducing additional mutations or modifying the protease cleavage site [27-32]. Despite only approximately 35% sequence identity, the structures of both the stabilized PreF and PostF proteins between RSV F and hMPV F are remarkably conserved [27-31, 33, 34].

[0439] Over the past few years, numerous monoclonal antibodies (mAbs) targeting the RSV F antigen have been isolated from the human B cell repertoire and characterized using various approaches [33,35-47]. These results have defined six major antigenic sites on RSV F (?, I, II, III, IV, and V). PreF-specific antibodies targeting sites ? and V have been shown to be immunodominant and account for the majority of neutralizing activity in human serum and the antigen-specific memory B cell repertoire of adults [48,49], suggesting that PreF is a better vaccine candidate for RSV, eliciting higher neutralizing antibody (nAb) responses than PostF. In contrast, the immunogenic potential of stabilized hMPV PreF and PostF appears to be comparable, likely due to the large glycan shield observed only at site φ of the hMPV F protein

[28] . Although a limited number of hMPV-F-specific mAbs have been discovered from mouse immunization [ 50 ], phage display [ 51 ], and human B cells [ 52 ], a comprehensive understanding of human antibody recognition against hMPV F during natural infection remains elusive.

[0440] In this study, we report a large panel of hMPV-F-specific mAbs isolated from memory B cells of multiple healthy human donors. Several isolated mAbs demonstrated ultrahigh neutralization potency in vitro. The researchers characterized one of the most potent mAbs, which recognizes a PreF-specific epitope on site V. Further characterization of the isolated mAbs revealed diverse antigenic sites on the hMPV F antigen, including overlapping but distinct epitopes on previously defined sites II and V, as well as four novel antigenic sites not previously reported. See also Huang et al., 2019 Front. Immunol, pp. 1–8; Rossey et al., 2018 Trends in Microbiology, vol. 26, issue 3, pp. 209–219. See also Antigenic sites of hMPV.

[0441] Site φ - amino acids 54 to 84; 165 to 197 of SEQ ID NO: 1

[0442] Site I - amino acids 19-36; 283-289; 347-358 of SEQ ID NO: 1

[0443] Site II—amino acids 156 and 224-247 of SEQ ID NO: 1

[0444] Site III—amino acids 37-45; 271-282; 312-320; and 335-346 of SEQ ID NO:1;

[0445] Competitive binding to the hMPV F antigen protein by site III'-site III and DS7 site antibodies (M2B6, DS7) does not compete with site II antibodies (M1D2 and M1C7s).

[0446] Region IV—amino acids 386 to 439 of SEQ ID NO: 1

[0447] Position IV' - amino acids 294 to 298 of SEQ ID NO: 1

[0448] Site V—amino acids 46-53; 116-164; 233 and 257-270 of SEQ ID NO: 1

[0449] Position α - amino acids 82, 198, 202, 207, 211, 214-216, 218, 219, 220, 224-229, 233, 257, 258, 261, 262, 338 and 405 of SEQ ID NO: 1

[0450] Site β - not well characterized. Most of the mAbs in this group only compete with themselves

[0451] DS7 site (reference DOI: 10.1038 / nsmb.2250): amino acids 20 to 26, 31, 33 to 34, 282, 284, 312, 345, 348 to 349, 351, 353, 356, 358, 411, 413 and 414 of SEQ ID NO: 1.

[0452] Unlike RSV, antibody responses to hMPV F appear to be less prevalent against apical antigens. Furthermore, a panel of mAbs that specifically bind to uncleaved PreF has been identified, suggesting potential immunogenic differences between unprocessed and processed F antigens. This is the first disclosure to reveal the global antigenic epitopes of hMPV F-specific mAbs elicited from natural infection.

[0453] definition

[0454] As used herein, "human metapneumovirus" or "hMPV" refers to a single-stranded, negative-sense ribonucleic acid (RNA) virus of the Pneumoviridae family with a genome of approximately 13 kb. As used herein, "hMPV F protein" refers to the fusion glycoprotein F0 having the amino acid sequence set forth in UniProtKB-G3KCK8 (G3KCK8_9MONO; SEQ ID NO: 1).

[0455] 10 20 30 40 50

[0456] MSWKVMIIIS LLITPQHGLK ESYLEESCST ITEGYLSVLR TGWYTNVFTL

[0457] 60 70 80 90 100

[0458] EVGDVENLTC TDGPSLIKTE LDLTKSALRE LKTVSADQLA REEQIENPRQ

[0459] 110 120 130 140 150

[0460] SRFVLGAIAL GVATAAAVTA GIAIAKTIRL ESEVNAIKGA LKTTNEAVST

[0461] 160 170 180 190 200

[0462] LGNGVRVLAT AVRELKEFVS KNLTSAINKN KCDIADLKMA VSFSQFNRRF

[0463] 210 220 230 240 250

[0464] LNVVRQFSDN AGITPAISLD LMTDAELARA VSYMPTSAGQ IKLMLENRAM

[0465] 260 270 280 290 300

[0466] VRRKGFGILI GVYGSSVIYM VQLPIFGVID TPCWIIKAAP SCSEKDGNYA

[0467] 310 320 330 340 350

[0468] CLLREDQGWY CKNAGSTVYY PNDKDCETRG DHVFCDTAAG INVAEQSREC

[0469] 360 370 380 390 400

[0470] NINISTTNYP CKVSTGRHPI SMVALSPLGA LVACYKGVSC SIGSNRVGII

[0471] 410 420 430 440 450

[0472] KQLPKGCSYI TNQDADTVTI DNTVYQLSKV EGEQHVIKGR PVSSSFDPIK

[0473] 460 470 480 490 500

[0474] FPEDQFNVAL DQVFESIENS QALVDQSNKI LNSAEKGNTG FIIVIILIAV

[0475] 510 520 530

[0476] LGLTMISVSI IIIIKKTRKP TGAPPELNGV TNGGFIPHS

[0477] (SEQ ID NO: 1)

[0478] As used herein, the term "antibody" refers to any form of immunoglobulin molecule that exhibits a desired biological activity or binding activity. An antibody that "specifically binds" to hMPV is one that binds preferentially to hMPV over other proteins, although 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 without generating undesirable results, such as false positives. An antibody or a binding fragment thereof binds to a target protein with an affinity that is at least 2-fold greater, preferably at least 10-fold greater, more preferably at least 20-fold greater, and most preferably at least 100-fold greater than its affinity for a non-target protein. Thus, the term "antibody" is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized fully human antibodies, and chimeric antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as humanizing the antibody for use as a human therapeutic.

[0479] Generally, a basic antibody structural unit comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain typically contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Human heavy chains are further typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody 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 or more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

[0480] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies) encoded by heavy chain constant region genes.

[0481] Antibodies are typically -5 ~10 -12 The dissociation constant (K D ) specifically bind to their cognate antigen with high affinity, reflected by approximately 10 -4 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen and a substantially identical antigen with high affinity, which is generally considered to indicate non-specific binding. -7 M or less, preferably 10 -8 M or less, and even more preferably 5×10 -9M or less, most preferably 10 -8 M~10 -10 K below M D This means that the antibody has a high affinity for the target antigen but does not bind with high affinity to unrelated antigens.

[0482] As used herein, the term "antibody" includes not only intact polyclonal or monoclonal antibodies, but, unless otherwise specified, also any antigen-binding portion thereof that competes with the intact antibody for specific binding, fusion proteins containing an antigen-binding portion, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site.

[0483] 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 specifically bind to an antigen, e.g., a fragment that retains one or more CDR regions and the ability to specifically bind to an antigen.

[0484] Antigen-binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, fragments containing the CDRs, and single-chain variable fragment antibodies (scFv), as well as polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to an antigen (e.g., hMPV).

[0485] Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof); antibodies need not be of a particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0486] The antigen-binding fragments within the scope of the present invention also include F(ab')2 fragments, which can be generated, for example, by enzymatic cleavage of IgG with pepsin. Fab fragments can be generated, for example, by reduction of F(ab')2 with dithiothreitol or mercaptoethylamine. The Fab fragment is a VL-CL chain attached to a VH-CH1 chain by a disulfide bridge. The F(ab')2 fragment is two Fab fragments attached by two disulfide bridges. The Fab portion of an F(ab')2 molecule contains a portion of the Fc region between which the disulfide bridges are located.

[0487] The term "acceptor human framework" refers to a light chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework. L ) framework or heavy chain variable domain (V H ) framework. A human immunoglobulin framework or human consensus framework "derived from" an acceptor human framework may have the same amino acid sequence as a naturally occurring human immunoglobulin framework or human consensus framework, or may have altered amino acid sequence compared to a wild-type naturally occurring human immunoglobulin framework or human consensus framework. In some embodiments, the number of amino acid changes is 10, 9, 8, 7, 6, 5, 4, 3, or 2, or 1. In some embodiments, a V L The acceptor human framework is V L The V is identical in sequence to a human immunoglobulin framework sequence or a human consensus framework sequence. H The acceptor human framework is V H The sequence is identical to a human immunoglobulin framework sequence or a human consensus framework sequence.

[0488] As used herein, the term "binding protein" also refers to a non-natural (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 multispecific molecule (e.g., a multispecific antibody) that comprises an anti-hMPV antibody or antigen-binding fragment thereof and a second molecule (e.g., a second antibody or antigen-binding fragment thereof).

[0489] A "multispecific antibody" is an antibody that recognizes two or more different antigens or epitopes (e.g., bispecific antibodies, trispecific antibodies). In various embodiments, multispecific antibodies typically bind to at least two antigens or epitopes (i.e., bispecific antibodies, BsAbs) or three or more antigens. In various embodiments, multispecific antibodies with additional specificities, such as trispecific antibodies, are encompassed herein. An example of a BsAb is one in which one arm is directed against viral antigen #1 and the other arm is directed against viral antigen #2. For example, BsAbs may bind to hMPV strain A and hMPV strain B.

[0490] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (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). Multispecific antibodies have also been developed by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004), by 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); by using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553), by using "diabody" technology to create bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and by using single-chain Fv (scFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 148 (1992) 1547-1553). al., J. Immunol. 152 (1994) 5368-5374); and, for example, by preparing trispecific antibodies as described 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., U.S. Patent Application Publication No. 2006 / 0025576 and Proc Natl Acad Sci USA. 2011 Jul 5;108(27):11187-92).

[0491] 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, for example, 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, each with a different specificity), 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, inter alia, those generated by quadroma technology (Milstein and Cuello (1983) Nature 305(5934):537-40), chemical conjugation of two different monoclonal antibodies (Staerz et al. (1985) Nature 314(6012):628-31), or knob-into-hole or similar approaches introducing mutations into the Fc region (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14):6444-6448).

[0492] The term "recombinant antibody" refers to an antibody that is prepared, expressed, generated, or isolated by recombinant means, such as (a) an antibody 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) an antibody isolated from a host cell, e.g., a transfectoma, that has been transformed to express the antibody; (c) an antibody isolated from a recombinant combinatorial antibody library (e.g., containing human antibody sequences) using phage display; and (d) an antibody prepared, expressed, generated, or isolated by any other means, including splicing immunoglobulin gene sequences (e.g., human immunoglobulin genes) into other DNA sequences. Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis, thus modifying the V and V regions of the recombinant antibody. H Area and V L The amino acid sequence of the region is human germline V H Sequence and V L Derived from human germline V sequences H Sequence and human germline V L These are sequences that are related to, but may not naturally occur within, the human antibody germline repertoire in vivo.

[0493] "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, and the remainder of the chain(s) is derived from another species (e.g., mouse) or belongs to another antibody class or subclass, as well as fragments of such antibodies, so long as the desired biological activity is exhibited.

[0494] A "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, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences or derivatives thereof, respectively.

[0495] "Humanized antibody" refers to forms of antibodies containing sequences derived from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. When it is necessary to distinguish a humanized antibody from its parent rodent antibody, the prefixes "hum," "hu," or "h" can be added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequences as the parent rodent antibody but may include certain amino acid substitutions to enhance affinity, increase the stability of the humanized antibody, or for other reasons.

[0496] As used herein, "monoclonal antibody" or "mAb" or "Mab" 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 minor, naturally occurring mutations. In contrast, conventional (polyclonal) antibody preparations typically include a large number 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 antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, 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). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0497] Antigen-binding fragments of such immunoglobulins (including scFvs) 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 an antigen, each monoclonal antibody is directed against a single epitope. Monoclonal antibodies can be prepared using any art-recognized technique and those described herein, e.g., hybridoma techniques, transgenic animals, recombinant DNA techniques (see, e.g., U.S. Pat. No. 4,816,567), or using phage antibody libraries using, e.g., the techniques described in U.S. Pat. No. 7,388,088 and PCT Publication WO 00 / 31246. Monoclonal antibodies include chimeric, human, and humanized antibodies, and may be naturally occurring or recombinantly produced.

[0498] A "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The two V domains of a bivalent domain antibody are covalently linked with a peptide linker to create a bivalent domain antibody. H The regions may target the same or different antigens.

[0499] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific (see below).

[0500] As used herein, the term "single chain Fv" or "scFv" antibody refers to the V of an antibody. H and V LFv refers to an antibody fragment containing domains, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker. For a review of scFvs, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

[0501] 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 incorporated herein by reference in their entireties). In one embodiment, the present invention provides a method for producing single-domain antibodies by combining two V-domain antibodies with modifications such that single-domain antibodies are formed. H A single domain antibody comprising a domain is provided.

[0502] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L or V L -V H ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H). 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 complementary domains on another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, 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, see generally Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0503] The antibodies of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Patent No. 5,624,821; WO 2003 / 086310; WO 2005 / 120571; WO 2006 / 0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modifications can be used to enhance or suppress various immune system responses, which may have beneficial effects in diagnosis and therapy. Alterations in the Fc region include amino acid changes such as substitutions, deletions, and insertions, glycosylation or deglycosylation, and the addition of multiple Fc regions. Fc alterations can be used to alter the half-life of therapeutic antibodies; a longer half-life can result in less frequent administration, with attendant increased convenience and reduced material usage. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

[0504] The term "fully human antibody" refers to an antibody that comprises only human immunoglobulin protein sequences. 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, a "murine antibody" refers to an antibody that comprises only mouse immunoglobulin sequences.

[0505] As used herein, "variable region" or "V region" or "V chain" refers to the segment of an IgG chain that is variable in sequence among different antibodies. The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, alone or in combination. The variable region of a heavy chain is referred to in this disclosure as a "heavy chain variable region," "heavy chain variable domain," "VH," or "V H The variable region of a light chain is sometimes referred to in this disclosure as a "light chain variable region," "heavy chain variable domain," "VL," or "V L ". Typically, both the heavy and light chain variable regions contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. Generally, from N- to C-terminus, both the light chain variable domain and the heavy chain variable domain contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain generally follows the definitions in 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.

[0506] "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) in the non-framework region of an antibody VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) in the non-framework region of an antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences. CDR regions are well known to those skilled in the art and are defined, for example, by Kabat, as the most hypervariable regions within an antibody variable domain. CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved b-sheet framework and can therefore adopt different conformations. Both terms are well recognized in the art. CDR region sequences have also been defined by AbM, Contact, and IMGT. The positions of CDRs within canonical antibody variable regions 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 the hypervariable regions varies among different antibodies, additional residues relative to their canonical positions are conventionally numbered with a, b, c, etc. next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra). Such nomenclature is also well known to those of skill in the art. For example, correspondences between numbering systems, including the Kabat numbering system and the IMGT-specific numbering system, are well known to those of skill in the art and are shown in Table 1 below. 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 yet other embodiments, the CDRs are as defined by the Chothia numbering system, and 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 residues 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).

[0507] The constant region of an antibody can 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 amino acids in the heavy chain constant domain begins at number 118 according to the Eu numbering scheme. The Eu numbering scheme is based on the amino acid sequence of human IgG1 (Eu), which has a constant domain starting at amino acid position 118 of the IgG1 amino acid sequence set forth in Edelman et al., Proc. Natl. Acad. Sci. USA. 63:78-85 (1969), and the IgG1, IgG2, IgG3, and IgG4 constant domains set forth in Beranger, et al., ibid.

[0508] The variable regions of the heavy and light chains contain binding domains that comprise CDRs that interact with antigen. Numerous methods for defining CDR sequences of antibody variable domains are available in the art (see Dondelinger et al., Frontiers in Immunol. 9:Article 2278 (2018)). Common numbering schemes include:

[0509] The Kabat numbering scheme is based on sequence variability and is the most commonly used (see Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (defining the CDR regions of an antibody by sequence);

[0510] The Chothia numbering scheme is based on the location of structural loop regions (see Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997));

[0511] The AbM numbering scheme is a compromise between the two used by Oxford Molecular's AbM antibody modeling software (see Karu et al., ILAR Journal 37:132-141 (1995));

[0512] The contact numbering scheme is based on the analysis of available complex crystal structures (see www.bioinf.org.uk: Prof. Andrew C. Martin's Group; Abhinandan & Martin, Mol. Immunol. 45:3832-3839 (2008)).

[0513] The IMGT (ImMunoGeneTics) numbering scheme is a standardized numbering system for all protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains and T-cell receptor chains from different species, counting consecutive residues from 1 to 128 based on germline V sequence alignment (see Giudicelli et al., Nucleic Acids Res. 25:206-11 (1997); Lefranc, Immunol Today 18:509 (1997); Lefranc et al., Dev Comp Immunol. 27:55-77 (2003)).

[0514] The general rules disclosed in www.bioinf.org.uk: Prof. Andrew C. Martin's Group and reproduced below in Table 1 can be used to define CDRs in an antibody sequence that contain amino acids that interact specifically with amino acids that comprise the epitope in the antigen to which the antibody binds. Although there are rare instances where these generally constant features do not occur, Cys residues are the most conserved features.

[0515] [Table 1]

[0516] As used herein, the terms "framework" or "FR" residues refer to variable domain residues other than the hypervariable region residues defined herein as CDR residues. The residue numbering above refers to the Kabat numbering system and does not necessarily correspond precisely to the sequence numbering in the accompanying sequence listing. Amino acid residues in antibodies may 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.

[0517] As used herein, the term "isolated" when used in the context of a polypeptide or polynucleotide refers to a polypeptide or polynucleotide that is at least partially free from other biological molecules from the cell or cell culture in which it is produced. Such biological molecules include other nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth medium. It may also be at least partially free from expression system components, such as biological molecules from a host cell or its growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of a pharmaceutical formulation that includes the polypeptide or polynucleotide.

[0518] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities.

[0519] As used herein, "isotype" refers to the antibody class (e.g., IgG (including IgG1, IgG2, IgG3, IgG4), IgM, IgA (including IgA1 and IgA2), IgD, and IgE antibodies) encoded by the antibody's heavy chain constant region genes.

[0520] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event resulting therefrom. Exemplary "effector functions" include Clq 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 cell surface receptors (e.g., B cell receptors; BCRs). Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain).

[0521] "Fc region," "Fc domain," or "Fc" refers to the C-terminal region of an antibody heavy chain. The Fc region thus comprises the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL).

[0522] 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 linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of a protein (usually conformational epitopes). Epitopes formed from adjacent amino acids are typically, but not always, retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon 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.

[0523] The term "epitope mapping" refers to the process of identifying molecular determinants on an antigen involved in antibody-antigen recognition. Methods for determining which epitopes are bound by a given antibody are well known in the art and include, for example, immunoblotting and immunoprecipitation assays (e.g., overlapping or adjacent peptides derived from hMPV are tested for reactivity with a given antibody (e.g., anti-hMPV antibodies)); X-ray crystallography; antigenic mutation 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, GE Morris, Ed. (1996)). See also Champe et al. (1995) J. Biol. Chem. 270:1388-1394.

[0524] The term "binds to the same epitope" in reference to two or more antibodies means that the antibodies bind to the same segment or the same segment of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the "same epitope on hMPV" using the antibodies described herein include, for example, epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope, and HDX-MS. Other methods monitor antibody binding to fragments (e.g., proteolytic fragments) of the antigen, or to mutant forms of the antigen, where loss of binding due to alteration of amino acid residues within the antigen sequence is often considered indicative of epitope content, such as alanine scanning mutagenesis (Cunningham & Wells (1985) Science 244:1081), yeast display of mutated target sequence variants, or chimera analysis. Additionally, 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-displayed peptide libraries. The same V H and V L Alternatively, antibodies with the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

[0525] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to a 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, can be determined using known binding competition experiments, such as BIACORE® surface plasmon resonance (SPR) analysis. In certain embodiments, an antibody competes with another antibody and inhibits the binding of the other antibody to a target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is a "blocking antibody" (i.e., an antibody that, when combined with an antigen, prevents another immunological reaction with the antigen). Competition assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harbor. 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, overlapping epitopes, or adjacent epitopes (e.g., as evidenced by steric hindrance). Two antibodies "cross-compete" when they block each other by at least 50% in both ways, i.e., regardless of whether one antibody or the other first contacts the antigen in a competition experiment.

[0526] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include competition for binding to cells expressing hMPV by, for example, flow cytometry. Other methods include SPR (e.g., BIACORE®), solid-phase direct or indirect radioimmunoassays (RIAs); biolayer interferometry (BLI) assays (e.g., OCTET assays using BLI); solid-phase direct or indirect enzyme immunoassays (EIAs); sandwich competition assays (see Stahl 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 label assays; solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using 1-125 labels (Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al. al., Virology 176:546 (1990)); and direct-labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0527] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to an antibody that binds to an epitope on a predetermined antigen. Typically, an antibody (i) binds to an epitope of about 10 as determined, for example, by SPR using the predetermined antigen as the analyte and the antibody as the ligand, or by Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than M, e.g., about 10 -8 M, 10 -9 M or 10 -10and (ii) binds to a given antigen with an equilibrium dissociation constant (KD) of less than M or even lower, and (iii) binds to a given antigen with an affinity at least two-fold higher than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). -4 Any K greater than M D is generally considered to represent non-specific binding.

[0528] As used herein, the term "kassoc" or "ka" is intended to refer 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. D " The term kd vs k a The term "dissociation constant" is intended to refer to the ratio of the dissociation constant of an antibody or antigen-binding fragment thereof, expressed as a molar concentration (M), obtained from the ratio of the K D The K value of an antibody or antigen-binding fragment thereof can be determined using methods well established in the art. D A preferred method for determining is by using SPR, preferably by using a biosensor system such as a Biacore® system or flow cytometry and Scatchard analysis or biolayer interferometry.

[0529] 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 antibody or antigen-binding fragment thereof that induces 50% of the maximal response, i.e., a response halfway between the maximal response and baseline.

[0530] As used herein, the term "cross-reactive" refers to the ability of an antibody or antigen-binding fragment thereof described herein to bind to a metapneumovirus of a different species. For example, an antibody or antigen-binding fragment thereof described herein that binds to hMPV may also bind to other family members of the Pneumoviridae family (e.g., human RSV, bovine RSV, or pneumonia virus of mice) or to another species of MPV (e.g., avian MPV). Cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA, biolayer interferometry) or by binding to or otherwise functionally interacting with cells that physiologically express the antigen (e.g., HT1080 cells overexpressing hMPV). Methods for determining cross-reactivity include standard binding assays described herein, e.g., by biolayer interferometry or flow cytometry techniques.

[0531] As used herein, the term "linked" refers to the association of two or more molecules. The association can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinantly fused). Such linkage can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0532] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0533] An antibody or an antibody-binding fragment thereof (e.g., V H , V L The term "isolated nucleic acid molecule," as used herein with respect to a nucleic acid encoding a CDR3), is intended to refer to a nucleic acid molecule whose nucleotide sequence is essentially free of other genomic nucleotide sequences, e.g., those encoding antibodies or antibody-binding fragments thereof that bind to antigens other than hMPV, which other sequences may naturally flank the nucleic acid in human genomic DNA.

[0534] As used herein, the term "vector" 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 can be ligated. Another type of vector is a viral vector, in which additional DNA segments can 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 operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0535] "Conservative sequence modifications" of the sequences described herein, i.e., amino acid sequence modifications that do not abrogate antigen binding of the antibody or antigen-binding fragment thereof encoded by the nucleotide sequence or containing the amino acid sequence, are also provided. 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 the sequences in the tables herein (e.g., Table 3B, Table 3C, Table 3D, or Examples 1-6) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an 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 for identifying conservative nucleotide and amino acid substitutions that 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 randomly introduced along all or part of the anti-hMPV antibody coding sequence or anti-hMPV antigen-binding fragment thereof coding sequence, for example by saturation mutagenesis, and the resulting modified anti-hMPV antibodies can be screened for binding activity.

[0536] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, usually at least about 80%-85%, 85%-90%, or 90%-95%, and more preferably at least about 98%-99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

[0537] In the context of polypeptides, the term "substantial homology" indicates that two polypeptides, or designated sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the amino acids, usually at least about 80%-85%, 85%-90%, 90%-95% of the amino acids, more preferably at least about 98%-99.5% of the amino acids, or more preferably at least about 98%-99.9% of the amino acids, with appropriate amino acid insertions or deletions.

[0538] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). 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.

[0539] 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 the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights 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)) 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. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm as 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 gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0540] The nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example, to 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, word length = 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, word length = 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) Nucleic Acids 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.

[0541] As used herein, the term "variant" in the context of a recombinant hMPV F protein refers to a molecule whose amino acid or nucleic acid sequence differs compared to a native or reference sequence. Sequence variants can have substitutions, deletions, insertions, or a combination of any two or three of the foregoing at specific positions within the sequence compared to the native or reference sequence. Typically, variants share at least 50% identity to the native or 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 the native or reference sequence. In some embodiments, a variant shares at least 90% to 99%, 91% to 99%, 92% to 99%, 93% to 99%, 94% to 99%, 95% to 99%, 96% to 99%, 97% to 99%, or 98% to 99% identity with a native or reference sequence, hi some embodiments, a variant differs from the native sequence by 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid or nucleic acid.

[0542] As used herein, the term "analog" is meant to include polypeptide variants that differ by one or more amino acid changes, e.g., substitution, addition, or deletion of an amino acid residue, that still retain one or more of the properties of the parent or starting polypeptide.

[0543] The present disclosure provides several types of polynucleotide- or polypeptide-based compositions, including variants and derivatives. These include, for example, substitutional, insertional, deletional, 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 altered in some way relative to a reference or starting molecule.

[0544] Thus, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly the polypeptide sequences disclosed herein, are within the scope of this disclosure. For example, sequence tags or amino acids such as one or more lysines can be added to a peptide sequence (e.g., at the N- or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysines can be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of a peptide or protein amino acid sequence can be deleted to provide a cleavage sequence. Depending on the use of the sequence, for example, expression of the sequence as part of a larger sequence that is soluble or linked to a solid support, specific amino acids (e.g., C- or N-terminal residues) can be deleted instead.

[0545] "Substitutional variants," when referring to polypeptides, are those in which at least one amino acid residue in a native or starting sequence has been removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or multiple, where two or more (e.g., 3, 4, or 5) amino acids have been substituted in the same molecule.

[0546] A polypeptide or polynucleotide molecule of the present disclosure may share a degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or reference polynucleotide), for example, with a molecule described in the art (e.g., an engineered or designed molecule or a wild-type molecule). 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 or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) that are addressed by a particular mathematical model or computer program (e.g., an "algorithm"). The identity of related peptides can be readily calculated by known methods.

[0547] The term "percent identity" or "% identity" as applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid 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 alignment are well known in the art. Identity is dependent on the calculation of percent identity, but values ​​may vary due to gaps and penalties introduced into the calculation. Generally, a variant of a particular polynucleotide or polypeptide will 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 with that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. The percent identity of two polynucleic acid sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the 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. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.The comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms in alignment tools (eg, the Needleman-Wunsch algorithm, an online tool).

[0548] As used herein, the term "global alignment" refers to the alignment of residues between two amino acid or nucleic acid sequences along their entire length, introducing gaps as necessary if the two sequences are not the same length, to achieve the maximum percent identity. Global alignments can be generated 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_DEFAULTS=on&BLAST_INIT=GlobalAln&BLAST_SPEC=GlobalAln&BLAST_PROGRAMS=blastn). 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, global alignment of nucleotide sequences using BLAST global alignment uses the following default parameters: match score=2; mismatch score=-3; gap cost extension score=5; gap cost extension score=2. In a preferred embodiment, global alignment of protein sequences using BLAST global alignment uses the following default parameters: gap cost extension=11; gap cost extension=1.

[0549] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that is not naturally occurring within the cell, and may be a cell into which a recombinant expression vector has been introduced. It is 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 successive 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.

[0550] As used herein, the term "inhibition" refers to any statistically significant decrease in biological activity, including partial and complete blocking of 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).

[0551] For example, as used herein, the term "inhibiting hMPV activity" includes any measurable decrease in hMPV activity compared to a control (e.g., a control antibody), e.g., at least about 10%, e.g., 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% inhibition of hMPV activity. Inhibition can be specific to a single mechanism of hMPV activity or can be generalizable to all mechanisms of hMPV activity.

[0552] The term "multivalent," as used herein in the context of immunogenic compositions (e.g., "multivalent immunogenic compositions"), refers to a pharmaceutical preparation that contains two or more active agents that provide active immunity to a disease or pathological condition caused by more than one pathogen strain. For example, a multivalent immunogenic composition against hMPV may protect against more than one strain of hMPV.

[0553] As used herein, an "adjuvant" is a substance that serves to enhance the immunogenicity of the composition of the present disclosure. An adjuvant can enhance the immune response to an antigen that is weakly immunogenic when administered alone, for example, can induce no or weak antibody titer or cell-mediated immune response, increase the antibody titer against the antigen, and / or reduce the effective dose of the antigen to achieve an immune response in an individual. Thus, adjuvants are often given to enhance the immune response and are well known to those skilled in the art.

[0554] As used herein, the terms "prevent" or "prevention" refer to administering a prophylactic agent, such as a composition containing any of the polypeptides of the present invention, to a subject or patient at risk of infection with human metapneumovirus (hMPV). Prevention includes reducing the likelihood or severity of subsequent hMPV infection, ameliorating symptoms associated with 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 to prevent infection. The amount of prophylactic agent effective to ameliorate any particular disease symptom may vary depending on factors such as the patient's age and weight and the ability of the agent to elicit a desired response in the subject. Whether disease symptoms have been ameliorated can be assessed by any clinical measurement routinely used by a physician or other skilled health care provider to assess the severity or progression of the condition, or, in certain cases, ameliorating the need for hospitalization.

[0555] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures described herein. A method of "treatment" involves administering an anti-hMPV antibody (e.g., an anti-human hMPV antibody) or antigen-binding fragment thereof described herein to a subject having a tumor or cancer, or a subject predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate the disease or disorder, or one or more symptoms of a recurrent disease or disorder, or to prolong the survival of the subject beyond that expected in the absence of such treatment.

[0556] "Immunotherapy" refers to the treatment of a subject suffering from, or at risk of contracting, a disease, or a recurrence of a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying the immune response.

[0557] "Immunostimulatory therapy" or "immunostimulating therapy" refers to a therapy that results in increasing (inducing or enhancing) the immune response in a subject, for example, to treat hMPV or a condition associated with hMPV.

[0558] As used herein, "immune cells" refers to a subset of blood cells known as leukocytes, which include mononuclear cells such as lymphocytes, monocytes, macrophages, and granulocytes.

[0559] As used herein, "administration" refers to the physical introduction of a molecule (e.g., an antibody or antigen-binding fragment thereof that binds hMPV) or a composition comprising a therapeutic agent (e.g., an anti-hMPV antibody or antigen-binding fragment thereof) into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration for the 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, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein can be administered parenterally, e.g., by topical, epidermal, or mucosal routes of administration, e.g., intranasally, orally, intravaginally, rectally, sublingually, or topically. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0560] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject, or administering an active agent (e.g., an anti-hMPV antibody or antigen-binding fragment thereof) to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease. Treatment can be treatment of a subject with a disease or a subject not having a disease (e.g., for prophylaxis).

[0561] As used herein, "adjunctive" or "combined" administration (coadministration) includes simultaneous administration of agents and / or compounds in the same or different dosage forms, 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 administered simultaneously in a single formulation. Alternatively, the first and second (or more) antibodies or antigen-binding fragments thereof can be formulated for separate administration, administered simultaneously or sequentially.

[0562] As used herein, "combination" therapy refers to the coordinated administration of two or more therapeutic agents, including, but not limited to, simultaneous administration. Specifically, combination therapy encompasses both simultaneous administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, so long as the administration of one therapeutic agent is coordinated in some way with the 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 predetermined period of time (see, e.g., Kohrt et al. (2011) Blood 117:2423). For example, an anti-hMPV antibody may be administered first, followed (e.g., immediately after) by 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 before the administration of the second antibody or antigen-binding fragment thereof. In another embodiment, the anti-hMPV antibody or antigen-binding fragment thereof is administered, e.g., within minutes (e.g., within about 30 minutes) or at least one hour, of the second antibody or antigen-binding fragment thereof. Such simultaneous or sequential administration preferably results in both antibodies or antigen-binding fragments thereof being present in the treated patient at the same time.

[0563] The term "effective amount" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect, such as virus neutralization and virus binding. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug (e.g., an anti-hMPV antibody or antigen-binding fragment thereof) is any quantity of a drug or therapeutic agent that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability resulting from disease affliction. A therapeutically effective amount or dosage of a drug or therapeutic agent includes a "prophylactically effective amount" or "prophylactically effective dosage," which is any amount of a drug or therapeutic agent that, when administered alone or in combination with another therapeutic agent, inhibits the onset or recurrence of disease when administered to a subject at risk of developing the disease or suffering from a recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the onset or recurrence of disease can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0564] By way of example, for the treatment of a viral infection such as hMPV, a therapeutically effective amount or dosage of a drug or therapeutic agent (e.g., an anti-hMPV antibody or antigen-binding fragment thereof) inhibits viral activity, viral load, or viral disease symptoms by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least more than 70%, at least about 80%, or at least about 90% compared to untreated subjects. In some embodiments, a therapeutically effective amount or dosage of a 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 assessed using the assays described herein. Alternatively, this property of a composition containing a compound or therapeutic agent can be assessed by examining the composition's ability to inhibit viral activity, which can be measured in vitro by assays known to those of skill in the art.

[0565] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0566] 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 with 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.

[0567] The term "sample" refers to tissue, fluid, or cells (or a fraction of any of the foregoing) taken from a patient or subject. Typically, the tissue or cells are removed from the patient, although in vivo diagnosis is also contemplated. Other samples, including urine, tears, serum, plasma, cerebrospinal fluid, feces, sputum, cell extracts, etc., may also be useful for certain cancers.

[0568] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes elements, steps, or ingredients 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 replaced with either of the other two terms, thus describing alternative aspects of the subject matter scope. The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0569] 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, the use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0570] As used herein, the term "about" when referring to a measurable value, such as an amount, duration, etc., encompasses a variation of up to ±10% from the specified value. Unless otherwise specified, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, etc. used herein should be understood to be modified by the term "about."

[0571] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" used in phrases such as "A and / or B" includes "A and B," "A or B," "A" only, and "B" only. Similarly, the term "and / or" used in phrases such as "A, B, and / or C" encompasses A, B, and C, respectively; 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.

[0572] As used herein, the terms "ug" and "microgram" are used interchangeably with "μg" in the following section. As used herein, the terms "uM" and "micromolar" are used interchangeably with "μM" in the following section.

[0573] The various aspects described herein are described in further detail in the following subsections.

[0574] I. Anti-hMPV antibody

[0575] In one aspect, provided herein is an isolated anti-hMPV antibody (ie, an antibody that binds to hMPV) or antigen-binding fragment thereof.

[0576] In one aspect, provided herein is an isolated anti-hMPV antibody (e.g., a recombinant humanized antibody, a chimeric antibody, or a human antibody) or an antigen-binding fragment thereof, comprising:

[0577] The functional characteristics of the anti-hMPV antibodies or antigen-binding fragments thereof provided herein are described in more detail below.

[0578] In some embodiments, the anti-hMPV antibodies or antigen-binding fragments thereof described herein bind to a portion of hMPV (e.g., site α, site β, site II, site III, site IV, and site V) with a KD as described in the Examples below.

[0579] An antibody or antigen-binding fragment thereof exhibiting one or more of the above functional properties (e.g., biochemical, immunochemical, cellular, physiological, or other biological activity), as determined using methods known in the art and described herein, is understood to be associated with a statistically significant difference in the particular activity compared to that seen in the absence of the antibody (e.g., or in the presence of a control antibody of irrelevant specificity). Preferably, the increase in the measured parameter induced by the anti-hMPV antibody results in a statistically significant increase of at least 10%, more preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold in the measured parameter. Conversely, an anti-hMPV antibody-induced decrease in a measured parameter (e.g., hMPV activity) results in a statistically significant decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.

[0580] Also provided herein are anti-hMPV antibodies that bind to the same epitope on hMPV as any of the antibodies described herein, and have the ability to cross-compete with any of the antibodies described herein for binding to hMPV.

[0581] The 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.

[0582] In some embodiments, the antibody is a bispecific antibody comprising a first and a 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.

[0583] The antibody can also be a Fab, F(ab')2, scFv, AFFIBODY, avimer, nanobody, single chain antibody, or domain antibody. The antibody can also 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 engineered using standard recombinant DNA techniques and nucleic acids encoding desired constant region sequences operably linked to variable region sequences. H and V L It can be prepared from the sequence.

[0584] In certain embodiments, the antibodies described herein may have effector function, reduced effector function, or no effector function. In certain embodiments, the antibodies comprise an effector-less or largely effector-less Fc, such as IgG2 or IgG4. In general, the variable regions described herein may be linked to an Fc that typically includes one or more modifications 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, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or 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.

[0585] 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 the variant) to provide desired structural characteristics and / or biological activity. For example, modifications can be made to the Fc region to generate an Fc variant with (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors compared to the parent Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications may be particularly desirable. For example, a variant Fc region may contain two, three, four, five, etc., substitutions therein, e.g., at specific Fc region positions identified herein.

[0586] The variant Fc region may also contain sequence changes in which 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 cells used to produce the antibodies described herein. Even with the cysteine ​​residues removed, the single-chain Fc domains can still form dimeric Fc domains 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, the PA sequence near the N-terminus of a typical native Fc region, which can be recognized by E. coli digestive enzymes such as proline iminopeptidase, may be removed. In other embodiments, one or more glycosylation sites within the Fc domain may be removed. Typically, glycosylated residues (e.g., asparagine) may confer a 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, the EKK sequence of human IgG1 may be deleted or substituted. In certain embodiments, sites affecting binding to Fc receptors, preferably sites other than the salvage receptor binding site, may be removed. In other embodiments, the Fc region may be modified to remove the ADCC site. ADCC sites are known in the art; see, for example, Molec. Immunol. 29(5):633-9 (1992) for the ADCC site of IgG1. Specific examples of variant Fc domains are disclosed, for example, in PCT Publication Nos. WO 97 / 34631 and WO 96 / 32478.

[0587] In one embodiment, the hinge region of an Fc is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent 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 such that the antibody has impaired Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is further described in U.S. Patent No. 6,165,745 by Ward et al.

[0588] In yet other embodiments, the Fc region is altered by substituting 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 altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260 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. Patent 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, thereby altering the ability of the antibody to fix complement. This approach is described further in PCT Publication No. WO 94 / 29351 by Bodmer et al.

[0589] In yet another example, the Fc region may be modified with any of 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, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 347, 248, 249, 252, 254, 255, 256, 258, 262, 2 The modification may be by modifying one or more amino acids at 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 to enhance FcR and complement interactions include, but are not limited to, the substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0590] Fc modifications that increase binding to Fcγ receptors include those at 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, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 39 and 439, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (PCT Patent Publication No. WO 00 / 42072).

[0591] Other Fc modifications that can be made to the Fc are those to reduce or eliminate binding to FcγRs and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions, such as ADCC, ADCP, and CDC. Exemplary alterations include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, numbering according to the EU index. Exemplary substitutions include, but are not limited to, 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, numbering according to the EU index. An Fc variant may include 236R / 328R. Other modifications to reduce FcγR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of glycosylation at position 297 by mutational or enzymatic means or production in an organism such as a bacterium that does not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0592] The Fc region may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; WO 00 / 42072; WO 01 / 58957; WO 02 / 0691). 9; 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).

[0593] Fc variants that enhance affinity for the inhibitory receptor FcγRIIb may also be used. Such variants enhance affinity for FcγRIIb, including, for example, B cells and monocytes. +This can provide an Fc fusion protein with immunomodulatory activity associated with cells. In one embodiment, the Fc variant provides selectively enhanced affinity for FcγRIIb relative to one or more activating receptors. Modifications to alter binding to FcγRIIb include one or more modifications at positions 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 FcγRllb 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.

[0594] In certain embodiments, antibodies are modified to increase their biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn. For example, as described in U.S. Pat. No. 6,277,375, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, and 436. Specific exemplary substitutions include one or more of T252L, T254S, and / or T256F. Alternatively, to increase biological half-life, antibodies can be altered within the CH1 or CL regions to contain salvage receptor binding epitopes from two loops in the CH2 domain of the IgG Fc region, 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, 259I, 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, 311A, 312A, 376A, 378Q, 380A, 382A, and 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 30 9P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S(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 specific biological characteristics can be used. For example, IgG1 / IgG3 hybrid variants can be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies can be constructed that contain one or more substitutions, such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F.In other embodiments described herein, IgG1 / IgG2 hybrid variants can be constructed by substituting IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgG1 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies can be constructed that contain one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to the insertion of a glycine at position 236), and 327A.

[0595] Furthermore, 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, RR et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Furthermore, the following combination mutants, which have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, have been shown to improve FcγRIII binding (Shields et al., 2001). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with the S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of 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 / I332E variant showed enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). Furthermore, IgG1 mutants containing the L235V, F243L, R292P, Y300L, and P396L mutations have been identified that exhibit enhanced binding to FcγRIIIa and consequently enhanced ADCC activity in transgenic mice expressing human FcγRIIIa in models of B-cell malignancies and breast cancer (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that can be used include S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0596] When an IgG4 constant domain is used, it is usually preferred to include the substitution S228P, which mimics the hinge sequence of IgG1, thereby stabilizing the IgG4 molecule.

[0597] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by 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 the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are 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 at N297 can be prevented by mutating the N297 residue to another residue, e.g., N297A, and / or by mutating an adjacent amino acid, e.g., 298, thereby reducing glycosylation at N297.

[0598] Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by 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 to express the recombinant antibodies described herein, thereby producing antibodies with altered glycosylation. For example, European Patent No. 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication No. WO 03 / 035835 by Presta describes a variant CHO cell line, Lec13 cells, which has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, and also results in hypofucosylation of antibodies expressed in the host cells (see also Shields, RLet et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication No. WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), and 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).

[0599] 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 is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Preferably, 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 form of PEG that has 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 No. 0 154 316 to Nishimura et al. and European Patent No. 0 401 384 to Ishikawa et al.

[0600] The affinity and binding properties of an Fc region for its ligand can 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), as well as 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 can utilize one or more labels of the component being tested and / or can use a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0601] II. Antibodies that bind to the same epitope as or cross-compete with anti-hMPV antibodies

[0602] Anti-hMPV antibodies that bind to the same or similar epitopes as (and thus cross-compete with) the antibodies disclosed herein can be produced using immunization protocols. The resulting antibodies can be screened for high-affinity binding to hMPV. Selected antibodies can then be studied, for example, in yeast display assays in which sequence variants of hMPV are displayed on the surface of yeast cells, or by hydrogen-deuterium exchange experiments to determine the precise epitope bound by the antibody.

[0603] The epitope to which an antibody binds can be determined using art-recognized methods. An hMPV antibody is considered to bind the same epitope as a reference anti-hMPV antibody if, for example, it contacts one or more of the same residues on hMPV as a reference antibody, contacts the same residues as one or more reference antibodies in at least one region of hMPV, contacts a majority of the residues in at least one region of hMPV as the reference antibody, contacts a majority of the same residues as the reference antibody in each region of hMPV, contacts a majority of the same residues as the reference antibody along the entire length of hMPV, contacts all of the same different regions of hMPV as the reference antibody, contacts all of the same residues as the reference antibody in any one region on hMPV, or contacts all of the same residues as the reference antibody in all of the same regions of hMPV as the reference antibody.

[0604] Techniques for determining antibodies that bind to the "same epitope on hMPV" using the hMPV antibodies described herein include X-ray analysis of crystals of the antigen:antibody complex, which provides atomic resolution of the epitope. Other methods monitor antibody binding to antigen-binding fragments or mutant forms of the antigen, where loss of binding due to amino acid modifications within the antigen sequence indicates the epitope component. Methods can also rely on the ability of the antibody of interest to affinity isolate specific short peptides (either in their native three-dimensional form or in their denatured form) from combinatorial phage-display peptide libraries or protease digests of the target protein. The peptides are then considered leads for defining the epitope corresponding to the antibody used to screen the peptide library. Computational algorithms have also been developed for epitope mapping, which have been shown to map conformational discontinuous epitopes.

[0605] Epitopes or regions containing epitopes 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 can be used to guide the selection of antibodies with the same epitope and therefore similar properties to the hMPV antibodies described herein. Using phage display, the heavy chain of an anti-hMPV antibody is first paired with a repertoire of (e.g., human) light chains to select for hMPV-binding antibodies, and then the new light chains are paired with a repertoire of (e.g., human) heavy chains to select for (e.g., human) hMPV-binding antibodies with the same epitope or epitope region as the anti-hMPV antibodies described herein. Alternatively, variants of the antibodies described herein can be obtained by mutagenesis of the cDNA sequences encoding the heavy and light chains of the antibodies.

[0606] Alanine scanning mutagenesis, as described by Cunningham & Wells (1989) Science 244:1081, or any other form of point mutagenesis of amino acid residues in hMPV, can also be used to determine functional epitopes of anti-hMPV antibodies.

[0607] The epitope or epitope region bound by a specific antibody (an "epitope region" is a region that includes or overlaps with the epitope) can also be determined by assessing antibody binding to peptides comprising hMPV fragments. A series of overlapping peptides encompassing hMPV sequences can be synthesized and screened for binding, for example, by direct ELISA, competitive ELISA (in which peptides are assessed for their ability to prevent antibody binding to hMPV bound to wells of a microtiter plate), or on a chip. Such peptide screening methods may not detect some discontinuous functional epitopes.

[0608] Epitopes can 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 crystallography, molecular modeling, and nuclear magnetic resonance spectroscopy.

[0609] Single-particle cryo-electron microscopy (SP-Cryo-EM) can also be used to identify epitopes bound by antibodies or their antigen-binding fragments. SP-Cryo-EM is a technique for macromolecular structural analysis that uses a high-intensity electron beam to image biological specimens in their native environment at cryogenic temperatures. In recent years, SP-cryo-EM has emerged as a complementary technique to crystallography and NMR for determining near-atomic structures suitable for drug discovery applications (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 added advantage of enabling access to larger, more complex biological systems, potentially characterizing multiple conformational or compositional solution states from the same sample, providing insight into more biologically relevant states of macromolecules. For imaging, a small amount of sample (e.g., 3 microliters, 3 μl, aliquot) is applied to a grid and flash-frozen in a liquid ethane bath. The frozen grid is then loaded onto a microscope, and hundreds to thousands of images of different regions of the grid are collected. These images contain two-dimensional projections of biological macromolecules (particles): using mathematical tools and GPU-driven algorithms, particles are identified, extracted, and classified. In a subsequent step, the different classes are used to calculate one or more 3D reconstructions corresponding to different conformations, oligomerization, or binding states when coexisting in the same sample. Individual reconstructions can then be refined to high resolution.

[0610] III. Nucleic acid molecules

[0611] Also provided herein are nucleic acid molecules encoding anti-hMPV antibodies or antigen-binding fragments thereof, as well as the hMPV F protein and variants thereof described herein. The nucleic acids may be present in whole cells, cell lysates, or in a partially purified or substantially pure form. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, the nucleic acids are cDNA molecules. 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 harboring human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the library.

[0612] In some embodiments, provided herein are nucleic acid molecules encoding the VH and / or VL sequences, or the heavy and / or light chain sequences, of any anti-hMPV antibody or antigen-binding fragment thereof described herein. Host cells comprising the nucleotide sequences (e.g., nucleic acid molecules) described herein are encompassed herein. Once DNA fragments encoding the 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 into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0613] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification.

[0614] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably 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, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment 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 constant region or a lambda constant region.

[0615] Also provided herein are nucleic acid molecules with conservative substitutions that do not change the resulting amino acid sequence upon translation of the nucleic acid molecule.

[0616] IV. Manufacturing method

[0617] Monoclonal antibodies that bind to 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.

[0618] Various methods for producing the monoclonal antibodies described herein are available in the art. For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or any subsequent developments thereof, or by recombinant DNA methods (U.S. Patent No. 4,816,567). For example, monoclonal antibodies can be produced using hybridoma techniques known in the art, including those taught in, for example, 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, NY, 1981) (these references are incorporated by reference in their entireties). Methods for producing and screening 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 (e.g., 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 described chain shuffling as a strategy for constructing very large phage libraries (Marks et al., Bio / Technology, 10:779-783 (1992)), and the production of high affinity (e.g., nM range) human antibodies by combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)).Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0619] Human antibodies can be made by a variety of methods known in the art, including the above-mentioned phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and 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 incorporated herein by reference in their entireties. Human antibodies can also be produced using transgenic mice expressing human immunoglobulin genes, which, upon immunization, can produce 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)) can also be used to produce human antibodies and antigen-binding fragments thereof in vitro from immunoglobulin variable (V) domain gene repertoires derived 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 incorporated herein by reference in their entireties.) Fully human antibodies that recognize selected epitopes can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, such as a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., 1994, Bio / technology 12:899-903).

[0620] Chimeric antibodies can be prepared based on the sequences of mouse monoclonal antibodies. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, mouse variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.).

[0621] Humanized forms of anti-hMPV antibodies (e.g., humanized forms of murine anti-hMPV antibodies) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are typically human immunoglobulins (recipient antibodies) in which residues from the recipient's CDR or hypervariable regions are replaced by residues from a CDR or hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human 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 include residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies 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 human immunoglobulin consensus sequences. The humanized antibody may also 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).

[0622] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor antibody CDR or consensus framework can be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue such that the CDR or framework residue at that position 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 a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (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 that occurs most frequently at that position in the family. If two amino acids occur equally frequently, both can be included in the consensus sequence. As used herein, "Vernier zone" refers to a subset of framework residues that can adjust CDR structure and fine-tune fit to the antigen, as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and can influence the structure of the CDRs and the affinity of the antibody. Human immunoglobulin (Ig) sequences that can be used as recipients are well known in the art.

[0623] Framework residues in the human framework regions can be substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling the interactions between CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at specific 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., analysis of residues that influence the ability of the candidate immunoglobulin to bind to 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 may be used in a variety of applications, 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. USA 89:4285 (1992), Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4 / 5):489-498 (1991), and Studnicka et al., Protein Engineering, each of which is fully incorporated herein by reference. 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994); PCT Publication Numbers WO 91 / 09967, PCT / US Patent No. 98 / 16280, US Patent No. 96 / 18978, US Patent No. 91 / 09630, US Patent No. 91 / 05939, US Patent No. 94 / 01234, UK Patent No. 89 / 01334, UK Patent No. 91 / 01134, UK Patent No. 92 / 01755, WO 90 / 14443, WO 90 / 14424, WO 90 / 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, and 4,816,567.

[0624] Anti-hMPV antibodies generated using the methods described above can be tested for desired functionality (e.g., specific binding specificity, binding affinity, targeted cell population, etc.) using methods known in the art and described in the Examples (e.g., art-recognized protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays). One aspect of the invention provides molecules that can be used to screen for antibodies or antigen-binding fragments thereof that bind to hMPV. Exemplary assays include, but are not limited to, immunoprecipitation or in vitro binding assays, such as radioimmunoassays (RIA), FACS, enzyme-linked immunosorbent assays (ELISA), biolayer interferometry (e.g., ForteBio assays), and Scatchard analysis.

[0625] antibody manipulation

[0626] Further included are embodiments in which the anti-hMPV antibodies or antigen-binding fragments thereof described herein (e.g., the antibodies or antigen-binding fragments thereof in Examples 1-6) are antibodies that have been engineered to include modifications to framework residues within the variable domains of a parent monoclonal antibody, e.g., to improve the properties of the antibody or antigen-binding fragment. Typically, such framework modifications are made to reduce the immunogenicity of the antibody or antigen-binding fragment. This is usually achieved by replacing non-CDR residues in the variable domains (i.e., framework residues) of a parent (e.g., rodent) antibody or antigen-binding fragment thereof with analogous residues from the immune repertoire of the species in which the antibody will be used, e.g., human residues in the case of a human therapeutic. Such antibodies or antigen-binding fragments thereof are referred to as "humanized" antibodies or antigen-binding fragments 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, antibodies or antigen-binding fragments thereof that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequence of an antibody or antigen-binding fragment thereof to the germline sequence from which the antibody or antigen-binding fragment is derived. Another approach is to restore the original parental (e.g., rodent) residue at one or more positions in an engineered (e.g., humanized) antibody, e.g., to restore binding affinity that may have been lost in the process of substituting framework residues. (See, e.g., U.S. Pat. Nos. 5,693,762, 5,585,089, and 5,530,101.)

[0627] In certain embodiments, anti-hMPV antibodies and antigen-binding fragments thereof are engineered to improve their properties (e.g., modifications in the framework and / or CDRs). Such engineered changes can be based on molecular modeling. Molecular models of the variable regions of the parent (non-human) antibody sequence can be constructed to understand the structural features of the antibody and can be used to identify potential regions on the antibody that can interact with the antigen. Traditional CDRs are based on the alignment of immunoglobulin sequences and the identification of variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 thed.; 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 the conformations of loops in antibody crystal structures and proposed hypervariable loops. Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There is variation between the regions classified as "CDRs" and "hypervariable loops." A later study (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 closely match the "CDR" residues or "hypervariable" loops. A molecular model of the variable region of a non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen. In reality, potential antigen-binding regions based on the model differ from traditional "CDR" or "hypervariable" loops. Commercially available scientific software, such as MOE (Chemical Computing Group), can be used for molecular modeling. Human frameworks can be selected based on the best match with the non-human sequence in both the framework and CDR. For FR4 (framework 4) in the VH, the VJ region of the human germline is compared with the corresponding non-human region. For FR4 (framework 4) in the VL, the J-kappa and J-lambda regions of the human germline sequence are compared with the corresponding non-human region. Once a suitable human framework is identified, the CDRs are grafted onto the selected human framework. In some cases, certain residues at the VL-VH interface can be retained as in the non-human (parent) sequence. Molecular models can also be used to identify residues that can potentially alter the CDR conformation and thus antigen binding. In some cases, these residues are retained as in the non-human (parent) sequence. Molecular models can also be used to identify solvent-exposed amino acids that may cause undesirable effects such as glycosylation, deamidation, and oxidation.To eliminate / minimize these potential problems, a developability filter can be introduced early in the design phase.

[0628] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T-cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach, also known as "deimmunization," is described in further detail in U.S. Patent No. 7,125,689.

[0629] In certain embodiments, it may be desirable to change certain amino acids containing exposed side chain(s) to other amino acid residues to avoid deamidation or isomerization and provide greater chemical stability to the final antibody. Deamidation of asparagine can occur at NG, DG, NG, NS, NA, NT, QG, or QS sequences, resulting in the generation of isoaspartic acid residues (the isoaspartic acid effect), which introduces a kink into the polypeptide chain and reduces its stability. Isomerization can occur at DG, DS, DA, or DT sequences. In certain embodiments, antibodies of the present disclosure do not contain deamidation or asparagine isomerization sites. For example, asparagine (Asn) residues can be changed to Gln or Ala to reduce the likelihood of isoaspartic acid formation at any Asn-Gly sequences, particularly within the CDRs.

[0630] Similar problems can occur with Asp-Gly sequences. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Formation of isoaspartate can abolish or completely abolish binding of an antibody to its target antigen. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In various embodiments, asparagine is changed to glutamine (Gln). It may also be desirable to change the amino acid adjacent to an asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation, which occurs when a small amino acid is present adjacent to an asparagine or glutamine. See Bischoff & Kolbe (1994) J. Chromatog. 662:261. Additionally, any methionine residues in the CDRs (typically solvent-exposed Met) may be altered to Lys, Leu, Ala, or Phe, or other amino acids, to reduce the likelihood of oxidation of the methionine sulfur, which can reduce antigen-binding affinity and contribute to molecular heterogeneity in the final antibody preparation. Id. Additionally, it may be desirable to alter any Asn-Pro combinations found in the CDRs to Gln-Pro, Ala-Pro, or Asn-Ala to prevent or minimize potentially cleavable Asn-Pro peptide bonds. Antibodies with such substitutions are then screened to ensure that the substitutions do not unacceptably reduce the antibody's affinity or specificity for hMPV or other desired biological activity.

[0631] Antibody engineering of the Fc region

[0632] The antibodies (i.e., anti-hMPV antibodies) and antigen-binding fragments thereof disclosed herein also can be engineered to contain 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 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.

[0633] 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. Patent No. 5,624,821; and PCT Publication Nos. 2003 / 086310; WO 2005 / 120571; and WO 2006 / 0057702. Such modifications can be used to enhance or suppress various immune system responses, potentially with beneficial effects in diagnosis and therapy. Alterations to the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fc regions. Fc modifications can also alter the half-life of therapeutic antibodies, allowing for less frequent administration and therefore improved convenience and reduced use of materials. See Presta (2005) J. Allergy Clin. Immunol. 116:731, pp. 734-35.

[0634] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is an IgG4 isotype antibody or antigen-binding fragment thereof, which comprises 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 heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al., supra; position 241 is based on the Kabat numbering system).

[0635] In one embodiment of the present invention, the hinge region of CH1 is modified to increase or decrease the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Patent 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.

[0636] In another embodiment, the Fc-hinge region of the antibody or antigen-binding fragment thereof of the present 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 Staphylococcus aureus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745.

[0637] In another embodiment, the antibody or antigen-binding fragment thereof of the present invention is modified to extend 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. Patent No. 6,277,375. Alternatively, to increase biological half-life, the antibody can be modified within the CH1 or CL region to contain salvage receptor binding epitopes taken from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022.

[0638] 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 altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for 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. Patent Nos. 5,624,821 and 5,648,260.

[0639] 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. Patent No. 6,194,551.

[0640] 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 further described in PCT Publication No. WO 1994 / 29351.

[0641] In yet another example, the Fc region may be modified to modify the Fc region at any of 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, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 343, 348, 349, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, , 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 further described in PCT Publication No. WO 00 / 42072. Furthermore, 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).

[0642] In one embodiment of the invention, the Fc region is modified to reduce the ability of the antibody of the invention to mediate effector function and / or 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 reduce the ability of the antibody or antigen-binding fragment thereof to mediate effector function and / or increase anti-inflammatory properties by modifying residues 243, 264, 267, and 328.

[0643] Altered effector functions

[0644] In some embodiments, the Fc region of an anti-hMPV antibody is modified to increase or decrease the ability of the antibody or its antigen-binding fragment to mediate effector functions and / or to increase / decrease their binding to Fcγ receptors (FcγRs).

[0645] Interactions between the constant regions of antigen-binding proteins and various Fc receptors (FcRs), including FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), are thought to mediate effector functions of antigen-binding proteins, such as ADCC and CDC. Fc receptors are also important for antibody cross-linking, which may be important for anti-tumor immunity.

[0646] Effector function can be measured in a number of ways, including, for example, measuring ADCC effector function via FcγRIII binding to natural killer cells or FcγRI binding to monocytes / macrophages. For example, antigen binding proteins of the 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, pp. 6591-6604; Chappel et al., 1993 J. Biol. Chem., Vol. 268, pp. 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-4010.

[0647] 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).

[0648] In one embodiment of the invention, such mutations are at one or more of positions selected from 239, 332 and 330 (IgG1), or 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 described herein is mutated at positions 239 and 332, for example S239D and 1332E, or in a further embodiment, mutated at three or more positions selected from 239, 332 and 330, for example S239D and 1332E and A330L. (EU index numbering).

[0649] 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, the antibody has enhanced ADCC or enhanced CDC, or has both enhanced ADCC and CDC effector function. Examples of suitable methodologies for generating antigen-binding proteins with altered glycosylation profiles are described in PCT Publication Nos. WO 2003011878 and WO 2006014679, and EP 1229125.

[0650] In a further aspect, the present invention provides "non-fucosylated" or "fucosylated" antibodies. Non-fucosylated antibodies have the tri-mannosyl core structure of the complex N-glycans of Fc without the fucose residue. These glycoengineered antibodies, lacking the core fucose residue from the Fc N-glycan, may exhibit stronger ADCC than their fucosylated counterparts due to their enhanced FcγRIIIa binding ability.

[0651] The present invention also provides a method for producing an antibody according to the invention, comprising the steps of: a) culturing a recombinant host cell containing an expression vector comprising an isolated nucleic acid described herein, wherein the recombinant host cell does not contain alpha-1,6-fucosyltransferase; and b) recovering the antigen-binding protein. The recombinant host cell typically does not contain a gene encoding alpha-1,6-fucosyltransferase (e.g., yeast host cells such as Pichia species) or may be genetically modified to inactivate alpha-1,6-fucosyltransferase. Recombinant host cells genetically modified to inactivate the FUT8 gene encoding alpha-1,6-fucosyltransferase are available. See, for example, the POTELLIGENT™ technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity compared to the same monoclonal antibody produced in cells with a functional FUT8 gene. Aspects of the POTELLIGENT™ technology system are described in U.S. Pat. Nos. 7,214,775 and 6,946,292, and PCT Publication Nos. WO 0061739 and WO 0231240. Those of skill in the art will recognize other suitable systems.

[0652] It will be apparent to one skilled in the art that such modifications can be used alone as well as in combination with each other to further enhance or decrease effector function.

[0653] Generation of modified glycosylated antibodies

[0654] In yet another embodiment, the antibodies or antigen-binding fragments thereof of the present invention comprise a particular glycosylation pattern. For example, an afucosylated or aglycosylated antibody or antigen-binding fragment thereof can be generated (i.e., the antibody lacks fucose or glycosylation, respectively). The glycosylation pattern of an antibody or antigen-binding fragment thereof can be altered to, for example, increase the affinity or avidity of the antibody or fragment for hMPV. Such modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody or antigen-binding fragment sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity or avidity of the antibody or antigen-binding fragment thereof for the antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.

[0655] The antibodies and antigen-binding fragments thereof disclosed herein can further include those produced in lower eukaryotic host cells, particularly those produced in fungal host cells such as yeast or filamentous fungi that have been genetically engineered to produce glycoproteins with mammalian- or human-like glycosylation patterns (see, e.g., 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. Oct;18(5):387-92 (2007)). A particular advantage of these genetically engineered host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins produced in the cells, such that compositions of glycoproteins can be produced in which specific N-glycan structures predominate (see, e.g., U.S. Pat. Nos. 7,029,872 and 7,449,308). These genetically engineered host cells have primarily been used to produce antibodies with specific N-glycan structures (see, e.g., Li et al., (2006) Nat. Biotechnol. 24:210-215).

[0656] In certain embodiments, the antibodies and antigen-binding fragments thereof disclosed herein further include those produced in lower eukaryotic host cells, which contain fucosylated and non-fucosylated hybrid and complex N-glycans (including bisected and multiantennary species, such as GlcNAc (1-4) Man3GlcNAc2;Gal (1-4) GlcNAc (1- 4) Man3GlcNAc2; NANA (1-4) Gal (1-4) GlcNAc (1-4) N-glycans such as, but not limited to, Man3GlcNAc2.

[0657] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein may include antibodies or antigen-binding fragments thereof having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In certain aspects, the hybrid N-glycan is the predominant N-glycan species in the composition.

[0658] In certain embodiments, antibodies and antigen-binding fragments thereof provided herein include 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 certain aspects, the complex N-glycan is the predominant N-glycan species in the composition. In a further aspect, a complex N-glycan is a particular N-glycan species that constitutes 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 antibodies 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, where such structures are fucosylated. Such structures can be produced, for example, in engineered Pichia pastoris host cells.

[0659] In certain embodiments, the N-glycan is fucosylated. Generally, fucose is linked via an α1,3-linkage to GlcNAc at the reducing end of the N-glycan, an α1,6-linkage to GlcNAc at the reducing end of the N-glycan, an α1,2-linkage to Gal at the non-reducing end of the N-glycan, an α1,3-linkage to GlcNAc at the non-reducing end of the N-glycan, or an α1,4-linkage to GlcNAc at the non-reducing end of the N-glycan. Thus, in certain embodiments of the glycoprotein composition, the glycoform is α1,3-linked or α1,6-linked fucose, and is selected from the group consisting of Man5GlcNAc2(Fuc), GlcNAcMan5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2 ... and producing glycoforms selected from the group consisting of GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc), and the α1,3-linked or α1,4-linked fucose is selected from the group consisting of GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc) 1- 2) Man3GlcNAc2, GalGlcNAc2 (Fuc 1-2 )Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc 1-2 )Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc 1-2 )Man3GlcNAc2, or the α1,2-linked fucose produces a glycoform selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc 1-2 )GlcNAc2Man3GlcNAc2, NANAGal2(Fuc 1- 2) GlcNAc2Man3GlcNAc2 and NANA2Gal2(Fuc 1-2 ) GlcNAc2Man3GlcNAc2.

[0660] In a further aspect, the antibody (e.g., a humanized antibody) or antigen-binding fragment thereof comprises high mannose N-glycans, such as, but not limited to, N-glycans consisting of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or Man3GlcNAc2 N-glycan structures.

[0661] In a further aspect of the above, the complex N-glycans further comprise fucosylated and non-fucosylated biantennary and hyperantennary species.

[0662] As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to N-linked oligosaccharides, e.g., those attached to asparagine residues of polypeptides via an asparagine-N-acetylglucosamine linkage. N-linked glycoproteins contain N-acetylglucosamine residues attached to the amide nitrogen of asparagine residues 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)). Processing of sugar groups occurs cotranslationally in the lumen of the ER and, for N-linked glycoproteins, continues posttranslationally in the Golgi apparatus. N-glycans share a common pentasaccharide core of Man3GlcNAc2 ("Man" refers to mannose, "Glc" refers to glucose, "NAc" refers to N-acetyl, and GlcNAc refers to N-acetylglucosamine). Typically, N-glycan structures are presented with the non-reducing end on the left and the reducing end on the right. The reducing end of an N-glycan is the end attached to an Asn residue that contains the glycosylation site on the protein. N-glycans differ in the number of branches (antennae) containing peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid) attached to the Man3GlcNAc2 ("Man3") core structure, also referred to as the "trimannose core," "pentasaccharide core," or "paucimannose core." N-glycans are classified according to their branching components (e.g., high mannose, complex, or hybrid). "High mannose" N-glycans have five or more mannose residues. "Complex" type N-glycans typically have 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 can also have galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues that may be 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 including a "bisected" GlcNAc and a core fucose ("Fuc"). Complex N-glycans may also have multiple antennae on the "trimannose core," often referred to as "multiple antennary glycans." "Hybrid" N-glycans have at least one GlcNAc at 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."

[0663] With respect to complex N-glycans, the terms "G-2", "G-1", "G0", "G1", "G2", "A1" and "A2" mean the following: The term "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 "A1" 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 "F," the "F" indicates that the N-glycan species includes 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 typically produce N-glycans that produce fucose.

[0664] With respect to multiantennary N-glycans, the term "multiantennary N-glycan" refers to an N-glycan further comprising a GlcNAc residue on a 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 mannose residue comprising the non-reducing end of the 1,6-arm and the 1,3-arm of the N-glycan. Thus, multiantennary N-glycans can be characterized by the formula 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 an N-glycan in which a GlcNAc residue is attached to a mannose residue at the reducing end of the N-glycan. A bisected N-glycan can be characterized by the formula GlcNAc3Man3GlcNAc2, where each mannose residue is linked to a GlcNAc residue at its non-reducing end. In contrast, when a multiantennary N-glycan is characterized as GlcNAc3Man3GlcNAc2, the formula indicates that two GlcNAc residues are linked to mannose residues at the non-reducing end of one of the two arms of the N-glycan and one GlcNAc residue is linked to a mannose residue at the non-reducing end of the other arm of the N-glycan.

[0665] Antibody physical properties

[0666] The antibodies and antigen-binding fragments thereof disclosed herein may further comprise one or more glycosylation sites in either the light chain immunoglobulin variable region or the heavy chain immunoglobulin variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or antigen-binding fragment thereof or altered 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 is known to occur at motifs containing NXS / T sequences.

[0667] Each antibody or antigen-binding fragment thereof has a characteristic melting temperature, with a higher melting temperature indicating a higher overall stability in vivo (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). M1 The initial unfolding temperature can 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 that do not rapidly degrade are selected. Degradation of antibodies or antigen-binding fragments thereof can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0668] In further embodiments, antibodies and antigen-binding fragments thereof are selected that have minimal aggregation effects that may result in the induction of unwanted immune responses 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.

[0669] V. Multispecific antibodies

[0670] The multispecific antibodies (e.g., bispecific antibodies) provided herein contain at least one binding region for a specific epitope on hMPV described herein and at least one other binding region (e.g., a viral antigen). Multispecific antibodies can be prepared as full-length antibodies or antigen-binding fragments thereof (e.g., F(ab')2 antibodies).

[0671] Methods for making multispecific antibodies are well known in the art (see, e.g., PCT Publication Nos. WO 05117973 and WO 06091209). For example, the production of full-length multispecific antibodies can be based on the co-expression of two pairs of immunoglobulin heavy chain-light chains, where the two chains have different specificities. Various techniques have also been described for making and isolating multispecific antibody fragments directly from recombinant cell culture. For example, leucine zippers can be used to make multispecific antibodies. Another strategy for making multispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported.

[0672] Examples of suitable multispecific molecular 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 (MedImmune / 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 Nanobody (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.

[0673] In certain embodiments, a multispecific antibody comprises a first antibody (or binding portion thereof) that binds to hMPV that has been derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a multispecific molecule that binds to hMPV and non-hMPV target molecules. An antibody may be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds to three or more different binding sites and / or target molecules. To generate a multispecific molecule, an antibody disclosed herein may be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent bonding, or other methods) 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 multispecific molecule results.

[0674] Thus, multispecific molecules, such as bispecific and bifunctional antibodies, are contemplated, which comprise at least one first binding specificity for a particular epitope on hMPV and a second binding specificity for a second target. In some embodiments, the second target is a second binding region that specifically binds to a virus-associated antigen. Virus-associated antigens are well known in the art.

[0675] 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., the 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), such as the targets described above.

[0676] In one embodiment, the multispecific molecule comprises as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab'), Fv, or single-chain Fv. The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof, such as the Fv or single-chain constructs described in U.S. Patent No. 4,946,778 to Ladner et al.

[0677] Multispecific molecules can be prepared by conjugating component binding specificities, such as anti-FcR and anti-hMPV binding specificities, using methods known in the art. For example, each binding specificity of the multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, various 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)cyclohexane-1-carboxylate (sulfo-SMCC). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0678] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge regions are modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0679] 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 when the multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. Multispecific molecules can be single-chain molecules containing one single-chain antibody and a binding determinant, or single-chain bispecific molecules containing two binding determinants. Multispecific molecules can contain at least two single-chain molecules. Methods for preparing multispecific molecules are described, for example, in U.S. Patent 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.

[0680] Binding of multispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS) analysis, bioassays (e.g., growth inhibition), or Western blot analysis. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, FcR-antibody complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, antibodies can be radioactively labeled and used in a radioimmunoassay (RIA). Radioisotopes can be detected using an αγ-β counter or a scintillation counter, or by autoradiography.

[0681] VI. Antigens / Antigenic Polypeptides

[0682] 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.

[0683] Cleavable affinity tags can be added to the sequences of 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 a recombinant hMPV F polypeptide, as described herein for enzymatic cleavage sequences and polypeptide sequences that can be used to purify the recombinant hMPV F protein polypeptide by affinity chromatography. Such tags and cleavage sequences are described in 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 referred to as 6xHis, 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 thioredoxins (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 acetyltransferase (CAT); horseradish peroxidase (HRP); modified haloalkane dehalogenase (HaloTag™); and modified serine protease subtilisin (Profinity eXact™).

[0684] Exemplary cleavage sequences for removing affinity tags include, but are not limited to, the thrombin protease cleavage sequence (LVPRGS, SEQ ID NO: 1013); the factor Xa cleavage sequence (SEQ ID NO: 1016, IEGR; or SEQ ID NO: 1017, IDGR); the PreScission™ protease cleavage site (SEQ ID NO: 1018, LEVLFQGP); the enterokinase cleavage sequence (SEQ ID NO: 1019, DDDDK); the genease I cleavage sequence (SEQ ID NO: 1020, TQLAYFTDSKNPGAAHYDTFADSLR); and the TEV protease cleavage sequence (SEQ ID NO: 1021, ENLYFQ).

[0685] In some embodiments, the tag can be directly recognized by a protease, resulting in cleavage of the affinity tag from the remainder of the polypeptide. For example, the tag small ubiquitin-like modifier (SUMO) is recognized by a SUMO protease via the tertiary structure of the SUMO tag. 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, an intein-chitin-binding domain tag (intein-CBD; IMPACT™ system, New England Biolabs, Beverley, MA) can self-cleave upon activation with a denaturant such as dithiothreitol.

[0686] Trimerization domain sequences can also be added to the hMPV F polypeptides and variants described herein. Such sequences can enable trimerization of the antigenic polypeptide. Exemplary trimerization domain sequences can include, but are not limited to, coiled-coil trimerization domains, such as the GCN4 domain (ARMKQIEDKIEEILSKIYHIENEIARIKKLIGEAGSG; SEQ ID NO: 1022); the Foldon domain from T4 fibritin (GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO: 1012); the human type XV collagen domain (SEQ ID NO: 1023); and the human type XVIII collagen domain (SEQ ID NO: 1024).

[0687] Human type XV collagen domain: GSRNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPADS (SEQ ID NO: 1023)

[0688] Human collagen XVIII domain (SEQ ID NO: 1024):

[0689] GVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEARTPLPRGTDNE

[0690] The protease cleavage site at residues 99-102 of SEQ ID NOs:910 and 911 can be replaced with a furin cleavage sequence. Furin cleavage sequences are conventionally described by the consensus sequences RXRR (SEQ ID NO:1025) or RXKR (SEQ ID NO:1026), where X is any amino acid. In various embodiments, residues 99-102 of SEQ ID NO:910 are replaced with SEQ ID NO:1025 or 1026. In various embodiments, residues 99-102 of SEQ ID NO:910 are selected from the group consisting of 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:10 46 (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).

[0691] VII. Assay

[0692] The anti-hMPV antibodies or antigen-binding fragments thereof disclosed herein can be tested for desired properties, such as those described herein, using a variety of assays known in the art.

[0693] In one embodiment, antibodies or antigen-binding fragments thereof are tested for specific binding to hMPV. Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-hMPV antibodies or antigen-binding fragments thereof include standard assays known in the art, such as Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or biolayer interferometry (e.g., ForteBio assays), as described in the Examples.

[0694] In one embodiment, antibodies or antigen-binding fragments thereof are tested for their ability to bind to cells transfected with hMPV.

[0695] In one embodiment, antibodies or antigen-binding fragments thereof are screened for the ability to bind to the surface of hMPV-coated beads.

[0696] In one embodiment, antibodies or antigen-binding fragments thereof are tested for their ability to bind to or affect hMPV, hi another embodiment, antibodies or antigen-binding fragments thereof are tested for their effect on hMPV (e.g., inhibition or no effect).

[0697] VIII. Composition

[0698] Also provided herein are compositions (e.g., pharmaceutical compositions) comprising an anti-hMPV antibody or antigen-binding fragment thereof, an immunoconjugate comprising the same, or a bispecific antibody comprising the same, as described herein, and a carrier (e.g., a pharmaceutically acceptable carrier). Such compositions are useful for a variety of therapeutic applications.

[0699] In some embodiments, the pharmaceutical compositions disclosed herein can include other compounds, drugs, and / or agents used to treat various diseases (e.g., respiratory diseases). Such compounds, drugs, and / or agents can include, for example, antiviral agents and / or anti-inflammatory agents. Exemplary compounds, drugs, and agents that can be formulated together or separately with the anti-hMPV antibodies or antigen-binding fragments thereof described herein.

[0700] 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.

[0701] 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, SM, 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 non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorus acid, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, and non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0702] The pharmaceutical compositions described herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0703] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), 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 the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the composition. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0704] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is contemplated.The pharmaceutical composition may contain or be free of preservatives.A supplementary active compound can be incorporated into the composition.

[0705] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions 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 (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. 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 dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0706] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the ingredients listed above as needed, and then sterilizing by microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other necessary ingredients listed herein.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which can obtain the powder of active ingredient and any other desired ingredients from its solution that has been previously sterilized and filtered.

[0707] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99% of the active ingredient, in combination with a pharmaceutically acceptable carrier, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30%.

[0708] Dosage regimens are adjusted to provide the optimum desired response (e.g., 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 particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for dosage unit forms described herein are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of formulating such active compounds for the treatment of susceptibility in individuals.

[0709] For administration of antibodies or antigen-binding fragments thereof, dosages range from about 0.0001 to 100 mg / kg, more usually 0.01 to 5 or 10 mg / kg, of the host body weight. For administration of antibodies or antigen-binding fragments thereof, dosages range from about 0.0001 to 100 mg. Exemplary treatment regimens entail administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every three months, or once every three to six months.

[0710] The antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are 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, relatively high dosages may be required at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete improvement in the symptoms of the disease. The patient can then be administered a preventive regime.

[0711] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition described herein, or its ester, salt, or amide, used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0712] In various embodiments, a therapeutically effective dose of an anti-hMPV antibody or antigen-binding fragment thereof results in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability resulting from disease affliction. In the context of cancer, a therapeutically effective dose preferably results in an increase in survival rate and / or prevention of further deterioration of physical symptoms associated with cancer. A therapeutically effective dose may prevent or delay the onset of cancer, as may be desired when early or preliminary signs of disease are present.

[0713] The compositions 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 those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Preferred routes of administration for the 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, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human metapneumovirus (hMPV), the antibody comprising: (i) three heavy chain complementarity determining regions (CDRs), including CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 257, 258 and 259, respectively; and three light chain CDRs comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 596, 597 and 598, respectively; or (ii) three heavy chain complementarity determining regions (CDRs), including CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively; and three light chain CDRs comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 359, 360 and 361, respectively; or (iii) three heavy chain complementarity determining regions (CDRs), including CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 29, 30 and 31, respectively; and three light chain CDRs comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 368, 369 and 370, respectively; or (iv) three heavy chain complementarity determining regions (CDRs), including CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 107, 108 and 109, respectively; and CDR1 comprising the amino acid sequences of SEQ ID NOs: 446, 447 and 448, respectively; three light chain CDRs, including CDR2 and CDR3 regions; or (v) three heavy chain complementarity determining regions (CDRs), including CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 182, 183 and 184, respectively; and three light chain CDRs comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 521, 522 and 523, respectively; or (vi) three heavy chain complementarity determining regions (CDRs), including CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 191, 192 and 193, respectively; and three light chain CDRs comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 530, 531 and 532, respectively; or (vii) three heavy chain complementarity determining regions (CDRs), including CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 86, 87 and 88, respectively; and three light chain CDRs comprising CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 425, 426 and 427, respectively; or (viii) three heavy chain complementarity determining regions (CDRs), comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 320, 321, and 322, respectively, and three light chain CDRs, comprising CDR1, CDR2, and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 659, 660, and 661, respectively; An antibody or an antigen-binding fragment thereof comprising:

2. (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 904 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 905; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 848 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 849; or (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 892 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 893; or (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 894 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 895; or (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 872 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 873; or (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 886 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 887; or (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:682, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:683; or (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:744, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:745; The antibody or antigen-binding fragment thereof described in claim 1.

3. 1. A pharmaceutical composition for use in preventing, treating or detecting a virus, comprising: The antibody or antigen-binding fragment of claim 1 or 2, and Pharmaceutically acceptable carriers 13. A pharmaceutical composition comprising:

4. 1. An isolated hMPV F polypeptide, comprising: (i) an isolated hMPV F polypeptide comprising an isolated hMPV F polypeptide that does not include residues 491-539 of SEQ ID NO:1 and comprises residues 1-490 of SEQ ID NO:911, or a variant thereof, wherein said variant has 95-99% identity to residues 1-490 of SEQ ID NO:911, and wherein the isolated hMPV F polypeptide comprising residues 1-490 of SEQ ID NO:911 has a mutation at residue 185; or (ii) 913. An 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 a variant thereof, wherein: 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 1-518 of SEQ ID NO:912, or 95-99% identity to residues 1-521 of SEQ ID NO:913, and wherein: The variant includes residues 1-521 of SEQ ID NO: 910, with no change in residues 486-517, The variant includes residues 1-518 of SEQ ID NO:912, with no changes in residues 102-105 and 490-516, and wherein: The variant containing residues 1-521 of SEQ ID NO:913 has no changes in residues 99-102, 185, and 485-521; An isolated hMPV F polypeptide.

5. The structure (i) is 1. An isolated hMPV F polypeptide comprising 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, wherein: The isolated hMPV F polypeptide of residues 1-490 of SEQ ID NO:911 has a mutation at residue 185, Where: The trimerization domain may be a GCN4 domain, a T4 Foldon domain, a human collagen XV domain, or a human collagen XVIII domain; wherein the GCN4 domain may have the amino acid sequence of SEQ ID NO: 1022, the T4 Foldon domain may have the amino acid sequence of SEQ ID NO: 1012, the human collagen XV domain may have the amino acid sequence of SEQ ID NO: 1023, and the human collagen XVIII domain may have the amino acid sequence of SEQ ID NO: 1024; 5. The isolated hMPV F polypeptide of claim 4.

6. 6. The isolated hMPV of claim 4 or 5, wherein in configuration (i), the protease cleavage sequence at residues 99-102 is replaced with a furin cleavage sequence.

10. An isolated hMPV F polypeptide, wherein the furin cleavage sequence is SEQ ID NO: 1025, 1026, 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.

7. 6. The isolated hMPV F polypeptide of claim 5, wherein the mutation is A185P.

8. 5. The isolated hMPV F polypeptide of claim 4, wherein component (ii) consists of residues 1-521 of SEQ ID NO:

913.

9. 9. The isolated hMPV F polypeptide of any one of claims 5 to 8, comprising a cleavage sequence and at least one affinity tag sequence, 10. The isolated hMPV F polypeptide of claim 9, wherein the cleavage sequence is a thrombin cleavage sequence, An isolated hMPV F polypeptide, wherein the affinity tag sequence may be a 6xHis sequence (SEQ ID NO: 1014) or a strep tag II sequence.

10. 7. The isolated hMPV F polypeptide of claim 6, wherein the isolated hMPV F polypeptide comprises the amino acid sequence of SEQ ID NO:

913.

11. 11. The isolated hMPV F polypeptide of claim 10, wherein the isolated hMPV F polypeptide consists of the amino acid sequence of SEQ ID NO:

913.

12. 12. An isolated nucleic acid encoding the isolated hMPV F polypeptide of any one of claims 5 to 11, comprising the nucleotide sequence of SEQ ID NO: 1010 or a variant thereof, or SEQ ID NO: 1011 or a variant thereof, wherein the variant encodes the amino acid sequence of SEQ ID NO:

913. Isolated nucleic acid.

13. An immunogenic composition comprising the isolated antigenic polypeptide of any one of claims 5 to 11 and a pharma- ceutically acceptable carrier, The immunogenic composition may be multivalent; It may further comprise an adjuvant, or It may not contain an adjuvant, Immunogenic composition.

14. For use in therapy, An antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, or an isolated hMPV F polypeptide according to any one of claims 4 to 11, or an immunogenic composition according to claim 13.

15. 13. The isolated hMPV F polypeptide of any one of claims 4 to 11, or the isolated nucleic acid of claim 12, for use in the prevention or treatment of human metapneumovirus (hMPV) infection in a subject.

16. The immunogenic composition of claim 13 for use in the prevention or treatment of human metapneumovirus (hMPV) infection in a subject.

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