KR20260007578A - Anti-human M-cadherin (CDH15) antibodies, conjugates, and their use for delivering gene payloads to muscle cells - Google Patents

Abstract

Provided herein are antibodies, antibody portions, and drug conjugates thereof directed to cadherin 15. Also provided are nucleic acid sequences encoding the antibodies, antibody portions, and drug conjugates thereof; viral particles comprising the antibodies, antibody portions, and drug conjugates thereof, e.g., for retargeting viral particles to muscle cells; compositions comprising the antibodies, antibody portions, and drug conjugates thereof, and methods of using the same, for treating a subject in need thereof, e.g., a skeletal muscle disorder (e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), facioscapulohumeral muscular dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, dystroglycanopathy, etc.) or rhabdomyosarcoma.

Description

Anti-human M-cadherin (CDH15) antibodies, conjugates, and their use for delivering gene payloads to muscle cells

Cross-reference to related applications

This application claims priority to U.S. Provisional Patent Application Nos. 63/499,531 and 63/499,527, filed May 2, 2023, the disclosures of which are incorporated herein by reference in their entirety.

Technology field

The present application relates generally to human antibodies and antigen-binding fragments of human antibodies that bind to human cadherin 15 (hM-cadherin, hCDH15), and methods of using them, for example, in treating a patient in need thereof. The present application also relates to antigen-binding molecules comprising at least an antigen-binding fragment of an anti-hCDH15 antibody, wherein complexing the antigen-binding molecule to CDH15 mediates internalization of the antigen-binding molecule/CDH15 complex and/or blocks activity of CDH15. In addition, the present application relates to conjugates comprising an anti-hCDH15 antibody (or an antigen-binding molecule comprising an antigen-binding fragment of an anti-hCDH15 antibody) and a therapeutic agent, wherein the conjugates can be useful for treating a disease. Additionally, the present disclosure relates to methods for producing and using, in vitro or in vivo, recombinant viral particles, e.g., recombinant AAV particles, comprising a capsid protein retargeted to Cadherin 15 (CDH15) that is useful for transforming muscle cells, such as muscle stem cells.

Sequence list

The sequence listing in XML format, named "11486WO01 Sequence Listing XML.xml", created on April 30, 2024 and having a size of 787 Kb, is incorporated herein by reference in its entirety.

Gene delivery into specific target cells has become one of the most important technologies in modern medicine, potentially treating a variety of chronic genetic diseases. Ideally, a gene delivery vehicle would stably introduce genetic material into the desired cells while preventing it from being introduced into non-target cells.

Viral particles as gene delivery vehicles, particularly those based on adeno-associated virus (AAV), have been the focus of much research because AAV can transduce a wide range of primate species and tissues in vivo (Muzyczka et al. [(1992) Current Topics in Microbiology and Immunology , 158:97-129]). Furthermore, AAV transduces tissues reliably after division. Although the virus can occasionally integrate into the host chromosome, integration occurs very rarely within the safe-harbor locus on human chromosome 19 and only when the replication (Rep) protein is supplied in trans. The AAV genome rapidly circulates and concatemerizes in infected cells, remaining in a stable episomal state there, enabling long-term, stable expression of its payload.

Furthermore, it has only been in the past few years that AAV infection has been manipulated and redirected to specific cells. Many advances in targeted gene therapy using viral particles can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of viral particles, which lead to pseudotyping, propagation, and/or retargeting of the natural tropism of the viral particle. (Reviewed in Nicklin and Baker [(2002) Curr. Gene Ther. 2:273-93]; Verheiji and Rottier [(2012) Advances Virol 2012:1-15]) .

In direct recombinant targeting approaches, the targeting ligand is directly inserted into or incorporated into the viral capsid. That is, the viral capsid protein gene is modified to express a capsid protein containing a heterologous targeting ligand. The targeting ligand then re-induces (e.g., binds to) a receptor or marker preferentially or exclusively expressed on the target cell. (See also Stachler et al. [(2006) Gene Ther . 13:926-931]; White et al. [(2004) Circulation 109:513-519]; Park et al. [(2007) Frontiers in Bioscience 13:2653-59]; Girod et al. [(1999) Nature Medicine 5:1052-56]; Grifman et al. [(2001) Molecular Therapy 3:964-75]; Shi et al. [(2001) Human Gene Therapy 12:1697-1711]; Shi and Bartlett [(2003) Molecular Therapy 7:515-525]).

In the indirect recombination approach, the viral capsid is modified using a heterologous “scaffold” and then linked to an adapter containing a targeting ligand. The adapter binds to the scaffold and to the target cell (see also Arnold et al. [(2006) Mol. Ther . 5:125-132]; Ponnazhagen et al. [(2002) J. Virol . 76:12900-907]; and WO 97/05266). The following scaffolds have been described for various viral particles: (1) Fc binding molecules (e.g., Fc receptors, protein A, etc.) that bind to the Fc of an antibody adapter, (2) (strept)avidin that binds to a biotinylated adapter, (3) biotin that binds to an adapter fused to (strept)avidin, (4) detectable labels useful for detection and/or isolation of viral particles, including bispecific adapters capable of non-covalently binding to a detectable label and a target molecule, and more recently (5) protein:protein binding pairs that form isopeptide bonds. (See, for example, Gigout et al. [(2005) Molecular Therapy 11:856-865]; Stachler et al. [(2008) Molecular Therapy 16:1467-1473]; Quetglas et al. [(2010) Virus Research 153:179-196]; Ohno et al. [(1997) Nature Biotechnology 15:763-767]; Klimstra et al. [(2005) Virology 338:9-21]).

Skeletal muscle is the largest organ in the body, accounting for approximately 40% of total body mass, and is one of the three major muscle tissues in the human body. When skeletal muscle is damaged, muscle stem cells (MuSCs) are activated, proliferate, and differentiate into functional muscle fibers, repairing the damaged muscle. However, MuSC-mediated muscle regeneration is delayed in aging subjects, which may be partly related to changes in muscle stem cell-specific markers.

Therefore, anti-human antibodies capable of binding to muscle stem cell-specific markers may be particularly useful in therapies targeting aging subjects (e.g., stimulating muscle repair) and/or in treating muscle-related cancers. Furthermore, anti-human antibodies as described herein may be used in conjunction with recombinant viral (e.g., AAV) particles to target and introduce nucleic acids of interest into cells expressing muscle stem cell-specific markers. Furthermore, some muscle-related cancers, including rhabdomyosarcoma, may also benefit from therapeutics as described herein that target muscle stem cell-specific markers, such as antibody-drug conjugates, retargeted viral particles, and the like.

Described herein are antigen binding proteins that bind to human cadherin 15 (CDH15). In some embodiments, the antigen binding protein comprises a set of three heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) amino acid sequences selected from Table 1 below. In some embodiments, the antigen binding protein comprises a set of three light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) amino acid sequences selected from Table 1 below. In some embodiments, the antigen binding protein comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the antigen binding protein comprises a sequence selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311-313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392-396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490-494-496-498, 502-504-506-510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634-638-640-642, Selected from the group consisting of 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730-734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and 766-768-770-774-776-778 It comprises a set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3.

Table 1 sets forth the amino acid and nucleic acid sequence identifiers of each of the HCVRs and LCVRs of the exemplary anti-hCDH15 antibodies of the present disclosure, as well as the HCDR1, HCDR2, and HCDR3 within each HCVR and the LCDR1, LCDR2, and LCDR3 within each LCVR.

anti-hCDH15 sequence identifier Sequence number: Antibody name HCVR
NA
(AA) HCDR1
NA
(AA) HCDR2
NA
(AA) HCDR3
NA
(AA) LCVR
NA
(AA) LCDR1
NA
(AA) LCDR2
NA
(AA) LCDR3
NA
(AA) 9271 1
(2) 3
(4) 5
(6) 7
(8) 9
(10) 11
(12) 13
(14) 15
(16) 9272 21
(22) 23
(24) 25
(26) 27
(28) 29
(30) 31
(32) 33
(34) 35
(36) 9273 41
(42) 43
(44) 45
(46) 47
(48) 49
(50) 51
(52) 33
(34) 53
(54) 9274 59
(60) 61
(62) 63
(64) 65
(66) 49
(50) 51
(52) 33
(34) 53
(54) 9275 69
(70) 71
(72) 73
(74) 75
(76) 49
(50) 51
(52) 33
(34) 53
(54) 9276 79
(80) 81
(82) 83
(84) 85
(86) 49
(50) 51
(52) 33
(34) 53
(54) 9277 89
(90) 91
(92) 93
(94) 95
(96) 97
(98) 99
(100) 33
(34) 101
(102) 9290 107
(108) 109
(82) 110
(111) 112
(113) 114
(115) 116
(117) 33
(34) 118
(119) 9291 124
(125) 126
(127) 128
(129) 130
(131) 132
(133) 134
(135) 136
(137) 138
(139) 9292 144
(145) 146
(147) 148
(149) 150
(151) 152
(153) 154
(155) 156
(157) 158
(159) 9293 164
(165) 166
(167) 168
(169) 170
(171) 172
(173) 174
(175) 176
(177) 178
(179) 9294 184
(185) 186
(187) 188
(189) 190
(191) 192
(193) 194
(52) 33
(34) 195
(196) 9295 201
(202) 203
(204) 205
(206) 207
(208) 209
(210) 211
(212) 136
(137) 213
(214) 9296 219
(220) 221
(222) 223
(224) 225
(226) 49
(50) 51
(52) 33
(34) 53
(54) 9297 229
(230) 231
(232) 233
(234) 235
(236) 49
(50) 51
(52) 33
(34) 53
(54) 9298 239
(240) 241
(242) 243
(244) 245
(246) 49
(50) 51
(52) 33
(34) 53
(54) 9299 249
(250) 251
(82) 252
(253) 254
(255) 49
(50) 51
(52) 33
(34) 53
(54) 9300 258
(259) 260
(261) 262
(263) 264
(265) 266
(267) 268
(269) 270
(271) 272
(273) 9316 278
(279) 280
(281) 282
(283) 284
(285) 286
(287) 288
(289) 290
(291) 292
(293) 9317 298
(299) 300
(301) 302
(303) 304
(305) 306
(307) 308
(309) 310
(311) 312
(313) 9318 318
(319) 320
(321) 322
(323) 324
(325) 326
(327) 328
(329) 330
(331) 332
(333) 9319 338
(339) 340
(341) 342
(343) 344
(345) 346
(347) 348
(349) 350
(14) 351
(352) 9320 357
(358) 359
(360) 361
(362) 363
(364) 365
(366) 367
(368) 369
(370) 371
(372) 9399 377
(378) 186
(187) 379
(380) 381
(382) 49
(50) 51
(52) 33
(34) 53
(54) 9400 385
(386) 387
(388) 389
(390) 391
(392) 393
(394) 395
(396) 13
(14) 397
(398) 9402 403
(404) 405
(406) 407
(408) 409
(410) 411
(412) 99
(100) 33
(34) 413
(414) 8674 419
(420) 421
(422) 423
(424) 425
(426) 427
(428) 429
(430) 431
(432) 433
(434) 8675 435
(436) 437
(438) 439
(440) 441
(442) 443
(444) 445
(446) 447
(448) 449
(450) 8676 451
(452) 453
(454) 455
(456) 457
(458) 459
(460) 461
(462) 463
(464) 465
(466) 8677 467
(468) 469
(470) 471
(472) 473
(474) 475
(476) 477
(478) 479
(480) 481
(482) 8678 483
(484) 485
(486) 487
(488) 489
(490) 491
(492) 493
(494) 495
(496) 497
(498) 8679 499
(500) 501
(502) 503
(504) 505
(506) 507
(508) 509
(510) 511
(512) 513
(514) 8681 515
(516) 517
(518) 519
(520) 521
(522) 523
(524) 525
(526) 527
(528) 529
(530) 8682 531
(532) 533
(534) 535
(536) 537
(538) 539
(540) 541
(542) 543
(544) 545
(546) 8683 547
(548) 549
(550) 551
(552) 553
(554) 555
(556) 557
(558) 559
(560) 561
(562) 8684 563
(564) 565
(566) 567
(568) 569
(570) 571
(572) 573
(574) 575
(576) 577
(578) 8685 579
(580) 581
(582) 583
(584) 585
(586) 587
(588) 589
(590) 591
(592) 593
(594) 8686/8687 595
(596) 597
(598) 599
(600) 601
(602) 603
(604) 605
(606) 607
(608) 609
(610) 8784 611
(612) 613
(614) 615
(616) 617
(618) 619
(620) 621
(622) 623
(624) 625
(626) 8785 627
(628) 629
(630) 631
(632) 633
(634) 635
(636) 637
(638) 639
(640) 641
(642) 8786 643
(644) 645
(646) 647
(648) 649
(650) 651
(652) 653
(654) 655
(656) 657
(658) 8787/8796 659
(660) 661
(662) 663
(664) 665
(666) 667
(668) 669
(670) 671
(672) 673
(674) 8788 675
(676) 677
(678) 679
(680) 681
(682) 683
(684) 685
(686) 687
(688) 689
(690) 8789 691
(692) 693
(694) 695
(696) 697
(698) 699
(700) 701
(702) 703
(704) 705
(706) 8790 707
(708) 709
(710) 711
(712) 713
(714) 715
(716) 717
(718) 719
(720) 721
(722) 8791 723
(724) 725
(726) 727
(728) 729
(730) 731
(732) 733
(734) 735
(736) 737
(738) 8792 739
(740) 741
(742) 743
(744) 745
(746) 683
(684) 685
(686) 687
(688) 689
(690) 8793 747
(748) 749
(750) 751
(752) 753
(754) 755
(756) 757
(758) 759
(760) 761
(762) 8795 763
(764) 765
(766) 767
(768) 769
(770) 771
(772) 773
(774) 775
(776) 777
(778) 8680 779
(780) 781
(782) 783
(784) 785
(786) NA
(NA) NA
(NA) NA
(NA) NA
(NA)

In some embodiments, the antigen binding protein comprises a heavy chain variable region (HCVR or VH). In some embodiments, the HCVR comprises a set of HCDR1-HCDR2-HCDR3 amino acid sequences selected from Table 1. In some embodiments, the HCVR comprises an amino acid sequence selected from any one of the HCVR amino acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In some embodiments, the antigen binding protein comprises a light chain variable region (LCVR or VL). In some embodiments, the LCVR comprises a set of LCDR1-LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the LCVR comprises an amino acid sequence selected from any one of the LCVR amino acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In some embodiments, the antigen-binding domain comprises an anti-hCDH15 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-hCDH15 antibody or antigen-binding fragment thereof comprises a human or humanized antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a trispecific antibody, or a chemically modified derivative thereof. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), where n is 1 to 10.

In some embodiments, the antigen binding protein, e.g., an antibody or antigen binding fragment thereof, binds to hCDH15 with an affinity KD of about 1X10 ^-7 M or stronger. In some embodiments, the antigen binding protein binds to hCDH15 with a KD of about 10X10 ^-8 to about 1X10 ^-10 . In some embodiments, the antigen binding protein binds to hCDH15 with a KD of about 5X10 ^-9 to about 1X10 ^-10 .

In some embodiments, the antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, comprises an HCVR and LCVR amino acid sequence pair (HCVR/LCVR) comprising any one of the HCVR amino acid sequences listed in Table 1 paired with an LCVR amino acid sequence listed in Table 1. In some embodiments, the antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, as described herein, comprises an HCVR/LCVR amino acid sequence pair contained in any one of the exemplary anti-hCDH15 antibodies listed in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pairs are selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772 It is selected from the group formed.

Also described herein are nucleic acid molecules, i.e., polynucleotides, encoding antigen binding proteins described herein, e.g., anti-hCDH15 antibodies or antigen binding fragments thereof. In some embodiments, a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding a set of HCDR1-HCDR2-HCDR3 amino acid sequences listed in Table 1. In some embodiments, a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding any one of the HCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCVR amino acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In some embodiments, a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding a set of LCDR1-LCDR2-LCDR3 amino acid sequences listed in Table 1. In some embodiments, a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding any one of the LCVR amino acid sequences listed in Table 1. In some embodiments, a nucleic acid molecule comprises a polynucleotide sequence selected from any one of the LCVR amino acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

Also described herein is a nucleic acid molecule encoding HCVR, wherein the HCVR comprises three sets of CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any one of the exemplary anti-hCDH15 antibodies listed in Table 1.

Also described herein is a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises three sets of CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any one of the exemplary anti-hCDH15 antibodies listed in Table 1.

Also described herein are nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any one of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises the amino acid sequence of any one of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCVR nucleic acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any one of the LCVR nucleic acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-hCDH15 antibody listed in Table 1.

Also described herein are pharmaceutical compositions comprising an antigen binding protein described herein, e.g., a recombinant human antibody or fragment thereof that binds human CDH15, and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition as described herein comprises a combination of an anti-hCDH15 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that advantageously combines with the anti-hCDH15 antibody. Additional combination therapies and co-formulations comprising an anti-hCDH15 antibody are described herein.

Also described herein is a method of inhibiting the activity of a cell expressing CDH15, e.g., in vivo, in vitro, or ex vivo, comprising contacting the cell expressing CDH15 with an antigen binding protein that binds human CDH15 or a pharmaceutical composition thereof as described herein. In some embodiments, the cell expressing CDH15 is a muscle stem cell, a myoblast, or a myocyte.

Also described herein is a method of accelerating the transition from mitotic arrest to activation of muscle cells, for example in vivo, in vitro, or ex vivo, comprising contacting muscle stem cells with an antigen binding protein that binds human CDH15 or a pharmaceutical composition thereof as described herein.

Also described herein is a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein, the pharmaceutical composition comprising an antigen binding protein that binds human CDH15. In some embodiments, the condition is muscle damage. In some embodiments, the condition is cancer, such as rhabdomyosarcoma. In some embodiments, the antigen binding protein is conjugated to a therapeutic agent, such as an antibody-drug conjugate (ADC). In some embodiments, the therapeutic agent comprises a cytotoxic chemotherapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, liposomal cytarabine, dacarbazine, dactinomycin (actinomycin D), daunorubicin, docetaxel, doxorubicin, liposomal doxorubicin, epirubicin, eribulin, etoposide, etoposide phosphate, fludarabine, fluorouracil, fotemustine, ganciclovir, gemcitabine, gemtuzumab ozogamicin Ozogamicin), Hydroxyurea, Idarubicin, Ifosfamide, Inotuzumab ozogamicin, Irinotecan, Ixazomib, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitozantrone, Nab-paclitaxel, Oxaliplatin, Paclitaxel, Pemetrexed, Pegaspargase, Polatuzumab vedotin), Pralatrexate, Procarbazine, Raltitrexed, Romidepsin, Sacituzumab govitecan, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Trabectedin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trifluridine/tipiracil, Valganciclovir, Vinblastine, Vincristine, Vindesine, vinflunine, vinorelbine, or vismodegib. In some embodiments, the antigen binding protein is conjugated to the therapeutic agent via a valine-citrulline (VC) linker. In some embodiments, the antigen binding protein is conjugated to the therapeutic agent via a para-aminobenzyl (PAB) linker. In some embodiments, the pharmaceutical composition is administered intravenously or subcutaneously to the subject.

Also described herein is a method of restoring muscle regenerative capacity in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein, the pharmaceutical composition comprising an antigen binding protein that binds human CDH15. In some embodiments, the subject is an elderly subject. In some embodiments, the muscle regenerative capacity of the elderly subject is restored to the functional state of a control subject or close to it. In some embodiments, the pharmaceutical composition is administered intravenously or subcutaneously to the subject.

Also described herein is a method of imaging muscle cells in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition as described herein comprising an antigen binding protein that binds human CDH15, wherein the antigen binding protein is conjugated to a detectable moiety. In some embodiments, the muscle cells comprise one or more selected from the group consisting of muscle stem cells, myoblasts, and myocytes. In some embodiments, the detectable moiety comprises a radionuclide. In some embodiments, the pharmaceutical composition is administered intravenously or subcutaneously to the subject.

Also described herein is the use of an antigen binding protein that binds human CDH15, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating a condition, for example, as described herein. Further described herein is an antigen binding protein that binds human CDH15, or a pharmaceutical composition thereof, for use in a therapy for treating a condition, for example, as described herein, and/or for use in restoring muscle regenerative capacity in a subject.

Also described herein are antibodies or antigen-binding fragments that compete for binding to human CDH15 with a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1. In some embodiments, the antibody or antigen-binding fragment as described herein binds to human CDH15 by binding to a sequence selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and Competes with a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of 764 and 772.

Also described herein is an antibody or antigen-binding fragment thereof that binds to an epitope on human CDH15 identical to a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1. In some embodiments, the antibody or antigen-binding fragment as described herein comprises a sequence selected from SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772 Binds to an epitope on human CDH15 identical to a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of:

Also described herein is an isolated antibody or antigen-binding fragment thereof that binds to human CDH15, wherein the antibody or antigen-binding fragment comprises: a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 1; and a CDR of a light chain variable region (LCVR) having an amino acid sequence as set forth in Table 1. In some embodiments, the isolated antibody or antigen-binding fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772 In another embodiment, the isolated antibody or antigen binding protein comprises heavy and light chain CDRs of an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311-313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392-396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490-494-496-498, 502-504-506-510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730-734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and Each of the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains selected from the group consisting of 766-768-770-774-776-778. Also described herein is an isolated antibody or antigen-binding fragment thereof that binds to human CDH15, comprising: (a) SEQ ID NO: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, A heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of 676, 692, 708, 724, 740, 748, 764, and 780; and/or (b) an amino acid selected from the group consisting of SEQ ID NOs: 10, 30, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 287, 307, 327, 347, 366, 50, 394, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 756, and 772 Light chain variable region (LCVR) with sequence. In some embodiments, the isolated antibody or antigen-binding fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772 Contains a pair of HCVR/LCVR amino acid sequences selected from the group consisting of:

Further described herein are viral particles specifically suited for targeting and introducing nucleotides of interest into muscle cells (e.g., muscle stem cells). The viral capsids or viral capsid protein(s) described herein are particularly suitable because they comprise a targeting ligand that binds to a muscle cell-specific surface protein (e.g., an antigen binding protein as described herein that binds human CDH15). In some embodiments, the viral capsids or viral capsid protein(s) described herein comprise direct insertion(s) of the targeting ligand (e.g., the targeting ligand is directly coupled to, fused to, or optionally coupled or fused via a linker to the viral capsid or viral capsid protein), e.g., the viral capsid gene is modified to express a capsid protein comprising the targeting ligand. In some embodiments, the viral capsid or viral capsid protein comprises a targeting ligand via a scaffold or adaptor, e.g., a first member of a protein:protein binding pair capable of binding to a cognate second member of the protein:protein binding pair, wherein the second member is linked (e.g., fused) to a targeting ligand that binds to a muscle cell-specific surface protein (e.g., an antigen binding protein such as those described herein that bind human CDH15). In some embodiments, the targeting ligand is operably linked (e.g., fused to) the second member, optionally via a linker. In some embodiments, the targeting ligand can be a binding moiety, e.g., a natural ligand, an antibody, a multispecific binding molecule, or the like. In some embodiments, the targeting ligand is an antibody or a portion thereof. In some embodiments, the targeting ligand is an antibody comprising a variable domain (e.g., a variable domain of an antigen binding protein described herein that binds human CDH15) that binds to a surface protein on a non-terminally differentiated muscle cell (e.g., a muscle stem cell, a myoblast, a muscle cell, any combination thereof, etc.) and a heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain (e.g., a variable domain of an antigen binding protein described herein that binds human CDH15) that binds to a non-terminally differentiated muscle cell surface protein on a target cell (e.g., a muscle stem cell, a myoblast, a muscle cell, any combination thereof, etc.) and, optionally, an IgG heavy chain constant domain. In some embodiments, the targeting ligand comprises a variable domain that binds to a terminally differentiated muscle cell surface protein on a target cell (e.g., a muscle stem cell, a myoblast, a muscle cell, any combination thereof, etc. ) (e.g., a variable domain of an antigen binding protein as described herein that binds human CDH15) and an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked, e.g., directly or via a linker, to a capsid protein. In some embodiments, the targeting ligand is an antibody comprising (i) a variable domain that binds to a terminally differentiated muscle cell surface (e.g., a variable domain of an antigen binding protein as described herein that binds human CDH15), and (ii) an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked (optionally via a linker) to a protein that forms an isopeptide covalent bond with a cognate first member of a protein:protein binding pair (e.g., a second member of the protein:protein binding pair). In some embodiments, a capsid protein described herein comprises a first member of a protein:protein binding pair operably linked to a viral capsid protein, wherein the first member of the protein:protein binding pair comprises, for example, SpyTag (SEQ ID NO: 815) or a biologically equivalent variant thereof, wherein the SpyTag or a biologically equivalent variant thereof is operably covalently linked (e.g., via an isopeptide bond) to a second cognate protein:protein binding member, for example, SpyCatcher (SEQ ID NO: 816) or a biologically equivalent variant thereof, and wherein the SpyCatcher or a biologically equivalent variant thereof can in turn be linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein the SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, for example, GSGESG (SEQ ID NO: 828). In some embodiments, the terminally differentiated muscle cell surface protein comprises CDH15. In some embodiments, the targeting ligand binds CDH15, e.g., human CDH15. In some embodiments, the targeting ligand comprises a variable domain of an antigen binding protein as described herein that binds human CDH15. In some embodiments, the targeting ligand comprises an antibody variable domain comprising CDRs of an HCVR and/or LCVR sequence as set forth in Table 1, e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3. In some embodiments, the targeting ligand comprises an antibody variable domain comprising CDRs of an HCVR and/or LCVR sequence as set forth in Table 1, e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3. In some embodiments, the targeting ligand comprises a set of three CDRs of an HCVR and/or LCVR sequence as set forth in Table 1, e.g., HCDR1, HCDR2, ?? An antibody variable domain comprising HCDR3, and/or LCDR1, LCDR2, and LCDR3. In some embodiments, the targeting ligand comprises an antibody variable domain comprising a set of three CDRs, e.g., HCDR1, HCDR2, ?? HCDR3, and/or LCDR1, LCDR2, and LCDR3, as set forth in Table 1. In some embodiments, the targeting ligand comprises an antibody variable domain comprising an HCVR and/or an LCVR as set forth in Table 1.

The patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the required fee.
Figure 1 depicts a schematic representation of the myogenesis of terminally undifferentiated muscle cells. Muscle stem cells (MuSCs; also known as satellite cells) are mitotically quiescent and non-proliferating under quiescent conditions in adult muscle tissue. However, MuSCs actively divide in response to injury, producing daughter cells. Some daughter cells become quiescent once more to replenish the MuSC pool, while others continue to proliferate as myoblasts, which align and fuse with each other during differentiation to form mature myotubes/myofibers that constitute muscle fibers. Quiescent muscle stem cells can be characterized by Pax7 expression, proliferating myoblasts can be characterized by Pax7 and myogenic differentiation factor 1 (MyoD) expression, terminally differentiating myoblasts (i.e., myocytes) can be characterized by MyoD and myogenin expression, and myotubes can be characterized by myosin heavy chain (MyHC) expression.
Figures 2a–d demonstrate accelerated muscle regeneration in mice with knockout CDH15 expression. Figure 2a depicts a schematic of an exemplary experiment. Muscle damage was induced by intramuscular injection of cardiotoxin (CTX) into wild-type (WT) or homozygous CDH15 knockout mice (CDH15-/-) on day 0. Histological analysis was then performed on muscle samples harvested at 5, 15, and 25 days post-injury (dpi). Figure 2b shows examples of immunohistochemical sections of WT and CDH15−/− muscle samples stained with DAPI (blue; light gray on a gray scale), laminin (white), and embryonic myosin heavy chain (eMyHC; green; dark gray on a gray scale), which are transiently upregulated in immature myofibers but downregulated as myofibers mature (Rodgers, Growth Hormone & IGF Research, 2005, 15(6): 377-383). Sections were visualized by fluorescence microscopy. Figure 2c shows the muscle fiber cross-sectional area (CSA) of WT and CDH15−/− mice at the indicated time points after injury, as quantified from histological images (see, e.g., Figure 2b). Figure 2d shows the percentage of centrally nucleated myofibers of WT and CDH15−/− mice at day 15 post-injury, as quantified from histological images (see, e.g., Figure 2b). Data are reported as mean +/- SEM, *p<0.05, **p<0.01.
Figures 3a-3b demonstrate improved functional recovery from injury in mice with knockout of CDH15 expression. Figure 3a depicts a schematic of an exemplary experiment. On day 0, WT or CDH15-/- mice were injected with CTX into the extensor digitorum longus (EDL) muscle. At 15 dpi, the contractile force of the EDL muscle was measured ex vivo. Figure 3b depicts the loss of maximal stiffness in WT or CDH15-/- muscles compared to muscles isolated from intact EDL. Data are reported as means +/- SEM, *p<0.05.
Figures 4a-4b demonstrate that CDH15-depleted MuSCs undergo accelerated exit from quiescence in vitro. Figure 4a shows examples of immunohistochemical images of WT and CDH15−/− MuSCs on single myofibers after 48 h of culture. Arrows in the Pax7 and merge columns indicate individual cells within multicellular clusters. Figure 4b shows the percentage of Pax7+ single-cell and multicellular clusters (top) and the number of Pax7+ cells per cluster (bottom) for WT and CDH15−/− MuSCs, as quantified from histological images (see, e.g., Figure 4a). Data are reported as mean +/- SEM, **p<0.01.
Figures 5a-5b illustrate the upregulation of early response genes containing the serum response factor (SRF) motif. Figure 5a depicts a volcano map of genes downregulated or upregulated in FACS-isolated CDH15-/- MuSCs compared to WT MuSCs. Figure 5b depicts a transcription factor motif analysis, with several relevant transcription factors highlighted in red.
Figures 6a–6d demonstrate that knocking out CDH15 expression in aged mice rescues the age-related decline in muscle regeneration. Figure 6a depicts a schematic of an exemplary experiment. On day 0, aged (23 months old) wild-type (WT) or homozygous CDH15 knockout mice (CDH15-/-) were injected with cardiotoxin (CTX) to induce muscle damage in the tibialis anterior muscle. Histology was then performed on muscle samples harvested 15 days post-injury (dpi). Figure 6b depicts examples of immunohistochemical sections of aged WT and CDH15-/- muscle samples stained with DAPI (blue; grayscale) and laminin (white) for nuclei and visualized by fluorescence microscopy. Figure 6c depicts the muscle fiber cross-sectional area (CSA) of aged WT and CDH15-/- mice at 15 dpi, as quantified from histological images (see, e.g., Figure 6b). The dashed line depicts the mean CSA for young mice at 15 dpi. Figure 6d depicts the percentage of centrally nucleated muscle fibers containing three or more central nuclei for aged WT and CDH15-/- mice at 15 dpi, as quantified from histological images (see, e.g., Figure 6b). Data are reported as mean +/- SEM, *p<0.05.
Figures 7a–c show that CDH15 protein is localized to the apical surface of most wild-type (WT) muscle stem cells (MuSCs) and is not detected in CDH15−/− MuSCs. Figure 7a shows an immunohistochemical cross-section of a WT single myofiber with bound WT MuSCs, in which CDH15 protein is present at the apical surface of MuSCs. Anti-CDH15 staining is shown in red and is indicated by arrowheads and dashed outlines, and DAPI staining is shown in blue and is indicated by arrowheads and dashed outlines. Figure 7b shows an immunohistochemical cross-section of a WT mouse muscle, in which Pax7-positive MuSCs are observed at the periphery of the myofibers, and CDH15 is also present at the apical surface of MuSCs. In the enlarged inset, anti-CDH15 is depicted in red and indicated by arrows and dashed outlines, DAPI-stained nuclei are depicted in blue, Pax7 is depicted in green, and laminin is depicted in white. Overlap of DAPI and Pax7 is indicated by arrowheads and dashed outlines in the enlarged inset. Figure 7c depicts the percentage of Pax7-positive MuSCs that are also CDH15-positive in WT and CDH15-/- mouse muscle, as quantified from histological images (see, e.g., Figures 7a and 7b). CDH15 protein is detected in the majority (approximately 95%) of WT Pax7-positive muscle cells, whereas CDH15 is not detected in CDH15-/- Pax7-positive cells, as expected.
Figure 8 shows the specific binding of anti-hCDH15 antibodies to human rhabdomyosarcoma cells but not to glioblastoma cells. Prior to visualization by fluorescence microscopy, alveolar cells, germ cells, and glioblastoma cells were live-stained with the indicated anti-hCDH15 antibodies (REGN8787 and REGN9295) for 30 minutes, washed, stained with anti-human IgG Alexa Fluor™ 647-conjugated secondary antibody (red), washed again, fixed, and stained for myogenin (green) and nuclei (DAPI; blue). Cells incubated with murine IgG2a and human IgG4 were also used as controls. The last row depicts the integration of the three markers.
Figure 9 shows the specific binding of anti-hCDH15 antibodies to human rhabdomyoblasts grown in 3D cultures. Embryonic rhabdomyosarcoma tumor spheres ( i.e. , rhabdomyoblasts) were incubated with anti-hCDH15 or human IgG4 as a negative control for 30 minutes, washed, stained with anti-human IgG Alexa Fluor™ 647-conjugated secondary antibody (white), washed again, fixed, and stained for myogenin (red) and nuclei (DAPI; blue). The last column depicts the integration of the three markers.
Figure 10 shows retargeting of AAV9 to C2C12 mouse myoblasts mediated by the anti-CDH15 antigen-binding domain. Representative immunofluorescence images showing transduction efficiency via GFP fluorescence of C2C12 myoblasts transduced with 2.5 x 10 ⁵ vg/cell of AAV9 +/- plasmids encoding the anti-CDH15 antigen-binding domain or a control antigen-binding domain (anti-ASGR1) expressing gGFP and various mosaic capsid ratios are shown. DAPI-stained nuclei are shown in blue (top row), and eGFP is shown in green (bottom row). The ratios provided represent the ratio of the amount of transfected plasmid encoding SpyTag-conjugated AAV9 capsid to unconjugated N272A detargeted AAV9 capsid. When the antigen binding domain is represented by, for example, a mAb or Fab, the AAV9 capsid comprises a SpyTag inserted at position 453 and attached via a 10 amino acid linker, and the antigen binding domain comprises a SpyCatcher fused to the C-terminus of the heavy chain construct.
Figure 11 shows retargeting of AAV9 to human skeletal myoblasts mediated by the anti-CDH15 antigen-binding domain. Representative immunofluorescence images showing transduction efficiency via GFP fluorescence of human skeletal myoblasts transduced with 2.5 x 10 ⁵ vg/cells of AAV9 +/- plasmids encoding the anti-CDH15 antigen-binding domain or a control antigen-binding domain (anti-ASGR1) expressing eGFP and varying mosaic capsid ratios are shown. DAPI-stained nuclei are shown in blue (top row), and eGFP is shown in green (bottom row). The ratios provided represent the ratio of the amount of transfected plasmid encoding SpyTag-conjugated AAV9 capsid to unconjugated N272A detargeted AAV9 capsid. When the antigen binding domain is represented by, for example, a mAb or Fab, the AAV9 capsid comprises a SpyTag inserted at position 453 and attached via a 10 amino acid linker, and the antigen binding domain comprises a SpyCatcher fused to the C-terminus of the heavy chain construct.

Each individual skeletal muscle is composed of thousands of muscle fibers wrapped together by a connective tissue sheath. Individual bundles of muscle fibers within a skeletal muscle are known as fasciculi. The outermost connective tissue sheath surrounding the entire muscle is known as the epimysium. The connective tissue sheath covering each fascicle is known as the perimysium, and the innermost sheath surrounding each muscle fiber is known as the endomysium. Each muscle fiber comprises a myofibril containing numerous myofilaments.

When muscle fibers are bundled together, each fiber is arranged in a unique striated pattern to form a sarcomere, the fundamental contractile unit of skeletal muscle. The two most important myofilaments are actin filaments, which are arranged in distinct bands on skeletal muscle, and myosin filaments.

The primary function of skeletal muscle occurs through a unique excitation-muscle contraction coupling process. Because muscles are attached to bone tendons, muscle contraction leads to movement of the corresponding bone, enabling specific movements. Skeletal muscle also provides structural support and helps maintain body posture. Skeletal muscle also serves as a storage source for amino acids, which can be used by various organs to synthesize organ-specific proteins. Skeletal muscle also serves as a site for glucose disposal in the form of muscle glycogen. Skeletal muscle also plays a central role in maintaining thermoregulation and serves as an energy source during times of hunger. Therefore, skeletal muscle plays a crucial role in movement, thermoregulation, and the regulation of systemic metabolism.

In many muscle diseases, as well as during normal aging, skeletal muscle tissue decreases in size and function, impairing functional mobility; in severe muscle diseases, this results in long-term disability and premature death.

Treatment of muscle wasting diseases and inherited muscle disorders generally consists of broad-spectrum therapies: testosterone therapy for muscle wasting diseases, glucocorticoids for muscular dystrophies, and systemic AAV delivery for muscle disorders (e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), capsulohumeral dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, and dystroglycanopathies, etc.). Non-targeted delivery of these therapies reduces uptake efficiency in specific muscles, while also causing significant detrimental off-target effects in other organs.

Muscle stem cells (MuSCs) are crucial for skeletal muscle regeneration. MuSCs are normally quiescent in adult muscle, but upon injury, they become activated, proliferate, and differentiate into functional muscle fibers. However, MuSCs from aged subjects exhibit delayed activation and reduced motility in vitro, leading to impaired MuSC-mediated repair in vivo.

Mitotically quiescent muscle stem cells (MuSCs, also called satellite cells) and proliferating myoblasts are embryonic precursors of muscle cells (also called muscle cells) that have not yet fused together to form myotubes or myofibrils that later become muscle fibers. Muscle stem cells and myoblasts differentiate into muscle cells through a process called myogenesis, which is schematically illustrated in Figure 1 (not to scale).

Normally, after exposure to signals from a damaging environment, muscle stem cells emerge from their quiescent state, re-enter the cell cycle, and begin to proliferate as myoblasts. Some daughter cells continue to differentiate, while others revert to quiescence to replenish the reserve population of muscle stem cells. During the differentiation phase, specific genes (such as the striated alpha-actin gene) are expressed, and myoblasts align with one another. Myoblasts then fuse to form muscle fibers, and actin is recruited to the plasma membrane.

Muscle stem cells can be characterized by a combination of genetic markers, such as Pax7 and muscle regulatory proteins. Pax7 is a paired homeobox transcription factor that specifies the myogenic nature of precursor muscle cells. Therefore, muscle stem cells can be characterized as Pax7+, regardless of whether they are quiescent or proliferating. See, for example, Figure 1. Furthermore, myogenesis relies on the precise and dynamic integration of multiple muscle regulatory factors, such as myogenic factor 5 (MYF5), myogenic differentiation factor 1 (MYOD), myogenin (MYOG), and embryonic myosin heavy chain (MyHC), enabling the characterization of myogenic cell lineages. For example, MYOD is expressed in myogenic cells but not in quiescent, resting muscle stem cells, and thus can be used to identify proliferating muscle stem cells, myoblasts, or other differentiated muscle cells. See, for example, Figure 1. Myogenin appears to be expressed by myoblasts committed to differentiation into muscle fibers. For example, see Figure 1. Additional markers that can be used to identify which stage of myogenesis a cell is undergoing include, but are not limited to, embryonic myosin heavy chain (eMyHC), which is transiently upregulated in immature muscle fibers but downregulated as the muscle fibers mature. See, for example, Figure 1.

Myoblasts can be classified into skeletal muscle myoblasts, smooth muscle myoblasts, and cardiac muscle myoblasts, depending on the type of muscle cell they differentiate into. Therefore, muscle stem cells, myoblasts, myocytes, and undifferentiated myotubes or myofibers can all be considered terminally undifferentiated muscle cells.

Many pathological conditions, such as muscular dystrophy (MD) or muscle wasting, may not provide sufficient signals to muscle stem cells, or satellite cells may have inherent defects in these conditions, which would impair their regenerative capacity. Therefore, the ability to specifically target non-terminally differentiated muscle cells, such as muscle stem cells, myoblasts, and myotubes, could be useful in the treatment of skeletal muscle disorders.

An exemplary cell surface protein expressed in terminally differentiated muscle cells is cadherin 15 (CDH15). Cadherins are a family of calcium-dependent transmembrane proteins involved in cell-cell adhesion. Classical cadherins consist of an extracellular domain containing five repeats of an immunoglobulin-like cadherin domain, a single transmembrane region, and a cytoplasmic domain. Cadherin 15 (also known as M-cadherin) is expressed on the apical surface of muscle stem cells and is thought to regulate their attachment to muscle myofibers. Cadherin 15 is encoded by the CDH15 gene, located on the long arm of chromosome 16 (16q24.3). CDH15 contains 14 exons and is approximately 23,745 bases in length. An exemplary sequence for the human CDH15 gene has the NCBI Accession Number NM_004933.3 (SEQ ID NO: 787). An exemplary human CDH15 protein has the NCBI Accession Number NP_004924.1 and/or UniProt Accession Number P55291 (SEQ ID NO: 788).

Genetically modified animal models may prove particularly useful for studying the function of CDH15 in muscle. For example, although deletion of Cdh15 in genetically modified animals does not alter the quiescence, proliferation, myogenic lineage commitment, or differentiation capacity of isolated MuSCs, deletion of Cdh15 in these mice accelerates regeneration after injury ( Figures 2A–D , 3A–3B ). This may be due, at least in part, to accelerated exit from quiescence following deletion of CDH15 expression ( Figures 4A–4B ), which is associated with the induction of SRF-regulated early response genes ( Figures 5A–5B ), ultimately leading to decreased Rho/Rac signaling. Interestingly, Cdh15 ^−/− MuSCs are slightly larger than wild-type control MuSCs, indicating that these cells are better primed for activation. Similar to the effects observed in young mice, Cdh15 deletion also rescues age-related defects in muscle regeneration ( Figures 6A–D ). Therefore, antagonistic or CDH15-blocking antibodies may be useful for enhancing muscle regeneration in both young and aged subjects following insults (e.g., arthroplasty).

Additionally, certain muscle-related cancers, including rhabdomyosarcoma, may benefit from therapies targeting muscle-specific markers. Rhabdomyosarcoma (RMS) is characterized by the expression of myogenic genes, but multiple subtypes exist. For example, embryonal RMS, the most prevalent subtype, generally has a better prognosis and is thought to be driven by loss of tumor suppressor genes or gain-of-function of proto-oncogenes. Alveolar RMS is less common but generally has a much worse prognosis. Alveolar RMS is thought to be caused by chromosomal translocations or alternative gene fusions in the Pax3 or Pax7 genes. Additional, rarer subtypes include spindle cell/sclerotic RMS and pleomorphic RMS. Rhabdomyosarcoma accounts for approximately 3% of all childhood cancers, with approximately 400-500 new cases diagnosed annually in the United States (American Cancer Society, 2021). The prognosis is generally good in children (e.g., approximately 70% survival), but varies by risk category. For example, one-third of patients with localized RMS and two-thirds of patients with metastatic RMS experience recurrent disease. If RMS relapses, the estimated five-year survival rate for most patients is approximately 10%. Furthermore, the prognosis is generally much worse in adults (overall survival rate of 20-50%).

Therefore, because high CDH15 expression is observed in all rhabdomyosarcomas (RMS) and may be associated with chemoresistance and poor prognosis, anti-CDH15 antibodies may be useful for delivering therapeutics (e.g., cytotoxic agents) to RMS tumors.

Described herein are antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that bind to human CDH15. The antibodies described herein may be particularly useful for specifically inducing internalization of agents (e.g., drug conjugates, etc.) to muscle cells and/or muscle-associated cancer cells, blocking the activity of CDH15, and/or specifically stimulating muscle regeneration. Likewise, antibodies or antigen-binding fragments thereof that bind to human CDH15 are also described herein; these include antibody-protein fusion constructs comprising antibodies or antigen-binding fragments thereof that bind to human CDH15; and antibody drug conjugates comprising antibodies or antigen-binding fragments thereof that bind to human CDH15. Furthermore, to enhance muscle delivery of therapeutic payloads and reduce off-target effects, viral particles, such as AAV viral particles, that can target terminally undifferentiated muscle cell surface proteins, such as mammalian CDH15 are described herein.

The subject matter described herein is not limited to the specific embodiments, compositions, methods, and experimental conditions described, as such embodiments, compositions, methods, and conditions may vary. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing described herein, certain preferred methods and materials are now described. All publications cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes one or more methods and/or steps of the type described herein and/or as would become apparent to one of ordinary skill in the art upon reading this disclosure.

When the term "about" is used in reference to a specific quoted value, it means that the value may vary by less than 1% from the quoted value. For example, the expression "about 100" includes 99 and 101, and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

"Percent identity (%)" and the like can be readily determined for amino acid or nucleotide sequences across the full length of a protein or a portion thereof. The "portion" can be at least about 5 amino acids or 24 nucleotides in length, respectively, and can be up to about 700 amino acids or 2100 nucleotides in length, respectively. Generally, when referring to "identity," "homology," or "similarity" between two different adeno-associated viruses, the "identity," "homology," or "similarity" is determined with reference to "aligned" sequences. An "aligned" sequence or "alignment" often refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that contain corrections for missing or added bases or amino acids compared to a reference sequence.

Alignments can be performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs such as “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs are available for amino acid sequences. Generally, all of these programs are used with default settings, but those skilled in the art can modify these settings as needed. Alternatively, those skilled in the art can use other algorithms or computer programs that provide at least the same level of identity or alignment as those provided by the referenced algorithms and programs. See, for example, J. D. Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

Nucleic acid sequence alignment programs are also available. Examples of such programs include “Clustal W,” “CAP Sequence Assembly,” “MAP,” and “MEME,” which are accessible via web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utilities can be used. Numerous algorithms known in the art can also be used to determine nucleotide sequence identity, including those included in the aforementioned programs. As another example, polynucleotide sequences can be compared using FASTA™, a program within GCG version 6.1. Fasta™ provides alignments of the most overlapping regions between the query and search sequences and percent sequence identity. For example, as provided in GCG version 6.1, which is incorporated herein by reference, percent sequence identity between nucleic acid sequences can be determined using FASTA™ with its default parameters (a word size of 6 and a NOPAM factor for the scoring matrix).

“Substantial identity” includes amino acid or nucleic acid sequence alignments that are at least 90%, such as at least 93%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as at least 100% identical.

The term “chimera” includes functional genes or polypeptides each comprising a nucleic acid sequence or an amino acid sequence derived from at least two different AAV serotypes, e.g., portions of genes or portions of polypeptides of at least a first and a second AAV, wherein at least the first and second portions are operably linked to form a functional chimeric AAV nucleic acid encoding a functional amino acid. Unless specified as chimeric, the nucleotide sequences, genes, polypeptides, and amino acids are considered non-chimeric in that the nucleotide sequences, genes, polypeptides, and amino acids comprise nucleic acid sequences or amino acid sequences that have substantial identity to a nucleic acid sequence or amino acid sequence of a single AAV serotype, respectively.

As used herein, the phrase “operably linked” includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact, directly or indirectly, with each other, or otherwise cooperate with each other to participate in a biological event, wherein such interaction and/or cooperation is achieved or permitted by the juxtaposition. As an example, a regulatory element (e.g., an expression control sequence) within a nucleic acid is said to be “operably linked” to a coding sequence if it is positioned relative to the coding sequence such that its presence or absence affects the expression and/or activity of the coding sequence. In many embodiments, an “operably linked” includes a covalent linkage between the relevant components or elements. Those skilled in the art will readily appreciate that, in some embodiments, a covalent linkage is not required to achieve an effective operably linked protein. For example, proteins that are operably linked together may be linked to each other, for example, via covalent or non-covalent bonds. As a non-limiting example, a capsid protein as described herein can be operably linked to a targeting ligand, wherein the capsid protein is non-covalently linked to the targeting ligand, or is non-covalently linked to the targeting ligand, optionally with or without a scaffold and/or adapter between the capsid protein and the targeting ligand. As another example, in some embodiments, a nucleic acid regulatory element operably linked to a coding sequence to be regulated is contiguous with a nucleotide of interest. Alternatively or additionally, in some embodiments, one or more such regulatory elements act in trans or at a regulatory distance from the coding sequence of interest. In some embodiments, the term “regulatory element,” as used herein, refers to a polynucleotide sequence that is necessary and/or sufficient to affect the expression and processing of a coding sequence to which the regulatory element is ligated. In some embodiments, a regulatory element is or may include: an appropriate transcription initiation sequence, termination sequence, promoter sequence, and/or enhancer sequence; efficient RNA processing signals, such as splicing and polyadenylation signals; A sequence that stabilizes cytoplasmic mRNA; a sequence that enhances translation efficiency (e.g., a Kozak consensus sequence); a sequence that enhances protein stability; and/or, in some embodiments, a sequence that enhances protein secretion. In some embodiments, one or more regulatory elements are preferentially or exclusively active in a particular host cell or organism or type thereof. For example, in prokaryotes, regulatory elements typically comprise a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, in many embodiments, regulatory elements typically comprise a promoter, an enhancer, and/or a transcription termination sequence. Those skilled in the art will appreciate that in many embodiments, the term "regulatory element" refers to a component whose presence is essential for expression and processing, and in some embodiments, to a component whose presence is advantageous for expression (e.g., including a leader sequence, a targeting sequence, and/or a fusion partner sequence).

The phrases “antibody that binds to CDH15,” “anti-CDH15 antibody,” and the like encompass antibodies and antigen-binding fragments thereof that specifically recognize a single CDH15 molecule. The antibodies and antigen-binding fragments thereof described herein can bind to soluble CDH15 and/or cell-surface expressed CDH15. Soluble CDH15 encompasses not only the native CDH15 protein but also recombinant CDH15 protein variants that lack the transmembrane domain or are otherwise not associated with the cell membrane.

The term “cell surface expressed CDH15” refers to one or more CDH15 protein(s) expressed on the surface of a cell, either in vitro or in vivo, such that at least a portion of the CDH15 protein is exposed on the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. “Cell surface expressed CDH15” may comprise or consist of a CDH15 protein expressed on the surface of a cell that normally expresses the CDH15 protein. Alternatively, “cell surface expressed CDH15” may comprise or consist of a CDH15 protein expressed on the surface of a cell that does not normally express human CDH15 on its surface, but has been artificially engineered to express CDH15 on its surface.

The term “antigen binding molecule” includes antibodies and antigen binding fragments of antibodies.

The term "antibody" refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CDH15). As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, namely, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term “high affinity” antibodies refers to antibodies having a binding affinity for their target of at least 10 ^-9 M, at least 10 -10 M; at least 10 ^-11 M; ^or at least 10 ^-12 M, as measured by surface plasmon resonance, e.g., ^BIACORE ™, or by solution-affinity ELISA. The term “antibody” can include any type of antibody, e.g., monoclonal or polyclonal. Furthermore, antibodies can be derived from, for example, mammalian or non-mammalian species. The antibody may be of any origin. In one embodiment, the antibody may be a mammalian or avian antibody. In a further embodiment, the antibody may be of human origin and may further be a human monoclonal antibody.

The term "antibody" also includes antigen-binding fragments of whole antibody molecules. Terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., encompass any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from whole antibody molecules using any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. Such DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in an appropriate configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapeutics (SMIPs), and shark variable IgNAR domains, are also encompassed by the term “antigen-binding fragment.”

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will typically comprise at least one CDR adjacent to or in frame with one or more framework sequences. In an antigen-binding fragment having a V _H domain associated with a V _L domain, the V _H and V _L domains may be positioned relative to each other in any suitable arrangement. For example, the variable domains may be dimeric and may comprise V _H -V _H , V _H -V _L or V _L -V _L dimers. Alternatively, the antigen-binding fragment of an antibody may comprise monomeric V _H or V _L domains.

In certain embodiments, an antigen-binding fragment of an antibody can comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody as disclosed herein include: (i) V _H -C _H 1; (ii) V _H -C _H 2; (iii) V _H -C _H 3; (iv) V _H -C _H 1-C _H 2; (v) V _H -C _H 1-C _H 2-C _H 3; (vi) V _H -C _H 2-C _H 3; (vii) V _H -C _L ; (viii) V _L -C _H 1; (ix) V _L -C _H 2; (x) V _L -C _H 3; (xi) V _L -C _H 1-C _H 2; (xii) _{V L} -C _H 1-C _H 2-C _H 3; (xiii) V _L -C _H 2 -C _H 3; and (xiv) V _L -C _L . In any of the configurations of variable and constant domains comprising any of the exemplary configurations enumerated above, such variable and constant domains may be directly connected to each other or may be connected in whole or in part by a hinge or linker region. The hinge region may be composed of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains of a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies as disclosed herein may comprise homo-dimers or hetero-dimers (or other multimers) of any of the variable and constant domain configurations enumerated above in non-covalent association (e.g., by disulfide bond(s)) with each other and/or with one or more monomeric V _H or V _L domains.

Like whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be modified for use in the context of antigen-binding fragments of antibodies described herein using routine techniques available in the art.

In certain embodiments, the anti-hCDH15 antibodies described herein are human antibodies. The term “human antibody” refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies as described herein may comprise amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-directed mutagenesis or in vivo by somatic mutation), for example, in the CDRs, particularly CDR3. However, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

Antibodies as disclosed herein may, in some embodiments, be recombinant human antibodies. The term “recombinant human antibody” is intended to include all human antibodies that are prepared, expressed, generated, or isolated by recombinant means, for example, antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) [Nucl. Acids Res. 20:6287-6295]), or antibodies prepared, expressed, generated, or isolated by any other means that involves grafting human immunoglobulin gene sequences to another DNA sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have undergone in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), such that the amino acid sequences of the V _H and V _L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V _H and V _L sequences, may not naturally exist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two general forms associated with hinge heterogeneity. In one general form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150 to 160 kDa, held together by interchain disulfide bonds. In the second form, the dimer is not linked by interchain disulfide bonds, resulting in a molecule of approximately 75 to 80 kDa (a half antibody) composed of covalently coupled light and heavy chains. This form is very difficult to isolate, even after affinity purification.

The frequency of occurrence of the second form in various intact IgG isotypes is due, but not limited to, to structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form to levels typically observed using the human IgG1 hinge (Angal et al. (1993) [Molecular Immunology 30:105]). Antibodies such as those described herein may have one or more mutations in the hinge, C _H 2 , or C _H 3 regions, which may be desirable, for example, to improve the yield of a desired antibody form in production.

An antibody as described herein may be an isolated antibody. An “isolated antibody” refers to an antibody that has been identified, separated, and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or an antibody that has been separated or removed from a tissue or cell in which the antibody naturally exists or is naturally produced, may be considered an “isolated antibody.” An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has undergone at least one purification or isolation step. In certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

Also described herein are single-arm antibodies that bind to CDH15. The term "single-arm antibody" refers to an antigen-binding molecule comprising one antibody heavy chain and one antibody light chain. A single-arm antibody as described herein may comprise any of the HCVR/LCVR or CDR amino acid sequences set forth in Table 1.

The anti-hCDH15 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains, as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. Also described herein are antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more of the framework regions and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibodies were derived, to the corresponding residue(s) of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as “germline mutations”). One skilled in the art can readily produce a number of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all of the framework and/or CDR residues within the V _H and/or V _L domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only specific residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the original germline sequence from which the antibody was derived). Additionally, antibodies as described herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated to corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, and the like. In some embodiments, antibodies or antigen-binding fragments as described herein are obtained in this general manner.

Also described herein are anti-hCDH15 antibodies comprising a variant of any one of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, some embodiments include anti-hCDH15 antibodies having an HCVR, LCVR, and/or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any one of the HCVR, LCVR, and/or CDR set forth in Table 1 herein.

The phrase "bispecific antibody" encompasses antibodies capable of selectively binding to two or more epitopes. Bispecific antibodies typically comprise two different heavy chains, each of which specifically binds to a different epitope on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). When a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will typically be at least several to tens or hundreds or thousands of times lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by the bispecific antibody may be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and these sequences can be expressed in cells that express immunoglobulin light chains. A typical bispecific antibody has two heavy chains and one immunoglobulin light chain, each heavy chain having (from N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain followed by three heavy chain CDRs; An immunoglobulin light chain may not confer antigen-binding specificity but may be capable of binding to each heavy chain, or may be capable of binding to each heavy chain and binding to one or more epitopes bound by the heavy chain antigen-binding domain, or may be capable of binding to each heavy chain and one or both of the heavy chains may participate in binding to one or both epitopes.

The phrase "heavy chain" or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain constant region sequence of any organism and, unless otherwise specified, a heavy chain variable domain. A heavy chain variable domain includes three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of a heavy chain include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain is capable of specifically recognizing an antigen that can be expressed and secreted by a cell (e.g., capable of recognizing the antigen with a KD in the micromolar, nanomolar, or picomolar range), and includes fragments that include at least one CDR.

The terms "heavy chain only antibody", "heavy chain only antigen binding protein", "single domain antigen binding protein", "single domain binding protein", and the like refer to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain, said immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, said variable domain being incapable of binding to a light chain because the heavy chain constant region generally lacks a functional _CH 1 domain. Thus, the terms "heavy chain-only antibody", "heavy chain-only antigen-binding protein", "single domain antigen-binding protein", "single domain binding protein", and the like encompass both (i) a _monomeric single domain antigen-binding protein comprising one of the immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region that lacks a functional C _H 1, or (ii) a homodimeric single domain antigen-binding protein comprising two immunoglobulin-like chains, each of which comprises a variable domain operably linked to a heavy chain constant region that lacks a functional C _{H 1. In various embodiments, the homodimeric single domain antigen-binding protein comprises two identical immunoglobulin-like chains, each of which comprises identical variable domains operably linked to identical heavy chain constant regions that lack a functional C H} 1. Additionally, each immunoglobulin-like chain of the single domain antigen binding protein comprises a variable domain that can be derived from a heavy chain variable region gene segment (e.g., V _H , D _H , J _H ), a light chain gene segment (e.g., V _L , J _L ), or a combination thereof, and that can be linked to a heavy chain constant region (C _H ) gene sequence comprising a deletion or inactivating mutation in the C _H 1 coding sequence (and optionally the hinge region) of a heavy chain constant region gene, e.g., IgG, IgA, IgE, IgD, or a combination thereof. A single domain antigen binding protein comprising a variable domain derived from a heavy chain gene segment may be referred to as a “V _H -single domain antibody” or a “V _H -single domain antigen binding protein”, as described, for example, in U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; See US Patent Publication No. 20150197554; US Patent Publication No. 20150197555; US Patent Publication No. 20150196015; US Patent Publication No. 20150197556; and US Patent Publication No. 20150197557, each of which is incorporated herein by reference in its entirety. A single domain antigen binding protein comprising a variable domain derived from a light chain gene segment may be referred to as a “V _L -single domain antigen binding protein” or is a “V _L -single domain antigen binding protein”; see, e.g., US Patent Publication No. 20150289489, which is incorporated herein by reference in its entirety.

The phrase "light chain" includes an immunoglobulin light chain constant region sequence of any organism, including human kappa and lambda light chains, unless otherwise specified. A light chain variable (VL) domain typically comprises three light chain CDRs and four framework (FR) regions, unless otherwise specified. Typically, a full-length light chain comprises a VL domain comprising, from amino-terminus to carboxyl-terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Useful light chains include, for example, those that do not selectively bind a first antigen or a second antigen that is selectively bound by an antigen binding protein. Suitable light chains include those that can be identified by screening the most commonly used light chains in existing antibody libraries (either wet libraries or in a virtual environment), wherein the light chain does not substantially interfere with the affinity and/or selectivity of the antigen binding domain of the antigen binding protein. Suitable light chains include those capable of binding to one or both epitopes bound by the antigen binding domain of an antigen binding protein.

The phrase "variable domain" includes an amino acid sequence of an immunoglobulin light or heavy chain (modified if desired) comprising, in order from N-terminus to C-terminus (unless otherwise specified), the following amino acid regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" includes an amino acid sequence capable of folding into a canonical domain (VH or VL) having a double beta sheet structure, wherein the beta sheets are linked by disulfide bonds between residues of the first beta sheet and the second beta sheet.

The phrase "complementarity determining region" or the term "CDR" generally refers to an amino acid sequence that occurs between two framework regions of the variable region of a light or heavy chain of an immunoglobulin molecule (e.g., an antibody or a T-cell receptor) (i.e., in a wild-type animal) and that is encoded by a nucleic acid sequence of an immunoglobulin gene of that organism. A CDR may be encoded, for example, by a germline sequence or by a rearranged or unrearranged sequence, and may be encoded, for example, by a naive or mature B cell or T cell. In some cases (e.g., for CDR3), a CDR may be encoded by two or more sequences (e.g., germline sequences) that are not contiguous in an unrearranged nucleic acid sequence, but are contiguous within a B cell nucleic acid sequence, for example, as a result of joining or linking of sequences (e.g., V-D-J recombination to form a heavy chain CDR3).

Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are known in the art and can be used to identify CDRs within the specific HCVR and/or LCVR amino acid sequences disclosed herein. Examples of definitions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 273 :927-948 (1997). USA 86 :9268-9272 (1989)]. Public databases can also be used to identify CDR sequences within antibodies.

The term "antibody fragment" means one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antibody fragment" include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment, which is composed of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of a VH domain (Ward et al. (1989) Nature 241:544-546), (vi) an isolated CDR, and (vii) an scFv fragment consisting of two domains, VL and VH, joined by a synthetic linker to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also encompassed under the term "antibody" (see, e.g., Holliger et al. [(1993) PNAS USA 90:6444-6448]; Poljak et al. [(1994) Structure 2:1121-1123]).

The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins comprising at least functional portions of immunoglobulin CH2 and CH3 domains. By "functional portion" is meant CH2 and CH3 domains capable of binding to an Fc receptor (e.g., FcγR; or FcRn, i.e., the neonatal Fc receptor) and/or participating in complement activation. If the CH2 and CH3 domains comprise deletions, substitutions, and/or insertions or other modifications that render them incapable of binding to any Fc receptor and incapable of activating complement, the CH2 and CH3 domains are not functional.

An Fc-containing protein may comprise a modification of an immunoglobulin domain, including where the modification affects one or more effector functions of the binding protein (e.g., a modification affecting FcyR binding, FcRn binding, and thus half-life and/or CDC activity). These modifications include, but are not limited to, the following modifications and combinations thereof, with reference to the EU numbering of immunoglobulin constant regions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

By way of example only, and not limitation, the binding protein is an Fc-containing protein that exhibits improved serum half-life (compared to the same Fc-containing protein without the modification(s) mentioned) and has a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at 428 and/or 433 (e.g., L/R/SI/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at 250 and/or 428; 307 or 308 (e.g., 308F, V308F), and 434. In another example, the variants may include the 428L (e.g., M428L) and 434S (e.g., N434S) variants; the 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) variants; the 433K (e.g., H433K) and 434 (e.g., 434Y) variants; the 252, 254, and 256 (e.g., 252Y, 254T, and 256E) variants; the 250Q and 428L variants (e.g., T250Q and M428L); and the 307 and/or 308 variants (e.g., 308F or 308P).

As used herein, the term "antigen binding protein" refers to a polypeptide or protein (one or more polypeptides complexed into a functional unit) that specifically recognizes an epitope on an antigen, such as a cell-specific antigen and/or a target antigen as described herein. An antigen binding protein may be multispecific. The term "multispecific" in relation to an antigen binding protein means that the protein recognizes different epitopes on the same antigen or on different antigens. A multispecific antigen binding protein as described herein may be a single multifunctional polypeptide or may be a multimeric complex comprised of two or more polypeptides that are covalently or noncovalently associated with each other. The term "antigen binding protein" includes an antibody or fragment thereof as described herein, which may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities, such as antibodies or antibody fragments, to generate bispecific or multispecific antigen-binding molecules having second binding specificities.

The term "protein" refers to any amino acid polymer having multiple amino acids covalently linked by amide bonds. A protein contains one or more amino acid polymer chains, commonly known in the art as "polypeptides." Thus, a polypeptide can be a protein, or a protein can contain multiple polypeptides to form a single functional biomolecule. Some proteins may contain disulfide bridges (cysteine-to-cystine bonds). These covalent linkages can exist within a single polypeptide chain or between two separate polypeptide chains. For example, disulfide bridges are essential for the proper structure and function of insulin, immunoglobulins, protamines, and others. For a recent review of disulfide bond formation, see Oka and Bulleid, "Forming disulfides in the endoplasmic reticulum," 1833(11) Biochim Biophys Acta 2425-9 (2013).

As used herein, “protein” includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins may also be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review of biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation,” 28 Biotechnol Genet Eng Rev. 147-75 (2012).

As used herein, the term "epitope" refers to a portion of an antigen recognized by a multispecific antigen-binding polypeptide. A single antigen (e.g., an antigen polypeptide) may have one or more epitopes. Epitopes may be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and are defined as residues that directly contribute to the affinity of the interaction between an antigen-binding polypeptide and the antigen. An epitope may also be a conformational (non-linear) amino acid sequence. In certain embodiments, an epitope may comprise a determinant, a chemically active surface group of molecules, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural properties and/or specific charge properties. Epitopes formed from consecutive amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.

The term "domain" refers to any portion of a protein or polypeptide having a specific function or structure. Preferably, the domain described herein binds to a cell-specific antigen or a target antigen. A cell-specific antigen-binding domain or a target antigen-binding domain, as used herein, includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen.

The terms "haploid" or "half antibody," used interchangeably, refer to one half of an antibody, essentially comprising one heavy chain and one light chain. Antibody heavy chains can form dimers, such that the heavy chain of one half can bind to a heavy chain associated with another molecule (e.g., the other half) or with another Fc-containing polypeptide. Two slightly different Fc domains can also "heterodimerize," such as in the formation of bispecific antibodies or other heterodimers, -trimers, -tetramers, etc. See Vincent and Murini, “Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies, and antibody drug conjugates,” 7 Biotechnol. J. 1444-1450 (20912); and Shimamoto et al., “Peptibodies: A flexible alternative format to antibodies,” 4(5) MAbs 586-91 (2012)).

The term “single-chain variable fragment” or “scFv” encompasses single-chain fusion polypeptides containing an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL). In some embodiments, the VH and VL are connected by a linker sequence of 10 to 25 amino acids. ScFv polypeptides may also include other amino acid sequences, such as a CL or CH1 region. ScFv molecules can be produced by phage display or by directly subcloning heavy and light chains from hybridomas or B cells. Ahmed et al., Clinical and Developmental Immunology, volume 2012, article ID 98025, is incorporated herein by reference for methods of producing scFv fragments by phage display and antibody domain cloning.

As used herein, the term "muscle-related cancer" refers to any cancerous cell defined by the expression of a myogenic gene, such as CDH15. "Muscle-related cancer" includes, but is not limited to, rhabdomyosarcoma (e.g., embryonal, alveolar, spindle cell/scirrhotic, pleomorphic, etc.).

The term "target cell" includes any cell in which expression of a nucleotide of interest is desired. Preferably, the target cell displays a receptor on its surface, which allows targeting of the cell using a targeting ligand, as described below. In certain embodiments, the target cell is a muscle cell. As used herein, "muscle cell" refers to any cell that expresses a myogenic marker and/or is involved in skeletal muscle regeneration, such as a muscle stem cell (also known as a "MuSC", satellite cell), which expresses Pax7 and is therefore considered a Pax7 ⁺ cell (e.g., Pax7 ⁺ /MyoD ^- in a quiescent state and Pax7 ⁺ /MyoD ⁺ in a proliferative state), a myoblast (e.g., MyoD ⁺ myogenin ⁺ cell), a myocyte, a myotube (also known as a muscle fiber, e.g., MyHC ⁺ cell), etc.

“Retargeting” or “redirecting” may include scenarios where a wild-type particle targets multiple cells within a tissue and/or multiple organs within an organism, wherein general targeting of the tissue or organ is reduced or eliminated by insertion of a heterologous amino acid, and retargeting to more specific cells within the tissue or specific organ within the organism is achieved with a targeting ligand that binds to a marker expressed by the specific cells (e.g., via a targeting ligand). Such retargeting or redirecting may also include scenarios where a wild-type particle targets a tissue, wherein targeting of the tissue is reduced or eliminated by insertion of a heterologous amino acid, and retargeting to an entirely different tissue is achieved with a targeting ligand.

“Specific binding pair”, “binding pair”, “protein:protein binding pair” includes two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non-covalent bond between a first member of an antibody that recognizes an epitope and a second member that is an antigen-binding portion; a covalent bond between proteins that can form isopeptide bonds; a split intein that recognizes each other and mediates ligation of flanking proteins through the process of protein trans-splicing and only then removes them). In some embodiments, the term “cognate” refers to components that function together. Epitopes and cognate antibodies thereto, particularly epitopes that can also act as detectable labels (e.g., c-myc), are well known in the art. Specific protein:protein binding pairs capable of interacting to form covalent isopeptide bonds are reviewed in Veggiani et al. [(2014) Trends Biotechnol . 32:506] and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002; SpyTag:KTag; isopeptag:pilin C, SnoopTag:SnoopCatcher, etc. and bioequivalent variants thereof, e.g., SpyTag003:SpyCatcher003. Generally, the first member of the protein:protein binding pair refers to a member of the protein:protein binding pair that is typically less than 30 amino acids in length and that forms a spontaneous covalent isopeptide bond with a second cognate protein, wherein the second cognate protein is typically larger, but may also be less than 30 amino acids in length, such as in the SpyTag:KTag system.

The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, at least one of which is not derived from the protein backbone or, alternatively, is not part of the protein backbone. Isopeptide bonds can form within a single protein, or between two peptides or a peptide and a protein. Thus, isopeptide bonds can be formed intramolecularly within a single protein, or intermolecularly, i.e., between two peptide/protein molecules, for example, between two peptide linkers. Typically, an isopeptide bond can form between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or the terminal carboxyl group of a protein or peptide chain, or between the alpha-amino terminus of a protein or peptide chain and asparagine, aspartic acid, glutamine, or glutamic acid. Each residue of a pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, the isopeptide bond may be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. Specifically, the isopeptide bond may be formed between the side chain amine of lysine and the carboxamide group of asparagine or the carboxyl group of aspartate.

The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zakeri et al. [(2012) PNAS 109:E690-E697] (each of which is incorporated herein by reference in its entirety) and is derived from the CnaB2 domain of the fibronectin-binding protein FbaB of Streptococcus pyogenes . By splitting the domain, Zakeri et al. obtained the peptide “SpyTag” having the sequence AHIVMVDAYKPTK (SEQ ID NO: 815), which forms an amide bond to its cognate protein “SpyCatcher,” a 112 amino acid polypeptide having the amino acid sequence set forth in SEQ ID NO: 816. (See Zakeri (2012) as supra.) An additional specific binding pair derived from the CnAb2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase. (Fierer et al. [(2014) PNAS 111:E1176-1181]) SpyLigase was engineered by cleaving the β-strand from SpyCatcher containing the reactive lysine, generating KTag, a 10-residue first member of the protein:protein binding pair having the amino acid sequence ATHIKFSKRD (SEQ ID NO: 817). The SpyTag002:SpyCatcher002 system is described in Keeble et al. [(2017) Angew Chem Int Ed Engl 56:16521-25], the entirety of which is incorporated herein by reference. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK as shown in SEQ ID NO: 821 and binds to SpyCatcher002. SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK as shown in SEQ ID NO: 822 and binds to SpyCatcher003.

The SnoopTag:SnoopCatcher system is described by Veggiani [(2016) PNAS 113:1202-07]. The D4 Ig-like domain of RrgA, the attachment site of Streptococcus pneumoniae , was split to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher generates specific, spontaneous isopeptide bonds between complementary proteins. See Veggiani (2016) above.

The isopeptag:pilin-C specific binding pair was derived from the major pilin protein Spy0128 of Streptococcus pyogenes (see Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, represented by SEQ ID NO: 820, and binds to pilin-C (residues 18-299 of Spy0128). Incubation of isopeptag and pilinC results in the formation of a specific spontaneous isopeptide bond between the complementary proteins. See Zakeir and Howarth (2010) above.

The terms "transduction" or "infection" and the like refer to the introduction of nucleic acids into target cells (e.g., muscle stem cells, myoblasts, muscle cells, any combination thereof, etc.) by viral particles. Terms related to transduction and the like, such as "transduction efficiency," refer to the fraction (e.g., percentage) of cells that express a nucleotide of interest after incubation with a set number of viral particles containing the nucleotide of interest. Well-known methods for determining transduction efficiency include flow cytometry of transduced cells using fluorescent reporter genes, PR-PCR for expression of the nucleotide of interest, etc.

Typically, a “reference” viral capsid protein/capsid/particle is identical to a test viral capsid protein/capsid/particle except for a change in the effect to be tested. For example, to determine the effect (e.g., transduction efficiency) of incorporating the first member of a specific binding pair into a test viral particle, the transduction efficiency of the test viral particle (with or without an appropriate targeting ligand) can be compared to the transduction efficiency of a reference viral particle (with or without an appropriate targeting ligand, as necessary) that is identical to the test viral particle in all respects (e.g., additional point mutations, nucleotides of interest, number of viral particles and target cells, etc.) except for the presence of the first member of the specific binding pair. In some embodiments, the reference viral capsid protein is a protein capable of forming a capsid with a second viral capsid protein modified to include at least a first member of a protein:protein binding pair, wherein the reference viral capsid protein does not include the first member of the protein:protein binding pair, and preferably, the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.

antigen binding protein

Described herein are antigen binding proteins, e.g., antibodies or antigen binding fragments thereof, comprising amino acids of HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCV1, LCVR2, and/or LCVR3 as set forth in Table 1.

In certain embodiments, an antigen binding protein, e.g., an antibody or antigen binding fragment thereof, as described herein comprises a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any one of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In certain embodiments, an antigen binding protein, e.g., an antibody or antigen binding fragment thereof, as described herein comprises a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any one of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In certain embodiments, an antigen binding protein, e.g., an antibody or antigen binding fragment thereof, as described herein comprises a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any one of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In certain embodiments, an antigen binding protein, e.g., an antibody or antigen binding fragment thereof, as described herein comprises a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any one of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In certain embodiments, an antigen binding protein, e.g., an antibody or antigen binding fragment thereof, as described herein comprises a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any one of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In certain embodiments, an antigen binding protein, e.g., an antibody or antigen binding fragment thereof, as described herein comprises a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any one of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a HCDR3 and LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any one of the HCDR3 amino acid sequences listed in Table 1 paired therewith and any one of the LCDR3 amino acid sequences listed in Table 1. In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises a HCDR3/LCDR3 amino acid sequence pair contained in any one of the exemplary anti-hCDH15 antibodies listed in Table 1. In some embodiments, the HCDR3/LCDR3 amino acid sequence pairs are selected from the group consisting of SEQ ID NOs: 8 and 16, 28 and 36, 48 and 54, 66 and 54, 76 and 54, 86 and 54, 96 and 102, 113 and 119, 131 and 139, 151 and 159, 171 and 179, 191 and 196, 208 and 214, 226 and 54, 236 and 54, 246 and 54, 255 and 54, 265 and 273, 285 and 293, 305 and 313, 325 and 333, 345 and 352, 364 and 372, 382 and 54, 392 and 398, 410 and 414, 426 and 434, 442 and 450, 458 and 466, 474 and 482, 490 and 498, 506 and 514, 522 and 530, 538 and 546, 554 and 562, 570 and 578, 586 and 594, 602 and 610, 618 and 626, 634 and 642, 650 and 658, 666 and 674, 682 and 690, 698 and 706, 714 and 722, 730 and 738, 746 and 690, 754 and 762, and 770 and Selected from the group consisting of 778.

Provided herein are antigen binding proteins, e.g., antibodies or antigen binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any one of the exemplary anti-hCDH15 antibodies listed in Table 1. In some embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set comprises SEQ ID NO: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311-313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392-396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490-494-496-498, 502-504-506-510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730-734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and Selected from the group consisting of 766-768-770-774-776-778.

Also described herein is an antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR/LCVR amino acid sequence pairs defined by any one of the exemplary anti-hCDH15 antibodies listed in Table 1. In some embodiments, the antibody or antigen-binding fragment as described herein comprises a sequence selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772 A set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within a HCVR/LCVR amino acid sequence pair selected from the group consisting of:

In some embodiments, the antigen binding protein comprises a heavy chain variable region (HCVR or VH). In some embodiments, the HCVR comprises a set of HCDR1-HCDR2-HCDR3 amino acid sequences selected from Table 1. In some embodiments, the HCVR comprises an amino acid sequence selected from any one of the HCVR amino acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In some embodiments, the antigen binding protein comprises a light chain variable region (LCVR or VL). In some embodiments, the LCVR comprises a set of LCDR1-LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the LCVR comprises an amino acid sequence selected from any one of the LCVR amino acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

In some embodiments, the antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, comprises an HCVR and LCVR amino acid sequence pair (HCVR/LCVR) comprising any one of the HCVR amino acid sequences listed in Table 1 paired with an LCVR amino acid sequence listed in Table 1. In some embodiments, the antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, as described herein, comprises an HCVR/LCVR amino acid sequence pair contained in any one of the exemplary anti-hCDH15 antibodies listed in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pairs are selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772 It is selected from the group formed.

nucleic acid molecules

Also described herein are nucleic acid molecules, e.g., polynucleotides, encoding antigen binding proteins, e.g., antibodies or antigen binding fragments thereof, as described herein.

Also provided herein are nucleic acid molecules encoding any one of the HCDR1 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCDR1 nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any one of the HCDR2 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCDR2 nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any one of the HCDR3 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCDR3 nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any one of the LCDR1 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the LCDR1 nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

Also provided herein is a nucleic acid molecule encoding any one of the LCDR2 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the LCDR2 nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

Also provided herein are nucleic acid molecules encoding any one of the LCDR3 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the LCDR3 nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. Also described herein are nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of any one of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises the amino acid sequence of any one of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCVR nucleic acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any one of the LCVR nucleic acid sequences listed in Table 1, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-hCDH15 antibody listed in Table 1.

Also provided herein are recombinant expression vectors capable of expressing polypeptides comprising the heavy and/or light chain variable regions of an anti-hCDH15 antibody. In some embodiments, the recombinant expression vector comprises any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any one of the HCVR, LCVR, and/or CDR sequences set forth in Table 1. Also described are methods for producing an antibody or a portion thereof by culturing a host cell into which such vector has been introduced under conditions that allow for production of the antibody or antibody fragment and recovering the antibody and antibody fragment thus produced.

multispecific antigen binding molecules

Anti-hCDH15 antibodies and antigen-binding fragments thereof described herein may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide, or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., [1991, J. Immunol. 147:60-69]; Kufer et al., [2004, Trends Biotechnol. 22:238-244]. Anti-hCDH15 antibodies and antigen-binding fragments thereof described herein may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody having a second or additional binding specificity.

The term "anti-hCDH15 antibody" used herein is intended to encompass not only monospecific anti-hCDH15 antibodies, but also bispecific antibodies comprising a CDH15-binding arm and a "target"-binding arm. Thus, bispecific antibodies are described herein in which one immunoglobulin arm binds human CDH15 and the other immunoglobulin arm is specific for another target molecule. The CDH15-binding arm may comprise any of the HCVR/LCVR or CDR amino acid sequences set forth in Table 1 herein.

In certain embodiments, the CDH15-binding arm binds to human CDH15 and induces internalization of CDH15 and the antibody bound thereto. In certain embodiments, the CDH15-binding arm weakly binds to human CDH15 and induces internalization of CDH15 and the antibody bound thereto. In certain embodiments, the CDH15-binding arm binds to human CDH15 and blocks the activity of CDH15.

In certain embodiments, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising first and second antigen-binding domains, the CDRs of the first antigen-binding domain may be designated with the prefix "A1", and the second antigen-binding domain may be designated with the prefix "A2." Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly linked to each other to form a bispecific antigen-binding molecule as described herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be linked to separate multimerization domains. The association of one multimerization domain with another multimerization domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. A "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid capable of associating with a second multimerization domain that is identical or similar in structure or composition. For example, the multimerization domain can be a polypeptide comprising an immunoglobulin C _H 3 domain. Non-limiting examples of multimerization domains are the Fc portion of immunoglobulins (comprising a C _H 2-C _H 3 domain), e.g., the Fc domain of IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

Bispecific antigen-binding molecules as described herein will generally comprise two multimerization domains, e.g., two Fc domains, each of which is a separate portion of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a peptide containing a cysteine residue or a short cysteine residue. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a di-coil motif.

Any bispecific antibody format or technology can be used to prepare a bispecific antigen-binding molecule as described herein. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical bonding, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific examples of bispecific formats include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMabs, CrossFabs, (SEED) bodies, leucine zippers, Duobodies, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. [2012, mAbs 4:6, 1-11], and references cited therein).

In the context of a bispecific antigen binding molecule as described herein, the multimerization domain (e.g., the Fc domain) can comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the Fc domain. For example, the bispecific antigen binding molecule can comprise one or more modifications in the Fc domain, which modifications result in a modified Fc domain having an altered (e.g., enhanced or attenuated) binding interaction between Fc and FcRn. In one embodiment, the bispecific antigen binding molecule comprises a modification in the C _H 2 or C _H 3 region, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g. , an endosome having a pH of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (e.g., an E or Q); Variations at 250 and 428 (e.g., L or F); Variations at 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or Variations at positions 428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or Variations at positions 250 and/or 428; or Variations at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the variants include 428L (e.g., M428L) and 434S (e.g., N434S) variants; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) variants; 433K (e.g., H433K) and 434 (e.g., 434Y) variants; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) variants; 250Q and 428L variants (e.g., T250Q and M428L); and 307 and/or 308 variants (e.g., 308F or 308P).

Also disclosed herein are bispecific antigen-binding molecules comprising a first C _H 3 domain and a second Ig C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one amino acid, and wherein said at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody without the amino acid difference. In one embodiment, the first Ig C _H 3 domain binds protein A, and the second Ig C _H 3 domain contains a mutation that reduces or eliminates protein A binding, such as a H95R modification (by IMGT exon numbering; which is H435R by EU numbering). The second C _H 3 can further comprise a Y96F modification (by IMGT numbering; which is Y436F by EU numbering). See, e.g., U.S. Patent No. 8,586,713. Additional modifications that can be found in the second C _H 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); N44S, K52N, and V82I for IgG2 antibodies (by IMGT; N384S, K392N, and V422I by EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In certain embodiments, the Fc domain can be a chimera, combining Fc sequences from two or more immunoglobulin isotypes. For example, a chimeric Fc domain can comprise part or all of a C _H 2 sequence from a human IgG1, human IgG2, or human IgG4 C _H 2 domain, and can comprise part or all of a C _H 3 domain from a human IgG1, human IgG2, or human IgG4. A chimeric Fc domain can also comprise a chimeric hinge region. For example, a chimeric hinge can comprise an "upper hinge" sequence from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence from a human IgG1, human IgG2, or human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any of the antigen binding molecules provided herein include, from N-terminal to C-terminal, the following: [IgG4 C _H 1] - [IgG4 upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen binding molecules provided herein includes, from N-terminal to C-terminal, the following: [IgG1 C _H 1] - [IgG1 upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any of the antigen binding molecules described herein are described in U.S. Patent Publication No. 2014/0243504, published August 28, 2014, which is incorporated herein by reference in its entirety. Chimeric Fc domains and variants thereof having this general structural arrangement can have altered Fc receptor binding, which in turn affects Fc effector function.

In certain embodiments, an antibody heavy chain as described herein comprises a heavy chain constant (CH) region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, or 802. In some embodiments, the heavy chain constant (CH) region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, or 802.

In some embodiments, an antibody heavy chain as described herein comprises an Fc domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, or 814. In some embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, or 814.

germline mutations

The anti-hCDH15 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy chain variable domain compared to the corresponding germline sequence from which the antibody is derived.

Anti-hCDH15 antibodies and antigen-binding fragments thereof as disclosed herein can be derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more of the framework regions and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as “germline mutations”), and have weak or undetectable binding to the hCDH15 antibody. Several such exemplary antibodies that recognize hCDH15 are described herein in Table 1.

Additionally, the anti-hCDH15 antibodies and antigen-binding domains thereof disclosed herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated to corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding domains comprising one or more germline mutations can be tested for one or more desirable properties, such as improved binding specificity, weaker or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. In some embodiments, antibodies or antigen-binding fragments as described herein are obtained in this general manner.

Also described herein are anti-hCDH15 antibodies and antigen-binding fragments thereof that comprise variants of any one of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, an anti-hCDH15 antibody or antigen-binding fragment thereof as described herein can comprise an HCVR, LCVR, and/or CDR amino acid sequence that has, for example, no more than 10, no more than 8, no more than 6, no more than 4, etc. conservative amino acid substitutions compared to the HCVR, LCVR, and/or CDR amino acid sequences set forth in Table 1 herein. The antibodies and antigen-binding fragments thereof as described herein can comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains while maintaining or improving, for example, desirable weak to undetectable binding to CDH15, compared to the corresponding germline sequence from which the individual antigen-binding domains were derived. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution will not substantially change the functional properties of the protein, and in the case of anti-hCDH15 binding molecules, the amino acid substitution maintains or improves the desired weak to undetectable binding affinity. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; And (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (see Gonnet et al. [(1992) Science 256: 1443-1445]). A “suitable conservative” substitution is any change that has a negative value in the PAM250 log-likelihood matrix.

Also described herein are anti-hCDH15 antibodies and antigen-binding fragments thereof comprising an antigen-binding domain having an HCVR and/or CDR amino acid sequence that is substantially identical to any one of the HCVR and/or CDR amino acid sequences disclosed herein, while maintaining or improving a desirable weak affinity for the CDH15 antigen. The term “substantial identity” or “substantially identical” when referring to amino acid sequences means that two amino acid sequences share at least 95% sequence identity, and more preferably at least 98% or 99% sequence identity, when optimally aligned, e.g., by the GAP or BESTFIT programs using default gap weights. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. When two or more amino acid sequences differ by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to compensate for the conservative nature of the substitutions. Means for making such adjustments are known to those skilled in the art. See, for example, Pearson [(1994) Methods Mol. Biol. 24: 307-331].

Sequence similarity (also referred to as sequence identity) for polypeptides is typically measured using sequence analysis software. Protein analysis software compares similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different organisms, or between a wild-type protein and its muteins. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments of the most overlapping regions between the query and search sequences and percent sequence identity (see Pearson (2000) above). Another preferred algorithm for comparing sequences described herein with databases containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) [J. Mol. Biol. 215:403-410] and Altschul et al. (1997) [Nucleic Acids Res. 25:3389-402].

After obtaining antigen-binding domains containing one or more germline mutations, they are tested for reduced binding affinity using one or more in vitro assays. Typically, antibodies recognizing a specific antigen are screened for this purpose by testing for high (i.e., strong) binding affinity to the antigen.

By further modifying the antibodies described herein by the methods described herein, unexpected advantages such as improved pharmacokinetic properties and lower toxicity to patients may be realized.

Binding properties of antibodies

For example, the term “binding” in the context of binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to a given antigen (e.g., a cell surface protein or fragment thereof) generally refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

For example, binding affinity corresponds to a K D value of about 10 -7 M or less, such as about 10 -8 M or less, such as about ^{10 -9 M} or less, when determined by surface plasmon resonance (SPR) technology, for example, on a BIAcore 3000 instrument, using an _antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or anti- ^ligand ). Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used to provide binding characterization data for cell surface expressed proteins. FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA [ J Immunol Methods . 1997, 201(2):223-31]; Geuijen, CA et al. [ J Immunol Methods . 2005, 302(1-2):68-77]).

Thus, an anti-hCDH15 antibody or antigen-binding fragment thereof as described herein binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K _D value that is at least 10-fold lower than its binding affinity to a non-specific antigen (e.g., BSA, casein). An affinity of the antibody corresponding to a K _D value that is 10-fold or lower than that for a non-specific antigen may be considered undetectable binding, but such an antibody may be paired with a second antigen-binding arm for the production of a bispecific antibody as described herein.

The term “K _D ” or “K D ” in molar units (M) refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, or the equilibrium dissociation constant of an antibody or antibody-binding fragment binding to an antigen. There is an inverse relationship between K _D and binding affinity, so that the lower the K _D value, the higher the affinity (i.e., the stronger the binding). Therefore, the term “higher affinity” or “stronger affinity” relates to a higher ability to form an interaction, and thus a lower K _D value; conversely, the term “lower affinity” or “weaker affinity” relates to a lower ability to form an interaction, and thus a higher K _D value. In some cases, a higher binding affinity (or K _{D ) of a particular molecule (e.g., an antibody) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity (or K D} ) of another interaction partner molecule (e.g., antigen Y) of the same molecule (e.g., an antibody) can be expressed as a binding ratio determined by dividing the higher K _D value (lower, weaker affinity) by the lower K _D (higher, stronger affinity), which can in some cases be expressed as, for example, a 5-fold or 10-fold higher binding affinity.

The term “k _d ” (seconds ^-1 or 1/second) refers to the dissociation rate constant for a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody binding fragment. This value is also referred to as the k _off value.

The term “k _a ” (M-1 x sec-1 or 1/M) refers to the association rate constant for a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

The term “K _A ” (M-1 or 1/M) refers to the binding equilibrium constant for a particular antibody-antigen interaction, or the binding equilibrium constant of an antibody or antibody-binding fragment. The binding equilibrium constant is calculated by dividing k _a by k _d .

The term “EC50” or “EC ₅₀ ” refers to the half-maximal effective concentration, which includes the concentration of an antigen-binding molecule that elicits a response that is half the maximum response after a specified exposure time compared to baseline. The EC ₅₀ essentially represents the concentration of an antibody at which 50% of the antibody’s maximal effect is observed. In certain embodiments, the EC ₅₀ value is equal to the concentration of an antibody as described herein that provides half-maximal binding to cells expressing CDH15, as determined, for example, by a FACS binding assay or an androgen receptor-activated luciferase assay. Thus, as the EC ₅₀ or half-maximal effective concentration value increases, decreased or weaker binding is observed.

In one embodiment, a decrease in binding may be defined as an increase in the EC ₅₀ antibody concentration that allows binding to a target cell at half-maximal amounts.

Also described herein are antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, that bind to CDH15-expressing cells with a KD in the single-digit nM or triple-digit pM range, as measured by surface plasmon resonance or an equivalent assay. Also described herein are antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, that bind to CDH15-expressing cells with an EC ₅₀ greater than 100 nM, as measured by FACS analysis. Also described herein are antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, that bind to CDH15-expressing cells and, after binding to CDH15, are internalized into the CDH15-expressing cells. Also described herein are antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, that bind to CDH15 and block its activity.

sequence variants

The anti-hCACNG1 antibodies and antigen-binding fragments described herein can comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains, as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules described herein can comprise an antigen-binding domain derived from any one of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more of the framework regions and/or CDR regions can be mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as “germline mutations”). One skilled in the art can readily produce a number of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all of the framework and/or CDR residues within the V _H and/or V _L domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was derived. In other embodiments, only specific residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the original germline sequence from which the antigen-binding domain was derived). Additionally, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated to corresponding residues in a different germline sequence. Once obtained, antigen-binding fragments comprising one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, and the like. Antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are described herein.

Also described herein are antigen binding molecules wherein one or both of the antigen binding domains comprises a variant of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, an antigen binding molecule as described herein can comprise an antigen binding domain having an HCVR, LCVR, and/or CDR amino acid sequence that has, for example, no more than 10, no more than 8, no more than 6, no more than 4, etc. conservative amino acid substitutions compared to the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. A “conservative amino acid substitution” is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution will not substantially alter the functional properties of the protein. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (see Gonnet et al. (1992) [Science 256: 1443-1445], which is incorporated herein by reference). A “proper conservative” substitution is any change that has a negative value in the PAM250 log-likelihood matrix.

An antigen-binding molecule as described herein may comprise an antigen-binding domain having an HCVR, LCVR, and/or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. The terms “substantial identity” or “substantially identical” when referring to amino acid sequences mean that two amino acid sequences share at least 95% sequence identity, and more preferably at least 98% or 99% sequence identity, when optimally aligned, e.g., by the GAP or BESTFIT programs using default gap weights. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. When two or more amino acid sequences differ by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to compensate for the conservative nature of the substitutions. Methods for making such adjustments are well known to those skilled in the art. See, e.g., Pearson [(1994) Methods Mol. Biol. Chem. 10:128-129]. 24: 307-331], which is incorporated herein by reference.

Sequence similarity (also referred to as sequence identity) for polypeptides is typically measured using sequence analysis software. Protein analysis software compares similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different organisms, or between a wild-type protein and its muteins. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments of the most overlapping regions between the query and search sequences and percent sequence identity (see Pearson (2000) above). Another preferred algorithm for comparing sequences against databases containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) [J. Mol. Biol. 215:403-410] and Altschul et al. (1997) [Nucleic Acids Res. 25:3389-402], each of which is incorporated herein by reference.

pH-dependent binding

Also described herein are anti-hCDH15 antibodies and antigen-binding fragments thereof having pH-dependent binding properties. For example, anti-hCDH15 antibodies as described herein may exhibit reduced binding to CDH15 at acidic pH compared to neutral pH. Alternatively, anti-hCDH15 antibodies as described herein may exhibit enhanced binding to CDH15 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or lower. The term "neutral pH" means a pH of from about 7.0 to about 7.4. The term "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding at acidic pH as compared to neutral pH” is expressed as a ratio of the K _D value of the antibody binding to its antigen at acidic pH to the K _D value of the antibody binding to its antigen at neutral pH (or vice versa). For example, if an antibody or antigen-binding fragment thereof exhibits an acidic K _D /neutral K _D ratio of about 3.0 or greater, the antibody or antigen-binding fragment thereof may be considered, for purposes of the present invention, to exhibit “reduced binding to CACNG1 at acidic pH as compared to neutral pH.” In certain embodiments, the acidic K _D /neutral K _D ratio for an antibody or antigen-binding fragment as described herein can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0, or more.

Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for antibodies that exhibit reduced (or increased) binding to a specific antigen at acidic pH compared to neutral pH. Additionally, antibodies with pH-dependent characteristics can be obtained by modifying the antigen-binding domain at the amino acid level. For example, substituting one or more amino acids in the antigen-binding domain (e.g., within a CDR) with a histidine residue can yield antibodies exhibiting reduced antigen binding at acidic pH compared to neutral pH.

Antibodies containing Fc variants

In some embodiments, anti-hCDH15 antibodies and antigen-binding fragments thereof (including multispecific antigen-binding molecules and multidomain therapeutic proteins comprising the anti-hCDH15 antibody or antigen-binding fragment thereof) are provided, comprising an Fc domain comprising one or more mutations that enhance or attenuate antibody binding to the FcRn receptor at acidic pH compared to, for example, neutral pH. For example, an antibody as described in Bonner can comprise a mutation in the C _H 2 or C _H 3 region of the Fc domain, wherein the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH in the range of about 5.5 to 6.0). Such mutations can increase the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (e.g., an E or Q); Variations at 250 and 428 (e.g., L or F); Variations at 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or Variations at positions 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or Variations at positions 250 and/or 428; or Variations at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the variants include 428L (e.g., M428L) and 434S (e.g., N434S) variants; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) variants; 433K (e.g., H433K) and 434 (e.g., 434Y) variants; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) variants; 250Q and 428L variants (e.g., T250Q and M428L); and 307 and/or 308 variants (e.g., 308F or 308P).

For example, the anti-hCDH15 antibodies and antigen-binding fragments described herein may comprise an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated to be encompassed by the present disclosure.

Biological properties of antibodies and bispecific antigen-binding molecules

Also described herein are antibodies and antigen-binding fragments thereof that bind human CDH15 with high, intermediate, or low affinity, depending on the therapeutic context and desired specific targeting characteristics. For example, in the context of a bispecific antigen-binding molecule in which one arm binds CDH15 and the other arm binds a target antigen (e.g., a tumor-associated antigen), it may be desirable for the target antigen-binding arm to bind the target antigen with high affinity, while the anti-hCDH15 arm binds CDH15 only with intermediate or low affinity. In this manner, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved, while avoiding general/untargeted CDH15 binding and consequently avoiding adverse adverse events associated therewith.

Also described herein are antibodies, antigen-binding fragments thereof, and bispecific antibodies that bind to human CDH15 with weak (low) or even undetectable affinity. In some embodiments, the antibodies and antigen-binding fragments thereof as described herein bind to human CDH15 with a K _D greater than about 100 nM (e.g., at 37°C) as measured by surface plasmon resonance. In some embodiments, the antibody or antigen-binding fragment described herein binds to CDH15 with a K D of greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 400 nM, greater than about 500 nM, greater than about 600 nM, greater than about 700 nM, greater than about 800 nM, greater than about 900 nM, or greater than about 1 μM, or with undetectable affinity, as measured by surface plasmon _resonance (e.g., in a mAb-capture or antigen-capture format) or a substantially similar assay.

Epitope mapping and related technologies

An epitope on CDH15 to which an anti-hCDH15 antibody and its antigen-binding domain as described herein binds can be comprised of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CDH15 protein. Alternatively, the epitope can be comprised of multiple non-contiguous amino acids (or amino acid sequences) of CDH15. The term “epitope” refers to an antigenic determinant in the variable region of an antibody molecule, known as a paratope, that interacts with a specific antigen-binding site. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on the antigen and have different biological effects. An epitope can be stereogenic or linear. Stereological epitopes are produced by spatially juxtaposed amino acids derived from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include moieties on the antigen, such as sugars, phosphoryl groups, or sulfonyl groups.

Various techniques known to those skilled in the art can be used to determine whether the antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described by Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY) in their description of Antibodies , alanine scanning mutagenesis, peptide blot analysis (see Reineke [2004, Methods Mol Biol 248:443-463]), and peptide cleavage assays. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (see Tomer [2000, Protein Science 9:487-496]). Another method that can be used to identify amino acids within a polypeptide that interact with the antigen-binding domain of an antibody is hydrogen/deuterium exchange, as detected by mass spectrometry. Generally speaking, the hydrogen/deuterium exchange method involves deuterium-labeling a protein of interest, followed by binding of an antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to water, allowing hydrogen-deuterium exchange at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring [(1999) Analytical Biochemistry 267(2):252-259]; Engen and Smith [(2001) Anal. Chem. 73:256A-265A]. X-ray crystallography of the antigen/antibody complex can also be used for epitope mapping purposes.

Also described herein are anti-hCDH15 antibodies that bind to the same epitope as any one of the specific exemplary antibodies described herein (e.g., antibodies comprising any one of the amino acid sequences set forth in Table 1 herein). Likewise, also described herein are anti-hCDH15 antibodies that compete for binding to CDH15 with any one of the specific exemplary antibodies described herein (e.g., antibodies comprising any one of the amino acid sequences set forth in Table 1 herein).

One skilled in the art can readily determine, using routine methods known in the art, whether a particular antigen-binding molecule (e.g., an antibody) or antigen-binding fragment thereof binds to the same epitope as a reference antigen-binding molecule, such as those described herein, or competes with the reference antigen-binding molecule for binding. For example, to determine whether a test antibody binds to the same epitope on CDH15 as a reference bispecific antigen-binding molecule, such as those described herein, the reference bispecific molecule is first bound to the CDH15 protein. Next, the ability of the test antibody to bind to the CDH15 molecule is assessed. If the test antibody can bind to CDH15 after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to an epitope on CDH15 that is different from the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to CDH15 after saturation binding with the reference bispecific antigen-binding molecule, the test antibody may bind to an epitope on CDH15 that is identical to the epitope bound by the reference bispecific antigen-binding molecule as described herein. Additional routine experiments (e.g., peptide mutagenesis and binding assays) can then be performed to determine whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. Such alignment experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to some embodiments described herein, two antigen binding proteins bind to the same (or overlapping) epitope if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antigen binding protein inhibits binding of the other antigen binding protein by at least 50%, preferably by 75%, 90%, or even 99% as measured in a competition binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen binding proteins are considered to bind to the same epitope if essentially every amino acid mutation in the antigen that reduces or eliminates binding of one antigen binding protein also reduces or eliminates binding of the other antigen binding protein. Two antigen binding proteins are considered to have “overlapping epitopes” if only a subset of amino acid mutations that reduce or eliminate binding of one antigen binding protein also reduce or eliminate binding of the other antigen binding protein.

To determine whether an antibody or its antigen-binding domain competes for binding with a reference antigen-binding molecule, the binding methodology described above is performed in two directions: In the first direction, a reference antigen-binding molecule is allowed to bind to the CDH15 protein under saturating conditions, and binding of the test antibody to the CDH15 molecule is then assessed. In the second direction, a test antibody is allowed to bind to the CDH15 molecule under saturating conditions, and binding of the reference antigen-binding molecule to the CDH15 molecule is then assessed. If, in both directions, only the first (saturating) antigen-binding molecule is able to bind to the CDH15 molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CDH15. As will be appreciated by those skilled in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antigen-binding molecule, but may sterically block binding of the reference antigen-binding molecule by binding to overlapping or adjacent epitopes.

Preparation of antigen-binding domains and construction of bispecific molecules

Antigen binding domains specific for a particular antigen can be produced by any antibody production technique known in the art. Once obtained, two different antigen binding domains specific for two different antigens (e.g., CDH15 and a target antigen) can be suitably arranged relative to each other using routine methods to produce a bispecific antigen binding molecule as described herein. (Discussion of exemplary bispecific antibody formats that can be used to construct bispecific antigen binding molecules as described herein is provided elsewhere herein.) In certain embodiments, the individual components of an antigen binding molecule as described herein (e.g., At least one of the heavy and light chains is derived from a chimeric antibody, a humanized antibody, or a fully human antibody. Methods for producing such antibodies are well known in the art. For example, at least one of the heavy and/or light chains of an antigen-binding molecule as described herein can be produced using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody production technology), a high-affinity chimeric antibody against a particular antigen (e.g., CDH15) having a human variable region and a mouse constant region is first isolated. The antibodies are characterized and selected for desirable properties, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with a desired human constant region to produce a fully human heavy and/or light chain that can be incorporated into an antigen-binding molecule as described herein.

Human bispecific antigen-binding molecules can be produced using genetically engineered animals. For example, a genetically modified mouse can be used that is unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically engineered mice can be used to isolate the heavy and light chain variable regions to produce a fully human bispecific antigen-binding molecule. Thus, a fully human bispecific antigen-binding molecule comprises two different heavy chains that bind to the same light chain. (See, e.g., US 2011/0195454). The term "fully human" refers to an antibody, antigen-binding fragment thereof, or immunoglobulin domain comprising an amino acid sequence encoded by DNA derived from a human sequence throughout the entire length of each polypeptide of the antibody, antigen-binding fragment thereof, or immunoglobulin domain. In some cases, the fully human sequence is derived from an endogenous human protein. In other cases, the fully human protein or protein sequence comprises a chimeric sequence in which each component sequence is derived from a human sequence. While not wishing to be bound by any one theory, it is believed that the chimeric protein or chimeric sequence is generally designed to minimize the generation of immunogenic epitopes at the junctions of the component sequences, for example, compared to any wild-type human immunoglobulin region or domain.

Bispecific antigen-binding molecules can be constructed with one heavy chain having a modified Fc domain that eliminates binding to protein A, thereby enabling a purification process to obtain heterodimeric proteins. See, e.g., U.S. Patent No. 8,586,713. As such, the bispecific antigen-binding molecule comprises a first C _H 3 domain and a second Ig C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody without the amino acid difference. In one embodiment, the first Ig C _H 3 domain binds protein A, and the second Ig C _H 3 domain contains a mutation/alteration that reduces or eliminates protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C _H 3 may additionally include the Y96F variant (by IMGT numbering; EU numbering is Y436F).

bioequivalent

Also described herein are antigen-binding molecules having amino acid sequences that differ from the amino acid sequences of the exemplary molecules disclosed herein, but retain the ability to bind to CDH15. Such variant molecules comprise one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit essentially equivalent biological activity to the bispecific antigen-binding molecules described herein.

Also described herein are antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins or antibodies are considered bioequivalent if they are pharmaceutically equivalent or pharmaceutical alternatives that do not significantly differ in rate and extent of absorption when administered, for example, at the same molar dose (single dose or multiple doses) under similar experimental conditions. If some antigen-binding proteins are equivalent in extent of absorption but differ in rate of absorption, they will be considered equivalent or pharmaceutical alternatives and still be considered bioequivalent because such differences in rate of absorption are intentional and reflected in the labeling, are not necessarily essential for achieving effective body drug concentrations, for example, when used chronically, and are not considered medically significant for the particular investigational drug product in question.

In one embodiment, two antigen binding proteins are bioequivalent if they have no clinically meaningful differences in safety, purity, and potency.

In one embodiment, two antibodies are bioequivalent if a patient can switch between treatment with the reference product and treatment with the biologic product at least once without any expected increase in risk of adverse events or decrease in efficacy, including a clinically significant change in immunogenicity, compared to continuous therapy without such switching.

In one embodiment, two antigen binding proteins are biologically equivalent if both act by a common mechanism or common mechanism(s) of action for the conditions of use to the extent known for that mechanism.

Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example: (a) in vivo studies in humans or other mammals, in which the concentration of the antibody or its metabolism is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with or are reasonably predictive of in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals, in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

Biologically equivalent variants of the exemplary bispecific antigen-binding molecules disclosed herein can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences not required for biological activity. For example, a cysteine residue essential for biological activity can be deleted or substituted with another amino acid to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon denaturation. In another context, a biologically equivalent antigen-binding protein can include variants of the exemplary bispecific antigen-binding molecules disclosed herein that include amino acid changes that alter the glycosylation characteristics of the molecule, for example, mutations that eliminate or eliminate glycosylation.

Also described herein are antigen binding proteins comprising altered glycosylation patterns, e.g., antibodies or antigen binding fragments thereof, such as anti-hCDH15 antibodies. In some embodiments, modifications that remove undesirable glycosylation sites may be useful, e.g., to enhance antibody-dependent cytotoxicity (ADCC) function, or antibodies lacking fucose residues present in the oligosaccharide chain may be useful, where cytotoxicity is desired (see Shields et al. (2002) JBC 277:26733). In other applications, galactosylation modifications may be made to alter complement-dependent cytotoxicity (CDC).

Species selectivity and species cross-reactivity

In some embodiments, an antigen-binding molecule as described herein binds to human CDH15 but not to CDH15 of another species. Also described herein are antigen-binding molecules that bind to human CDH15 and CDH15 of one or more non-human species.

In some embodiments, an antigen binding molecule as described herein that binds to human CDH15 may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus monkey, or chimpanzee CDH15.

Antibody-drug conjugate (ADC)

Also described herein are antibody-drug conjugates (ADCs) comprising an anti-hCDH15 antibody or antigen-binding fragment thereof conjugated to a payload, e.g., a drug or molecular cargo (e.g., a small molecule and/or a therapeutic moiety). Also provided are anti-hCDH15 antibodies, antigen-binding fragments thereof, and/or multispecific antigen-binding molecules thereof conjugated to a therapeutic moiety. Generally, the ADC comprises A - [L - P] _y , wherein A is an antigen-binding molecule, e.g., an anti-hCDH15 antibody, or a fragment thereof (e.g., a fragment comprising at least one HCDR3 selected from any one of the HCDR3 amino acid sequences listed in Table 1), L is a linker, P is a payload or molecular cargo, and y is an integer from 1 to 30.

In various embodiments, the ADC comprises a sequence ID set forth in Table 1 (e.g., SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 748, 764, and 780; or 10, 30, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 287, 307, 327, 347, 366, 50, 394, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 756, and CDRs of HCVR or LCVR having an amino acid sequence of 772), or a specific HCVR/LCVR pair (e.g., SEQ ID NOs: 2+10, 22+30, 42+50, 60+50, 70+50, 80+50, 90+98, 108+115, 125+133, 145+153, 165+173, 185+193, 202+210, 220+50, 230+50, 240+50, 250+50, 259+267, 279+287, 299+307, 319+327, 339+347, 358+366, 378+50, 386+394, An anti-hCDH15 antibody or an antigen-binding fragment thereof comprising CDRs of the following amino acids: 404+412, 420+428, 436+444, 452+460, 468+476, 484+492, 500+508, 516+524, 532+540, 548+556, 564+572, 580+588, 596+604, 612+620, 628+636, 644+652, 660+668, 676+684, 692+700, 708+716, 724+732, 740+684, 748+756, and 764+772. In some cases, the anti-hCDH15 antibody or fragment has a sequence number as set forth in Table 1 (e.g., SEQ ID NOs: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311-313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392-396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490-494-496-498, 502-504-506-510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730-734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and It comprises a CDR having an amino acid sequence of (766-768-770-774-776-778). In some cases, the anti-hCDH15 antibody or fragment has a sequence number set forth in Table 1 (e.g., SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 748, 764, and 780; and 10, 30, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 287, 307, 327, 347, 366, 50, 394, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 756, and 772), or specific amino acid sequence pairs (e.g., SEQ ID NOs: 2+10, 22+30, 42+50, 60+50, 70+50, 80+50, 90+98, 108+115, 125+133, 145+153, 165+173, 185+193, 202+210, 220+50, 230+50, 240+50, 250+50, 259+267, 279+287, 299+307, 319+327, 339+347, 358+366, 378+50, 386+394, 404+412, It includes HCVR and LCVR having amino acid sequences of 420+428, 436+444, 452+460, 468+476, 484+492, 500+508, 516+524, 532+540, 548+556, 564+572, 580+588, 596+604, 612+620, 628+636, 644+652, 660+668, 676+684, 692+700, 708+716, 724+732, 740+684, 748+756, and 764+772.

In some embodiments, the payload or molecular cargo comprises a small molecule as a therapeutic agent, such as a therapeutic agent useful for treating muscle wasting or genetic muscle disorders and/or muscle-related cancers. Small molecules (SMs) have low molecular weights (typically up to about 1 kDa), allowing them to readily enter cells. Once inside a cell, small molecules can affect other molecules, such as proteins, and, for example, can kill cancer cells. This differs from many high-molecular-weight molecules, such as antibodies. One example of a small molecule can be conjugated to an anti-CDH15 antigen-binding protein to form an anti-CDH15:SM conjugate.

Therapeutic agents that may be useful in treating muscle wasting or genetic muscle disorders include testosterone and its biologically active variants (e.g., dihydrotestosterone (DHT)), β2-adrenergic receptor agonists (e.g., clenbuterol), rapamycin or its analogs, MAPK inhibitors, or histone deacetylase inhibitors.

In some embodiments, the therapeutic payload is testosterone, or a biologically active derivative and/or portion thereof, such as dihydrotestosterone. In some embodiments, the therapeutic payload is rapamycin or an analog thereof, a MAPK inhibitor, a histone deacetylase inhibitor, or a Notch ligand. In some embodiments, the payload is a chemotherapeutic agent, such as a cytotoxic drug.

Therapeutic agents that may be useful in treating muscle-related cancers include any chemotherapeutic agent known to slow, arrest, and/or destroy cancer cells. Examples of therapeutic agents that may be useful in treating muscle-related cancers include, but are not limited to: Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Cytarabine liposomal, Dacarbazine, Dactinomycin (Actinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab Gemtuzumab ozogamicin, Hydroxyurea, Idarubicin, Ifosfamide, Inotuzumab ozogamicin, Irinotecan, Ixazomib, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitozantrone, Nab-paclitaxel, Oxaliplatin, Paclitaxel, Pemetrexed, Pegaspargase, Polatuzumab Polatuzumab vedotin, Pralatrexate, Procarbazine, Raltitrexed, Romidepsin, Sacituzumab govitecan, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Trabectedin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trifluridine/tipiracil, Valganciclovir, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, or Vismodegib.

Also provided herein are antibody-radionuclide conjugates (ARCs) comprising an anti-hCDH15 antibody conjugated to one or more radionuclides. Examples of radionuclides that may be used in the context of this aspect of the present disclosure include, but are not limited to, ²²⁵ Ac, ²¹² Bi, ²¹³ Bi, ¹³¹ I, ¹⁸⁶ Re, ²²⁷ Th, ²²² Rn, ²²³ Ra, ²²⁴ Ra, and ⁹⁰ Y.

In certain embodiments provided herein, an ADC is provided comprising an anti-hCDH15 antigen binding protein conjugated to a therapeutic agent (e.g., any of the therapeutic agents described above) via, for example, a linker molecule. A linker is any group or moiety that links, connects, or associates an antibody or antigen binding protein described herein with a therapeutic moiety, such as a cytotoxic agent. Suitable linkers can be found, for example, in Antibody-Drug Conjugates and Immunotoxins ; Phillips, GL, Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates ; Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates ; Wang, J., Shen, W.-C., and Zaro, J.L., Eds.; Springer International Publishing, 2015, the contents of each of which are incorporated herein by reference in their entirety. In general, suitable linkers for the antibody conjugates described herein are those that are sufficiently stable to utilize the circulating half-life of the antibody, while simultaneously being capable of releasing the payload following antigen-mediated internalization of the conjugate. The linker may be cleavable or non-cleavable. Cleavable linkers include linkers that are cleaved by intracellular metabolism after internalization, for example, by hydrolysis, reduction, or enzymatic cleavage. Non-cleavable linkers include linkers that release the attached payload through lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-reactive linkers, hydrolysis-reactive linkers, enzyme-cleavable linkers, reduction-reactive linkers, self-immolative linkers, and non-cleavable linkers. Suitable linkers include, but are not limited to, those that are or comprise peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.

Any linker molecule or linker technology known in the art can be used to produce or construct the ADCs of the present disclosure. In certain embodiments, the linker is a cleavable linker. In other embodiments, the linker is a non-cleavable linker. Examples of linkers that can be used in the context of the present disclosure include, for example, linkers comprising or consisting of MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), val-gly (valine-glycine), the dipeptide portion of a protease-cleavable linker, ala-phe (alanine-phenylalanine), the dipeptide portion of a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate), SIAB (N-succinimidyl (4-iodo-acetyl)aminobenzoate), and variants and combinations thereof. Additional examples of linkers that may be used in the context of the present disclosure are provided, for example, in U.S. Pat. No. 7,754,681 and Ducry, Bioconjugate Chem., 2010, 21 :5-13, and references cited herein, the contents of which are incorporated herein by reference in their entireties.

In certain embodiments, the linker is stable under physiological conditions. In certain embodiments, the linker is cleavable, for example, capable of releasing at least a portion of the payload in the presence of an enzyme or at a specific pH range or value. In some embodiments, the linker comprises an enzyme-cleavable moiety. Exemplary enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages. In some embodiments, the linker comprises a cathepsin-cleavable linker.

In some embodiments, the linker comprises a non-cleavable moiety.

Suitable linkers include, but are not limited to, those chemically bonded to two cysteine residues of a single binding agent (e.g., an antibody). Such linkers may serve to mimic the disulfide bonds in an antibody that are disrupted as a result of the conjugation process.

In some embodiments, the linker comprises one or more amino acids. Suitable amino acids include natural, unnatural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-α-amino acids. In some embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or combinations thereof. In certain embodiments, one or more side chains of the amino acids are linked to the side chain groups described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the linker comprises lysine, valine, and citrulline. In some embodiments, the linker comprises lysine, valine, and alanine. In some implementations, the linker comprises valine and alanine.

In some embodiments, the linker comprises a self-immolative group. The self-immolative group may be any such group known to those skilled in the art. In certain embodiments, the self-immolative group is p -aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p -aminobenzyloxycarbonyl (PABC). Those skilled in the art will recognize that the self-immolative group can undergo a chemical reaction that releases the remaining atoms of the linker from the payload.

In some implementations, the linker is:

Here is binding to an antibody or antigen binding protein (e.g., via a lysine residue), is a linker to a therapeutic payload (e.g., testosterone or a bioequivalent variant thereof). In some embodiments, the linker is:

Here is binding to an antibody or antigen binding protein (e.g., via a lysine residue), is a linker to a therapeutic payload (e.g., testosterone or a bioequivalent variant thereof). In certain embodiments, the linker is:

.

In a given implementation, the linker is:

.

In some embodiments, the linker is derived from maleimidylmethyl-4-trans-cyclohexanecarboxysuccinate:

.

In some implementations, the linker is:

Here is binding to an antibody or antigen binding protein (e.g., via a lysine residue), is a binding to a therapeutic payload (e.g., testosterone or a bioequivalent variant thereof).

In some implementations, the linker is:

Here is binding to an antibody or antigen binding protein (e.g., via a lysine residue), is a binding to a therapeutic payload (e.g., testosterone or a bioequivalent variant thereof).

The present disclosure encompasses ADCs in which a linker connects an anti-hCDH15 antigen binding protein as described herein to a therapeutic agent via a specific amino acid in the antibody or antigen binding molecule. Examples of amino acid attachment moieties that can be used in the context of the present embodiment include, for example, lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al. [ Bioconjugate Chem. , 2008, 19:358-361]; WO 2005/089808; US 5,714,586; and US 2013/0101546), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al. [ Proc. Natl. Acad. Sci., USA, 2008, 105 :12451-12456]), formyl glycine (see, e.g., Carrico et al. [ Nat. Chem. Biol ., 2007, 3:321-322]; Agarwal et al. [ Proc. Natl. Acad. Sci., USA, 2013, 110 :46-51], and Rabuka et al. [ Nat. Protocols , 2012, 10:1052-1067]), unnatural amino acids (see, e.g., WO 2013/068874, and WO 2012/166559), and acidic amino acids (see, e.g., WO 2012/05982). Linkers may also be conjugated to antigen binding proteins via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, and Ryan et al. [ Food & Agriculture Immunol. , 2001, 13 :127-130]) and disulfide linkers (see, e.g., WO 2013/085925, WO 2010/010324, WO 2011/018611, and Shaunt et al. [ Nat. Chem. Biol . , 2006, 2:312-313]). Site-specific conjugation techniques may also be used to direct conjugation to specific residues of an antibody or antigen binding protein (see, e.g., Schumacher et al. [ J Clin Immunol (2016) 36(Suppl 1): 100]). Site-specific conjugation techniques include, but are not limited to, glutamine conjugation via transglutaminase (see, e.g., Schibli, Angew Chemie Inter Ed. 2010, 49,9995). In some embodiments, residues of an antibody as described herein, e.g., residues within the heavy chain constant region of the antibody, may be substituted with glutamine to facilitate glutamine conjugation via transglutaminase. As a non-limiting example, a human heavy chain constant region may be modified by the N180Q substitution found in the sequence of a human IgG1 heavy chain constant region. This substitution provides a total of four glutamines for conjugation by a transglutaminase.

Antibody drug conjugates described herein can be prepared using conjugation conditions known to those of skill in the art (see, e.g., Doronina et al. [ Nature Biotechnology 2003, 21, 7, 778], which is incorporated herein by reference in its entirety). In some embodiments, anti-hCDH15 antigen binding protein drug conjugates are prepared by contacting an anti-hCDH15 antigen binding protein as described herein with a compound comprising a desired linker and a therapeutic agent, wherein the linker has a moiety that reacts with the antibody or antigen binding protein, e.g., at a desired residue of the antibody or antigen binding protein.

adeno-associated virus (AAV)

“AAV” is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. AAV is a small, non-enveloped, single-stranded DNA virus. Typically, the wild-type AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames (ORFs), rep and cap . The wild-type rep reading frame encodes four proteins with molecular weights of 78 kD (“Rep78”), 68 kD (“Rep68”), 52 kD (“Rep52”), and 40 kD (“Rep40”). Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. These proteins primarily function to regulate the transcription and replication of the AAV genome. The wild-type cap reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83 to 85 kD (VP1), 72 to 73 kD (VP2), and 61 to 62 kD (VP3). VP3 comprises over 80% of the total protein in an AAV virion (capsid); in mature virions, VP1, VP2, and VP3 are found in a relative abundance ratio of approximately 1:1:10, although a ratio of 1:1:8 has been reported. See Padron et al. [(2005) J. Virology 79:5047-58].

The genomic sequences of various AAV serotypes, as well as the sequences of the unique inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, are known in the art. These sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated herein by reference for the teachings of AAV nucleic acid and amino acid sequences. Also, for example, Srivistava et al. [(1983) J. Virology 45:555]; Chiorini et al. [(1998) J. Virology 71:6823]; Chiorini et al. [(1999) J. J. Virology 73: 1309]; Bantel-Schaal et al. [(1999) J. Virology 73:939]; Xiao et al. [(1999) J. Virology 73:3994]; Muramatsu et al. [(1996) Virology 221:208]; Shade et al. [(1986) J. Virol. 58:921]; Gao et al. [(2002) Proc. Nat. Acad. Sci. USA 99: 11854]; See Moris et al. [(2004) Virology 33:375-383]; US Patent Publication No. 20170130245; International Patent Publications WO 00/28061, WO 99/61601, WO 98/11244; and US Patent No. 6,156,303, each of which is incorporated by reference in its entirety. Table 2 herein provides sequences of various non-primate AAVs.

“AAV” includes all subtypes, both naturally occurring and modified forms well known in the art. AAV is primate AAV (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV rh10 (AAV rh10), AAV h10 (AAV h10), AAV hu11 (AAV hu11), AAV rh32.33 (AAV rh32.33), AAV retro (AAV retro), AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAV2/8, etc., non-primate animal AAV (e.g., avian AAV (AAAV)) and other non-primate animal AAV, such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, and ovine AAV, etc.), primates AAV (e.g., snake AAV, bearded dragon AAV), etc. “Primate AAV” generally refers to AAV isolated from a primate. Similarly, “non-primate animal AAV” refers to AAV isolated from a non-primate animal.

As used herein, “of [a specified] AAV” with respect to a gene (e.g., rep , cap , etc.), a capsid protein (e.g., VP1 capsid protein, VP2 capsid protein, VP3 capsid protein, etc.), a region of a capsid protein of a specified AAV (e.g., PLA2 region, VP1-u region, VP1/VP2 common region, VP3 region), a nucleotide sequence (e.g., ITR sequence), e.g., a cap gene or capsid protein of an AAV, etc., includes, in addition to a gene or polypeptide comprising a nucleic acid sequence or amino acid sequence set forth herein for a specified AAV, variants of the gene or polypeptide also include variants that include the minimum number of nucleotides or amino acids necessary to retain one or more biological functions. As used herein, a variant gene or variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for a specified AAV gene or polypeptide, wherein the difference(s) generally do not alter at least one biological function of the gene or polypeptide and/or the gene or polypeptide's phylogenetic characteristics, for example, where the difference(s) may be due to degeneracy of the genetic code, an isolated variant, the length of the sequence, etc. For example, a rep gene and a cap gene, as used herein, can comprise a rep gene and a cap gene that differ from the wild-type gene in that the genes can encode one or more Rep proteins and Cap proteins, respectively. In some embodiments, the Rep gene encodes at least Rep78 and/or Rep68. In some embodiments, the cap gene may differ from the wild type in that one or more alternative start codons or sequences between one or more alternative start codons are deleted such that the cap gene encodes only one Cap protein, for example, wherein the VP2 and/or VP3 start codons are deleted or substituted such that the cap gene encodes a functional VP1 capsid protein but not a VP2 capsid protein or a VP3 capsid protein. Thus, as used herein, a rep gene includes any sequence that encodes a functional Rep protein. A cap gene includes any sequence that encodes at least one functional cap gene.

It is well known that the wild-type cap gene expresses all three capsid proteins, VP1, VP2, and VP3, from a single open reading frame of the cap gene under the control of the p40 promoter found in the rep ORF. The terms "capsid protein,""Capprotein," and the like encompass proteins that are part of the viral capsid. In the case of adeno-associated virus, the capsid proteins are generally referred to as VP1, VP2, and/or VP3 and may be encoded by a single cap gene. In the case of AAV, although the three proteins share a common stop codon, the three AAV capsid proteins are naturally produced from the cap ORF in an overlapping manner through the use of alternative translation start codons. The ORF of the wild-type cap gene encodes three alternative start codons in the 5' to 3' direction: a "VP1 start codon," a "VP2 start codon," and a "VP3 start codon"; and one "common stop codon." VP1, the largest viral protein, is typically encoded from the VP1 start codon to the “common stop codon.” VP2 is typically encoded from the VP2 start codon to the common stop codon. VP3 is typically encoded from the VP3 start codon to the common stop codon. Therefore, VP1 contains a region in its N-terminal sequence, referred to as the VP1 unique region (VP1-u), which is not shared with VP2 or VP3. The VP1-u region is typically encoded by the sequence of the wild-type cap gene, which begins at the VP1 start codon and continues to the “VP2 start codon.” VP1-u also contains a phospholipase A2 domain (PLA ₂ ), which may be important for infectivity, as well as a nuclear localization signal that may help the virus target the nucleus for uncoating and genome release. The VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that constitutes the integrity of VP3, also referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the common stop codon. VP2 shares approximately 60 additional amino acids with VP1. This region is referred to as the VP1/VP2 common region.

In some embodiments, one or more of the Cap proteins of the invention can be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the invention can be expressed from two or more ORFs comprising nucleotide sequences encoding any combination of VP1, VP2, and/or VP3, by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in packaging cells, each producing one or more of the VP1, VP2, and/or VP3 capsid proteins of the invention. In some embodiments, the VP capsid proteins of the invention can be individually expressed from ORFs comprising nucleotide sequences encoding any one of VP1, VP2, or VP3, by using separate nucleotide sequences operably linked to one expression control sequence for expression in virus replicating cells, each producing only one of the VP1, VP2, or VP3 capsid proteins. In another embodiment, the VP proteins of the present invention can be expressed from an ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a virus replicating cell, each producing VP1, VP2, and VP3 capsid proteins. Thus, while the amino acid positions provided herein may be provided with respect to the VP1 capsid protein of a reference AAV, one of skill in the art will be able to readily determine the positions of corresponding identical amino acids within the VP2 and/or VP3 capsid proteins of an AAV, and the corresponding positions of amino acids in different AAVs, respectively.

The phrase "Inverted Terminal Repeat" or "ITR" contains symmetrical nucleic acid sequences in the genome of adeno-associated virus (AAV) that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as origins of replication for viral DNA synthesis and are essential cis components for the production of AAV particles, such as those required for packaging into AAV particles.

AAV ITRs contain recognition sites for the replication proteins Rep78 or Rep68. The “D” region of the ITR contains a DNA nick site that initiates DNA replication and provides directionality for the nucleic acid replication step. AAV replication in mammalian cells typically involves two ITR sequences.

A single ITR can be engineered by Rep binding sites on both strands of the "A" region and two symmetrical D regions on each side of the ITR palindrome. This engineered construct on a double-stranded circular DNA template allows Rep78 or Rep68 to initiate nucleic acid replication in both directions. A single ITR is sufficient for AAV circular particle replication. In the method for producing AAV viral particles of the present invention, the rep coding sequence encodes a Rep protein or a Rep protein equivalent capable of binding to the ITR contained on the transfer plasmid.

The Cap protein of the present invention, when expressed by a packaging cell together with an appropriate Rep protein, can encapsulate a transfer plasmid comprising a nucleotide of interest and two or more even ITR sequences. In some embodiments, the transfer plasmid comprises one ITR sequence. In some embodiments, the transfer plasmid comprises two ITR sequences.

Rep78 and/or Rep68 bind to unique and known sites on the sequence of the ITR hairpin and act to disrupt and unravel the hairpin structure at the end of the AAV genome, thereby allowing access to the replication machinery of the virus replicating cell. As is well known, the Rep proteins can be expressed from two or more ORFs comprising nucleotide sequences encoding any combination of Rep78, Rep68, Rep 52, and/or Rep40, by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in the virus replicating cell, each of which produces one or more of the Rep78, Rep68, Rep 52, and/or Rep40 Rep proteins. Alternatively, the Rep proteins can be expressed individually from ORFs comprising nucleotide sequences encoding any one of Rep78, Rep68, Rep 52, and/or Rep40, by using separate nucleotide sequences operably linked to an expression control sequence for expression in packaging cells, each producing only one Rep78, Rep68, Rep 52, and/or Rep40 Rep protein. In another embodiment, the Rep proteins can be expressed from a single ORF comprising nucleotide sequences encoding Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in virus replicating cells, each producing a Rep78 and a Rep52 Rep protein.

In a method for producing AAV virions, e.g., viral particles, of the present invention, the rep coding sequence and cap gene of the present invention may be provided in a single packaging plasmid. However, those skilled in the art will recognize that this provision is not necessary. Such viral particles may or may not contain a genome.

A “chimeric AAV capsid protein” comprises an AAV capsid protein that comprises amino acid sequences (e.g., portions) from two or more different AAVs and is capable of forming and/or forming an AAV viral capsid/viral particle. The chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide sequence comprising a plurality, e.g., at least two, nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a distinct AAV, which together encode a functional chimeric AAV capsid protein. Binding of the chimeric capsid protein to a specific AAV indicates that the capsid protein comprises at least one portion from a capsid protein of that AAV and at least one portion from a capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein comprises a capsid protein comprising one or more portions of the VP1, VP2, and/or VP3 capsid proteins of AAV2 and one or more portions of the VP1, VP2, and/or VP3 capsid proteins of a different AAV.

The term “portion” refers to a polypeptide or nucleic acid molecule that has at least 5 amino acids or at least 15 nucleotides, but less than full length, that has 100% identity to the sequence from which the portion is derived (see Penzes et al., (2015) J. General Virol. 2769). A “portion” includes any contiguous segment of amino acids or nucleotides that is sufficient to determine that the polypeptide or nucleic acid molecule form from which the portion is derived is “of [the specified] AAV” or has “substantial identity” to a particular AAV, e.g., a non-primate AAV or a remote AAV. In some embodiments, the portion comprises at least 5 amino acids or 15 nucleotides that have 100% identity to a sequence associated with a specified AAV. In some embodiments, the portion comprises at least 10 amino acids or 30 nucleotides that have 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 15 amino acids or 45 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 20 amino acids or 60 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 25 amino acids or 75 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 30 amino acids or 90 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 35 amino acids or 105 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 40 amino acids or 120 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 45 amino acids or 135 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 50 amino acids or 150 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 60 amino acids or 180 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 70 amino acids or 210 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 80 amino acids or 240 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 90 amino acids or 270 nucleotides having 100% identity to a sequence associated with a specified AAV. In some embodiments, the part comprises at least 100 amino acids or 300 nucleotides having 100% identity to a sequence associated with a specified AAV.

Modified viral capsid proteins, viral particles, and nucleic acids

In some embodiments, a Cap protein, e.g., a VP1 capsid protein as described herein, a VP2 capsid protein as described herein, and/or a VP3 capsid protein as described herein, is modified to include, for example, one or a combination of the following: insertion of a targeting ligand, a chemical modification, a member of a binding pair, a detectable label, a point mutation, etc.

In general, a modification of a gene or polypeptide of a specified AAV or variant thereof results in a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, wherein the modification alters, confers, or eliminates one or more biological functions, but does not change the phylogenetic characteristics of the gene or polypeptide as an AAV gene or AAV polypeptide. The modification may include any one or a combination of the following, such that the natural tropism of the capsid protein is reduced or eliminated, the tropism of the capsid protein is more readily re-inducible, and/or the capsid protein comprises a detectable label: substituting a sequence of a first AAV serotype for a sequence of a second AAV serotype to create a chimerism; chemical modification; insertion of a first member of a binding pair and/or a point mutation; etc. Modifications as described herein do not alter the modified capsid, and preferably do not reduce low or no recognition of said capsid by pre-existing antibodies found in the general population, produced during the course of infection with another AAV, for example, a serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions in currently used gene therapy methods, or a combination thereof.

targeting ligand

The modifications described herein may relate to associating (e.g., displaying, operably linking, binding) a targeting ligand with a modified capsid protein and/or a capsid comprising a modified capsid protein. Typically, a targeting ligand as described herein binds to a surface protein expressed by a mammalian muscle cell, such as a protein expressed on the surface of a mammalian muscle cell, such as a mammalian non-terminally differentiated muscle cell-specific surface protein. In some embodiments, the modified capsid protein and/or the modified capsid comprises a targeting ligand that binds to mammalian CDH15, such as human CDH15.

Table 1 provides a summary of the sequence numbers for each binding portion (e.g., heavy chain variable domain (HCVR), light chain variable domain (LCVR), and CDR1, CDR2, and CDR3) of non-limiting, exemplary anti-human-CDH15 monoclonal antibodies (mAb IDs) that can be used to redirect AAV capsids as described herein. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a set of heavy chain variable domains, light chain variable domains, heavy chain variable domain/light chain variable domain pairs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) that are at least 90% identical to any one of the amino acid sequences of the set of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a set of heavy chain variable domains, light chain variable domains, heavy chain variable domain/light chain variable domain pairs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are at least 95% identical to the amino acid sequence(s) of any one of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a set of heavy chain variable domains, light chain variable domains, heavy chain variable domain/light chain variable domain pairs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are at least 97% identical to the amino acid sequence(s) of any one of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a set of heavy chain variable domains, light chain variable domains, heavy chain variable domain/light chain variable domain pairs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) that is at least 98% identical to the amino acid sequence(s) of any one of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a set of heavy chain variable domains, light chain variable domains, heavy chain variable domain/light chain variable domain pairs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are 99% identical to the amino acid sequences of any one of SEQ ID NOs: 1-786. Also described herein is an antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR/LCVR amino acid sequence pairs defined by any one of the exemplary anti-CDH15 antibodies listed in Table 1. In some embodiments, the targeting ligand as described herein comprises a sequence selected from the group consisting of SEQ ID NOs: 2+10, 22+30, 42+50, 60+50, 70+50, 80+50, 90+98, 108+115, 125+133, 145+153, 165+173, 185+193, 202+210, 220+50, 230+50, 240+50, 250+50, 259+267, 279+287, 299+307, 319+327, 339+347, 358+366, 378+50, 386+394, 404+412, 420+428, A set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within a HCVR/LCVR amino acid sequence pair selected from the group consisting of 436+444, 452+460, 468+476, 484+492, 500+508, 516+524, 532+540, 548+556, 564+572, 580+588, 596+604, 612+620, 628+636, 644+652, 660+668, 676+684, 692+700, 708+716, 724+732, 740+684, 748+756, and 764+772.

Non-limiting examples of targeting ligands that bind to CDH15 include: (i) a Fab fragment; (ii) a F(ab') 2 fragment; (iii) a Fd fragment; (iv) an Fv fragment; (v) a single-chain Fv (scFv) molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapeutics (SMIPs), and shark variable IgNAR domains, are also encompassed by the term “targeting ligand” as used herein. In a non-limiting embodiment, an anti-CDH15 targeting ligand that binds to CDH15 useful for retargeting a viral capsid as described herein comprises an scFv. As a non-limiting example, a scFv sequence of the VL-(Gly4Ser)3-VH format useful for retargeting a viral capsid as described herein comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or an amino acid sequence of the set HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, which is 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOS: 1 to 786, respectively. It may include a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3.

Targeting ligands that bind to mammalian terminally differentiated muscle cell surface proteins can be associated with (e.g., displayed by, operably linked to, or bound to) modified AAV capsid proteins and the resulting AAV capsids by well-known methods, e.g., direct approaches in which the targeting ligand is directly inserted (e.g., using recombinant methods). See, e.g., Stachler et al., supra (2006); White et al., supra (2004); Girod et al., supra (1999); Grifman et al., supra (2001); Shi et al., supra (2001); Shi and Bartlett, supra (2003). A targeting ligand that binds to a mammalian terminally differentiated muscle cell surface protein can be linked to the modified AAV capsid protein and the resulting AAV capsid using well-known chemical linkers, for example, wherein the AAV capsid protein can be chemically modified to include a dibenzocyclooctyne group or an azide group, and optionally wherein a targeting ligand as described herein is attached to the dibenzocyclooctyne group or the azide group (see, e.g., U.S. 2022/028234, which is incorporated herein by reference in its entirety); and the targeting ligand is covalently linked to a primary amino acid group of the AAV capsid protein, for example, via a -CSNH- linkage. In some embodiments, the modified capsids as described herein comprise a targeting ligand, e.g., an anti-CDH15 antibody or binding portion thereof, that has been directly inserted or linked by well-known direct recombinant methods.

bonded pair

In some embodiments, a targeting ligand that binds to a mammalian terminally differentiated muscle cell surface protein can be associated with (e.g., represented by, operably linked to, or linked to) an AAV capsid protein and the resulting AAV capsid modified according to an indirect recombination approach, wherein the AAV capsid protein is modified to include a first member of a binding pair (e.g., a heterologous scaffold), optionally wherein the first member of the binding pair is linked (e.g., covalently or non-covalently linked) to a second cognate member of the binding pair (e.g., an adaptor), and further optionally wherein the second cognate member of the binding pair is fused to the targeting ligand. Non-limiting exemplary binding pairs are listed in Buning and Srivastava [(2019) Mol. Ther. Methods Clin Dev 12:248-265].

Thus, in some embodiments, modifications of the capsid protein as described herein include those that are generally due to modifications at the genetic level for display by the Cap protein, for example, modifications of the cap gene, such as those that insert the first member of a binding pair (e.g., protein:protein binding pair, protein:nucleic acid binding pair), a detectable label, etc.

In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member forms a binding pair with a metal ion, such as Ni ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ³⁺ , etc. In some embodiments, the first member is selected from the group consisting of streptavidin, strep II, HA, L14, 4C-RGD, LH, and protein A.

In some embodiments, the binding pair comprises an enzyme:nucleic acid binding pair. In some embodiments, the first member comprises a HUH-endonuclease or HUH-tag, and the second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises a HUH tag. See, e.g., U.S. 2021/0180082, the entirety of which is incorporated herein by reference.

In some embodiments, the capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

In some embodiments, each of the first and second members of a peptide:peptide binding pair comprises an intein. See, e.g., Wagner et al. [(2021) Adv. Sci. 8: 2004018 (1/22)]; Muik et al. [(2017) Biomaterials 144: 84], each of which is incorporated herein by reference in its entirety.

In some embodiments, the first member is a B cell epitope, e.g., from about 1 amino acid to about 35 amino acids in length, and forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, the capsid proteins of the invention are modified to include a detectable label as the first member of the binding pair. Many detectable labels are known in the art. (See, e.g., Nilsson et al. [(1997) "Affinity fusion strategies for detection, purification, and immobilization of modified proteins" Protein Expression and Purification 11: 1-16], Terpe et al. [(2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60: 523-533], and references therein.) Detectable labels include, but are not limited to: a polyhistidine detectable label (e.g., His-6, His-8, or His-10) that binds to an immobilized divalent cation (e.g., Ni ²⁺ ); a biotin moiety (e.g., on a polypeptide sequence biotinylated in vivo) that binds to immobilized avidin; a GST (glutathione S-transferase) sequence that binds to immobilized glutathione; an S tag that binds to an immobilized S protein; an antigen that binds to an immobilized antibody or domain or fragment thereof (e.g., T7, myc, FLAG, and B tags that bind to corresponding antibodies); a FLASH tag (a highly detectable label that binds to a specific arsine moiety); a receptor or receptor domain that binds to an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds to an immobilized IgG; maltose binding protein (MBP) that binds to immobilized amylose; An albumin binding protein that binds to immobilized albumin; a chitin binding protein that binds to immobilized chitin; a calmodulin binding peptide that binds to immobilized calmodulin; and a cellulose binding domain that binds to immobilized cellulose. Another exemplary detectable label is a SNAP-tag, commercially available from Covalys (www.covalys.com). In some embodiments, the detectable labels disclosed herein comprise a detectable label recognized by an antibody paratope, wherein the detectable label and the antibody paratope form a protein:protein binding pair.

In some embodiments, the capsid proteins of the invention comprise a first member of a protein:protein binding pair comprising a detectable label, wherein the detectable label can also be used to detect and/or isolate the Cap protein and/or can also be used as the first member of the protein:protein binding pair. In some embodiments, the detectable label acts as the first member of the protein:protein binding pair for binding of a targeting ligand comprising a multispecific binding protein capable of binding both a target expressed by a cell of interest and a detectable label. In some embodiments, the Cap proteins of the invention comprise a first member of a protein:protein binding pair comprising c-myc (SEQ ID NO: 818). The use of a detectable label as the first member of a protein:protein binding pair is described, for example, in WO2019006043, which is incorporated herein by reference in its entirety.

In some embodiments, the detectable label comprises a B1 epitope (SEQ ID NO: 819). In some embodiments, the capsid protein is modified to comprise the B1 epitope in the VP3 region. In some embodiments, the first member is selected from the group consisting of FLAG, HA, and c-myc (SEQ ID NO: 818).

In some embodiments, the capsid protein comprises a first member of a protein:protein binding pair, wherein the protein:protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of the peptide:peptide binding pair is covalently linked to a cognate second member of the peptide:peptide binding pair via an isopeptide bond, optionally wherein the cognate second member of the peptide:peptide binding pair is fused to a targeting ligand, and wherein the targeting ligand binds to a target expressed by a cell of interest. In some embodiments, the protein:protein binding pair can be selected from the group consisting of SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag003:SpyCatcher003, SpyTag:KTag, Isopeptag:pilin-C, and SnoopTag:SnoopCatcher. In some embodiments, the first member is a SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is a SpyCatcher (or a biologically active portion or variant thereof). In some embodiments, the first member is a SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is a KTag (or a biologically active portion or variant thereof). In some embodiments, the first member is a KTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is a SpyTag (or a biologically active portion or variant thereof). In some embodiments, the first member is a SnoopTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is a SnoopCatcher (or a biologically active portion or variant thereof). In some embodiments, the first member is Isopeptag (or a biologically active portion or variant thereof) and the protein (second cognate member) is Pilin-C (or a biologically active portion or variant thereof). In some embodiments, the first member is SpyTag002 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion or variant thereof). In some embodiments, the first member is SpyTag003 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher003 (or a biologically active portion or variant thereof). In some embodiments, the Cap protein of the invention comprises a SpyTag, or a biologically active portion or variant thereof. The use of the first member of a protein:protein binding pair is described in WO2019006046, which is incorporated herein in its entirety.

In some embodiments, the first member of the protein:protein binding pair and/or the detectable label are operably linked to a Cap protein of the invention (translated in-frame with the Cap protein, chemically attached to the Cap protein, and/or represented by the Cap protein) via a first and/or second linker, e.g., an amino acid spacer that is at least one amino acid in length. In some embodiments, the first member of the protein:protein binding pair is flanked by a first and/or second linker (e.g., a first and/or second amino acid spacer, each of which is at least one amino acid in length).

In some embodiments, the first and/or second linker are not identical. In some embodiments, the first and/or second linker are each independently 1 or 2 amino acids long. In some embodiments, the first and/or second linker are each independently 1, 2, or 3 amino acids long. In some embodiments, the first and/or second linker are each independently 1, 2, 3, or 4 amino acids long. In some embodiments, the first and/or second linker are each independently 1, 2, 3, 4, or 5 amino acids long. In some embodiments, the first and/or second linker are each independently 1, 2, 3, 4, or 5 amino acids long. In some embodiments, the first and/or second linker are each independently 1, 2, 3, 4, 5, or 6 amino acids long. In some embodiments, the first and/or second linker are each independently 1, 2, 3, 4, 5, 6, or 7 amino acids in length. In some embodiments, the first and/or second linker are each independently 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length. In some embodiments, the first and/or second linker are each independently 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length. In some embodiments, the first and/or second linker are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In some embodiments, the first and/or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length.

In some embodiments, the first and second linkers are identical in sequence and/or length and are each one amino acid long. In some embodiments, the first and second linkers are identical in length and are each one amino acid long. In some embodiments, the first and second linkers are identical in length and are each two amino acids long. In some embodiments, the first and second linkers are identical in length and are each three amino acids long. In some embodiments, the first and second linkers are identical in length and are each four amino acids long (e.g., the linker is GLSG (SEQ ID NO: 823)). In some embodiments, the first and second linkers are identical in length and are each five amino acids long. In some embodiments, the first and second linkers are the same length and are each 6 amino acids long (e.g., the first and second linkers each comprise the sequence of GLSGSG (SEQ ID NO: 824) or GSGESG (SEQ ID NO: 828)). In some embodiments, the first and second linkers are the same length and are each 7 amino acids long. In some embodiments, the first and second linkers are the same length and are each 8 amino acids long (e.g., the first and second linkers each comprise the sequence of GLSGLSGS (SEQ ID NO: 825)). In some embodiments, the first and second linkers are the same length and are each 9 amino acids long. In some embodiments, the first and second linkers are the same length and are each 10 amino acids long (e.g., the first and second linkers each comprise the sequence GLSGLSGLSG (SEQ ID NO: 826) or GLSGGSGLSG (SEQ ID NO: 827)). In some embodiments, the first and second linkers are the same length and are each more than 10 amino acids long.

Generally, the first member of a protein:protein binding pair amino acid sequence as described herein (e.g., comprising the first member of the specific binding pair itself or in combination with one or more linkers) is from about 5 amino acids to about 50 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is at least 5 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 6 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 7 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 8 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 9 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 10 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 11 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 12 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 13 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 14 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 15 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 16 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 17 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 18 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 19 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 20 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 21 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 22 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 23 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 24 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 25 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 26 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 27 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 28 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 29 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 30 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 31 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 32 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 33 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 34 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 35 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 36 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 37 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 38 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 39 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 40 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 41 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 42 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 43 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 44 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 45 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 46 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 47 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 48 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 49 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 50 amino acids in length.

In some embodiments of the invention comprising a detectable label, the targeting ligand comprises a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor that can be conjugated to the surface of a bead (e.g., for purification) or expressed by a target cell (e.g., a muscle stem cell, a myoblast, a muscle cell, any combination thereof, etc.). Thus, the multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor targets viral particles. Such “targeting” or “directing” may include a scenario where a wild-type virus particle targets multiple cells within a tissue and/or multiple organs within an organism, where broad targeting of the tissue or organ is reduced or eliminated by insertion of a detectable label, and where retargeting to more specific cells within the tissue or more specific organs within the organism is achieved with a multispecific binding molecule. Such retargeting or redirecting may also include a scenario where a wild-type virus particle targets a tissue, where targeting to the tissue is reduced or eliminated by insertion of a detectable label, and where retargeting to an entirely different tissue is achieved with a multispecific binding molecule. An antibody paratope as described herein generally comprises at least a complementarity determining region (CDR) that specifically recognizes a detectable label, e.g., a CDR3 region of the heavy and/or light chain variable domains. In some embodiments, the multispecific binding molecule comprises an antibody (or portion thereof) comprising an antibody paratope that specifically binds to a detectable label. For example, the multispecific binding molecule can comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or the single domain light chain variable region comprises an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecule can comprise an Fv region, for example, the multispecific binding molecule can comprise an scFv comprising an antibody paratope that specifically binds to a detectable label. In some embodiments, a multispecific binding molecule as described herein comprises an antibody paratope that specifically binds c-myc (SEQ ID NO: 818).

Modified capsid comprising a modified capsid protein

In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein is a mosaic capsid, e.g., comprising at least two sets of VP1, VP2, and/or VP3 proteins, each set encoded by a different cap gene. A mosaic capsid herein generally refers to a mosaic comprised of a first viral capsid protein modified to include a first member of a binding pair and a corresponding second viral capsid protein lacking the first member of the binding pair. In the context of a mosaic capsid, the second viral capsid protein lacking the first member of the binding pair may be referred to as a reference capsid protein encoded by a reference cap gene. In some embodiments of the mosaic capsid, preferably when the VP1, VP2, and/or VP3 capsid protein modified as the first member of the protein:protein pair is not a chimeric capsid protein, the VP1, VP2, and/or VP3 reference capsid protein may comprise an amino acid sequence that is identical to the amino acid sequence of a viral VP1, VP2, and/or VP3 capsid protein modified as the first member of the binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments of the mosaic capsid, the VP1, VP2, and/or VP3 reference capsid protein corresponds to a viral VP1, VP2, and/or VP3 capsid protein modified as the first member of the binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP1 reference capsid protein corresponds to a viral VP1 capsid protein modified with the first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP2 reference capsid protein corresponds to a viral VP2 capsid protein modified with the first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with the first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments of a mosaic capsid comprising chimeric VP1, VP2, and/or VP3 capsid proteins further modified to include a first member of a binding pair, the reference protein may be a corresponding capsid protein, a portion of which forms a portion of the chimeric capsid protein. As a non-limiting example, in some embodiments, a mosaic capsid comprising a chimeric AAV2/AAAV VP1 capsid protein modified to include a first member of a binding pair can further comprise, as a reference capsid protein, an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2/AAAV VP1 capsid protein lacking the first member. Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2/AAAV VP2 capsid protein modified to include a first member of a binding pair can further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2/AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid comprising a chimeric AAV2/AAAV VP3 capsid protein modified to include a first member of a binding pair can further comprise a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2/AAAV VP3 capsid protein lacking the first member. In some embodiments of the mosaic capsid, the reference capsid protein can be any capsid protein that can form a capsid with a first capsid protein that lacks the first member of the binding pair and is modified to include the first member of the binding pair.

In general, mosaic particles can be generated by transfecting a mixture of modified Cap genes and a reference Cap gene into a production cell at a indicated ratio. The protein subunit ratio, e.g., the modified VP protein:unmodified VP protein ratio within the particle, can, but does not necessarily, stoichiometrically reflect the ratio of at least two species (e.g., the modified cap gene transfected into the packaging cell:the reference cap gene(s)) consisting of a cap gene encoding a first capsid protein modified as the first member of the binding pair and one or more reference cap genes. In some embodiments, the protein subunit ratio within the particle does not stoichiometrically reflect the modified cap gene:reference cap gene(s) ratio transfected into the packaging cell.

In some embodiments of the mosaic virus particle, the protein subunit ratio is in the range of about 1:59 to about 59:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:1 (e.g., the mosaic virus particle comprises about 30 modified capsid proteins and about 30 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:2 (e.g., the mosaic virus particle comprises about 20 modified capsid proteins and about 40 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 3:5. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:3 (e.g., the mosaic virus particle comprises about 15 modified capsid proteins and about 45 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:4 (e.g., the mosaic virus particle comprises about 12 modified capsid proteins and about 48 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:5 (e.g., the mosaic virus particle comprises about 10 modified capsid proteins and about 50 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:6. In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:7. In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:8. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:9 (e.g., the mosaic virus particle comprises about 6 modified capsid proteins and about 54 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:10. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:11 (e.g., the mosaic virus particle comprises about 5 modified capsid proteins and about 55 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:12. In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:13. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:14 (e.g., the mosaic virus particle comprises about 4 modified capsid proteins and about 56 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:15. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:19 (e.g., the mosaic virus particle comprises about 3 modified capsid proteins and about 57 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 1:29 (e.g., the mosaic virus particle comprises about 2 modified capsid proteins and about 58 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 1:59. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 2:1 (e.g., the mosaic virus particle comprises about 40 modified capsid proteins and about 20 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 5:3. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 3:1 (e.g., the mosaic virus particle comprises about 45 modified capsid proteins and about 15 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 4:1 (e.g., the mosaic virus particle comprises about 48 modified capsid proteins and about 12 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 5:1 (e.g., the mosaic virus particle comprises about 50 modified capsid proteins and about 10 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 6:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is about 7:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is about 8:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 9:1 (e.g., the mosaic virus particle comprises about 54 modified capsid proteins and about 6 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 10:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 11:1 (e.g., the mosaic virus particle comprises about 55 modified capsid proteins and about 5 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 12:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is about 13:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 14:1 (e.g., the mosaic virus particle comprises about 56 modified capsid proteins and about 4 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 15:1. In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 19:1 (e.g., the mosaic virus particle comprises about 57 modified capsid proteins and about 3 reference capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is at least about 29:1 (e.g., the mosaic virus particle comprises about 58 modified capsid proteins and about 2 standard capsid proteins). In some embodiments of the mosaic virus particle, the protein subunit ratio is about 59:1.

In some embodiments of the non-mosaic virus particle, the protein subunit ratio can be 1:0, wherein the capsid protein of each non-mosaic virus particle is modified with the first member of the binding pair. In some embodiments of the non-mosaic virus particle, the protein subunit ratio can be 0:1, wherein the capsid protein of each non-mosaic virus particle is not modified with the first member of the binding pair.

Also provided herein is a nucleic acid encoding a VP3 capsid protein of the present invention. AAV capsid proteins may, but are not necessarily, encoded by overlapping reading frames of the same gene with staggered start codons. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention does not encode either a VP2 capsid protein or a VP1 capsid protein of the present invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention may encode a VP2 capsid protein of the present invention but does not encode a VP1 capsid protein of the present invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention may encode both a VP2 capsid protein of the present invention and a VP1 capsid protein of the present invention.

One embodiment of the present invention is a multimeric structure comprising a modified viral capsid protein of the present invention. The multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 modified viral capsid proteins comprising a first member of a specific binding pair as described herein. These can form regular viral capsids (empty viral particles) or viral particles (capsids encapsulating nucleotides of interest). Formation of viral particles comprising the viral genome is a highly desirable feature for use with the modified viral capsids described herein.

A further embodiment of the present invention is the use of at least one modified viral capsid protein and/or a nucleic acid encoding the same, preferably in the preparation of a nucleotide of interest, and the use of at least one multimeric structure (e.g., a viral particle) for use in delivering the nucleotide of interest to a target cell (e.g., a muscle stem cell, a myoblast, a muscle cell, any combination thereof, etc.).

Insertion site

Because at least many of the extension regions and many members of closely related family members are highly conserved, corresponding insertion sites for AAVs other than those listed can be identified by performing amino acid alignments or comparing capsid structures. See, e.g., Rutledge et al. [(1998) J. Virol. 72:309-19]; Mietzsch et al. [(2019) Viruses 11, 362, 1-34]; and U.S. Patent No. 9,624,274, each of which is incorporated herein by reference in its entirety. For example, Mietzsch et al. (2019) provides an overlay of ribbons from different defendoparvoviruses in Figure 7, depicting the variable regions VR I through VR IX. Using such structural and sequence analyses as described herein, one of skill in the art can determine which amino acids within the variable region correspond to amino acid sequences of AAVs capable of accepting insertion of, for example, a targeting ligand, a first member of a binding pair, and/or a detectable label as described herein.

In general, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into a variable region or variable loop of an AAV capsid protein, a GH loop of an AAV capsid protein, or the like. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into a variable region or variable loop VRI of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into a variable region or variable loop VRII of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into a variable region or variable loop VRIII of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into a variable region or variable loop VRIV of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into the variable region or variable loop VRV of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into the variable region or variable loop VRV of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into the variable region or variable loop VRVI of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into the variable region or variable loop VRVII of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label can be inserted into the variable region or variable loop VRIII of an AAV capsid protein. In some embodiments, the targeting ligand, the first member of the binding pair, and/or the detectable label may be inserted within the variable region or variable loop VRIX of the AAV capsid protein.

In some embodiments, the first member of the binding pair and/or the detectable label is inserted into the VP1 capsid protein of the non-primate AAV at an amino acid position corresponding to an amino acid position selected from the group consisting of G453 of the AAV2 capsid protein VP1, N587 of the AAV2 capsid protein VP1, G453 of the AAV9 capsid protein VP1, and A589 of the AAV9 capsid protein VP1. In some embodiments, the first member of the binding pair and/or the detectable label is inserted into the VP1 capsid protein of the non-primate animal AAV between amino acids corresponding to N587 and R588 of the AAV2 VP1 capsid.

The nomenclature I-###, I#, etc. herein refers to an insertion site (I) having ### designating the amino acid number for the VP1 protein of the AAV capsid protein, but such insertion can be located immediately N-terminus or C-terminus, preferably C-terminus of one of the five amino acids that are N-terminal or C-terminal to a given amino acid, preferably C-terminus of one of the three, more preferably two, amino acids that are N-terminal or C-terminal to a given amino acid, and in particular C-terminus of one amino acid. Furthermore, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and point mutations thereof) can be readily identified for the VP2 and VP3 capsid proteins encoded by the capsid gene by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid gene.

Additional suitable insertion sites for non-primate VP1 capsid proteins include those corresponding to I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716 of the VP1 capsid protein of AAV2 (see Wu et al. [(2000) J. Virol. 74:8635-8647]). A modified viral capsid protein as described herein may be a non-primate animal capsid protein comprising a first member of a binding pair and/or a detectable label inserted into a position corresponding to a position of an AAV2 capsid protein selected from the group consisting of I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, I-716, and combinations thereof. Additional suitable insertion sites for non-primate human AAV include those corresponding to I-587 or I-590 of AAV1, I-589 of AAV1, I-585 of AAV3, I-584 or I-585 of AAV4, and I-575 or I-585 of AAV5. In some embodiments, the modified viral capsid protein as described herein can be a non-primate animal capsid protein comprising a targeting ligand, a first member of a binding pair, and/or a detectable label inserted into a position corresponding to a position selected from the group consisting of I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5), and combinations thereof.

In some embodiments, the first member of the binding pair and/or the detectable label is inserted into the VP1 capsid protein of a non-primate animal AAV at an amino acid position selected from the group consisting of: I444 of avian AAV capsid protein VP1, I580 of avian AAV capsid protein VP1, I573 of bearded lizard AAV capsid protein VP1, I436 of bearded lizard AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I433 of sea lion AAV capsid protein VP1, I434 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I433 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I434 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I433 of sea lion AAV capsid protein VP1, I434 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I429 of sea lion AAV caps I437, and I565 of the sea lion AAV capsid protein VP1.

Thus, inserting the cap gene into the corresponding position in the coding nucleic acid of one of these sites results in insertion into VP1, VP2, and/or VP3, since the capsid proteins are encoded by staggered start codons and overlapping reading frames of the same gene. Thus, for example, in the case of AAV2 according to the present nomenclature, insertion between amino acids 1 and 138 is inserted only into VP1, insertion between 138 and 203 is inserted into VP1 and VP2, and insertion between 203 and the C-terminus is inserted into VP1, VP2, and VP3, which of course also applies to insertion site I-587. Accordingly, the present invention encompasses structural genes of AAV having insertions corresponding to VP1, VP2, and/or VP3 proteins.

Transduction efficiency

In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is capable of infecting a specified cell and has an enhanced ability to target and bind to the specified cell compared to the ability of a control viral capsid that is identical to the modified viral capsid protein, except that it lacks one or both of the first and second members of the binding pair, and, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to an undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 80% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.

In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is capable of infecting a specified cell and has an enhanced ability to target and bind to the specified cell compared to the ability of a control viral capsid that is identical to the modified viral capsid protein, except that it lacks one or both of the first and second members of the binding pair, and, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to an undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 80% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 99% greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 1.5 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 2 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency at least three times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency at least four times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency at least five times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 6-fold greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 7-fold greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 8-fold greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 9 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 10 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 20 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 30 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 40 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 50 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 60 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 70 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and a second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 80 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 90 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100 times greater than a transduction efficiency of a control viral capsid. In some embodiments, a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof, and optionally comprising first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.), can better evade neutralization by pre-existing antibodies in a human patient isolated from a human patient, as compared to a suitable control viral particle (e.g., comprising a viral capsid of an AAV serotype of which a portion of a viral capsid is incorporated, e.g., comprising as a portion of a viral capsid protein the amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof), optionally also comprising first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.). In some embodiments, a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof requires at least twice as much total IVIG or IgG for neutralization (e.g., greater than 50% inhibition of infection) as compared to a suitable control viral particle (e.g., the viral particle of the invention has an IC50 value at least twice that of the control viral particle).

Therapeutic formulations and administration of antigen-binding molecules

Also described herein are pharmaceutical compositions comprising antigen binding molecules as described herein. In some embodiments, the pharmaceutical compositions may be formulated with suitable carriers, excipients, and other agents that improve transport, delivery, tolerability, etc. Many suitable formulations can be found in the following formularies known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such formulations include, for example, powders, plasters, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption ointments, oil-in-water and water-in-oil emulsifiers, carbowax emulsifiers (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. ["Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311].

The dosage of the antigen-binding molecule administered to a patient may vary depending on the patient's age and size, target disease, condition, route of administration, etc. Preferred dosages are generally calculated based on body weight or body surface area. When an antigen-binding molecule as described herein is used therapeutically in an adult patient, it may be advantageous to administer the antigen-binding molecule as described herein intravenously in a single dose of generally about 0.01 to about 20 mg/kg of body weight, more preferably about 0.02 to about 7 mg/kg, about 0.03 to about 5 mg/kg, or about 0.05 to about 3 mg/kg. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. An effective dosage and schedule for administration of the bispecific antigen-binding molecule can be empirically determined; for example, the subject's progress can be monitored through periodic evaluations and the dosage adjusted accordingly. Additionally, interspecies scaling of dosage can be performed using methods well known in the art (see, e.g., Mordenti et al., Pharmaceut. Res. 8:1351 (1991)).

Various delivery systems are known and can be used to administer pharmaceutical compositions as described herein, such as encapsulated liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through the lining of epithelia or mucosa (e.g., oral mucosa, rectal, and intestinal mucosa), and may be administered together with other biologically active agents. Administration can be systemic or topical.

The pharmaceutical compositions described herein can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, for subcutaneous delivery, pen delivery devices are readily adapted to deliver the pharmaceutical compositions described herein. These pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition in the cartridge has been dispensed and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. This allows the pen delivery device to be reused. Disposable pen delivery devices do not have replaceable cartridges. Rather, disposable pen delivery devices are provided with the pharmaceutical composition pre-stored in a reservoir within the device. Once the pharmaceutical composition in the reservoir is depleted, the entire device is discarded.

A number of reusable pen delivery devices and self-injector delivery devices are applicable to the subcutaneous delivery of pharmaceutical compositions as described herein. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices adapted for subcutaneous delivery of pharmaceutical compositions as described herein include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name a few.

In certain circumstances, the pharmaceutical composition may be delivered using a controlled-release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987)). In another embodiment, polymeric materials may be used (see Langer and Wise (eds.), Medical Applications of Controlled Release, 1974, CRC Press., Boca Raton, Florida). In yet another embodiment, the controlled-release system may be positioned proximate the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, supra, 1984, Medical Applications of Controlled Release, vol. 2, 115-138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, dropwise infusion, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending or emulsifying the antibody or its salt as described above in a sterile aqueous medium or oily medium conventionally used for injection. Examples of aqueous media for injection include isotonic solutions containing physiological saline, glucose and other adjuvants, etc., which may be used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like. As a solubilizing medium, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc., are used. Injections prepared in this manner are preferably filled into appropriate ampoules.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms of unit dosages appropriately adjusted to the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the antibody described above is generally about 5 to about 500 mg per unit dosage form; particularly, when in the form of an injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and when in other dosage forms, the antibody is preferably contained in an amount of about 10 to about 250 mg.

Pharmaceutical compositions, dosage forms, and administrations for viral particles

A further embodiment provides a medicament comprising at least one modified viral capsid protein according to the present invention and a suitable targeting ligand and/or a nucleic acid according to the present invention. Preferably, such a medicament is useful as a gene delivery particle.

Pharmaceutical compositions comprising the virus particles described herein and pharmaceutically acceptable carriers and/or excipients are also disclosed herein. Furthermore, pharmaceutical dosage forms comprising the virus particles described herein are also disclosed herein.

As discussed herein, the viral particles described herein can be used for a variety of therapeutic applications (in vivo and ex vivo) and as research tools.

Pharmaceutical compositions based on the viral particles disclosed herein may be formulated in any conventional manner using one or more physiologically acceptable carriers and/or excipients. The viral particles may be formulated, for example, for administration by injection, inhalation, or aspiration (via the mouth or nose), for oral, buccal, parenteral, or rectal administration, or for direct administration to a tumor.

Pharmaceutical compositions can be formulated for various modes of administration, including systemic, topical, or localized administration. Techniques and formulations can be found, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injections, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections, are preferred. For injection, the pharmaceutical composition may be formulated as a liquid solution, preferably in a physiologically compatible buffer such as Hank's solution or Ringer's solution. The pharmaceutical composition may also be formulated in solid form, which can be reconstituted or suspended immediately prior to use. Lyophilized forms of the pharmaceutical composition are also suitable.

For oral administration, the pharmaceutical composition may take the form of a tablet or capsule prepared by conventional means together with pharmaceutically acceptable excipients such as, for example, a binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); a filler (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); a lubricant (e.g., magnesium stearate, talc, or silica); a disintegrant (e.g., potato starch or sodium starch glycolate); or a wetting agent (e.g., sodium lauryl sulfate). The tablets may also be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product for mixing with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., oils, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid). The preparations may also contain appropriate amounts of buffering salts, flavoring agents, coloring agents, and sweeteners.

The pharmaceutical composition may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. The injectable formulation may be presented in unit dosage form, for example, in ampoules, or in multi-dose containers, optionally with a preservative. The pharmaceutical composition may be further formulated as a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain other agents, including suspending agents, stabilizers, and/or dispersing agents.

Pharmaceutical compositions may also be formulated as depot preparations. These long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the compound may be formulated with a suitable polymer or hydrophobic material (e.g., an emulsion in an acceptable oil) or an ion-exchange resin, or as a sparingly soluble derivative, such as a soluble salt. Other suitable delivery systems include microspheres, which enable non-invasive local delivery of the drug over an extended period of time. This technology may include microspheres having a precapillary size, which can be injected into any selected portion of an organ via a coronary artery catheter without causing inflammation or ischemia. The administered therapeutic agent is released from the microspheres and taken up by surrounding cells in the selected tissue.

Systemic administration may also be via mucosal or transdermal routes. For mucosal or transdermal administration, penetrants appropriate for the target barrier are used in the formulation. Such penetrants are generally known in the art and include, for example, bile salts and fusidic acid derivatives for mucosal administration. Detergents that facilitate penetration may also be used. Mucosal administration may be achieved using nasal sprays or suppositories. For topical administration, the viral particles described herein may be formulated as ointments, plasters, gels, or creams, as generally known in the art. Topical solutions for treating injuries or inflammation may be used to accelerate healing.

Pharmaceutical forms suitable for injection may include: sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, these forms must be sterile and must be fluid. They must be stable under the conditions of manufacture and specified storage parameters (e.g., refrigeration and freezing), and must be preserved against the contaminating action of microorganisms such as bacteria and molds.

When the formulations disclosed herein are used as therapeutic agents that enhance an immune response in a subject, the therapeutic agents may be formulated as compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), such as those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

The carrier may be a solvent, or a dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents known in the art. In many cases, it will be desirable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using absorption delaying agents such as aluminum stearate and gelatin in the composition.

Sterile injectable solutions can be prepared by incorporating the active compound or composition in the required amount in an appropriate solvent with various other ingredients enumerated above, as required, followed by sterile filter mixing.

When formulated, the solution can be administered in a manner similar to a dosage form, and in a therapeutically effective amount. The formulation can be readily administered in a variety of dosage forms, such as the aforementioned injectable solutions, but sustained-release capsules, microparticles, and microspheres can also be used.

For example, when administering aqueous solutions parenterally, the solution must be appropriately buffered, if necessary, and the liquid diluent must first be isotonicized with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration. Suitable sterile aqueous media for use in this context will be known to those skilled in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous fluid or injected into the intended injection site.

In all cases, the appropriate dosage for an individual subject will be determined by the administering physician. For example, depending on the need for exposure to pathogenic organisms or the subject's medical condition (e.g., cancer), the subject may be administered the viral particles described herein daily, weekly, monthly, biennially, or annually for a period of time.

In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, intradermal, or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; liposomal formulations; sustained-release capsules; biodegradable forms, and any other forms currently in use.

Intranasal solutions, sprays, aerosols, or inhalers may also be used. Nasal solutions may be aqueous solutions designed to be administered by instillation or spray into the nasal passages. Nasal solutions can be formulated to mimic nasal secretions in many respects. Therefore, aqueous nasal solutions are typically isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. Antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if necessary, may also be included in the formulation. A variety of commercial nasal preparations are known, including, for example, antibiotics and antihistamines, which are used to prevent asthma.

Oral dosage forms may include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained-release dosage forms, or powders. In certain defined embodiments, the oral pharmaceutical compositions may include inert diluents or assimilable edible carriers, may be enclosed in hard or soft-shell gelatin capsules, may be compressed into tablets, or may be incorporated directly into food in the diet. For oral therapeutic administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

Tablets, troches, pills, capsules, and the like may also contain: a binder such as gum tragacanth, acacia, corn starch, or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, or alginic acid; a lubricant such as magnesium stearate; and a sweetener such as sucrose, lactose, or saccharin. In addition, flavorings such as peppermint, oil of wintergreen, or cherry flavor may also be added. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the types of materials described above. Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Elixir syrups may contain active compounds, such as sucrose as a sweetener, methyl and propylparamene as preservatives, and coloring and flavoring agents such as cherry or orange flavoring.

Additional embodiments disclosed herein may include kits for use with the methods and compositions. The kits may also include suitable containers, such as vials, tubes, mini- or microfuge tubes, test tubes, flasks, bottles, syringes, or other containers. If additional components or agents are provided, the kits may include one or more additional containers into which such agents or components may be placed. The kits herein will also generally include means for containing the viral particles and a sealed reagent container for commercial sale. Such containers may include injection-molded or blow-molded containers that retain the desired vial. Optionally, one or more additional active agents may be required for the described compositions, such as, for example, anti-inflammatory, antiviral, antifungal, antibacterial, or anti-tumor agents.

The compositions disclosed herein may be administered by any means known in the art. For example, the compositions may be administered to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion, via catheter, by lavage, as a cream, or as a lipid composition.

Any method known to those skilled in the art can be used for the large-scale production of the viral particles, packaging cells, and particle constructs described herein. For example, master seed stocks and working seed stocks can be prepared from validated primary CEFs under GMP conditions or by other methods. Packaging cells can be plated in large-surface-area flasks, grown to near-confluency, and viral particles purified. Cells can be harvested, releasing viral particles into isolated and purified culture medium, or intracellular viral particles can be mechanically disrupted to release viral particles (cell debris removed by large-pore depth filtration, and host cell DNA digested with endonucleases). The viral particles can be subsequently purified and concentrated by tangential flow filtration followed by diafiltration. The resulting concentrated bulk can be formulated by diluting with a buffer containing a stabilizer, filling vials, and lyophilizing. The compositions and formulations can be stored for later use. For use, the lyophilized virus particles can be reconstituted by adding a diluent.

Any additional agent used in combination therapy may be formulated and administered by any means known in the art.

Compositions disclosed herein may include adjuvants, such as aluminum salts and other mineral supplements, tensoactive agents, bacterial derivatives, vehicles, and cytokines. Adjuvants may also possess antagonistic immunomodulatory properties. For example, adjuvants may stimulate Th1 or Th2 immunity. Compositions and methods disclosed herein may also include adjuvant therapy.

Therapeutic and diagnostic uses of antigen-binding molecules

For example, disclosed herein is a method of inhibiting the activity of CDH15 in a cell. The method may comprise contacting a cell expressing CDH15 (e.g., in vitro, ex vivo, or in vivo) with an anti-hCDH15 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof of the present disclosure. The activity of CDH15 includes, but is not limited to, inhibiting muscle regeneration by inhibiting the activation of CDH15-expressing muscle stem cells.

In various embodiments, the cell expressing CDH15 is a muscle cell. In some embodiments, the cell is a mammalian muscle cell. In some embodiments, the cell is a mammalian skeletal muscle cell. In some embodiments, the cell is a non-terminally differentiated mammalian skeletal muscle cell. In some embodiments, the cell is a terminally differentiated mammalian skeletal muscle cell. In some embodiments, the cell is a muscle stem cell, a myoblast, or a myocyte. In some embodiments, the cell is a human rhabdomyosarcoma cell.

Also disclosed herein is a method of accelerating the transition of muscle stem cells from a quiescent state to an activated state, comprising contacting the muscle stem cells (e.g., in vitro, ex vivo, or in vivo) with an anti-hCDH15 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof of the present disclosure. As used herein, “quiescence” refers to an inactive, non-proliferative state of a cell. For example, quiescent stem cells do not proliferate or differentiate into muscle fibers. As used herein, “activation” with respect to muscle stem cells refers to the ability of the muscle stem cells to proliferate and differentiate into muscle fibers, thereby promoting, for example, the regrowth and/or repair of damaged muscle.

Also disclosed herein is a method comprising administering to a subject in need thereof a composition (e.g., a therapeutic composition) comprising an anti-hCDH15 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody (e.g., an anti-hCDH15 antibody, or an ADC comprising any of the HCVR/LCVR or CDR sequences set forth in Table 1 herein). The therapeutic composition can comprise any of the anti-hCDH15 antibodies, antigen-binding fragments thereof, or ADCs as disclosed herein, and a pharmaceutically acceptable carrier or diluent.

Also disclosed herein are uses of compositions (e.g., therapeutic compositions) comprising an anti-hCDH15 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody for treating one or more conditions as disclosed herein.

Also disclosed herein are compositions (e.g., therapeutic compositions) comprising an anti-hCDH15 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody for use as a therapeutic agent, for example, for use in treating one or more conditions as disclosed herein.

Also disclosed herein is the use of an anti-hCDH15 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody in the manufacture of a medicament for treating one or more conditions as disclosed herein.

Antibodies, antigen-binding fragments thereof, or antibody-drug conjugates comprising an anti-hCDH15 antibody as described herein may be particularly useful for treating, preventing, and/or ameliorating one or more conditions, e.g., any disease or disorder associated with skeletal muscle tissue. For example, antibodies and ADCs as described herein may be useful for treating muscle wasting disorders (e.g., cachexia, glucocorticoid-induced muscle wasting, heart failure-induced muscle wasting, HIV wasting, disuse, aging, etc.), muscular dystrophies/myopathies, and/or muscle-associated cancers (e.g., rhabdomyosarcoma).

In another embodiment, a method for treating a disease, such as a muscle wasting disorder, is provided. The method may comprise providing a subject in need of such treatment with an antibody or CDH15 antigen-binding fragment thereof, as described above.

In another embodiment, a method for treating muscle-related cancer is provided. The method may comprise providing a subject in need thereof with an antibody or CDH15 antigen-binding fragment thereof, as described above. Non-limiting examples of muscle-related cancers include rhabdomyosarcoma, such as embryonal, alveolar, pleomorphic, staphylococcal, and spindle/sclerotic rhabdomyosarcoma.

In another embodiment, a method for treating muscle injury is provided. The method may comprise administering an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject in need of such treatment, e.g., a subject who has experienced and/or is expected to experience muscle injury. Thus, the antibody or CDH15 antigen-binding fragment thereof may be administered prior to injury, e.g., prior to surgery that injures a muscle, or after injury, e.g., after an unexpected muscle injury.

In another embodiment, a method of improving muscle regeneration after muscle injury in a subject is provided. The method may comprise administering to a subject in need thereof an antibody or CDH15 antigen-binding fragment thereof as described above. As used herein, “improvement” in relation to muscle regeneration refers to any measurable increase in muscle repair. For example, muscle regeneration may be considered “improved” in a subject if muscle regeneration occurs at an accelerated rate and/or to a greater extent compared to a control subject, e.g., a subject not administered the anti-CDH15 antibody or antigen-binding fragment thereof. “Improved” muscle regeneration may be an increase in rate or degree of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%.

In another embodiment, a method of restoring muscle regenerative capacity in an elderly subject is provided. The method may comprise providing to a subject in need thereof an antibody or CDH15 antigen-binding fragment thereof as described above. As used herein, “restoration” refers to the ability to return to a previous functional state. For example, an elderly subject’s muscle regenerative capacity may be considered “restored” if the muscle regenerative capacity can be returned to that of a control subject, e.g., the same subject at a younger age and/or a separate subject representing a younger version of the elderly subject. A functional state may be considered “restored” if the functional state is similar to (e.g., not statistically different from) the previous functional state or the functional state of a subject(s) representing the previous functional state. A functional status may be considered “restored” if it differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% from the previous functional status or from the functional status of the subject(s) representing the previous functional status.

Anti-hCDH15 antibodies as described herein have various utilities. For example, in some embodiments, anti-hCDH15 antibodies as described herein can be used in diagnostic assays for CDH15, e.g., to detect CDH15 expression in specific cells, tissues, etc., and as reagents for identifying/labeling skeletal muscle fibers and/or muscle stem cells. Various diagnostic and prognostic assay techniques known in the art can be used, such as competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays performed in either heterogeneous or homogeneous phases (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. pp. 147-1581). Antibodies used in assays can be labeled with a detectable moiety. The detectable moiety must be capable of producing a detectable signal, either directly or indirectly. Any method known in the art for conjugating an antibody to a detectable moiety can be used.

Methods of using and manufacturing virus particles

A further embodiment of the modified viral capsids described herein is the use of the modified viral capsids to deliver a nucleotide of interest, e.g., a reporter gene or a therapeutic gene, to a target cell (e.g., a muscle stem cell, a myoblast, a muscle cell, any combination thereof, etc.). Generally, packaging the nucleotide of interest involves replacing the AAV genome between the AAV ITR sequences with the gene of interest to create a transfer plasmid, which is then encapsulated in an AAV capsid according to well-known methods. Thus, a modified viral capsid as described herein can encapsulate the nucleotide of interest and/or the transfer plasmid, which can generally comprise 5' and 3' inverted terminal repeat (ITR) sequences flanking the gene of interest, e.g., reporter gene(s) or therapeutic gene(s), or a portion of the gene of interest (which can be regulated by a viral or non-viral promoter). According to well-known methods for packaging AAV viral particles, the modified viral capsid, 5' ITR, and 3' ITR do not need to be of the same AAV serotype. In one embodiment, the transfer plasmid and/or nucleotides of interest comprise, in the 5' to 3' direction: a 5' ITR, a promoter, a gene (e.g., a reporter and/or therapeutic gene), and a 3' ITR.

A consideration for AAV transfer plasmid design is that the wild-type AAV genome is approximately 4.7 kb. Therefore, well-known strategies that enable packaging of nucleotides of interest that exceed the packaging capacity of individual AAVs are encompassed herein. These strategies include, but are not limited to, dual vector strategies that utilize ITR-mediated recombination to express genes of interest larger than the wild-type AAV genome via transcriptional splicing across intermolecularly recombined ITRs from two complementary vector genomes, vector recombination by homology, RNA trans-splicing, and/or protein “trans-splicing” via split intein design. See, e.g., Nakai, H. et al. [(2000) Nat. Biotechnol. 18:527-532]; Sun, L. [(2000) Nat. Med. 6:599-602 (2000)]; See Ghosh, A. et al. [(2008) Mol. Ther. 16:124-130 (2008)]; Lai, Y. [(2005) Nat. Biotechnol. 23:1435-1439]; Chew, WL et al. [(2016) Nat. Methods 13:868-874]; Li, J. [(2008) Hum. Gene Ther. 19:958-964], each of which is incorporated herein by reference in its entirety.

Dual AAV vector strategies for delivering large genes into target cells have been described, relying on different mechanisms, including, but not limited to, trans-splicing involving overlapping regions within the dual vector, and hybridization of the two. See Tornabene and Trapani [(2020) Human Gene Ther. 31:47-56]; U.S. Patent No. 8,236,557, each of which is incorporated herein by reference in its entirety.

The trans-splicing approach exploits the ability of conjugated AAV ITR sequences to reconstitute a full-length genome, wherein two or more viral capsids each encapsulate one of two or more transfer plasmids, each containing a portion of the gene of interest. For example, in a dual vector approach, the two transfer plasmids can be designed as follows: the 5' transfer plasmid contains a promoter, the 5' portion of the coding sequence of the gene of interest, and a splicing donor (SD) signal; and the 3' transfer plasmid contains a splicing acceptor (SA) signal, the 3' portion of the gene of interest, and a polyA signal. After tail-head ITR-mediated conjugation of the two AAV genomes, the SD and SA signals allow splicing of the recombined genomes.

When choosing an overlapping region approach, even a large gene of interest is fragmented. In the overlapping region approach, the 5' and 3' segments (and thus the 5' transfer plasmid and 3' transfer plasmid) share recombinant sequences, such as regions of homology, and each segment contains overlapping sequences. The gene of interest is then produced entirely in the targeted cell through homologous recombination mediated by the recombinogenic sequences, such as the homologous/overlapping regions.

In the hybrid approach, the 5'-transfer plasmid and the 3'-transfer plasmid each contain highly recombinant sequences, wherein the recombinant sequences are located downstream of the SD signal at the 5' end of the coding sequence of the gene of interest and upstream of the SA signal at the 3' end of the coding sequence of the gene of interest. In this hybrid system, the gene of interest can be produced entirely through ITR-mediated joining and splicing and/or by homologous recombination.

Trans-splicing can also be used at the RNA or protein level. In the RNA trans-splicing approach, two transfer plasmids encode the 5' and 3' segments of the pre-mRNA of a large gene, respectively, and share an intronic hybridization domain that favors trans-splicing, allowing the two halves of the transcripts to be joined into a complete full-length mRNA.

Protein trans-splicing occurs post-translationally and is catalyzed by intervening proteins called split inteins. Split inteins are expressed as two independent polypeptides (N-intein and C-intein) at the most distal ends of two host proteins. The N-intein and C-intein polypeptides remain catalytically inactive until they encounter each other. Upon encounter, each intein precisely excises itself from the host protein, mediating the linkage of the N- and C-host polypeptides via a peptide bond. Split inteins have been used for AAV-based delivery of therapeutic genes of interest in muscle, liver, and retinal diseases. For example, co-delivery of two halves of a mini-dystrophin cDNA fused to the N- and C-intein coding sequences resulted in efficient production of both polypeptides. Li et al. [(2008) Hum Gene Ther 19:958-64]. Similarly, AAV-split-inteins have been widely used for expression and ligation of clustered regularly spaced short palindromic repeats (CRISPR)-Cas9 nuclease.

The above dual vector approach is well known in the art. See, e.g., Tornabene and Trapani (2020) as cited above; U.S. Patent No. 8,236,557. Accordingly, in some embodiments, the modified viral capsids described herein encapsulate a nucleotide of interest comprising a portion of a gene of interest. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest further comprises a splicing donor signal, a splicing acceptor signal, and/or a recombination sequence. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest comprises an intronic hybridization domain encoding sequence. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest comprises an N-intein or C-intein encoding sequence.

The design of the transfer plasmid/nucleotide of interest includes including one or more regulatory elements, such as promoter and/or enhancer elements, that control the expression of the gene of interest. Non-limiting examples of useful promoters include, for example, the cytomegalovirus (CMV) promoter, the spleen foci-forming virus (SFFV) promoter, the transcription elongation factor 1 alpha (EF1a) promoter (either the 1.2 kb EFla promoter or the 0.2 kb EFla promoter), the chimeric EF1a/IF4 promoter, and the phospho-glycerate kinase (PGK) promoter. Internal enhancers may also be present within the viral construct to increase expression of the gene of interest. For example, the CMV enhancer (see Karasuyama et al., 1989. J. Exp. Med. 169:13, incorporated herein by reference in its entirety) may be used. In some embodiments, the CMV enhancer can be used in combination with the chicken β-actin promoter. In some embodiments, tissue-specific regulatory elements, such as muscle-specific promoters and/or regulatory elements, can be used to drive expression of the gene of interest. For example, the use of muscle-specific regulatory elements based on the muscle creatine kinase gene has been used in muscle gene therapy treatments such as Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy (LGMD). See, e.g., Salva, MZ et al. [(2007) Mol. Ther. 15: 320-329], which is incorporated herein by reference in its entirety. In some embodiments, the transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter of muscle creatine kinase (MCK), wherein the enhancer and/or promoter of MCK drives expression of the gene of interest. In some embodiments, the transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter element that recruits RNA polymerase II, wherein the enhancer and/or promoter of MCK drives expression of the gene of interest. In some embodiments, the transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter element that recruits RNA polymerase III, wherein the enhancer and/or promoter of MCK drives expression of the gene of interest.

In some embodiments, bidirectional promoter vectors have also been used to deliver dual therapeutic gene cassettes. An example is the bidirectional chicken β-actin ubiquitous promoter, which drives the simultaneous expression of the hexosaminidase α- and β-subunits of the HexA enzyme, two genes implicated in Tay-Sachs disease and Sandhoff disease, respectively. See Lahey et al. (2020) Mol. Ther . 28: 2150-2160, which is incorporated herein by reference in its entirety. In some embodiments, the transfer plasmid and/or the nucleotide of interest herein comprises a bidirectional promoter, wherein the bidirectional promoter drives the expression of two different genes of interest.

Various reporter genes (or detectable moieties) can be encapsulated within the multimeric structure comprising the modified viral capsid proteins described herein. Exemplary reporter genes include, for example, b-galactosidase (encoded by the lacZ gene), Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (eGFP), MmGFP, Blue Fluorescent Protein (BFP), Enhanced Blue Fluorescent Protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (eYFP), Emerald, CyPet, Cyan Fluorescent Protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or combinations thereof. Although the methods described herein demonstrate the construction of targeting particles using a reporter gene encoding green fluorescent protein, those skilled in the art, upon reading this disclosure, will appreciate that the viral capsids described herein can be produced without a reporter gene or using any reporter gene known in the art.

Various therapeutic genes may also be encapsulated in multimeric structures comprising the modified viral capsid proteins described herein, for example, as part of a delivery particle. Non-limiting examples of therapeutic genes include therapeutic genes encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, the CRISPR/Cas system or portion(s) thereof, antisense RNA, siRNA, shRNA, and the like.

A further embodiment of the present invention is a method for producing a modified capsid protein, the method comprising:

a) expressing a nucleic acid encoding a modified capsid protein under appropriate conditions, and

b) a step of isolating the expressed capsid protein of step a).

In some embodiments, a viral particle as described herein comprises a mosaic capsid, e.g., a capsid comprising a genetically modified capsid protein as described herein (with or without covalent linkage to a targeting ligand), in a specific ratio with a reference capsid protein. Methods for producing such mosaic viral particles include:

a) under suitable conditions, expressing a nucleic acid encoding a modified capsid protein and a nucleotide encoding a reference capsid protein in a ratio of at least about 60:1 to about 1:60, for example, 2:1, 1:1, 3:5, 1:2, 1:3, 1:8, etc. (wild type/wild type), and

b) a step of isolating the expressed capsid protein of step a).

In some embodiments, the compositions described herein, or the methods described herein, combine the modified cap gene:reference cap gene (or combination of reference cap genes) in a ratio ranging from at least about 1:60 to about 60:1, for example, 2:1, 1:1, 3:5, 1:2, 1:3, 1:8, etc. In some embodiments, the ratio is at least about 1:2. In some embodiments, the ratio is at least about 1:3. In some embodiments, the ratio is at least about 1:4. In some embodiments, the ratio is at least about 1:5. In some embodiments, the ratio is at least about 1:6. In some embodiments, the ratio is at least about 1:7. In some embodiments, the ratio is at least about 1:8. In some embodiments, the ratio is at least about 1:9. In some embodiments, the ratio is at least about 1:10. In some embodiments, the ratio is at least about 1:11. In some embodiments, the ratio is at least about 1:12. In some embodiments, the ratio is at least about 1:13. In some embodiments, the ratio is at least about 1:14. In some embodiments, the ratio is at least about 1:15. In some embodiments, the ratio is at least about 1:16. In some embodiments, the ratio is at least about 1:17. In some embodiments, the ratio is at least about 1:18. In some embodiments, the ratio is at least about 1:19. In some embodiments, the ratio is at least about 1:20. In some embodiments, the ratio is at least about 1:25. In some embodiments, the ratio is at least about 1:30. In some embodiments, the ratio is at least about 1:35. In some embodiments, the ratio is at least about 1:40. In some embodiments, the ratio is at least about 1:45. In some embodiments, the ratio is at least about 1:50. In some embodiments, the ratio is at least about 1:55. In some embodiments, the ratio is at least about 1:60. In some embodiments, the ratio is at least about 2:1. In some embodiments, the ratio is at least about 3:1. In some embodiments, the ratio is at least about 4:1. In some embodiments, the ratio is at least about 5:1. In some embodiments, the ratio is at least about 6:1. In some embodiments, the ratio is at least about 7:1. In some embodiments, the ratio is at least about 8:1. In some embodiments, the ratio is at least about 9:1. In some embodiments, the ratio is at least about 10:1. In some embodiments, the ratio is at least about 11:1. In some embodiments, the ratio is at least about 12:1. In some embodiments, the ratio is at least about 13:1. In some embodiments, the ratio is at least about 14:1. In some embodiments, the ratio is at least about 15:1. In some embodiments, the ratio is at least about 16:1. In some embodiments, the ratio is at least about 17:1. In some embodiments, the ratio is at least about 18:1. In some embodiments, the ratio is at least about 19:1. In some embodiments, the ratio is at least about 20:1. In some embodiments, the ratio is at least about 25:1. In some embodiments, the ratio is at least about 30:1. In some embodiments, the ratio is at least about 35:1. In some embodiments, the ratio is at least about 40:1. In some embodiments, the ratio is at least about 45:1. In some embodiments, the ratio is at least about 50:1. In some embodiments, the ratio is at least about 55:1. In some embodiments, the ratio is at least about 60:1.

In some embodiments, the ratio of VP protein subunits within a mosaic virus particle may, but does not necessarily, stoichiometrically reflect the ratio of the modified cap gene to the reference cap gene. As a non-limiting example, the mosaic capsid formed according to the method may, but does not necessarily, be considered to have a modified capsid protein to reference capsid protein ratio similar to the ratio of the nucleic acids encoding the modified capsid proteins (wild-type:wild-type) used to produce the mosaic capsid. In some embodiments, the mosaic capsid comprises a protein subunit ratio of about 1:59 to about 59:1.

A further embodiment of the present invention is a method of altering the tropism of a virus, the method comprising: (a) inserting a nucleic acid encoding an amino acid sequence into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the amino acid sequence, and/or (b) incubating packaging cells under conditions sufficient to produce virus particles, wherein the packaging cells comprise the nucleic acid. A further embodiment of the present invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising: (a) expressing, under suitable conditions, a nucleic acid encoding a viral capsid protein modified as described herein (and optionally nucleotides encoding a reference capsid protein), wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair, (b) isolating the expressed capsid protein comprising the first member of the specific binding pair of step (a) or a capsid comprising it, and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under suitable conditions that allow formation of an isopeptide bond between the first and second members, wherein the second cognate member of the specific binding pair is fused to a targeting ligand.

In some embodiments, the packaging cells further comprise a helper plasmid and/or a transfer plasmid comprising the nucleotide of interest. In some embodiments, the method further comprises isolating self-complementary adeno-associated virus particles from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating single-stranded adeno-associated virus particles from the cell lysate. In some embodiments, the method further comprises (a) removing cell debris, (b) treating the supernatant containing the viral particles with a nuclease (e.g., DNase I) and MgCl ₂ , (c) concentrating the viral particles, (d) purifying the viral particles, and (e) any combination of (a) to (d).

Packaging cells useful for producing the virus particles described herein include, for example, animal cells that are permissive to the virus, or cells modified to be permissive to the virus; or packaging cell constructs that utilize, for example, a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing the viral particles described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] no. CRL-1573), HEK-293 cells containing SV40 large T-antigen (HEK-293T or 293T), HEK293T/17 cells, the human sarcoma cell line HT-1080 (CCL-121), the lymphoblast-like cell line Raji (CCL-86), the glioblastoma-astrocytoma epithelial-like cell line U87-MG (HTB-14), the T-lymphoma cell line HuT78 (TIB-161), NIH/3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC no. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC no. CCL-2), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells, etc.

L929 cells, a FLY virus packaging cell system outlined in Cosset et al. [(1995) J Virol 69,7430-7436], include NS0 (murine myeloma) cells, human amniotic fluid cells (e.g., CAP, CAP-T), yeast cells (e.g., S. cerevisiae, Pichia pastoris), plant cells (e.g., but not limited to, S. cerevisiae, Pichia pastoris), plant cells (e.g., but not limited to, tobacco NTI, BY-2), insect cells (e.g., but not limited to, SF9, S2, SF21, Tni (e.g., High 5)), or bacterial cells (e.g., but not limited to, Escherichia coli).

For additional packaging cells and systems, packaging techniques and particles for packaging nucleic acid genomes into pseudotyped viral particles, see, e.g., Polo et al., Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express viral components, or transiently transfecting cells with plasmids.

Additional embodiments include methods comprising contacting a Cap protein with a targeting vector under conditions sufficient to operably link a modified Cap protein as described herein to the targeting vector, e.g., via chemical linkage and/or association of the first and second members of a specific binding pair, wherein the first member is inserted into the modified Cap protein, and the first member and the targeting vector are fused to the second member of the specific binding pair.

Additional embodiments include methods of redirecting a virus into a target cell (e.g., a muscle stem cell, a myoblast, a muscle cell, any combination thereof, etc.) and/or methods of delivering a reporter gene or a therapeutic gene to such a target cell, the methods including methods of transducing a target cell in vitro (e.g., ex vivo) or in vivo, the methods including the step of contacting a target cell with a viral particle comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the target cell is in vitro (e.g., ex vivo). In other embodiments, the target cell is in vivo in a subject, e.g., a human.

target cells

A wide variety of cells can be targeted for delivery of a nucleotide of interest using modified viral particles as disclosed herein. Target cells are generally selected based on the nucleotide of interest and the desired effect.

In some embodiments, the nucleotide of interest can be delivered to a target cell (e.g., a muscle stem cell, a myoblast, a muscle cell, or any combination thereof) such that the target cell produces a protein that compensates for an enzyme deficiency or an immune deficiency, such as X-linked severe combined immunodeficiency, in the organism. Thus, in some embodiments, cells that normally produce the protein in the animal are targeted. In other embodiments, cells within the region where the protein would be most beneficial are targeted.

In another embodiment, a nucleotide of interest, such as a gene encoding siRNA, can inhibit the expression of a specific gene in a target cell (e.g., muscle stem cells, myoblasts, muscle cells, or any combination thereof). The nucleotide of interest can, for example, inhibit the expression of a gene involved in the pathogen life cycle. Thus, cells susceptible to pathogen infection or cells infected with a pathogen can be targeted. In another embodiment, the nucleotide of interest can inhibit the expression of a gene responsible for toxin production in a target cell.

In another embodiment, the nucleotide of interest may encode a toxic protein that kills cells expressing the toxic protein. In this case, tumor cells or other unwanted cells may be targeted.

In another embodiment, there is a nucleotide of interest that encodes a therapeutic protein. In some embodiments, the nucleotide of interest encodes a therapeutic protein comprising an antigen binding protein or an antigen binding portion thereof. In some embodiments, the nucleotide of interest encodes a therapeutic protein comprising a human or humanized antibody or antigen binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a trispecific antibody, or a chemically modified derivative thereof. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), wherein n is 1 to 10.

Once a specific population of target cells is identified where expression of the nucleotide of interest is desired, a target that is specifically expressed in that population of target cells is selected. The target may be expressed exclusively in that cell population, or may be expressed to a greater extent in that cell population than in other cell populations. The more specific the expression, the more specific the delivery to the target cells. Depending on the situation, the desired amount of marker specificity (and therefore, the desired amount of gene delivery) may vary. For example, when introducing a toxic gene, high specificity is most desirable to avoid killing non-targeted cells. For expression of a harvest protein or secreted product, where a global impact is desired, lower marker specificity may be necessary.

As described above, the target can be any cell surface moiety, such as a protein, from which a targeting ligand can be identified or generated. Preferably, the target is a peptide or polypeptide, such as a receptor. However, in other embodiments, the target can be a carbohydrate or other molecule that can be recognized by the binding partner. If the binding partner (e.g., ligand) of the target is already known, it can be used as the affinity molecule. However, if the binding molecule is not known, an antibody against the target can be generated using standard procedures. The antibody can then be used as the targeting ligand.

Thus, target cells can be selected based on a variety of factors, including, for example, (1) the application (e.g., treatment, expression of the protein to be harvested, and conferring disease resistance) and (2) expression of a marker with a desired amount of specificity.

The target cell is not limited in any way and includes both germline cells and cell lines, as well as somatic cells and cell lines. If the target cell is a germline cell, the target cell is preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES cells).

In various embodiments, the target cell is a cell expressing a terminally undifferentiated muscle cell surface protein. In some embodiments, the target cell is a cell expressing a mammalian terminally undifferentiated muscle cell surface protein. In some embodiments, the target cell is a cell expressing a human terminally undifferentiated muscle cell surface protein. In some embodiments, the target cell is a cell expressing CDH15, e.g., mammalian CDH15, e.g., human CDH15.

In various embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a mammalian muscle cell. In some embodiments, the target cell is a mammalian muscle cell. In some embodiments, the target cell is a non-terminal differentiated mammalian muscle cell. In some embodiments, the target cell is a terminally differentiated mammalian muscle cell. In some embodiments, the target cell is a muscle stem cell (also known as a satellite cell), a myoblast, a myocyte, or an undifferentiated muscle fiber. In some embodiments, the target cell is a mammalian rhabdomyosarcoma cell.

Skeletal muscle disorders

Also provided herein are methods for treating skeletal muscle related disorders, such as muscle wasting diseases and/or genetic muscle diseases, such as X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), myosophic brachial dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, dystroglycanosis, muscular atrophy, metabolic diseases, and the like.

Generally, these methods comprise administering to a patient suffering from or at risk for such a skeletal disorder a viral particle or pharmaceutical composition as described herein, wherein the viral particle comprises:

(i) a viral capsid modified to include the first member of a protein:protein binding pair;

(ii) a second member of a protein:protein binding pair, wherein the second member of the protein:protein binding pair comprises a targeting ligand that binds to a terminally differentiated muscle cell surface protein expressed on the surface of muscle cells (e.g., CDH15);

The first member of the protein:protein binding pair and the second member of the protein:protein binding pair combine to direct the tropism of the viral capsid to the patient's muscle cells), and

(iii) a nucleotide of interest encapsulated within the viral capsid.

In some embodiments, the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR/Cas system or portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. For example, in some embodiments, the nucleotide of interest may encode a growth factor, a neurotrophic factor, a disease-modifying muscle protein, such as a metabolic protein for muscular dystrophy or a metabolic disease.

muscle-related cancers

Also provided herein are methods for treating muscle-associated cancers, such as cancers expressing a terminally undifferentiated muscle cell protein, such as CDH15. Non-limiting examples of muscle-specific cancers include rhabdomyosarcomas, such as embryonal, alveolar, pleomorphic, staphylococcal, and spindle/sclerotic rhabdomyosarcomas.

Generally, such methods comprise administering to a patient suffering from or at risk for such skeletal cancer a viral particle or pharmaceutical composition as described herein, wherein the viral particle comprises:

(i) a viral capsid modified to include a targeting ligand, wherein the targeting ligand is inserted directly into the viral capsid or is inserted, for example, via a first member of a protein:protein binding pair and a cognate second member of the protein:protein binding pair, wherein the second member of the protein:protein binding pair comprises a targeting ligand that binds to a muscle-specific surface protein expressed on the surface of non-terminally differentiated muscle cells (e.g., CDH15), and wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair combine to direct tropism of the viral capsid to the muscle cells of the patient, and

(ii) a nucleotide of interest encapsulated within the viral capsid.

In some embodiments, the nucleotide of interest comprises or encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR/Cas system or portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule capable of treating a muscle-associated cancer, e.g., slowing and/or stopping the growth of and/or destroying a muscle-associated cancer. Exemplary, non-limiting embodiments of the nucleotide of interest include a therapeutic protein that targets and inhibits an oncogenic intracellular signaling pathway, a suicide gene that induces a cell to undergo apoptosis, and an RNAi molecule that targets and inhibits an oncogenic transcript.

Exemplary sequences of non-primate adeno-associated viruses. explanation Deposit number/GI number ITR Region encoding nonstructural/rep proteins
(Protein Accession Number/GI Number) The region that encodes the capsid
Protein (Protein Accession Number/GI Number) avian adeno-associated virus Avian adeno-associated virus isolate YZ-1, complete genome GQ368252.1/ GI: 255710032 243-2237 (ACU30841.1/ GI: 255710033) 2255-4471 (ACU30842.1/ GI: 255710034) Avian adeno-associated virus isolate ZN1, complete genome KF937794.1/ GI: 588284633 1-142
4540-4682 244-2235 (AHK22792.1/ GI: 588284634) 2253-4469 (AHK22793.1/ GI: 588284635) Avian adeno-associated virus ATCC VR-865 Avian adeno-associated virus ATCC VR-865, complete genome AY186198.1/ GI: 31414777 1-142

4552-4694 244-2232 (AAO32086.1/ GI: 31414778) 2250-4481 (AAO32087.1/ GI: 31414779) Avian adeno-associated virus ATCC VR-865, complete genome NC_004828.1/ GI: 31543992 1-142
4552-4694 244-2232 (NP_852780.1/ GI: 31543993) 2250-4481 (NP_852781.1/ GI: 31543994) Avian adeno-associated virus ATCC VR-865, complete genome AY629582.1/ GI: 48996102 1-142
4552-4694 244-223 2 (AAT48612.1/ GI: 48996103) 2250-4481 (AAT48613.1/ GI: 48996104) Avian adeno-associated virus strain DA-1 Avian adeno-associated virus strain DA-1, complete genome AY629583.1/ GI: 48996105 1-142

4540-4682 244-2235 (AAT48614.1/ GI: 48996106) 2253-4469 (AAT48615.1/ GI: 48996107) Avian adeno-associated virus strain DA-1, complete genome NC_006263.1/ GI: 51949968 1-142
4540-4682 244-2235 (YP_077182.1/ GI: 51949969) 2253-4469 (YP_077183.1/ GI: 51949970) Bat adeno-associated virus YNM Bat adeno-associated virus YNM, complete sequence GU226971.1/ GI: 28971900 8 200-2041 (ADD17085.1/ GI: 289719009) 2059-4233 (ADD17086.1/ GI: 289719010) Bat adeno-associated virus YNM, complete genome NC_014468.1 / GI: 30442291 6 200-2041 (YP_003858571.1/ GI: 304422917) 2059-4233 (YP_003858572.1/ GI: 304422918) bovine adeno-associated virus Bovine adeno-associated virus, complete genome AY388617.1/
GI: 38679253 368-2200 (AAR26464.1/ GI: 38679254) 2216-4426 (AAR26465.1/ GI: 38679255) Bovine adeno-associated virus, complete genome NC_005889.1 / GI: 48696557 368-2200 (YP_024970.1/ GI: 48696558) 2216-4426 (YP_024971.1/ GI: 48696559) bovine parvovirus-2 Putative nonstructural protein and putative capsid protein genes of bovine parvovirus 2, complete cds. AF406966.1/
GI: 15825370 306-1919 (AAL09671.1/ GI: 15825371) 2268-5384 (AAL09672.1/ GI: 15825372) Bovine parvovirus 2, complete genome NC_006259.1 / GI: 51949957 306-1919 (YP_077175.1/ GI: 51949958) 2268-5384 (YP_077176.1/ GI: 51949959) California sea lion adeno-associated virus 1 California sea lion adeno-associated virus 1 isolate 1136 Rep78 and VP1 genes, complete cds. JN420371.1/ GI: 34345896 6 284-2086 (AEM37641.1/ GI: 343458967) 2117-4273 (AEM37642.1/ GI: 343458968) California sea lion adeno-associated virus 1 isolate 1187 Rep78 and VP1 genes, complete cds. JN420372.1/ GI: 343458969 291-2093 (AEM37643.1/ GI: 343458970) 2124-4280 (AEM37644.1/ GI: 343458971) goose parvovirus Goose parvovirus virulent B strain, complete genome (U25749.1/ GI 1113795) 1-444

4663-5106 537-2420 (AAA83229.1/ GI: 1113796) 2439-4637 (AAA83230.1/ GI: 1113797) 2874-4637 (AAA83231.1/ GI: 1113798)

3033-4637 (AAA83232.1/ GI: 1113799) Goose parvovirus, complete genome (NC_001701.l/ GI: 9628649) 1-444
4663-5106 537-2420 (NP_043514.1/ GI: 9628650) 2439-4637 (NP_043515.1/ GI: 9628651)

2874-4637 (NP_043516.1/ GI: 9628652)

3033-4637 (NP_043517.1/ GI: 9628653) Goose parvovirus strain 82-0321V, complete genome (EU583389.1/ GI: 19088818 8) 1-381
4600-4980 474-2357 (ACE95848.1/ GI: 190888189) 2376-4574 (ACE95849.1/ GI: 190888190) Goose parvovirus strain 82-0321, complete genome (EU583390.1/ GI: 19088819 1) 1-416
4635-5050 509-2392 (ACE95850.1/ GI: 190888192) 2411-4609 (ACE95851.1/ GI: 190888193) Goose parvovirus strain 06-0329, complete genome (EU583391.1/GI: 190888194) 1-418

4637-5054 511-2394 (ACE95852.1/ GI: 190888195) 2413-4611 ACE95853.1/
GI: 190888196) Goose parvovirus strain VG32/1, complete genome (EU583392.1/ GI: 190888197) 1-443
4662-5104 536-2419 (ACE95854.1/ GI: 190888198) 2438-4636 (ACE95855.1/ GI: 190888199) Goose parvovirus strain SH, complete genome (JF333590.1/ GI: 343113618) 1-444
4663-5106 537-2420 (AEL87777.1/ GI: 343113619) 2439-4637 (AEL87778.1/ GI: 343113620)

2874-4637 (AEL87779.1/ GI: 343113621)

3033-4637 (AEL87780.1/ GI: 343113622) Goose parvovirus strain GDaGPV, complete genome (HQ891825.1/ GI: 359843307) 1-444
4663-5106 537-2420 (AEV89789.1/ GI: 359843308) 2439-4637 (AEV89790.1/ GI: 359843309)

2874-4637 (AEV89791.1/ GI: 359843310)

3033-4637 (AEV89792.1/ GI: 359843311) Goose parvovirus strain SHFX1201, complete genome (KC478066.1 / GI: 459360514) 1-416 509-23 92 (AGG56527.1/ GI: 459360515) 2411-4609 (AGG56528.1/ GI: 459360516) Goose parvovirus strain Y, complete genome (KC178571.1/ GI: 513129761) 1-444 537-2420 (AGO 17637.1/ GI: 513129762) 2439-4637 (AGO17638.1/ GI: 513129763) Goose parvovirus strain E, complete genome (KC184133.1 / GI: 513129764) 1-443 536-2419 (AGO17639.1/ GI: 513129765) 2438-46.36 (AGO17640.1/ GI: 513129766) Goose parvovirus strain SYG61v, complete genome (KC996729.1 / GI: 531997217) 1-442 535-2418 (AGT62580.1/ GI: 531997218) 243 7-4635 (AGT62581.1/ GT: 531997219) Goose parvovirus strain YZ99-6, complete genome (KC996730.1 / GI: 531997220) 1-414 507-23 90 (AGT62582.1/ GI: 531997221) 2409-4607 (AGT62583.1/ GI: 531997222) Goose parvovirus nonstructural protein NS (ns) and capsid protein VP (vp) genes, complete cds. (AF416726.1/ GI: 17226299) 1-1884 (AAL37721.1/ GI: 17226300)529-1884 (ABP93843.1/ GI: 145694447) 1903-4101 (AAL37722.1/ GI: 17226301) Goose parvovirus strain DY NS1 (NS1), NS2 (NS2), VP1 (VP1), VP2 (VP2), and VP3 (VP3) genes, complete cds. (EF515837.1/ GI: 145694445) 1-1884 (ABP93842.1/ GI: 145694446) 1903-4101 (ABP82770.1/ GI: 145573169)
2338-4101 (ABP93844.1/ G 1: 145694448)

2497-4101 (ABP93845.1/ GI: 145694449) Goose parvovirus strain PT NS1 protein and VP1 protein genes, complete cds. (JF926695.1/ GI: 354463162) 9-1892 (AER25356.1/ GI: 354463163) 1911-4109 (AER25357.1/ GI: 354463164) Goose parvovirus strain D NS1 protein and VP1 protein genes, complete cds. (JF926696.1/ GI: 354463165) 6-1889 (AER25358.1/ GI: 354463166) 1908-4106 (AER25359.1/ GI: 354463167) mouse adeno-associated virus 1 Mouse adeno-associated virus 1 rep gene, partial cds; and VP1 capsid, VP2 capsid, and VP3 capsid genes, complete cds. DQ100362.1/ GI: 73665994 1-327 (AAZ79671.1/ GI: 73665995) 344-2485 (AAZ79672.1/ GI: 73665996)

731-2485 (AAZ79673.1/ GI: 73665997)

911-2485 (AAZ79674.1/ GI: 73665998) Muscovy duck parvovirus Wild duck parvovirus REP protein (rep) and three capsid protein VP (vp) genes, complete cds U22967.1/ GI: 1113784 1-457

4678-5132 548-2431 (AAA83224.1/ GI: 1113785) 2450-4648 (AAA83225.1/ GI: 1113786)

2885-4648 (AAA83226.1/ GI: 1113787)

3044-4648 (AAA83227.1/ GI: 1113788) Muscovy duck parvovirus, complete genome NC_006147.2/ GI: 51593841 1-457
4678-5132 548-2431 (YP_068410.1/ GI: 51593 842) 2450-4648 (YP_068411.1/ GI: 51593843)

2885-4648 (YP_068412.1; GI: 51593844)

3044-4648 (YP_068413.1/ GI: 51593845) Muscovy duck parvovirus strain P REP protein and VP1 protein genes, complete cds. JF926697.1/ GI: 354463168 61-1944 (AER25360.1/ GI: 354463169) 1963-4161 (AER25361.1/ GI: 354463170) Muscovy duck parvovirus strain P1 REP protein and VP1 protein genes, complete cds. JF926698.1/ GI: 354463171 61-1944 (AER25362.1/ GI: 354463172) 1963-4161 (AER25363.1/ GI: 354463173) Muscovy duck parvovirus isolate SAAS-SHNH, complete genome KC171936.1/ GI: 459256867 1-381
4681-5061 512-2395 (AGG53766.1/ GI: 459256868) 2414-4612 (AGG53768.1/ GI: 459256870)

2849-4612 (AGG53769.1/ GI: 459256871)

3008-4612 (AGG53767.1/ GI: 459256869) snake adeno-associated virus 2 Large snake adeno-associated virus 2 nonstructural protein 1 and capsid protein genes, partial cds EU872429.1/ GI: 215401981 1-642 (ACJ66590.1/ GI: 215401982) 661-1297 (ACJ66591.1/ GI: 215401983) Snake parvovirus 1 Snake parvovirus 1 nonstructural protein 1 (NS1) and capsid protein (VP1) genes, complete cds. AY349010.1/ GI: 38017148 324-2012 (AAR07954.1/ GI: 38017149) 2030-4210 (AAR07955.1/ GI: 38017150) Snake parvovirus 1, complete genome NC_006148.1/ GI: 51555744 324-2012 (YP_068093.1/ GI: 51555745) 2030-4210 (YP_068094.1/ GI: 51555746)

A brief description of the sequences in the sequence list Sequence number Brief explanation 1-786 anti-human CDH15 antibody or binding portion thereof 787 human CDH15 CDS 788 human CDH15 protein 789 GGGGS linker 790-802 synthetic heavy chain constant domain 803-814 synthetic Fc domain 815 SpyTag protein 816 SpyCatcher protein 817 KTag protein 818 c-myc protein 819 B1 epitope protein 820 isopeptidyl peptide-tagged protein 821 SpyTag002 protein 822 SpyTag003 protein 823 GLSG protein linker 824 GLSGSG protein linker 825 GLSGLSGS protein linker 826 GLSGLSGLSG protein linker 827 GLSGGSGLSG protein linker 828 GSGESG protein linker 829 Artificial hGH pA-specific primer 830 Artificial 5' barcode primer 831 Artificial 3' barcode primer 832 9295 mAb HC hIgG4US SpyCatcher DNA 833 9295 mAb HC hIgG4US SpyCatcher AA 834 9295 mAb/Fab LC DNA 835 9295 mAb/Fab LC AA 836 9295 Fab HC SpyCatcher Fab DNA 837 9295 Fab HC SpyCatcher Fab AA

Example

Example 1: Cadherin 15 regulates the transition of MuSCs from a quiescent to an activated state.

Muscle stem cells (MuSCs) are mitotically quiescent and non-proliferating under quiescent conditions in adult muscle tissue. However, MuSCs actively divide in response to injury, producing daughter cells. Some daughter cells become quiescent once more to replenish the MuSC pool, while others continue to proliferate as myoblasts, which align and fuse with each other during differentiation to form mature myotubes/myofibers that constitute muscle fibers. Quiescent muscle stem cells can be characterized by Pax7 expression, proliferating myoblasts can be characterized by Pax7 and myogenic differentiation factor 1 (MyoD) expression, terminally differentiating myoblasts (i.e., myocytes) can be characterized by MyoD and myogenin expression, and myotubes can be characterized by myosin heavy chain (MyHC) expression.

Cadherin 15 (CDH15) mRNA is highly expressed in MuSCs, and expression increases after muscle injury. To determine the role CDH15 plays in skeletal muscle biology, homozygous CDH15 knockout mice (CDH15 ^-/- ) were generated. Although deleting CDH15 expression did not appear to affect MuSC quiescence, proliferation, or differentiation, CDH15 ^-/- mice exhibited accelerated muscle regeneration after injury. As shown in Figures 2C-2D , CDH15 ^-/- mice exhibited increased muscle fiber size at days 5 and 15 after injury induced by cardiotoxin (CTX) injection into the tibialis anterior muscle, and an increase in the number of central myonuclei in muscle fibers at day 15 post-injury. Furthermore, CDH15 ^-/- mice exhibited improved functional recovery after CTX injury, as evidenced by normal stiffness measurements in ex vivo isolated extensor digitorum longus muscles 15 days after CTX injury (see Figure 3B ). Notably, WT mice exhibited approximately 40% loss of stiffness in ex vivo isolated muscles 15 days after injury.

To determine how deleting CDH15 in mice improves skeletal muscle regeneration, MuSCs derived from WT or CDH15 ^-/- mice were cultured ex vivo with their associated myofibers for 48 h. As shown in Figures 4A-4B , a greater percentage of multicellular clusters and a greater number of Pax7 ⁺ cells per cluster were observed in cultured CDH15 ^-/- MuSCs compared to WT MuSCs. These results suggest that deleting CDH15 expression in MuSCs accelerates the transition from a quiescent to an activated state.

To further explore how deleting CDH15 expression in MuSCs affects the expression of other genes involved in muscle regeneration, RNA-seq was performed on FACS-isolated MuSCs from intact, healthy mice. As shown in Figure 5A , 132 genes were downregulated and 270 genes were upregulated in CDH15 ^−/− MuSCs compared to WT MuSCs. Among these upregulated genes, a gene cluster containing serum response factor (SRF) binding motifs was identified (see Figure 5B ), suggesting that the accelerated activation of MuSCs in the absence of CDH15 may be due to altered SRF signaling.

Example 2: Deleting CDH15 Expression in Aged Mice Restores Muscle Regeneration

Muscle stem cells (MuSCs) from aged subjects exhibit delayed activation and reduced motility in vitro, which leads to impaired MuSC-mediated repair in vivo. Therefore, we hypothesized that CDH15 may be involved in the decreased muscle regenerative capacity in aged subjects. To test this, we generated homozygous CDH15 knockout mice (CDH15 ^-/- ) up to 23 months of age, and induced muscle injury by intramuscular injection of cardiotoxin into the tibialis anterior muscle (day 0). At 15 days post-injury (dpi), histology was performed on tibialis anterior muscle samples from aged (23 months of age) CDH15 ^-/- mice and aged (23 months of age) WT controls (see Figures 6a-6b ). As shown in Figures 6c-6d , at 15 dpi, CDH15 ^-/- mice exhibited an increase in muscle fiber size ( Figure 6c ) and an increase in the number of muscle fibers with three or more central nuclei ( Figure 6d ) compared to WT mice of the same age. Notably, the muscle fiber size of old CDH15 ^-/- mice was similar to that of young WT mice ( Figure 6c , see dotted line), suggesting that eliminating CDH15 expression can restore the muscle regeneration capacity of old mice to that of young mice.

Example 3: Example of CDH15 antibody

Generation of anti-human CDH15 antibodies

Anti-human CDH15 antibodies are obtained by immunizing a mouse with human CDH15 (see, e.g., engineered mice comprising DNA encoding human immunoglobulin heavy chain and human kappa light chain variable regions, U.S. Patent No. 7,105,348; U.S. Patent No. 8,642,835; and U.S. Patent No. 9,622,459, each incorporated herein by reference).

After immunization, splenocytes were harvested from each mouse and either (1) fused with mouse myeloma cells to maintain viability and form hybridoma cells that were screened for human CDH15 specificity, or (2) bound to reactive antibodies (antigen-positive B cells) and sorted using human CDH15 fragments as sorting reagents to identify these B cells (as described in US 2007/0280945A1).

For example, chimeric antibodies to human CDH15 having human variable regions and mouse constant regions were first isolated using VELOCIMMUNE technology, as described in U.S. Patent No. 7,105,348; U.S. Patent No. 8,642,835; and U.S. Patent No. 9,622,459 (each of which is incorporated herein by reference).

In some antibodies for testing purposes, the mouse constant region was replaced with a desired human constant region, such as a wild-type human CH or a modified human CH (e.g., IgG1, IgG2, or IgG4 isotype) and a light chain constant region (CL), to generate a complete anti-hCDH15 antibody or antigen-binding portion thereof. While the constant region selected can vary widely depending on the specific application, the high-affinity antigen-binding properties and target specificity properties reside in the variable region.

Certain biological characteristics of exemplary anti-human CDH15 antibodies generated according to the methods of the present invention are described in detail in the Examples presented below.

Heavy and light chain variable region amino acid and nucleic acid sequences of anti-hCDH15 antibodies

Table 1 sets forth the sequence identifiers of nucleic acid (NA) sequences encoding the heavy or light chain variable region (HCVR or LCVR, respectively) or the heavy or light chain CDRs (HCDR and LCDR, respectively) of selected anti-hCDH15 antibodies used to generate the therapeutic anti-hCDH15 proteins disclosed herein, with the sequence identifiers of the amino acid (AA) sequences of said HCVR or LCVR or HCDR and LCDR set forth in parentheses. These sequences are also included below.

Sequence number 1

CAGGTGCAGCTGGTGGAGTCGGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTATCTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTTATATCACATGATGGAAATAATAGATAC TATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAAAGGGGTAGCAGTGACCCCCTTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 2

QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYVMHWVRQAPGKGLEWVAVISHDGNNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGVAVTPFDHWGQGTLVTVSS

Sequence number 3

GGATTCACCTTCAGTATCTATGTC

Sequence number 4

GFTFSIYV

Sequence number 5

ATATCACATGATGGAAATAATAGA

Sequence number 6

ISHDGNNR

Sequence number 7

GCGAAAGGGGTAGCAGGTGACCCCCTTTGACCAC

Sequence number 8

AKGVAVTPFDH

Sequence number 9

GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCCTACACAGTGATGGAAACACCTACTTGAGTTGGCTTCAGCAGCGGCCAGGCCAGCCTCCAAGACTCCTAATTTATAAGACT TCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGTTGGAAGCTGAGGATGTCGGCATTTATTACTGCATGCAAGCTACACAATTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Sequence number 10

DIVMTQTPLSSPVTLGQPASISCRSSQSLLHSDGNTYLSWLQQRPGQPPRLLIYKTSNRFSGVPDRFSGSGAGTDFTLKISRLEAEDVGIYYCMQATQFPWTFGQGTKVEIK

Sequence number 11

CAAAGCTCCTACACAGTGATGGAAACACCTAC

Sequence number 12

QSLLHSDGNTY

Sequence number 13

AAGACTTCT

Sequence number 14

KTS

Sequence number 15

ATGCAAGCTACACAATTTCCGTGGACG

Sequence number 16

MQATQFPWT

Sequence number 17

CAGGTGCAGCTGGTGGAGTCGGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTATCTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTTATATCACATGATGGAAA TAATAGATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAAAGGGGTAGCAGTGACCCCCTTTGACCACTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGAGTTGAGTCCAAATATGGTCCCCCA TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACTGGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 18

QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYVMHWVRQAPGKGLEWVAVISHDGNNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGVAVTPFDHWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 19

GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCCTACACAGTGATGGAAACACCTACTTGAGTTGGCTTCAGCAGCGGCCAGGCCAGCCTCCAAGACTCCTAATTTATAAG ACTTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGTTGGAAGCTGAGGATGTCGGCATTTATTACTGCATGCAAGCTACACAATTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAA ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 20

DIVMTQTPLSSPVTLGQPASISCRSSQSLLHSDGNTYLSWLQQRPGQPPRLLIYKTSNRFSGVPDRFSGSGAGTDFTLKISRLEAEDVGIYYCMQATQFPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 21

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCATCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGATTGGGTGGCAGTTATTGCATATGACGGAAACAGTAAATACTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTATATCTACAAATGAACGGCCTGAGAGCTGAGGACACGGCTCTGTATTACTGTGCGAGAATCTATTATGGGTCGGGACTTATCTATACGGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCTCCTCA

Sequence number 22

QVQLVESGGGVVQPGRSLRLSCEASGFIFSSYGMHWVRQAPGKGLDWVAVIAYDGNSKYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTALYYCARIYYGSGLIYTDFWGQGTLVTVSS

Sequence number 23

GGATTCATCTTCAGTAGTTATGGC

Sequence number 24

GFIFSSYG

Sequence number 25

ATTGCATATGACGGAAACAGTAAA

Sequence number 26

IAYDGNSK

Sequence number 27

GCGAGAATCTATTATGGGTCGGGACTTATCTATACGGACTTC

Sequence number 28

ARIYYGSGLIYTDF

Sequence number 29

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGTATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGACATTAGCAATTATTTAGCCTGGTATCAACAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTT TGCAATCAGGGGTCCCATCTCGGATCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTTTAACAGTGCCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

Sequence number 30

DIQMTQSPSSLSVSVGDRVTITCRASQDISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRISGSGSGTDFTLTISSLQPEDVATYYCQKFNSAPFTFGPGTKVDIK

Sequence number 31

CAGGACATTAGCAATTAT

Sequence number 32

QDISNY

Sequence number 33

GCTGCATCC

Sequence number 34

AAS

Sequence number 35

CAAAAGTTTAACAGTGCCCCATTCACT

Sequence number 36

QKFNSAPFT

Sequence number 37

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCATCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGATTGGGTGGCAGTTATTGCATATGACGGAAAC AGTAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTATATCTACAAATGAACGGCCTGAGAGCTGAGGACACGGCTCTGTATTACTGTGCGAGAATCTATTATGGGTCGGGACTTATCTATACGGACTTCTGGGGC CAGGGAACCCTGGTCACCGTCTCTCCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC CCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGG ATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 38

QVQLVESGGGVVQPGRSLRLSCEASGFIFSSYGMHWVRQAPGKGLDWVAVIAYDGNSKYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTALYYCARIYYGSGLIYTDFWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 39

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGTATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGACATTAGCAATTATTTAGCCTGGTATCAACAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATGCTGCATCCACTTT GCAATCAGGGGTCCCATCTCGGATCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTTTAACAGTGCCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 40

DIQMTQSPSSLSVSVGDRVTITCRASQDISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRISGSGSGTDFTLTISSLQPEDVATYYCQKFNSAPFTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 41

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGTTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAGATCAGTCATACTGGAAGGACCAACTAC AACCCGTCCCTCAAGAGTCGAGTCACCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCGTGTGGCAGCAGCGGGGTTGGACGTCTGGGGCCTAGGGACCACGGTCACCGTCTCCTCA

Sequence number 42

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSVYYWSWIRQPPGKGLEWIGEISHTGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARGRVAAAGLDVWGLGTTVTVSS

Sequence number 43

GGTGGGTCCTTCAGTGTTTACTAC

Sequence number 44

GGSFSVYY

Sequence number 45

ATCAGTCATACTGGAAGGACC

Sequence number 46

ISHTGRT

Sequence number 47

GCGAGAGGCCGTGTGGCAGCAGCGGGGTTGGACGTC

Sequence number 48

ARGRVAAAGLDV

Sequence number 49

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

Sequence number 50

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

Sequence number 51

CAGAGCATTAGCAGCTAT

Sequence number 52

QSISSY

Sequence number 53

CAACAGAGTTACAGTACCCCTCCGATCACC

Sequence number 54

QQSYSTPPIT

Sequence number 55

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGTTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAGATCAGTCATACTGGAAG GACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCGTGTGGCAGCAGCGGGGTTGGACGTCTGGGGCCTAGGGA CCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGAGTTGAGTCCAAATATGGTCCCCCA TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACTGGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 56

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSVYYWSWIRQPPGKGLEWIGEISHTGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARGRVAAAGLDVWGLG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 57

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 58

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 59

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAAAATCTCCTGTGCAACTTCTGGATTCACCTTCGGTGGCTCTGATATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTTGGCCGCATTTCAAAGAATGCAAATACTCACGGCGGCAGAATATGGT GCGTCGGTGAAAGGCAGATTCACCATCTCCAGAGATGATTCAAAGAACACGGCGTTTCTGCAATTGAGCAGCCTGAAAACCGAGGACACGGCCGTCTATTATTGTACTAGAGAATATAGTGGGAGTTACCCCCTGTACTTCTACGGTATGGACTTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

Sequence number 60

EVQLVESGGGLVQPGGSLKISCATSGFTFGGSDMHWVRQASGKGLEWVGRISKNANTHAAEYGASVKGRFTISRDDSKNTAFLQLSSLKTEDTAVYYCTREYSGSYPLYFYGMDFWGQGTTVTVSS

Sequence number 61

GGATTCACCTTCGGTGGCTCTGAT

Sequence number 62

GFTFGGSD

Sequence number 63

ATTTCAAAGAATGCAAATACTCACGGCGGCA

Sequence number 64

ISKNANTHAA

Sequence number 65

ACTAGAGAATATAGTGGGAGTTACCCCCTGTACTTCTACGGTATGGACTTC

Sequence number 66

TREYSGSYPLYFYGMDF

Sequence number 67

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAAAATCTCCTGTGCAACTTCTGGATTCACCTTCGGTGGCTCTGATATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTTGGCCGCATTTCAAAGAATGCAAATAC TCACGCGGCAGAATATGGTGCGTCGGTGAAAGGCAGATTCACCATCTCCAGAGATGATTCAAAGAACACGGCGTTTCTGCAATTGAGCAGCCTGAAAACCGAGGACACGGCCGTCTATTATTGTACTAGAGAATATAGTGGGAGTTACCCCCTGTACTTCTACGGTATGG ACTTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAA TATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTA CGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 68

EVQLVESGGGLVQPGGSLKISCATSGFTFGGSDMHWVRQASGKGLEWVGRISKNANTHAAEYGASVKGRFTISRDDSKNTAFLQLSSLKTEDTAVYYCTREYSGSYPLYFYGM DFWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 69

GAGGTGCAGCTGGTGGAGTCTGGGGGAGTCGTGGTCCAGCCTGGGGGATCCCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGATATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGATTATGTTTCAGCTATTACTACTACTGATGGGGGAAGTACTTTTTAT GCAGACTCTGTGAAGGGCAGATTCACCATTTCCAGAGACAATTCCAAGAATATGGTATATCTTCAAATGGGCAGCCTGAAACCTGAAGACATGGCTGTGTATTATTGTGCGAGAGATCGGCACTGGGGCTACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCCTCA

Sequence number 70

EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYAMHWVRQAPGKGLDYVSAITTDGGSTFYADSVKGRFTISRDNSKNMVYLQMGSLKPEDMAVYYCARDRHWGYWYFDLWGRGTLVTVSS

Sequence number 71

GGATTCACCTTCAGTAGATATGCT

Sequence number 72

GFTFSRYA

Sequence number 73

ATTACTACTGATGGGGGAAGTACT

Sequence number 74

ITTDGGST

Sequence number 75

GCGAGAGATCGGCACTGGGGCTACTGGTACTTCGATCTC

Sequence number 76

ARDRHWGYWYFDL

Sequence number 77

GAGGTGCAGCTGGTGGAGTCTGGGGGAGTCGTGGTCAGCCTGGGGGATCCCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGATATGCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGATTATGTTTCAGCTATTACTACTGATGGGGGA AGTACTTTTTATGCAGACTCTGTGAAGGGCAGATTCACCATTTCCAGAGACAATTCCAAGAATATGGTATATCTTCAAATGGGCAGCCTGAAACCTGAAGACATGGCTGTGTATTATTGTGCGAGAGATCGGCACTGGGGCTACTGGTACTTCGATCTCTGGGGCCGT GGCACCCTGGTCACCGTCTCTCCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCC CCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 78

EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYAMHWVRQAPGKGLDYVSAITTDGGSTFYADSVKGRFTISRDNSKNMVYLQMGSLKPEDMAVYYCARDRHWGYWYFDLWGR GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 79

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATGGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTAAAATGGGTCTCAATTATTAGTGCTAGTGGTGGTAGAACA TACTACGCAGACTCCGTGAAGGGCCGGTTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGGCGGGAATATAGTGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

Sequence number 80

EVQLVESGGGSVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLKWVSIISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGNIVAFDYWGQGTLVTVSS

Sequence number 81

GGATTCACCTTTAGTAGCTATGGC

Sequence number 82

GFTFSSYG

Sequence number 83

ATTAGTGCTAGTGGTGGTAGAACA

Sequence number 84

ISASGGRT

Sequence number 85

GCGGGGAATATAGTGGCCTTTGACTAC

Sequence number 86

AGNIVAFDY

Sequence number 87

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATGGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTAAAATGGGTCTCAATTATTAGTGCTAGTGGTG GTAGAACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGGGGAATATAGTGGCCTTTGACTACTGGGGCCAGGGAACCCTG GTCACTGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGC CCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 88

EVQLVESGGGSVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLKWVSIISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGNIVAFDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 89

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGAGTCCCTAAAACTCTCCTGTGCAGCCTCTGGGTTCGCCTTCAGTGTCTCTATTATACACTGGGTCCGCCAGAGTTCCGGGAAAGGGCTGGAGTGGATTGGCCGTATCAGAACCAAAACTAATAATTACGCGACAGGATATAGT GAGTCGATGAAGGGCAGGTTCACCATTTCCAGAGATGATTCAGAGAACACGGCGTATCTGCAAATGAACAGCCTCAAAACCGAAGACACGGCCGTATATTATTGTACTAGACATTATTATAGCAGCACCTTTTACTACTACTACGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

Sequence number 90

EVQLVESGGGLVQPGESLKLSCAASGFAFSVSIIHWVRQSSGKGLEWIGRIRTKTNNYATGYSESMKGRFTISRDDSENTAYLQMNSLKTEDTAVYYCTRHYYSSTFYYYYGMDVWGQGTTVTVSS

Sequence number 91

GGGTTCGCCTTCAGTGTCTCTATT

Sequence number 92

GFAFSVSI

Sequence number 93

ATCAGAACCAAAAACTAATAATTACGCGACA

Sequence number 94

IRTKTNNYAT

Sequence number 95

ACTAGACATTATTATAGCAGCACCTTTTACTACTACTACGGAATGGACGTC

Sequence number 96

TRHYYSSTFYYYYGMDV

Sequence number 97

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCATCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

Sequence number 98

DIQMTQSPSSLSASVGDRVIITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPITFGQGTRLEIK

Sequence number 99

CAGGGCATTAGAAATGAT

Sequence number 100

QGIRND

Sequence number 101

CTACAGCATAATAGTTACCCGATCACC

Sequence number 102

LQHNSYPIT

Sequence number 103

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGAGTCCCTAAAACTCTCCTGTGCAGCCTCTGGGTTCGCCTTCAGTGTCTCTATTATACACTGGGTCCGCCAGAGTTCCGGGAAAGGGCTGGAGTGGATTGGCCGTATCAGAACCAAAACTAATAA TTACGCGACAGGATATAGTGAGTCGATGAAGGGCAGGTTCACCATTTCCAGAGATGATTCAGAGAACACGGCGTATCTGCAAATGAACAGCCTCAAAACCGAAGACACGGCCGTATATTATTGTACTAGACATTATTATAGCAGCACCTTTTACTACTACTACGGAATGG ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAA TATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTA CGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 104

EVQLVESGGGLVQPGESLKLSCAASGFAFSVSIIHWVRQSSGKGLEWIGRIRTKTNNYATGYSESMKGRFTISRDDSENTAYLQMNSLKTEDTAVYYCTRHYYSSTFYYYYGM DVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 105

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCATCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTT GCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 106

DIQMTQSPSSLSASVGDRVIITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 107

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAATGGGTGGCAGTTACATCATATGATGGAGGTTATAAATTCTAT GCAGACTCCGTGAAGGACCGATTCATCATTTCCAGAGACAATTCCAAGAATACGCTGTATCTGCAAATGAACAGCCTGAGACCTGATGATACGGCTGTGTATTTCTGTGCGAAAGATCTGTGGATAAAGTTATGGTTACTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 108

QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVTSYDGGYKFYADSVKDRFIISRDNSKNTLYLQMNSLRPDDTAVYFCAKDLWIKLWLLDYWGQGTLVTVSS

Sequence number 109

GGATTCACCTTCAGCAGCTATGGC

Sequence number 110

ACATCATATGATGGAGGTTATAAA

Sequence number 111

TSYDGGYK

Sequence number 112

GCGAAAGATCTGTGGATAAAGTTATGGTTACTTGACTAC

Sequence number 113

AKDLWIKLWLLDY

Sequence number 114

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAACAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTACTAAGCTCCTGATCTACGCTGCATCCACTT TGCATTCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAGCTTATTACTGTCAAAAGTATAACAGTGCCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

Sequence number 115

DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVTKLLIYAASTLHSGVPSRFSGSGSGTDFTLTISSLQPEDVAAYYCQKYNSAPLTFGGGTKVEIK

Sequence number 116

CAGGGCATTAACAATTAT

Sequence number 117

QGINNY

Sequence number 118

CAAAAGTATAACAGTGCCCCGCTCACT

Sequence number 119

QKYNSAPLT

Sequence number 120

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAATGGGTGGCAGTTACATCATATGATGGAGGT TATAAATTCTATGCAGACTCCGTGAAGGACCGATTCATCATTTCCAGAGACAATTCCAAGAATACGCTGTATCTGCAAATGAACAGCCTGAGACCTGATGATACGGCTGTGTATTTCTGTGCGAAAGATCTGTGGATAAAGTTATGGTTACTTGACTACTGGGGCCAG GGAACCCTGGTCACCGTCTCTCCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCC CCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 121

QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVTSYDGGYKFYADSVKDRFIISRDNSKNTLYLQMNSLRPDDTAVYFCAKDLWIKLWLLDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 122

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAACAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTACTAAGCTCCTGATCTACGCTGCATCCACTTT GCATTCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAGCTTATTACTGTCAAAAGTATAACAGTGCCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 123

DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVTKLLIYAASTLHSGVPSRFSGSGSGTDFTLTISSLQPEDVAAYYCQKYNSAPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 124

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGG ACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACGGGTATAGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 125

EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGIGDYWGQGTLVTVSS

Sequence number 126

GGATTCACTTTCAGTAACGCCTGG

Sequence number 127

GFTFSNAW

Sequence number 128

ATTAAAAGCAAAACTGATGGTGGGACAACA

Sequence number 129

IKSKTDGGTT

Sequence number 130

ACCACGGGTATAGGGGACTAC

Sequence number 131

TTGIGDY

Sequence number 132

GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATATAGGTCCAACAATAAGAACAACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGATGCTCATTTACTGGG CATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

Sequence number 133

DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNNLAWYQQKPGQPPKMLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK

Sequence number 134

CAGAGTGTTTTATATAGGTCCAACAATAAGAACAAC

Sequence number 135

QSVLYRSNNKNN

Sequence number 136

TGGGCATCT

Sequence number 137

WAS

Sequence number 138

CAGCAATATTATAGTACTCCGCTCACT

Sequence number 139

QQYYSTPLT

Sequence number 140

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTG ATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACGGGTATAGGGGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGC CCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 141

EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGIGDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 142

GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATATAGGTCCAACAATAAGAACAACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGATGCTCATTTAC TGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGG AGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 143

DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNNLAWYQQKPGQPPKMLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 144

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGAGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAATGGATTGGGTACATCTATTACAGTGGGAGCACCAACTAC AACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTCGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCCGACACGGCCGTGTATTACTGTGGCCGAAATGGTGGATATAGTGGCTACGATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 145

QVQLQESGPGLVKPSETLSLTTCTVSGGSISSYYWSWIRQSPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARNGGYSGYDDYWGQGTLVTVSS

Sequence number 146

GGAGGCTCCATCAGTAGTTACTAC

Sequence number 147

GGSISSYY

Sequence number 148

ATCTATTACAGTGGGAGCACC

Sequence number 149

IYYSGST

Sequence number 150

GCGCGAAATGGTGGATATAGTGGCTACGATGACTAC

Sequence number 151

ARNGGYSGYDDY

Sequence number 152

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAGTAGTTATTTAAATTGGTATCAGCAGAAAGCAGGGAAGGCCCCTAAGCTCCTAATCTATTCTGCATCCAGTT TGCAAACTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTCTCAACTTACTACTGTCAACAGACTTACAGTGTCCCTCGGACGTTCGGCCAGGGGACCAAGGTGGAAATCAAA

Sequence number 153

DIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQKAGKAPKLLIYSASSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFSTYYCQQTYSVPRTFGQGTKVEIK

Sequence number 154

CAGACCATTAGTAGTTAT

Sequence number 155

QTISSY

Sequence number 156

TCTGCATCC

Sequence number 157

SAS

Sequence number 158

CAACAGACTTACAGTGTCCCTCGGACG

Sequence number 159

QQTYSVPRT

Sequence number 160

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGAGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAATGGATTGGGTACATCTATTACAGTGGGAG CACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTCGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCCGACACGGCCGTGTATTACTGTGCGCGAAATGGTGGATATAGTGGCTACGATGACTACTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGAGTTGAGTCCAAATATGGTCCCCCA TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACTGGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 161

QVQLQESGPGLVKPSETLSLTTCTVSGGSISSYYWSWIRQSPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARNGGYSGYDDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 162

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAGTAGTTATTTAAATTGGTATCAGCAGAAAGCAGGGAAGGCCCCTAAGCTCCTAATCTATTCTGCATCCAGTTT GCAAACTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTCTCAACTTACTACTGTCAACAGACTTACAGTGTCCCTCGGACGTTCGGCCAGGGGACCAAGGTGGAAATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 163

DIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQKAGKAPKLLIYSASSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFSTYYCQQTYSVPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 164

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACCTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGTTATAAATAC TATGCAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACACTTCCAAGAACACACTGTTTCTGCAAATGGACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCGGGACAGTAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

Sequence number 165

QVQLVESGGGVVQPGRSLRLSCAASGFTFTFGMHWVRQAPGKGLEWVALISYDGSYKYYAGSVKGRFTISRDTSKNTLFLQMDSLRAEDTAVYYCAKDRDSNYFDYWGQGTLVTVSS

Sequence number 166

GGATTCACCTTCAGTACCTTTGGC

Sequence number 167

GFTFSTFG

Sequence number 168

ATATCATATGATGGAAGTTATAAA

Sequence number 169

ISYDGSYK

Sequence number 170

GCGAAAGATCGGGACAGTAACTACTTTGACTAC

Sequence number 171

AKDRDSNYFDY

Sequence number 172

GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCCGGTCTAGTCAAAGCCTCGTACACAGTAATGGAAACACCTACTTGAGTTGGCTTCAGCAGAGGCCAGGCCAGCCTCCAAGACTCCTGATTTATAAGATC TCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGACAGGGACAGATTTCACACTGAAAATCAGCAGGGTGGAACCTGAGGATGTCGGGACTTATTACTGCATGCAAGCTACACATTTTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

Sequence number 173

DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGTGTDFTLKISRVEPEDVGTYYCMQATHFPLTFGGGTKVEIK

Sequence number 174

CAAAGCCTCGTACACAGTAATGGAAACACCTAC

Sequence number 175

QSLVHSNGNTY

Sequence number 176

AAGATCTCT

Sequence number 177

KIS

Sequence number 178

ATGCAAGCTACACATTTTCCTCTCACT

Sequence number 179

MQATHFPLT

Sequence number 180

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACCTTTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAG TTATAAATACTATGCAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACACTTCCAAGAACACACTGTTTCTGCAAATGGACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCGGGACAGTAACTACTTTGACTACTGGGGCCAGGGAA CCCTGGTCACTGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGAGTTGAGTCCAAATATGGTCCCCCA TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACTGGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 181

QVQLVESGGGVVQPGRSLRLSCAASGFTFTFGMHWVRQAPGKGLEWVALISYDGSYKYYAGSVKGRFTISRDTSKNTLFLQMDSLRAEDTAVYYCAKDRDSNYFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 182

GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCCGGTCTAGTCAAAGCCTCGTACACAGTAATGGAAACACCTACTTGAGTTGGCTTCAGCAGAGGCCAGGCCAGCCTCCAAGACTCCTGATTTATAAG ATCTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGACAGGGACAGATTTCACACTGAAAATCAGCAGGGTGGAACCTGAGGATGTCGGGACTTATTACTGCATGCAAGCTACACATTTTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAG ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 183

DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGTGTDFTLKISRVEPEDVGTYYCMQATHFPLTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 184

CAGGTGCAGCTGGTGGAGTCGGGGGGGGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATCTGGTATGATGGAAGTAATAAATACT ATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATTCCAAGAACACGCTGTATCTGCAAATGAATAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGAGTAGCAGCCCAATACTTTGAGTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

Sequence number 185

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWYDGSNKYYVDSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARDGVAAQYFEYWGQGTLVTVSS

Sequence number 186

GGATTCACCTTCAGTACCTATGGC

Sequence number 187

GFTFSTYG

Sequence number 188

ATCTGGTATGATGGAAGTAATAAA

Sequence number 189

IWYDGSNK

Sequence number 190

GCGAGAGACGGAGTAGCAGCCCAATACTTTGAGTAC

Sequence number 191

ARDGVAAQYFEY

Sequence number 192

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAGCAGAAACTAGGGAAAGCCCCTAAGCTCCTGATCTCTGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Sequence number 193

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKLGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK

Sequence number 194

CAGAGCATTAGCAGTTAT

Sequence number 195

CAACAGAGTTACAGTACCCCTCGGACG

Sequence number 196

QQSYSTPRT

Sequence number 197

CAGGTGCAGCTGGTGGAGTCGGGGGGGGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATCTGGTATGATGGAAG TAATAAATACTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATTCCAAGAACACGCTGTATCTGCAAATGAATAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGAGTAGCAGCCCAATACTTTGAGTACTGGGGCCAGGG AACCCTGGTCACTGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCC CATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 198

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWYDGSNKYYVDSVKGRFTISSRENSKNTLYLQMNSLRAEDTAVYYCARDGVAAQYFEYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 199

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAGCAGAAACTAGGGAAAGCCCCTAAGCTCCTGATCTCTGCTGCATCCAGTTT GCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 200

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKLGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 201

CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATTTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACATGTCCAAGTGGTATAAT GATTATGCAGTATCTGTGAAAGAGTCGAATAACCATCAACCCAGATACATCCAAGAACCATTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGGCTGTGTATTACTGTGCAAGAGATGGGGGGTGGGAGGTATTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 202

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYMSKWYNDYAVSVKSRITINPDTSKNHFSLQLNSVTPEDTAVYYCARDGGWEVFFDYWGQGTLVTVSS

Sequence number 203

GGGGACAGTGTCTCTAGCAACAGTGCTGCT

Sequence number 204

GDSVSSNSAA

Sequence number 205

ACATACTACATGTCCAAGTGGTATAAT

Sequence number 206

TYYMSKWYN

Sequence number 207

GCAAGAGATGGGGGGGTGGGAGGTATTCTTTGACTAC

Sequence number 208

ARDGGWEVFFDY

Sequence number 209

GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAAGAATCAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAAACTACTCATTTACTGGG CATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCGGCCTGCAGCCTGAAGATGTGGCAGTTTATTCCTGTCAGCAATATTATAGTATTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

Sequence number 210

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSKNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQPEDVAVYSCQQYYSIPYTFGQGTKLEIK

Sequence number 211

CAGAGTGTTTTATACAGCTCCAAGAATCAGAACTAC

Sequence number 212

QSVLYSSKNQNY

Sequence number 213

CAGCAATATTATAGTATTCCGTACACT

Sequence number 214

QQYYSIPYT

Sequence number 215

CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATTTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACATG TCCAAGTGGTATAATGATTATGCAGTATCTGTGGAAGAGTCGAATAACCATCAACCCAGATACATCCAAGAACCATTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGATGGGGGGGTGGGAGGTATTCTTTGACTACTGGG GCCAGGGAACCCTGGTCACCGTCTCTCTCAGGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGT CCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTG GATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 216

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYMSKWYNDYAVSVKSRITINPDTSKNHFSLQLNSVTPEDTAVYYCARDGGWEVFFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 217

GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAAGAATCAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAACTACTCATTTAC TGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCGGCCTGCAGCCTGAAGATGTGGCAGTTTATTCCTGTCAGCAATATTATAGTATTCCGTACACTTTTGGCCAGGGGACCAAGCTGG AGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 218

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSKNQNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQPEDVAVYSCQQYYSIPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 219

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGGTTATTACTACTGGAGTTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTTTAGAGGGAGCACTTAC TATGACCCGTCCCTCAAGAGTCGACTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGAGACGGAGCAGCTCGCCGGGGGCTTTCGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

Sequence number 220

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGYYYWSWIRQHPGKGLEWIGFIYFRGSTYYDPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARRSSSPGAFDIWGQGTMVTVSS

Sequence number 221

GGTGGCTCCATCAGTAGTGGTTATTACTAC

Sequence number 222

GGSISSGYYY

Sequence number 223

ATCTATTTTAGAGGGAGCACT

Sequence number 224

IYFRGST

Sequence number 225

GCGAGACGGAGCAGCTCGCCGGGGGCTTTCGATATC

Sequence number 226

ARRSSSPGAFDI

Sequence number 227

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCACAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGGTTATTACTACTGGAGTTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTTTAGA GGGAGCACTTACTATGACCCGTCCCTCAAGAGTCGACTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGAGACGGAGCAGCTCGCCGGGGGCTTTCGATATCTGGGGCCAA GGGACAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCC CCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 228

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGYYYWSWIRQHPGKGLEWIGFIYFRGSTYYDPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARRSSSPGAFDIWGQ GTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 229

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAATTATGAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTTTCATACATTGATAATAGTGGTAGTGCCATATATTACGCAGACTCT GTGAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTATTTTTTTCTGTGTGAGAGAAGGAGGATATGACAGCTCGTCCCGGACCTGGAGGGATGCTTTTGATATCTGGGGCCAGGGGACAATGGTCACCGTCTCTTCA

Sequence number 230

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMNWVRQAPGKGLEWVSYIDNSGSAIYYADSVKGRFTLSRDNAKNSLYLQMSSLRAEDTAIFFCVREGGYDSSSRTWRDAFDIWGQGTMVTVSS

Sequence number 231

GGATTCACCTTCAGTAATTATGAA

Sequence number 232

GFTFSNYE

Sequence number 233

ATTGATAATAGTGGTAGTGCCATA

Sequence number 234

IDNSGSAI

Sequence number 235

GTGAGAGAAGGAGGATATGACAGCTCGTCCCGGACCTGGAGGGATGCTTTTGATATC

Sequence number 236

VREGGYDSSSRTWRDAFDI

Sequence number 237

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAATTATGAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTTTCATACATTGATAATAGTGGTAGTGC CATATAATTACGCAGACTCTGTGAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTATTTTTTTCTGTGTGAGAGAAGGAGGATATGACAGCTCGTCCCGGACCTGGAGGGATGCTTTTG ATATCTGGGGCCAGGGGACAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAA TATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTA CGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 238

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMNWVRQAPGKGLEWVSYIDNSGSAIYYADSVKGRFTLSRDNAKNSLYLQMSSLRAEDTAIFFCVREGGYDSSSRTWRDAF DIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 239

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCCTCAGTAGGTACGACATGTACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGGGTCTCAGCTATCAATACTGCTGGTGACACATACTATCCAGGCTCC GTGAAGGGCCGATTCACCATCTCCAGAGAAAATGCCCAGAACTCCTTATATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTGTAAGAGAGGGGTACATTCGTACAACTGGAACGACGGGATTCTCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

Sequence number 240

EVQLVESGGGLVQPGGSLRLSCAASGFTLSRYDMYWVRQTTGKGLEWVSAINTAGDTYYPGSVKGRFTISRENAQNSLYLQMNSLRAGDTAVYYCVREGYIRTTGTTGFSFDYWGQGTLVTVSS

Sequence number 241

GGATTCACCCTCAGTAGGTACGAC

Sequence number 242

GFTLSRYD

Sequence number 243

ATCAATACTGCTGGTGACACA

Sequence number 244

INTAGDT

Sequence number 245

GTAAGAGAGGGGTACATTCGTACAACTGGAACGACGGGATTCTCCTTTGACTAC

Sequence number 246

VREGYIRTTGTTGFSFDY

Sequence number 247

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCCTCAGTAGGTACGACATGTACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGGTCTCAGCTATCAATACTGCTGGTGACA CATACTATCCAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATGCCCAGAACTCCTTATATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTGTAAGAGAGGGGTACATTCGTACAACTGGAACGACGGGATTCTCCTTTGACTAC TGGGGCCAGGGAACCCTGGTCACTGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATAT GGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACG TGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAGGGCCTCCCGTCCTCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 248

EVQLVESGGGLVQPGGSLRLSCAASGFTLSRYDMYWVRQTTGKGLEWVSAINTAGDTYYPGSVKGRFTISRENAQNSLYLQMNSLRAGDTAVYYCVREGYIRTTGTTGFSFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 249

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGATGTCAGTTATATCATATGATGGAAGTAATAAATACTATGCA GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCTTGAGACCTGAGGACACGGCTGTGTATTACTGTTCAATAATGAGACGGAACTCTGGTCTATACAGCTCGTTCGACCCCTGGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 250

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWMSVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCSIMRRNSGLYSSFDPWGQGTLVTVSS

Sequence number 251

GGATTCACCTTCAGTAGTTATGGC

Sequence number 252

ATATCATATGATGGAAGTAATAAA

Sequence number 253

ISYDGSNK

Sequence number 254

TCAATAATGAGACGGAACTCTGGGTCTATACAGCTCGTTCGACCCC

Sequence number 255

SIMRRNSGLYSSFDP

Sequence number 256

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGATGTCAGTTATATCATATGATGGAAGT AATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCTTGAGACCTGAGGACACGGCTGTGTATTACTGTTCAATAATGAGACGGAACTCTGGTCTATACAGCTCGTTCGACCCCTGGG GCCAGGGAACCCTGGTCACCGTCTCTCTCAGGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGT CCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTG GATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 257

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWMSVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCSIMRRNSGLYSSFDPW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 258

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGTCGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAACACCTGGATGAGCTGGGTCCGCCAGTCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAACCAAAACTGATGGTGGG ACATCGTACTACGGTTCACCCGTGAAAGACAGATTCACCATCTCAAGAGATGATTCAAAAACCACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAAGACACAGCCGTCTATTACTGTCTTACAGGACTGGGGGACTACTGGGGCCAGGGAACCCTGTCACCGTCTCCTCA

Sequence number 259

EVQLVESGGGLVKSGGSLRLSCAASGFTFTNTWMSWVRQSPGKGLEWVGRIKTKTDGGTSYYGSPVKDRFTISRDDSKTTLYLQMNSLRAEDTAVYYCLTGLGDYWGQGTLVTVSS

Sequence number 260

GGATTCACTTTCACTAACACCTGG

Sequence number 261

GFTFTNTW

Sequence number 262

ATTAAAACCAAAACTGATGGTGGGACATCG

Sequence number 263

IKTKTDGGTS

Sequence number 264

CTTACAGGACTGGGGGACTAC

Sequence number 265

LTGLGDY

Sequence number 266

GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG

Sequence number 267

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

Sequence number 268

CAGTCAGTCTCTAGCTCTTAT

Sequence number 269

QSVSSSY

Sequence number 270

GGGGCAAGT

Sequence number 271

GAS

Sequence number 272

CAACAGTACGGAAGCAGCCCGTGGACG

Sequence number 273

QQYGSSPWT

Sequence number 274

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGTCGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAACACCTGGATGAGCTGGGTCCGCCAGTCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAACCAAAACTG ATGGTGGGACATCGTACTACGGTTCACCCGTGAAAGACAGATTCACCATCTCAAGAGATGATTCAAAAACCACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAAGACACAGCCGTCTATTACTGTCTTACAGGACTGGGGGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGC CCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 275

EVQLVESGGGLVKSGGSLRLSCAASGFTFTNTWMSWVRQSPGKGLEWVGRIKTKTDGGTSYYGSPVKDRFTISRDDSKTTLYLQMNSLRAEDTAVYYCLTGLGDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 276

GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGGGAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCTTATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTGATCTACGGGGCAAGTTCC AGGGCCACCGGAATCCCCGACCGGTTCAGTGGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAGCCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCGTGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 277

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 278

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGATACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCTTTAGCAGCTATGCCATGGGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTACTAGTAGTGGTGGTAGCTCATACTTCG CACACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTTCTGTGCGAAAGATGGCCCTAATTACGATGTTTGGAGTGGTTATTACTGGGGCCAGGGAACCCTGTCACCGTCTCCTCA

Sequence number 279

EVQLVESGGGLIQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVSTITSSGGSSYFAHSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYFCAKDGPNYDVWSGYYWGQGTLVTVSS

Sequence number 280

GGCTTCACCTTTAGCAGCTATGCC

Sequence number 281

GFTFSSYA

Sequence number 282

ATTACTAGTAGTGGTGGTAGCTCA

Sequence number 283

ITSSGGSS

Sequence number 284

GCGAAAGATGGCCCTAATTACGATGTTTGGAGTGGTTATTAC

Sequence number 285

AKDGPNYDVWSGYY

Sequence number 286

GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACACCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGACAGTCTCCACAACTCCTGATCTATCTGGGT TCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGAGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGGCCAAGGGACCAAGGTGGAAATCAAA

Sequence number 287

DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFRGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK

Sequence number 288

CAGAGCCTCCTGTATAGTAATGGATACAACTAT

Sequence number 289

QSLLYSNGYNY

Sequence number 290

CTGGGTTCT

Sequence number 291

LGS

Sequence number 292

ATGCAAGCTCTACAAACTCCTCGGACG

Sequence number 293

MQALQTPRT

Sequence number 294

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGATACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCTTTAGCAGCTATGCCATGGGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTACTAGTAGTGGTGGT AGCTCATACTTCGCACACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTTCTGTGCGAAAGATGGCCCTAATTACGATGTTTGGAGTGGTTATTACTGGGGC CAGGGAACCCTGGTCACCGTCTCTCCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC CCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGG ATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 295

EVQLVESGGGLIQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVSTITSSGGSSYFAHSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYFCAKDGPNYDVWSGYYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 296

GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACACCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGACAGTCTCCACAACTCCTGATCCTATCTG GGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGAGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAA ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 297

DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFRGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 298

CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTTGAGTGGCTTGCACTCATTTATTGGAATGATGTTCAG CGCTTCAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTTGTCCTTTCAATGACCAATATGGACCCTCTAGACACAGCCACATATTACTGTGCACACACATTAACTAACTTTTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 299

QITLKESGPTLVKPTQTLTLTCTFSGFSLTSGVGVGWIRQPPGKALEWLALIYWNDVQRFSPSLKSRLTITKDTSKNQVVLSMTNMDPLDTATYYCAHTLTNFFDPWGQGTLVTVSS

Sequence number 300

GGGTTCTCACTCAGCACTAGTGGAGTGGGT

Sequence number 301

GFSLSTSGVG

Sequence number 302

ATTTATTGGAATGATGTTCAG

Sequence number 303

IYWNDVQ

Sequence number 304

GCACACACATTAACTAACTTTTTCGACCCC

Sequence number 305

AHTLTNFFDP

Sequence number 306

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAACTCCTGATCTATGCTGTTTCCACTT TGCAATCAGGGGTCCCTTCTCGGTTCAGTGGCCGTGGATCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATGTTGCAACTTATTACTGTCAAAAGTATTCCAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

Sequence number 307

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAVSTLQSGVPSRFSGRGSGADFTLTISSLQPEDVATYYCQKYSSTPLTFGGGTKVEIK

Sequence number 308

CAGGGCATTAGCAATTAT

Sequence number 309

QGISNY

Sequence number 310

GCTGTTTCC

Sequence number 311

AVS

Sequence number 312

CAAAAGTATTCCAGTACCCCGCTCACT

Sequence number 313

QKYSSTPLT

Sequence number 314

CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTTGAGTGGCTTGCACTCATTTATTGGAA TGATGTTCAGCGCTTCAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTTGTCCTTTCAATGACCAATATGGACCCTCTAGACACAGCCACATATTACTGTGCACACACATTAACTAACTTTTTCGACCCCTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGAGTTGAGTCCAAATATGGTCCCCCA TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACTGGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 315

QITLKESGPTLVKPTQTLTLTCTFSGFSLTSGVGVGWIRQPPGKALEWLALIYWNDVQRFSPSLKSRLTITKDTSKNQVVLSMTNMDPLDTATYYCAHTLTNFFDPWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 316

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAACTCCTGATCTATGCTGTTTCCACTTT GCAATCAGGGGTCCCTTCTCGGTTCAGTGGCCGTGGATCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATGTTGCAACTTATTACTGTCAAAAGTATTCCAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 317

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAVSTLQSGVPSRFSGRGSGADFTLTISSLQPEDVATYYCQKYSSTPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 318

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAATGGATCAGCACTTACAATGGTAACACAAACTATGCA CAGAAACTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGTACAGCCTCCATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGCGCGAGAGACTCGGGTATTGCAGCTCGTCCTAGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 319

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKLQGRVTTMTTDTSTSTASMELRSLRSDDTAVYYCARDSGIAARPRWFDPWGQGTLVTVSS

Sequence number 320

GGTTACACCTTTACCAGCTATGGT

Sequence number 321

GYTFTSYG

Sequence number 322

ATCAGCACTTACAATGGTAACACA

Sequence number 323

ISTYNGNT

Sequence number 324

GCGAGAGACTCGGGTATTGCAGCTCGTCCTAGGTGGTTCGACCCC

Sequence number 325

ARDSGIAARPRWFDP

Sequence number 326

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGACGAGTCAGGACATTAGCAACGATTTAACTTGGTATCAGCAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTTCGATGCTTTCCAATT TGTTAACAGGGGTCCCATCAAGGGTCAGTGGAAGTGGATCTGGGACAGATTTTACTCTCACCATCACCAGCCTGCAGCCTGAAGATGTTTCAACTTATTACTGTCAACACTATCATAGTCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

Sequence number 327

DIQMTQSPSSLSASVGDRVTITCQTSQDISNDLTWYQQKPGKAPKLLIFDASNLLTGVPSRVSGSGSGTDFTLTITSLQPEDVSTYYCQHYHSLPITFGQGTRLEIK

Sequence number 328

CAGGACATTAGCAACGAT

Sequence number 329

QDISND

Sequence number 330

GATGCTTCC

Sequence number 331

DAS

Sequence number 332

CAACACTATCATAGTCTCCCGATCACC

Sequence number 333

QHYHSLPIT

Sequence number 334

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGAATGGATCAGCACTTACAATGGT AACACAAACTATGCACAGAAACTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGTACAGCCTCCATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGCGCGAGAGACTCGGGTATTGCAGCTCGTCCTAGGTGGTTCGACCCCTGGG GCCAGGGAACCCTGGTCACCGTCTCTCTCAGGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGT CCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTG GATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 335

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKLQGRVTTMTTDTSTSTASMELRSLRSDDTAVYYCARDSGIAARPRWFDPW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 336

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGACGAGTCAGGACATTAGCAACGATTTAACTTGGTATCAGCAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTTCGATGCTTTCCAATTT GTTAACAGGGGTCCCATCAAGGGTCAGTGGAAGTGGATCTGGGACAGATTTTACTCTCACCATCACCAGCCTGCAGCCTGAAGATGTTTCAACTTATTACTGTCAACACTATCATAGTCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 337

DIQMTQSPSSLSASVGDRVTITCQTSQDISNDLTWYQQKPGKAPKLLIFDASNLLTGVPSRVSGSGSGTDFTLTITSLQPEDVSTYYCQHYHSLPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 338

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAGGGTCTCCTGTAAGGCTTCTGGATACACCTTCATCGCCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGGCACAGAC TATGCACAGAAGTTTCAGGGCAGGGTCTCCATGACCAGGGACACGTCCATCACCACAGCCTACATGGACCTGAGCAGCCTGAGATCTGACGACACGGCCGTATATTACTGTGCGAGAGGGGGTAGAGGCTATACTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

Sequence number 339

QVQLVQSGAEVKKPGASVRVSCKASGYTFIAYYMHWVRQAPGQGLEWMGWINPNSGGTDYAQKFQGRVSMTRDTSITTAYMDLSSLRSDDTAVYYCARGGGYTFDIWGQGTMVTVSS

Sequence number 340

GGATACACCTTCATCGCCTACTAT

Sequence number 341

GYTFIAYY

Sequence number 342

ATCAACCCTAACAGTGGTGGCACA

Sequence number 343

INPNSGGT

Sequence number 344

GCGAGAGGGGGGTAGAGGCTATACTTTTGATATC

Sequence number 345

ARGGRGYTFDI

Sequence number 346

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTGTTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGACGTCTAGTT TAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATCCACTCTCACCGTCAGCGGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATGATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Sequence number 347

DIQMTQSPSTLSASVGDRVTITCRASQSVSSWLAWYQQKPGKAPKLLIYKTSSLESGVPSRFSGSGSGTESTLTVSGLQPDDFATYYCQQYDSYSWTFGQGTKVEIK

Sequence number 348

CAGAGTGTTAGTAGCTGG

Sequence number 349

QSVSSW

Sequence number 350

AAGACGTCT

Sequence number 351

CAACAGTATGATAGTTATTCGTGGACG

Sequence number 352

QQYDSYSWT

Sequence number 353

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAGGGTCTCCTGTAAGGCTTCTGGATACACCTTCATCGCCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGG TGGCACAGACTATGCACAGAAGTTTCAGGGCAGGGTCTCCATGACCAGGGACACGTCCATCACCACAGCCTACATGGACCTGAGCAGCCTGAGATCTGACGACACGGCCGTATATTACTGTGCGAGAGGGGGTAGAGGCTATACTTTTGATATCTGGGGCCAAGGGA CAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGAGTTGAGTCCAAATATGGTCCCCCA TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACTGGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 354

QVQLVQSGAEVKKPGASVRVSCKASGYTFIAYYMHWVRQAPGQGLEWMGWINPNSGGTDYAQKFQGRVSMTRDTSITTAYMDLSSLRSDDTAVYYCARGGGRGYTFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 355

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTGTTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGACGTCTAGTTT AGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATCCACTCTCACCGTCAGCGGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATGATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 356

DIQMTQSPSTLSASVGDRVTITCRASQSVSSWLAWYQQKPGKAPKLLIYKTSSLESGVPSRFSGSGSGTESTLTVSGLQPDDFATYYCQQYDSYSWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 357

GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCGCTGGATTTAGTTTTAGTACCTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGGAGAGCTGGTTCCACA TTCTACGGTGACTCCGTGAAGGGCCGGTTCACCCTCTCCAGAGACACTTCCAGTAACACGGTGCATCTGCAAATGAATAGTCTGAGAGCCGAGGATACGGCCGTATATTACTGTGCGAAAGGAAATGGAGTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 358

EVQLVESGGGLVQPGGSLRLSCAAAGFSFSTYAMNWVRQAPGKGLEWVSTISGRAGSTFYGDSVKGRFTLSRDTSSNTVHLQMNSLRAEDTAVYYCAKGNGVFDYWGQGTLVTVSS

Sequence number 359

GGATTTAGTTTTAGTACCTATGCC

Sequence number 360

GFSFSTYA

Sequence number 361

ATTAGTGGGAGAGCTGGTTCCACA

Sequence number 362

ISGRAGST

Sequence number 363

GCGAAAGGAAATGGAGTCTTTGACTAC

Sequence number 364

AKGNGVFDY

Sequence number 365

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGACTATTACTAACTACTTGGCCTGGTATCAACAGAACCCAGGGAAACCCCCTAAGTTACTGATCTATAAGGCGTCTAGT TTAGAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGAATTCACTCTAACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAATTTTTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

Sequence number 366

DIQMTQSPSTLSASVGDRVTITCRASQTITNYLAWYQQNPGKPPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNFYTFGQGTKLEIK

Sequence number 367

CAGACTATTACTAACTAC

Sequence number 368

QTITNY

Sequence number 369

AAGGCGTCT

Sequence number 370

KAS

Sequence number 371

CAACAGTATAATAATTTTTACACT

Sequence number 372

QQYNNFYT

Sequence number 373

GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCGCTGGATTTAGTTTTAGTACCTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGGAGAGCTG GTTCCACATTCTACGGTGACTCCGTGAAGGGCCGGTTCACCCTCTCCAGAGACACTTCCAGTAACACGGTGCATCTGCAAATGAATAGTCTGAGAGCCGAGGATACGGCCGTATATTACTGTGCGAAAGGAAATGGAGTCTTTGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGC CCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 374

EVQLVESGGGLVQPGGSLRLSCAAAGFSFSTYAMNWVRQAPGKGLEWVSTISGRAGSTFYGDSVKGRFTLSRDTSSNTVHLQMNSLRAEDTAVYYCAKGNGVFDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 375

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCCAGTCAGACTATTACTAACTACTTGGCCTGGTATCAACAGAACCCAGGGAAACCCCCTAAGTTACTGATCTATAAGGCGTCTAGTT TAGAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGAATTCACTCTAACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAATTTTTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGA ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 376

DIQMTQSPSTLSASVGDRVTITCRASQTITNYLAWYQQNPGKPPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNFYTFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 377

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTCAGTACCTATGGCATGTTCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTGATAAATATTATACAGACTCT ATTAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAAAATACACTGTTTCTCCAAATGAACAGCCTGAGAGTAGAGGACACGGCTGTGTATTGGTTGCGAGGATGAAGGGGTATAGAAGTTCGTCCGCCCACCACTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGGTCACCGTCTCCTCA

Sequence number 378

QVQLVESGGGVVQPGRSLRLSCEASGTFFTYGMFWVRQAPGKGLEWVAVISYDGSDKYYTDSIKGRFTISRDNSKNTLFLQMNSLRVEDTAVYWCARMKGYRSSSAHHYYSMDVWGQGTTVTVSS

Sequence number 379

ATATCATATGATGGAAGTGATAAA

Sequence number 380

ISYDGSDK

Sequence number 381

GCGAGGATGAAGGGGTATAGAAGTTCGTCGCCCACCACTACTACAGTATGGACGTC

Sequence number 382

ARMKGYRSSSAHHYYSMDV

Sequence number 383

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTCAGTACCTATGGCATGTTCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTGA TAAATATTATACAGACTCTATTAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAAAATACACTGTTTCTCCAAATGAACAGCCTGAGAGTAGAGGACACGGCTGTGTATTGGTTGTGCGAGGATGAAGGGGTATAGAAGTTCGTCCGCCCACCACTACTACAGTATGG ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC GCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAA TATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTA CGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 384

QVQLVESGGGVVQPGRSLRLSCEASGFTFTTYGMFWVRQAPGKGLEWVAVISYDGSDKYYTDSIKGRFTISRDNSKNTLFLQMNSLRVEDTAVYWCARMKGYRSSSAHHYYSM DVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 385

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACTCCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTAGACAAGGACTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGACACAAACTATG TACAGAACTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGTCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTATTGTGCGAGACTGGAACTTGGCTACTATTACAACGATATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

Sequence number 386

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPRQGLEWMGWINPNSGDTNYVQNFQGRVTMTRDTSISTVYMELSRLRSDDTAVYYCARLELGYYYNDMDVWGQGTTVTVSS

Sequence number 387

GGATACTCCTTCACCAACTACTAT

Sequence number 388

GYSFTNYY

Sequence number 389

ATCAACCCTAACAGTGGTGACACA

Sequence number 390

INPNSGDT

Sequence number 391

GCGAGACTGGAACTTGGCTACTATTACAACGATATGGACGTC

Sequence number 392

ARLELGYYYNDMDV

Sequence number 393

GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAACACCTACTTGAGTTGGCTTCAGCAGACGCCAGGCCAGCCTCCAAGACTCCTAATTTATAAGACT TCTAACCGATTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGGTGGAAGCTGAGGATGTCGGGGTTTATTACTGCATGCAAGCTACACAATTTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

Sequence number 394

DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQTPGQPPRLLIYKTSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPLTFGGGTKVEIK

Sequence number 395

CAAAGCCTCGTACACAGTGATGGAAACACCTAC

Sequence number 396

QSLVHSDGNTY

Sequence number 397

ATGCAAGCTACACAATTTCCTCTCACT

Sequence number 398

MQATQFPLT

Sequence number 399

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACTCCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTAGACAAGGACTTGAGTGGATGGGATGGATCAACCCTAACAGTGGT GACACAAACTATGTACAGAACTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGTCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTATTGTGCGAGACTGGAACTTGGCTACTATTACAACGATATGGACGTCTGGGGC CAAGGGACCACGGGTCACCGTCTCTCCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC CCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGG ATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 400

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPRQGLEWMGWINPNSGDTNYVQNFQGRVTMTRDTSISTVYMELSRLRSDDTAVYYCARLELGYYYNDMDVWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 401

GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAACACCTACTTGAGTTGGCTTCAGCAGACGCCAGGCCAGCCTCCAAGACTCCTAATTTATAAG ACTTCTAACCGATTCTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGGTGGAAGCTGAGGATGTCGGGGTTTATTACTGCATGCAAGCTACACAATTTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAG ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 402

DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQTPGQPPRLLIYKTSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPLTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 403

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGGTTCACCTTCAGTGACTCTGCTATGCACTGGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTTGGCCGTATTAGAAGCAAAACTAACAGTTACG CGACAGCATATACTGCGTCGGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCGTATATTACTGTACTAGACTGGCAGTAACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

Sequence number 404

EVQLVESGGGLVQPGGSLKLSCAASGFTFSDSAMHWVRQASGKGLEWVGRIRSKTNSYATAYTASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRLAVTDYWGQGTLVTVSS

Sequence number 405

GGGTTCACCTTCAGTGACTCTGCT

Sequence number 406

GFTFSDSA

Sequence number 407

ATTAGAAGCAAAACTAACAGTTACGCGACA

Sequence number 408

IRSKTNSYAT

Sequence number 409

ACTAGACTGGCAGTAACTGACTAC

Sequence number 410

TRLAVTDY

Sequence number 411

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTTTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCA GTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACTGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAAAACTTACACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

Sequence number 412

DIQMTQSPSSLFASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHKTYTFGQGTKVEIK

Sequence number 413

CTACAGCATAAAACTTACACG

Sequence number 414

LQHKTYT

Sequence number 415

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGGTTCACCTTCAGTGACTCTGCTATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGGTTGGCCGTATTAGAAGCAAAACTA ACAGTTACGCGACAGCATATACTGCGTCGGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCGTATATTACTGTACTAGACTGGCAGTAACTGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCAT GCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGC GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

Sequence number 416

EVQLVESGGGLVQPGGSLKLSCAASGFTFSDSAMHWVRQASGKGLEWVGRIRSKTNSYATAYTASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRLAVTDYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*

Sequence number 417

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTTTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGT TTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACTGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAAAACTTACACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAA CTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Sequence number 418

DIQMTQSPSSLFASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHKTYTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

Sequence number 419

caggcgcacctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccctcagtaattactactggagctggatccggcagcccccagggaagggactggaatggattggctatatccattacagtgggcataccaagtaca attcctccctcaagagtcgagtcaccatatcagtagacacgtccaaaaaccagttctccctgatgctgacctctgtgaccgccgcagaacacggccctgtattattgtgcgagacatggtataactggaactacgtggttcgacccctggggccagggagccctggtcaccgtctcctca

Sequence number 420

QAHLQESGPGLVKPSETLSLTCTVSGGSLSNYYWSWIRQPPGKGLEWIGYIHYSGHTKYNSSLKSRVTISVDTSKNQFSLMLTSVTAADTALYYCARHGITGTTWFDPWGQGALVTVSS

Sequence number 421

ggtggctccctcagtaattactac

Sequence number 422

GGSLSNYY

Sequence number 423

atccattacagtgggcatacc

Sequence number 424

IHYSGHT

Sequence number 425

gcgagacatggtataactggaactacgtggttcgacccc

Sequence number 426

ARHGITGTTWFDP

Sequence number 427

gacatccagatgacccagtctccatcttccgtgtctgcatctgtaggggacagagtcaccatcacttgtcgggcgcgtcagggcattaactactggttagcctggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtt tgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttactattgtcaacaggctaacagtttccctccgacgttcggacaagggaccaaggtggaaatcaag

Sequence number 428

DIQMTQSPSSVSASVGDRVTITCRARQGINYWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKVEIK

Sequence number 429

cagggcattaactactgg

Sequence number 430

QGINYW

Sequence number 431

gctgcatcc

Sequence number 432

AAS

Sequence number 433

caacaggctaacagtttccctccgacg

Sequence number 434

QQANSFPPT

Sequence number 435

gaggtgcagctgttggagtctgggggaggcgtggtacagcctggggagtccctgagactctcctgtgcggcctctgaattttcctttagtagttatgccatgggctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtaggaca tactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtttctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgtgggggttatagtggcctttgactactggggccagggaaccctggtcaccgtctcctca

Sequence number 436

EVQLLESGGGVVQPGESLRLSCAASEFSFSSYAMGWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCVGVIVAFDYWGQGTLVTVSS

Sequence number 437

gaattttcctttagtagtagttatgcc

Sequence number 438

EFSFSSYA

Sequence number 439

attagtggtagtggtggtaggaca

Sequence number 440

ISGSGGRT

Sequence number 441

gtgggggttatagtggcctttgactac

Sequence number 442

VGVIVAFDY

Sequence number 443

gacatccacatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagtattagcagctatttaaattggtatcagcagaagccagggaaagcccctaaattcctgatctatactgcatccagtt tgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgtagattttgctacttactactgtcaacagagttacactaccccgtacacttttggccaggggaccaagctggagatcaca

Sequence number 444

DIHMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKFLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPVDFATYYCQQSYTTPYTFGQGTKLEIT

Sequence number 445

cagagtattagcagctat

Sequence number 446

QSISSY

Sequence number 447

actgcatcc

Sequence number 448

TAS

Sequence number 449

caacagagttacactaccccgtacact

Sequence number 450

QQSYTTPYT

Sequence number 451

caggtgcagctgcaggagtcgggcccaggactggtgaacccctcggagaccctgtccctcacctgcactgtctctggtggctccatcaatggttactactggaattggatccggcagtccccaggaaaggaacttgagtggattggctatatcttttatagtgggagcaccacctacagccct tccctcaggggtcgagtcaccatgtcggtagactcgtccaagaaccaattctctctgaggttggacgctgtgaccgccgcagactcggccctatattactgtgcgagacgctattacgatactttcactggttggggttactttgactcctggggccagggaaccctggtcaccgtctcctca

Sequence number 452

QVQLQESGPGLVNPSETLSLTFTGSGGSINGYYWNWIRQSPGKELEWIGYIFYSGSTTYSPSLRGRVTMSVDSKNQFSLRLDAVTAADSALYYCARRYYDTFTGWGYFDSWGQGTLVTVSS

Sequence number 453

ggtggctccatcaatggttactac

Sequence number 454

GGSINGYY

Sequence number 455

atcttttatagtgggagcacc

Sequence number 456

IFYSGST

Sequence number 457

gcgagacgctattacgatactttcactggttggggttactttgactcc

Sequence number 458

ARRYYDTFTGWGYFDS

Sequence number 459

gacatccagatgacccagtctccatcctccgtgtctgcatctgtgggagacagagtcaccgtcacttgtcgggcgagtcaggttattagcagatggttagcctggtatcagcagaagccagggaaagcccctaaactcctgatctatgctgcgtccagtt tacaaagtggggtcccatcacggttcagcggcagtggatctgggacagattttactctcaccatcagcggcctgcagcctgaagattttgcaatttacttttgtcaacaggctaacagtttcccattcactttcggccctgggaccaaagtggatagcaag

Sequence number 460

DIQMTQSPSSVSASVGDRVTVTCRASQVISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDFAIYFCQQANSFPFTFGPGTKVDSK

Sequence number 461

caggttattagcagatgg

Sequence number 462

QVISRW

Sequence number 463

gctgcgtcc

Sequence number 464

AAS

Sequence number 465

caacaggctaacagtttcccattcact

Sequence number 466

QQANSFPFT

Sequence number 467

catgtgcagttggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatctggctacaccttcaccagctactatctgcactgggtgcgacaggcccctggacaagggcttgagtggatgggagtcatcaaacctagtggtggtaacacaggt tacgcacagcagttccgggacagagtcaccatgaccagggacacgtccacgagcacagtctccatggaactgagcagcctgagatatgaagacacggccgtgtattattgtgtgagagataatgtggaactggaactagggtattggggccagggaaccctggtcaccgtctcctca

Sequence number 468

HVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYLHWVRQAPGQGLEWMGVIKPSGGNTGYAQQFRDRVTMTRDTSTSTVSMELSSLRYEDTAVYYCVRDNVELELGYWGQGTLVTVSS

Sequence number 469

ggctacaccttcaccagctactat

Sequence number 470

GYTFTSYY

Sequence number 471

atcaaacctagtggtggtaacaca

Sequence number 472

IKPSGGNT

Sequence number 473

gtgagagataatgtggaactggaactagggtat

Sequence number 474

VRDNVELELGY

Sequence number 475

gacatcgtgatgacccagtctccagactccctgtctatgtctctgggcgagaggaccaccatcaactgcaagtccagccagagtgttttatacagctccaacaataggaactacttagcttggtttccaacagaaaccaggacagcctcctaaacttctcatttactggg catctacccgggagtccggggtccctgaccgattccgtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgatgatgtggcagtttattactgtcagcaatattttgatactccgtacacttttggcctggggaccaagctggagatcaaa

Sequence number 476

DIVMTQSPDSLSMSLGERTTINCKSSQSVLYSSNNRNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFRGSGSGTDFTLTISSLQADDVAVYYCQQYFDTPYTFGLGTKLEIK

Sequence number 477

cagagtgttttatacagctccaacaataggaactac

Sequence number 478

QSVLYSSNNRNY

Sequence number 479

tgggcatct

Sequence number 480

WAS

Sequence number 481

cagcaatattttgatactccgtacact

Sequence number 482

QQYFDTPYT

Sequence number 483

gaggtacatctgttggagtctgggggaggcttagtacagcctggggggtccctgagactctcctgtgcagcctctggactcacctttagtaactatgccatgagctgggtccgccaggttccagggaaggggctggcgtgggtctcaggtattaatgatcgtggtagtggcacat actacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacactgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaagaggactacggtaactttgactattggggccagggaaccctggtcaccgtctcctca

Sequence number 484

EVHLLESGGGLVQPGGSLRLSCAASGLTFSNYAMSWVRQVPGKGLAWVSGINDRGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEDYGNFDYWGQGTLVTVSS

Sequence number 485

ggactcacctttagtaactatgcc

Sequence number 486

GLTFSNYA

Sequence number 487

attaatgatcgtggtagtggcaca

Sequence number 488

INDRGSGT

Sequence number 489

gcgaaagaggactacggtaactttgactat

Sequence number 490

AKEDYGNFDY

Sequence number 491

gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagaatattagtaactggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctataaggcgtctagttta gaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtataatagttattctatgtacacttttggccaggggaccaagctggagatcaaa

Sequence number 492

DIQMTQSPSTLSASVGDRVTITCRASQNISNWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSMYTFGQGTKLEIK

Sequence number 493

cagaatattagtaactgg

Sequence number 494

QNISNW

Sequence number 495

aaggcgtct

Sequence number 496

KAS

Sequence number 497

caacagtataatagttattctatgtacact

Sequence number 498

QQYNSYSMYT

Sequence number 499

gaggtacagctggtggagtctgggggaggcttggtacagcctggagggtccctgacactttcctgtgcagcctctggattcaccttcagtagttatgaaatgaattgggtccgccaggctccagggaaggggctggagtgggtttcatacattagtagtagtggtagtaccatatactacgcag actctgtgaagggccgattcaccatttatagagacaacgccaagaactcactgtatctgcaaatgaatagcctgagagccgaggacacggctgtttattactgtgcgagagatgaggattttggagtggcccactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca

Sequence number 500

EVQLVESGGGLVQPGGSLTLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTIYRDNAKNSLYLQMNSLRAEDTAVYYCARDEDFGVAHYYGMDVWGQGTTVTVSS

Sequence number 501

ggattcaccttcagtagttatgaa

Sequence number 502

GFTFSSYE

Sequence number 503

attagtagtagtggtagtaccata

Sequence number 504

ISSSGSTI

Sequence number 505

gcgagagatgaggattttggagtggcccactactacggtatggacgtc

Sequence number 506

ARDEDFGVAHYYGMDV

Sequence number 507

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagaagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcaaacagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccatccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 508

DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKLLIYAANSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTHPITFGQGTRLEIK

Sequence number 509

cagagcattagaagctat

Sequence number 510

QSIRSY

Sequence number 511

gctgcaaac

Sequence number 512

AAN

Sequence number 513

caacagagttacagtacccatccgatcacc

Sequence number 514

QQSYSTHPIT

Sequence number 515

gaggtgcaactgttggagtctgggggaggcttggtgcagccgggggggtccctgagactctcctgtgcagcctctgggttcatctttagtagttatgccatgtcctgggtccgccaggctccagggaaggggctggagtgggtctcaattattagtggaagtggtggtagaatat actacgcagagtccgtgaagggccggttcaccatctccagagacaattccaagaacacgttgtatctgcaaatgaaaagtgtgcgagccgaggacacggccgggatattactgtgcggggggaatagttacgctctttgactattggggccagggaaccctggtcaccgtctcctca

Sequence number 516

EVQLLESGGGLVQPGGSLRLSCAASGFIFSSYAMSWVRQAPGKGLEWVSIISGSGGRIYYAESVKGRFTISRDNSKNTLYLQMKSVRAEDTAGYYCAGGIVTLFDYWGQGTLVTVSS

Sequence number 517

gggttcatctttagtagttatgcc

Sequence number 518

GFIFSSYA

Sequence number 519

attagtggaagtggtggtagaata

Sequence number 520

ISGSGGRI

Sequence number 521

gcggggggaatagttacgctctttgactat

Sequence number 522

AGGIVTLFDY

Sequence number 523

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggcgacagagtcaccatcacttgccgggcaagtcagagcattagcagttatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttg catagtggggtcccatcaaggttcagtggcagtggatctgggacagagttcactctcaccatcagcagtctgcaacctgatgatttgcaacttactactgtcatcagagttacagtagtcctccgatcaccttcggccaagggacacgagtggacattaaa

Sequence number 524

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQSYSSPPITFGQGTRVDIK

Sequence number 525

cagagcattagcagttat

Sequence number 526

QSISSY

Sequence number 527

gctgcatcc

Sequence number 528

AAS

Sequence number 529

catcagagttacagtagtcctccgatcacc

Sequence number 530

HQSYSSPPIT

Sequence number 531

gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtagtagtacttacatat actacgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagaccggggctggaactttgactactggggccagggaaccctggtcaccgtctcctcc

Sequence number 532

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSTYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRGWNFDYWGQGTLVTVSS

Sequence number 533

ggattcaccttcagtagctatagc

Sequence number 534

GFTFSSYS

Sequence number 535

attagtagtagtagtacttacata

Sequence number 536

ISSSSTYI

Sequence number 537

gcgagagaccggggctggaactttgactac

Sequence number 538

ARDRGWNFDY

Sequence number 539

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagaccattaacagctatttaaattggtttcagcagaaaccagggaaagcccctaagctcctgatctatactgcatccagtt tgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctccgacgttcggccaagggaccaaggtggaaatcaaa

Sequence number 540

DIQMTQSPSSLSASVGDRVTITCRASQTINSYLNWFQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

Sequence number 541

cagaccattaacagctat

Sequence number 542

QTINSY

Sequence number 543

actgcatcc

Sequence number 544

TAS

Sequence number 545

caacagagttacagtacccctccgacg

Sequence number 546

QQSYSTPPT

Sequence number 547

gaggtgcagttggtggaatttgggggagacctggtcaagccgggggggtccctgagactctcctgtgcagcctctggattcgccttcagttcctatagcatgaactgggtccgccaggctccagggaagagcctggagtgggtctcctctatcactaatagtggtagtttca tatactacgcagactcagtgaagggccgattcaccatttccagagacaacgccaagagttcactgtttctgcaaatgaacagcctgagagtcgaggacacggctgtgtatttttgtgcgagtaagacttcacttgactattggggccagggaaccctggtcaccgtctcctca

Sequence number 548

EVQLVEFGGDLVKPGGSLRLSCAASGFAFSSYSMNWVRQAPGKSLEWVSSITNSGSFIYYADSVKGRFTISRDNAKSSLFLQMNSLRVEDTAVYFCASKTSLDYWGQGTLVTVSS

Sequence number 549

ggattcgccttcagttcctatagc

Sequence number 550

GFAFSSYS

Sequence number 551

atcactaatagtggtagtttcata

Sequence number 552

ITNSGSFI

Sequence number 553

gcgagtaagacttcacttgactat

Sequence number 554

ASKTSLDY

Sequence number 555

ggtattgtgatgacccagactccactctcctcacctgtcacccttggacagccggcctccatctcctgcaggtctagtcaaagcctcgtgcacagtgatggaaacacctacttgagttggcttcagcagaggccaggccagcctcctagactcctaatttctaaga tttctgaccggttatctggggtcccagacagattcactggcagtgggacagggacagatttcacactgaagatcagcagggtggaagttgaggatgtcggggtttattactgcatgcaaactacacaatttccgacgttcggccaagggaccaaggtggaaatcaaa

Sequence number 556

GIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLISKISDRLSGVPDRFTGSGTGTDFTLKISRVEVEDVGVYYCMQTTQFPTFGQGTKVEIK

Sequence number 557

caaagcctcgtgcacagtgatggaaacacctac

Sequence number 558

QSLVHSDGNTY

Sequence number 559

aagatttct

Sequence number 560

KIS

Sequence number 561

atgcaaactacacaatttccgacg

Sequence number 562

MQTTQFPT

Sequence number 563

gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgtagcctctggattcaatttcactaacacctggatgagctgggtccgccaggttccagggagggggctggagtgggttggccgtattaaaagcgaaactgatggtggg acaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattcaaaaaactcgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattcctgtaccacagggatcggggactactggggccagggaaccctggtcaccgtctcctca

Sequence number 564

EVQLVESGGGLVKPGGSLRLSCVASGFNFTNTWMSWVRQVPGRGLEWVGRIKSETDGGTTDYAAPVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYSCTTGIGDYWGQGTLVTVSS

Sequence number 565

ggattcaatttcactaacacctgg

Sequence number 566

GFNFTNTW

Sequence number 567

attaaaagcgaaactgatggtgggacaaca

Sequence number 568

IKSETDGGTT

Sequence number 569

accacagggatcggggactac

Sequence number 570

TTGIGDY

Sequence number 571

gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccagccagagtgttttatacagctccaacaataagaactacttagcttggtaccagcagaaaccaggacagcctcctaagctgctcatttactggg catctacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacaatattatagtactccgctcactttcggcggagggaccaaggtggagatcaaa

Sequence number 572

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK

Sequence number 573

cagagtgttttatacagctccaacaataagaactac

Sequence number 574

QSVLYSSNNKNY

Sequence number 575

tgggcatct

Sequence number 576

WAS

Sequence number 577

caacaatattatagtactccgctcact

Sequence number 578

QQYYSTPLT

Sequence number 579

gaggtgcaactggtggagtctgggggaggcctggtcaagcctggggggtccctaagactctcctgtgcagcctctggattcaccttcagttcttatagcatgaactgggtccgccagtctccagggaaggggctggagtgggtctcatccattagtagtagcagtaatttcatatac tacgcagactcagtgaagggccgcttcaccatctccagagacaacgccagaaactcactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgtgagaagtatcgcaacaactggctttgactattggggccagggaaccctggtcaccgtctcctcg

Sequence number 580

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQSPGKGLEWVSSISSSSNFIYYADSVKGRFTISRDNARNSLYLQMNSLRAEDTAVYYCVRSIATTGFDYWGQGTLVTVSS

Sequence number 581

ggattcaccttcagttcttatagc

Sequence number 582

GFTFSSYS

Sequence number 583

attagtagtagcagtaatttcata

Sequence number 584

ISSSSNFI

Sequence number 585

gtgagaagtatcgcaacaactggctttgactat

Sequence number 586

VRSIATTGFDY

Sequence number 587

gatattgtgatgacccagactccactctcctcacctgtcacccttggacagccggcctccatctcctgcaggtctagtcaaagcctcgtacacagtgatggaaatacgtacttgagttggcttcatcagaggccaggccagcctccaagactcctcatttataaga tttctaaccggttttctggggtcccagacagattcagtggcagtggggcaggggcagacttcacactgaaaatcagcagggtggaaactgaggatgtcggggtttattactgcgtgcaagctacacaaatttccgacgttcggccaagggaccaaggtggaaatcaaa

Sequence number 588

DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGADFTLKISRVETEDVGVYYCVQATQFPTFGQGTKVEIK

Sequence number 589

caaagcctcgtacacagtgatggaaatacgtac

Sequence number 590

QSLVHSDGNTY

Sequence number 591

aagatttct

Sequence number 592

KIS

Sequence number 593

gtgcaagctacacaatttccgacg

Sequence number 594

VQATQFPT

Sequence number 595

gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatacattagtagtagtagtagtttcatatatc acgcagactcagtgaagggccgattcaccatttccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagtcgaggacacggctgtgtattcctgtgcgagattccgaaccgggagagatgcttttgatatctggggccaagggacaatggtcaccgtctcttca

Sequence number 596

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSFIYHADSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYSCARFRTGRDAFDIWGQGTMVTVSS

Sequence number 597

ggattcaccttcagtagctatagc

Sequence number 598

GFTFSSYS

Sequence number 599

attagtagtagtagtagtttcata

Sequence number 600

ISSSSSFI

Sequence number 601

gcgagattccgaaccgggagagatgcttttgatatc

Sequence number 602

ARFRTGRDAFDI

Sequence number 603

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcttccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 604

DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

Sequence number 605

cagagcattaacagctat

Sequence number 606

QSINSY

Sequence number 607

gctgcttcc

Sequence number 608

AAS

Sequence number 609

caacagagttacagtacccctccgatcacc

Sequence number 610

QQSYSTPPIT

Sequence number 611

gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagttatagcatgaattgggtccgccaggctccagggaaggggctggagtgggtctcatacattagtagtagtagtagtttcatatatc acgcagactcagtgaagggccgattcaccatttccagagacaacgccaagaactcactgtatctgcaaatgaacagtctgagagtcgaggacacggctgtgtattcctgtgcgagattccgaaccgggagagatgcttttgatatctggggccaagggacaatggtcaccgtctcttca

Sequence number 612

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSFIYHADSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYSCARFRTGRDAFDIWGQGTMVTVSS

Sequence number 613

ggattcaccttcagtagttatagc

Sequence number 614

GFTFSSYS

Sequence number 615

attagtagtagtagtagtttcata

Sequence number 616

ISSSSSFI

Sequence number 617

gcgagattccgaaccgggagagatgcttttgatatc

Sequence number 618

ARFRTGRDAFDI

Sequence number 619

gacatccagatgacccagtctccatcctccctgtctgcatttgtaggagacagagtcaccatcacttgccgggcaagtcagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcttccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactggacattaaa

Sequence number 620

DIQMTQSPSSLSAFVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLDIK

Sequence number 621

cagagcattaacagctat

Sequence number 622

QSINSY

Sequence number 623

gctgcttcc

Sequence number 624

AAS

Sequence number 625

caacagagttacagtacccctccgatcacc

Sequence number 626

QQSYSTPPIT

Sequence number 627

cagctgcaactacaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacttgcactgtctctggtggctccatcaccaataataattactactactggggctggatccgccagcccccagggaagggactggaatggatagggattatctattatggtgggatcacctact acaatccgtccctcaagagtcgagtcagcatatccgtggacacgtccaagaatcagttctccctgaatctgagttctgtcaccgccgcagacacggctgtctattactgtgcgagactcgattttttggagtggttatctctttgactactggggccagggagccccggtcaccgtctcctca

Sequence number 628

QLQLQESGPGLVKPSETLSLTTCTVSGGSITNNNYYWGWIRQPPGKGLEWIGIIYYGGITYYNPSLKSRVSISVDTSKNQFSLNLSSVTAADTAVYYCARLDFWSGYLFDYWGQGAPVTVSS

Sequence number 629

ggtggctccatcaccaataataattactac

Sequence number 630

GGSITNNNYY

Sequence number 631

atctattatggtgggatcacc

Sequence number 632

IYYGGIT

Sequence number 633

gcgagactcgatttttggagtggttatctctttgactac

Sequence number 634

ARLDFWSGYLFDY

Sequence number 635

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagggtcaccatcacttgccgggcgagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcaacagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtagtcctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 636

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQSYSSPPITFGQGTRLEIK

Sequence number 637

cagagcattagcagctat

Sequence number 638

QSISSY

Sequence number 639

gctgcatcc

Sequence number 640

AAS

Sequence number 641

caacagagttacagtagtcctccgatcacc

Sequence number 642

QQSYSSPPIT

Sequence number 643

gaggtgcagctggtggagtctgggggaggcttggaacagcctggagggtccctgagactctcctgtgcagcctctggattcatttttcagtagttatgaaatgaactgggtccgccaggctccagggaaggggctggaatgggtttcatatattagtagtagtggtgataccataaactccgcagactctgtg aagggccgattcaccatctccagagacaacgccaagaaatcactgtatcttcaaatgaacagcctgagagccgaggacacggcagtctattattgtgtgagagggggatattgttctagtagcagttgctttatggcctactattacggcatggacgtctggggccaagggaccacggtcaccgtctcctca

Sequence number 644

EVQLVESGGGLEQPGGSLRLSCAASGFIFSSYEMNWVRQAPGKGLEWVSYISSSGDTINSADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCVRGGYCSSSSCFMAYYYGMDVWGQGTTVTVSS

Sequence number 645

ggattcattttcagtagttatgaa

Sequence number 646

GFIFSSYE

Sequence number 647

attagtagtagtggtgataccata

Sequence number 648

ISSSGDTI

Sequence number 649

gtgagagggggatattgttctagtagcagttgctttatggcctactattacggcatggacgtc

Sequence number 650

VRGGYCSSSSCFMAYYYGMDV

Sequence number 651

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccggacaagtcagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 652

DIQMTQSPSSLSASVGDRVTITCRTSQSINSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

Sequence number 653

cagagcattaacagctat

Sequence number 654

QSINSY

Sequence number 655

gctgcatcc

Sequence number 656

AAS

Sequence number 657

caacagagttacagtacccctccgatcacc

Sequence number 658

QQSYSTPPIT

Sequence number 659

gaggtgcaactggtggagtctgggggaggcccggtcaagcctggggggtccctgagactctcctgtgcaaccgctggattcaccttcagtagttatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtcgtagtagtttcatttact acgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaattcactgtttctgcaaatgaacagcctgagagtcgaggacacggctgtatatttctgtgtgagattccgaatcgggagggatgcttttgacatctggggccaagggacaatggtcaccgtctcttca

Sequence number 660

EVQLVESGGGPVKPGGSLRLSCATAGFTFSSYSMNWVRQAPGKGLEWVSSISSRSSFIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYFCVRFRIGRDAFDIWGQGTMVTVSS

Sequence number 661

ggattcaccttcagtagttatagc

Sequence number 662

GFTFSSYS

Sequence number 663

attagtagtcgtagtagtttcatt

Sequence number 664

ISSRSSFI

Sequence number 665

gtgagattccgaatcgggagggatgcttttgacatc

Sequence number 666

VRFRIGRDAFDI

Sequence number 667

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacgtgccgggcaagtcagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctccttatctattctgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcatcatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagtgacagtacccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 668

DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLIISSLQPEDFATYYCQQSDSTPPITFGQGTRLEIK

Sequence number 669

cagagcattaacagctat

Sequence number 670

QSINSY

Sequence number 671

tctgcatcc

Sequence number 672

SAS

Sequence number 673

caacagagtgacagtacccctccgatcacc

Sequence number 674

QQSDSTPPIT

Sequence number 675

caggttcagctggtgcagtctgaagctgtggtgaagaagcctggggcctcagtgaaggtctcttgcaaggcttctggttacacctttaccaactatggtatcggctgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcagcggttacaatggtaacacaaactatgtac agaaactccagggcagagttaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtatattattgtgtgagaaataactgggaatatgacttccactattacggtatggacgtctggggccaagggaccacggtcaccgtctcctca

Sequence number 676

QVQLVQSEAVVKKPGASVKVSCKASGYTFTNYGIGWVRQAPGQGLEWMGWISGYNGNTNYVQKLQGRVTTMTTDTSTSTAYMELRSLRSDDTAVYYCVRNNWEYDFHYYGMDVWGQGTTVTVSS

Sequence number 677

ggttacacctttaccaactatggt

Sequence number 678

GYTFTNYG

Sequence number 679

atcagcggttacaatggtaacaca

Sequence number 680

ISGYNGNT

Sequence number 681

gtgagaaataactgggaatatgacttccactattacggtatggacgtc

Sequence number 682

VRNNWEYDFHYYGMDV

Sequence number 683

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagttatataaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgttgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttattactgtcaacagagttacagtacccctgcgatcaccttcggccaagggacacgactggagattaaa

Sequence number 684

DIQMTQSPSSLSASVGDRVTITCRASQSISSYINWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPAITFGQGTRLEIK

Sequence number 685

cagagcattagcagttat

Sequence number 686

QSISSY

Sequence number 687

gttgcatcc

Sequence number 688

VAS

Sequence number 689

caacagagttacagtacccctgcgatcacc

Sequence number 690

QQSYSTPAIT

Sequence number 691

caggtccatctggtggagtctgggggaggcttggtcaagcctggagggtccctgagactctcctgtgcagcctctggattcaccttcagtgactactatatgacctggatccgccaggctccagggaagggactggactgggtttcatatattagtggtggtggtagtaccgtaaactacgcagac tctgtgaagggccgcttcaccatctccagggacaacgccaagaatttaatgtctctgcaaatgaacagcctgggagtcgaggacacggccgtgtattattgtgcgagagaagggggacacagttatggtcgcttttccttcggtttggacgtctggggccaagggaccacggtcaccgtctcctca

Sequence number 692

QVHLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLDWVSYISGGGSTVNYADSVKGRFTISRDNAKNLMSLQMNSLGVEDTAVYYCAREGGHSYGRFSFGLDVWGQGTTVTVSS

Sequence number 693

ggattcaccttcagtgactactat

Sequence number 694

GFTFSDYY

Sequence number 695

attagtggtggtggtagtaccgta

Sequence number 696

ISGGGSTV

Sequence number 697

gcgagagaagggggacacagttatggtcgcttttccttcggtttggacgtc

Sequence number 698

AREGGHSYGRFSFGLDV

Sequence number 699

gacatccagatggcccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagatatttaaattggtatcagcacaaagcagggaaagcccctaaactcctgatctatgctgcatccagttta caaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagtctgcagcctgaagattttgcaacttactactgtcaacagagttacagtaaccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 700

DIQMAQSPSSLSASVGDRVTITCRASQSISRYLNWYQHKAGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPPITFGQGTRLEIK

Sequence number 701

cagagcattagcagatat

Sequence number 702

QSISRY

Sequence number 703

gctgcatcc

Sequence number 704

AAS

Sequence number 705

caacagagttacagtaaccctccgatcacc

Sequence number 706

QQSYSNPPIT

Sequence number 707

caggtacagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtggctccatcagcagtggtaattattactggagctggatccgccagcacccagggaagggcctggagtggattgggtacatcaactacagtggatacacct actacaatccgtccctcaagagtcgaattaacatatcagcagacacgtctaagaatcagttctccctgaaggtgagctctgtgacggccgcggacacggccgtgtattactgtgcgagacataactcgaattatgaaatggactactactggggccagggaaccctggtcaccgtctcttca

Sequence number 708

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGNYYWSWIRQHPGKGLEWIGYINYSGYTYYNPSLKSRINISADTSKNQFSLKVSSVTAADTAVYYCARHNSNYEMDYWGQGTLVTVSS

Sequence number 709

ggtggctccatcagcagtggtaattattac

Sequence number 710

GGSISSGNYY

Sequence number 711

atcaactacagtggatacacc

Sequence number 712

INYSGYT

Sequence number 713

gcgagacataactcgaattatgaaatggactac

Sequence number 714

ARHNSNYEMDY

Sequence number 715

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 716

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

Sequence number 717

cagagcattagcagctat

Sequence number 718

QSISSY

Sequence number 719

gctgcatcc

Sequence number 720

AAS

Sequence number 721

caacagagttacagtacccctccgatcacc

Sequence number 722

QQSYSTPPIT

Sequence number 723

gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggagtccctgaaactctcctgtgcagcctctggattcaccttcagtaattataacatgcactgggtccggcaggctccagggaaggggctgaagtgggtctcatccattagtggtagtagtagttacatat actacgcagactcagtgaagggccgattcaccatctccagagacaacaccaataaatcactatatctccaaatgaacagcctgagagccgaggacacggctgtctattactgtgcgagactctatagggggggtatggacgtctggggccaggggaccacggtcaccgtctcctca

Sequence number 724

EVQLVESGGGLVKPGESLKLSCAASGFTFSNYNMHWVRQAPGKGLKWVSSISGSSSYIYYADSVKGRFTISRDNTNKSLYLQMNSLRAEDTAVYYCARLYRGGMDVWGQGTTVTVSS

Sequence number 725

ggattcaccttcagtaattataac

Sequence number 726

GFTFSNYN

Sequence number 727

attagtggtagtagtagttacata

Sequence number 728

ISGSSSYI

Sequence number 729

gcgagactctatagggggggtatggacgtc

Sequence number 730

ARLYRGGMDV

Sequence number 731

caggtgcagctacaacagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtggttactactggagctggatccgccagcccccagggaaggggctggagtggattggggaaatcagtcatcgtggaagcaccaact acaatccgtccctcaagagtcgagtcaccatatcagtagacacgtccaggaaccagttctccctgaagctgagctctgtgaccgccgcggacacggctgtgtatttctgtgcgaattattttggggagatcctactttgacgactggggcccgggaaccctggtcaccgtctcctca

Sequence number 732

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEISHRGSTNYNPSLKSRVTISVDTSRNQFSLKLSSVTAADTAVYFCANYFGRSYFDDWGPGTLVTVSS

Sequence number 733

ggtgggtccttcagtggttactac

Sequence number 734

GGSFSGYY

Sequence number 735

atcagtcatcgtggaagcacc

Sequence number 736

ISHRGST

Sequence number 737

gcgaattattttgggagatcctactttgacgac

Sequence number 738

ANYFGRSYFDD

Sequence number 739

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagactcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaatctcctgatctatgctgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagtttcagtagccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 740

DIQMTQSPSSLSASVGDRLTITCRASQSISSYLNWYQQKPGKAPNLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSSPPITFGQGTRLEIK

Sequence number 741

cagagcattagcagctat

Sequence number 742

QSISSY

Sequence number 743

gctgcatcc

Sequence number 744

AAS

Sequence number 745

caacagagtttcagtagccctccgatcacc

Sequence number 746

QQSFSSPPIT

Sequence number 747

caggtgcagctacagcagtggggcgcaggactgttgaagccttcggataccctgtccctcacctgcgctgtctatggtgggtccttcagtgattactactggagttggatccgccagcccccagggaagggactggaatggattggggaaggcagtcatactggaagaacaaactac aacccgtccctcaagagtcgaatcaccatatcagtagacacgtccaagaaccagttctccctgaagctgaactctgtgaccgccgcggacacggctttatattactgtgcgagaggagcaccagctggtaagggcttcgacccctggggccagggaaccctggtcaccgtctcctca

Sequence number 748

QVQLQQWGAGLLKPSDTLSLTCAVYGGSFSDYYWSWIRQPPGKGLEWIGEGSHTGRTNYNPSLKSRITISVDTSKNQFSLKLNSVTAADTALYYCARGAPAGKGFDPWGQGTLVTVSS

Sequence number 749

ggtgggtccttcagtgattactac

Sequence number 750

GGSFSDYY

Sequence number 751

ggcagtcatactggaagaaca

Sequence number 752

GSHTGRT

Sequence number 753

gcgagaggagcaccagctggtaagggcttcgacccc

Sequence number 754

ARGAPAGKGFDP

Sequence number 755

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcaggtatttaaattggtatcagcagaaaccagggaaagcccctaagctcctcatctatgctgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 756

DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

Sequence number 757

cagagcattagcaggtat

Sequence number 758

QSISRY

Sequence number 759

gctgcatcc

Sequence number 760

AAS

Sequence number 761

caacagagttacagtacccctccgatcacc

Sequence number 762

QQSYSTPPIT

Sequence number 763

caggtacaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcactaactatggcatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatgaaacttatagattcagtgcag actccgtgaagggccgattcaccatctccagagacaactccaagaacacggtgtatctgcaaatgaacagcctgagagccgaggacacggctgtttattactgtgcgcgccatagtgggaattacttacgtgacttttacggtttggacgtctggggccaagggactacggtcaccgtctcctca

Sequence number 764

QVQLVESGGGVVQPGRSLRLSCAASGFTFTNYGIHWVRQAPGKGLEWVAVIWYDETYRFSADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARHSGNYLRDFYGLDVWGQGTTVTVSS

Sequence number 765

ggattcaccttcactaactatggc

Sequence number 766

GFTFTNYG

Sequence number 767

atatggtatgatgaaacttataga

Sequence number 768

IWYDETYR

Sequence number 769

gcgcgccatagtgggaattacttacgtgacttttacggtttggacgtc

Sequence number 770

ARHSGNYLRDFYGLDV

Sequence number 771

gacatccagatgacccagtctccttcctccctttctgcatctgttggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgttgcatccagtttg caaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacaatacccctccgatcaccttcggccaagggacacgactggagattaaa

Sequence number 772

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPPITFGQGTRLEIK

Sequence number 773

cagagcattagcagctat

Sequence number 774

QSISSY

Sequence number 775

gttgcatcc

Sequence number 776

VAS

Sequence number 777

caacagagttacaatacccctccgatcacc

Sequence number 778

QQSYNTPPIT

Sequence number 779

caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtttctatggcatgcactgggtccgccagactcctggcaaggggctggagtgggtggcagtcatatggtatgatggaagttctaaat actatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaactcgctgtatctgcaaatgagcagcctgagagccgaggacacggctctatattactgtgtcgctcacggcaccaacccttttgagtactggggccagggaaccctggtcaccgtctcctca

Sequence number 780

QVQLVESGGGVVQPGRSLRLSCAASGFTFSFYGMHWVRQTPGKGLEWVAVIWYDGSSKYYADSVKGRFTISRDNSKNSLYLQMSSLRAEDTALYYCVAHGTNPFEYWGQGTLVTVSS

Sequence number 781

ggattcaccttcagtttctatggc

Sequence number 782

GFTFSFYG

Sequence number 783

atatggtatgatggaagttctaaa

Sequence number 784

IWYDGSSK

Sequence number 785

gtcgctcacggcaccaacccttttgagtac

Sequence number 786

VAHGTNPFEY

Example 4: Binding parameters of exemplary CDH15 antibodies

The equilibrium dissociation constant (KD) for CADH15 binding to purified anti-CDH15 monoclonal antibody was determined using real-time surface plasmon resonance (SPR) based on a Biacore 4000 biosensor. All binding studies were performed at 25°C and 37°C in a running buffer containing 10 mM HEPES, 150 mM NaCl, 1 mM CaCl ₂ , 0.5 mM MgCl ₂ , 0.05% and 0.05% v/v surfactant Tween-20, pH 7.4 (HBS-ET). The Biacore CM5 sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567) or an anti-mouse Fc-specific antibody (GE Healthcare, # BR100838) to capture the anti-CDH15 monoclonal antibody. Different concentrations of CDH15 reagent, human CDH15 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hCDH15-MMH; REGN4409), human CDH15 (K584Q) extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hCDH15_ K584Q -MMH; REGN5943), monkey CDH15 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfCDH15-MMH; REGN4586), and mouse CDH15 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mCDH15-MMH; REGN4410) were added at concentrations ranging from 6.25 nM to 100 nM for 30 min in a 4-fold serial dilution prepared in HBS-P running buffer. The injections were performed at a flow rate of μL/min for 2.5 or 4 minutes. The dissociation of different CDH15 reagents bound to the anti-CDH15 monoclonal antibody was monitored for 7 or 10 minutes in HBS-P running buffer. After the end of each cycle, the anti-CDH15 monoclonal antibody capture surface was regenerated by injecting 20 mM H ₃ PO ₄ or glycine, pH 1.5, for 12 or 60 seconds.

Association rates (ka) and dissociation rates (kd) were determined by fitting real-time binding sensorgrams to a mass-transport-limited 1:1 binding model using Scrubber 2.0c curve-fitting software. Association-dissociation equilibrium constants (KD) and dissociation half-lives (t½) were calculated from the kinetic rates as follows:

The equilibrium and kinetic binding parameters for anti-CDH15 monoclonal antibodies binding to hCDH15-MMH, hCDH15(K584Q).MMH, mfCDH15-MMH, and mCDH15-MMH at 25°C and 37°C are presented in Tables 4 to 11, respectively.

result

As shown in Table 4 below, at 25°C, anti-CDH15 monoclonal antibodies bound to hCDH15-MMH with KD values ranging from 27.6 pM to 125 nM.

Kinetic binding parameters for the interaction of hCDH15-MMH and anti-CDH15 monoclonal antibodies at 25°C. Antibody name mAb capture level (RU) 100 nM Ag (RU) bound k _a k _d K _D t½ (1/Ms) (1/s) (M) (minute) 8674 384 ± 3.8 74 1.11E+05 1.45E-04 1.31E-09 79 8675 278 ± 0.6 77 2.09E+05 8.02E-04 3.83E-09 14 8676 305 ± 0.4 127 3.75E+05 5.55E-05 1.48E-10 208 8677 388 ± 1.2 111 4.91E+05 1.36E-05 2.76E-11 852 8678 212 ± 0.6 93 2.49E+05 1.00E-05 * 4.01E-11 1155 * 8679 358 ± 6.9 89 1.29E+05 2.43E-04 1.89E-09 48 8680 137 ± 1 16 1.24E+05 7.66E-04 6.20E-09 15 8681 323 ± 1.6 54 9.06E+04 1.68E-04 1.85E-09 69 8682 355 ± 1.2 1 NB NB NB NB 8683 284 ± 0.2 7 IC IC IC IC 8684 197 ± 0.6 6 IC IC IC IC 8685 233 ± 0.4 2 NB NB NB NB 8686 441 ± 0.7 3 NB NB NB NB 8687 509 ± 3.8 7 IC IC IC IC 8784 408 ± 0.7 4 NB NB NB NB 8785 269 ± 2.4 3 NB NB NB NB 8786 259 ± 1.1 53 3.49E+05 3.35E-02 9.61E-08 0.34 8787 587 ± 1.1 7 IC IC IC IC 8788 224 ± 1.3 44 2.46E+05 3.07E-02 1.25E-07 0.38 8789 349 ± 3.7 96 3.76E+05 4.03E-04 1.07E-09 29 8790 277 ± 0.6 49 2.29E+05 9.97E-03 4.35E-08 1.2 8791 461 ± 1 14 2.16E+05 1.79E-02 8.25E-08 0.6 8792 297 ± 1.8 81 2.39E+05 1.39E-03 5.81E-09 8 8793 345 ± 1.3 68 1.99E+05 1.53E-03 7.68E-09 8 8794 279 ± 5 6 NB NB NB NB 8795 381 ± 5.6 64 1.94E+05 3.75E-04 1.93E-09 31 8796 541 ± 4.8 12 IC IC IC IC 9271 489 ± 1.4 78 5.17E+04 7.89E-04 1.52E-08 15 9272 481 ± 1.8 78 4.05E+04 9.96E-04 2.46E-08 12 9273 497 ± 1.5 95 5.43E+04 2.36E-03 4.34E-08 4.9 9274 506 ± 2.1 190 1.44E+05 3.29E-03 2.28E-08 3.5 9275 499 ± 1.3 66 3.63E+04 4.38E-04 1.21E-08 26 9276 491 ± 0.9 37 3.97E+04 3.76E-03 9.47E-08 3.1 9277 502 ± 3.1 83 6.69E+04 3.85E-04 5.75E-09 30 9290 482 ± 1.1 124 5.58E+04 7.27E-04 1.30E-08 16 9291 515 ± 0.9 47 2.66E+04 2.57E-04 9.66E-09 45 9292 478 ± 1.1 138 4.61E+04 2.29E-04 4.95E-09 51 9293 490 ± 1.3 102 5.66E+04 2.02E-03 3.57E-08 6 9294 497 ± 1.7 131 4.91E+04 1.06E-04 2.17E-09 109 9295 501 ± 0.8 309 3.02E+05 9.35E-05 3.10E-10 124 9296 510 ± 1.3 119 5.79E+04 2.74E-04 4.73E-09 42 9297 475 ± 1.8 194 2.38E+05 2.75E-03 1.16E-08 4.2 9298 481 ± 0.8 60 4.98E+04 1.99E-04 3.99E-09 58 9299 488 ± 1.1 34 1.80E+04 1.33E-04 7.37E-09 87 9300 480 ± 1.7 35 1.74E+04 3.67E-04 2.10E-08 32 9316 542 ± 1.3 91 3.52E+04 7.39E-05 2.10E-09 156 9317 487 ± 6.8 152 7.17E+04 3.86E-04 5.38E-09 30 9318 480 ± 2.2 193 9.07E+04 1.26E-04 1.38E-09 92 9319 499 ± 0.6 91 1.82E+05 1.11E-02 6.09E-08 1.0 9320 466 ± 0.7 169 1.41E+05 1.00E-05 * 7.10E-11 1155 * 9399 492 ± 1.5 26 2.81E+04 7.59E-04 2.70E-08 15 9400 466 ± 0.8 37 1.27E+04 9.42E-04 7.44E-08 12 9402 486 ± 1.1 132 1.77E+05 2.28E-03 1.29E-08 5.1 Isotype control group 1 477 ± 2.1 4 NB NB NB NB Isotype control group 2 341 ± 1 0 NB NB NB NB * = No dissociation was observed under the current experimental conditions, and the k _a value was determined by manually fixing the k _d value to 1.00E-05 s ^-1 while fitting the real-time binding sensorgram; NB = No binding; IC = Not determined

As shown in Table 5 below, at 37°C, anti-CDH15 monoclonal antibodies bound to hCDH15-MMH with KD values ranging from 218 pM to 234 nM.

Kinetic binding parameters for the interaction of hCDH15-MMH and anti-CDH15 monoclonal antibodies at 37°C. Antibody name mAb capture level (RU) 100 nM Ag (RU) bound k _a
(1/Ms) k _d
(1/s) K _D
(M) t½
(minute) 8674 428 ± 4.7 86 1.43E+05 7.84E-04 5.49E-09 15 8675 318 ± 0.4 94 3.16E+05 2.14E-03 6.78E-09 5 8676 341 ± 1.6 144 5.89E+05 1.96E-04 3.33E-10 59 8677 371 ± 1.4 127 5.57E+05 1.46E-04 2.62E-10 79 8678 237 ± 1.1 104 2.87E+05 1.60E-04 5.57E-10 72 8679 386 ± 7.7 113 1.92E+05 1.22E-03 6.33E-09 9 8680 174 ± 3.9 19 IC IC IC IC 8681 405 ± 1.3 82 2.24E+05 5.97E-04 2.66E-09 19 8682 375 ± 0.9 0 NB NB NB NB 8683 340 ± 0.4 11 IC IC IC IC 8684 216 ± 0.9 9 NB NB NB NB 8685 260 ± 5.6 5 NB NB NB NB 8686 497 ± 2 4 NB NB NB NB 8687 562 ± 2.5 16 IC IC IC IC 8784 472 ± 0.5 9 NB NB NB NB 8785 322 ± 3.2 3 NB NB NB NB 8786 275 ± 1.3 31 4.71E+05 9.96E-02 2.12E-07 0.12 8787 652 ± 1.9 11 IC IC IC IC 8788 244 ± 1.5 20 4.42E+05 7.02E-02 1.59E-07 0.16 8789 363 ± 3.3 103 5.03E+05 7.93E-04 1.58E-09 15 8790 311 ± 0.4 46 3.21E+05 2.20E-02 6.85E-08 0.5 8791 505 ± 1.2 19 2.27E+05 5.60E-03 2.47E-08 2.1 8792 349 ± 0.9 90 3.54E+05 5.10E-03 1.44E-08 2 8793 406 ± 0.8 75 2.98E+05 7.21E-03 2.42E-08 2 8794 290 ± 4.6 6 NB NB NB NB 8795 418 ± 5.7 82 2.63E+05 1.48E-03 5.65E-09 8 8796 618 ± 8.8 3 NB NB NB NB 9271 578 ± 2.4 75 6.44E+04 4.46E-03 6.93E-08 3 9272 588 ± 2.7 72 6.02E+04 6.20E-03 1.03E-07 2 9273 602 ± 2.7 95 7.32E+04 8.07E-03 1.10E-07 1.4 9274 618 ± 7.1 160 2.18E+05 1.06E-02 4.85E-08 1.1 9275 588 ± 3.2 68 1.61E+05 3.36E-03 2.08E-08 3 9276 585 ± 4.2 46 7.33E+04 5.69E-03 7.76E-08 2.0 9277 591 ± 4.1 100 9.24E+04 2.48E-03 2.68E-08 5 9290 586 ± 3.3 153 1.35E+05 3.93E-03 2.91E-08 3 9291 606 ± 2.2 66 3.55E+04 2.02E-03 5.68E-08 6 9292 582 ± 5.4 218 8.68E+04 4.22E-04 4.86E-09 27 9293 592 ± 3.2 85 6.34E+04 8.05E-03 1.27E-07 1 9294 609 ± 5 211 8.27E+04 2.38E-04 2.88E-09 48 9295 585 ± 6.2 375 5.07E+05 3.38E-04 6.66E-10 34 9296 605 ± 4.3 182 1.04E+05 1.08E-03 1.04E-08 11 9297 565 ± 2.6 173 3.06E+05 9.14E-03 2.99E-08 1.3 9298 598 ± 2.1 180 9.17E+04 6.13E-04 6.69E-09 19 9299 575 ± 1.5 53 3.09E+04 1.01E-03 3.27E-08 11 9300 587 ± 1.3 38 2.02E+04 3.73E-03 1.85E-07 3 9316 644 ± 3.4 133 4.58E+04 4.97E-04 1.08E-08 23 9317 595 ± 4.7 214 1.36E+05 1.45E-03 1.06E-08 8 9318 579 ± 2.9 258 1.41E+05 9.43E-04 6.71E-09 12 9319 599 ± 4 51 1.15E+05 2.68E-02 2.34E-07 0.4 9320 557 ± 2.6 244 1.74E+05 3.79E-05 2.18E-10 305 9399 592 ± 3.2 40 3.70E+04 3.41E-03 9.21E-08 3 9400 551 ± 1.5 36 2.93E+04 6.12E-03 2.09E-07 2 9402 580 ± 2.1 111 1.07E+05 8.47E-03 7.93E-08 1.4 Isotype control group 1 562 ± 1.9 0 NB NB NB NB Isotype control group 2 352 ± 0.6 -1 NB NB NB NB NB = No binding; IC = Undetermined

As shown in Table 6 below, at 25°C, anti-CDH15 monoclonal antibodies bound to hCDH15(K584Q)-MMH with KD values ranging from 65.6 pM to 235 nM.

Kinetic binding parameters for the interaction of hCDH15(K584Q)-MMH and anti-CDH15 monoclonal antibodies at 25°C. Antibody name mAb capture level (RU) 100 nM Ag (RU) bound k _a
(1/Ms) k _d
(1/s) K _D
(M) t½
(minute) 8674 378 ± 1 52 7.53E+04 1.56E-04 2.07E-09 74 8675 278 ± 0.2 66 1.58E+05 7.47E-04 4.74E-09 15 8676 304 ± 1.1 112 2.30E+05 8.97E-05 3.90E-10 129 8677 388 ± 1.4 88 4.67E+05 3.06E-05 6.56E-11 378 8678 212 ± 0.5 76 1.48E+05 1.49E-05 1.01E-10 774 8679 348 ± 2.2 69 8.28E+04 2.84E-04 3.43E-09 41 8680 134 ± 0.7 24 1.22E+05 7.80E-04 6.37E-09 15 8681 323 ± 0.4 45 7.39E+04 1.30E-04 1.76E-09 89 8682 353 ± 0.3 -2 NB NB NB NB 8683 284 ± 0.2 3 NB NB NB 39 8684 196 ± 1.2 11 IC IC IC IC 8685 233 ± 0.3 -1 NB NB NB NB 8686 438 ± 1.2 2 NB NB NB NB 8687 513 ± 0.9 5 IC IC IC IC 8784 407 ± 1.2 9 IC IC IC IC 8785 265 ± 0.8 -1 NB NB NB NB 8786 259 ± 0.7 40 2.30E+05 3.15E-02 1.37E-07 0.37 8787 584 ± 0.7 3 NB NB NB 39 8788 222 ± 0.8 28 1.66E+05 3.90E-02 2.35E-07 0.30 8789 341 ± 1.5 75 2.66E+05 3.66E-04 1.38E-09 32 8790 277 ± 0.2 39 1.79E+05 9.71E-03 5.44E-08 1 8791 462 ± 0.5 9 IC IC IC IC 8792 296 ± 1.9 74 1.47E+05 1.47E-03 1.00E-08 8 8793 344 ± 0.7 51 1.22E+05 1.72E-03 1.41E-08 7 8794 270 ± 2 2 NB NB NB NB 8795 369 ± 2.6 45 1.02E+05 4.72E-04 4.63E-09 24 8796 541 ± 1 13 IC IC IC IC 9271 486 ± 0.8 53 3.03E+04 6.18E-04 2.04E-08 19 9272 477 ± 1 52 2.46E+04 8.63E-04 3.51E-08 13 9273 495 ± 1.2 91 4.38E+04 2.07E-03 4.73E-08 6 9274 500 ± 0.9 188 1.43E+05 2.54E-03 1.78E-08 5 9275 497 ± 0.3 61 2.83E+04 3.86E-04 1.36E-08 30 9276 490 ± 0.9 40 4.19E+04 3.34E-03 7.96E-08 3 9277 494 ± 2.4 60 5.32E+04 3.08E-04 5.78E-09 38 9290 480 ± 1.1 115 5.03E+04 6.11E-04 1.21E-08 19 9291 515 ± 0.8 34 2.25E+04 2.54E-04 1.13E-08 45 9292 476 ± 0.6 126 4.35E+04 2.56E-04 5.88E-09 45 9293 488 ± 2 93 4.38E+04 1.53E-03 3.50E-08 8 9294 492 ± 2.4 123 4.15E+04 1.05E-04 2.53E-09 110 9295 497 ± 1.5 270 2.72E+05 7.63E-05 2.80E-10 151 9296 507 ± 1.3 109 4.89E+04 2.48E-04 5.07E-09 47 9297 473 ± 0.7 170 1.93E+05 2.38E-03 1.23E-08 5 9298 479 ± 0.6 49 3.92E+04 1.64E-04 4.17E-09 71 9299 487 ± 0.5 23 1.42E+04 6.63E-05 4.68E-09 174 9300 477 ± 1.4 24 1.24E+04 2.96E-04 2.39E-08 39 9316 553 ± 1.4 94 3.19E+04 8.11E-05 2.54E-09 142 9317 480 ± 1.3 150 6.47E+04 3.12E-04 4.82E-09 37 9318 476 ± 1.2 166 1.09E+05 1.22E-04 1.11E-09 95 9319 501 ± 2.4 71 1.20E+05 9.17E-03 7.66E-08 1 9320 464 ± 0.9 138 1.24E+05 1.00E-05 * 8.08E-11 1155 * 9399 488 ± 0.6 21 2.52E+04 6.30E-04 2.50E-08 18 9400 465 ± 0.6 21 6.16E+03 8.87E-04 1.44E-07 13 9402 485 ± 1.6 132 1.70E+05 1.39E-03 8.19E-09 8 Isotype control group 1 475 ± 0.7 2 NB NB NB NB Isotype control group 2 340 ± 0.2 -4 NB NB NB NB * = No dissociation was observed under the current experimental conditions, and the k _a value was determined by manually fixing the k _d value to 1.00E-05 s ^-1 while fitting the real-time binding sensorgram; NB = No binding; IC = Not determined

As shown in Table 7 below, at 37°C, anti-CDH15 monoclonal antibodies bound to hCDH15(K584Q)-MMH with KD values ranging from 375 pM to 334 nM.

Kinetic binding parameters for the interaction of hCDH15(K584Q)-MMH and anti-CDH15 monoclonal antibodies at 37°C. Antibody name mAb capture level (RU) Combined 100 nM Ag (RU) k _a
(1/Ms) k _d
(1/s) K _D
(M) t½
(minute) 8674 419 ± 2.3 66 1.02E+05 7.71E-04 7.56E-09 15 8675 318 ± 2.7 82 2.24E+05 1.96E-03 8.74E-09 6 8676 339 ± 0.8 128 4.32E+05 2.37E-04 5.48E-10 49 8677 371 ± 0.8 102 4.78E+05 1.79E-04 3.75E-10 64 8678 234 ± 0.9 88 1.93E+05 1.61E-04 8.35E-10 72 8679 372 ± 2.9 95 1.85E+05 1.19E-03 6.43E-09 10 8680 167 ± 2 32 IC IC IC IC 8681 402 ± 0.7 72 1.14E+05 5.42E-04 4.75E-09 21 8682 373 ± 0.6 -1 NB NB NB NB 8683 338 ± 0.3 7 IC IC IC IC 8684 215 ± 0.9 11 IC IC IC IC 8685 262 ± 0.3 2 NB NB NB NB 8686 492 ± 1.7 2 NB NB NB NB 8687 565 ± 0.7 11 IC IC IC IC 8784 471 ± 0.8 21 IC IC IC IC 8785 315 ± 1.7 3 NB NB NB NB 8786 272 ± 0.8 21 4.45E+05 1.49E-01 3.34E-07 0.08 8787 646 ± 1.7 8 IC IC IC IC 8788 242 ± 0.8 11 IC IC IC IC 8789 354 ± 2.2 83 3.37E+05 9.06E-04 2.69E-09 13 8790 310 ± 0.3 37 2.66E+05 2.40E-02 9.02E-08 0.5 8791 505 ± 0.4 13 1.60E+05 9.85E-04 6.17E-09 11.7 8792 348 ± 0.4 89 2.93E+05 4.87E-03 1.66E-08 2 8793 404 ± 0.8 58 2.51E+05 7.65E-03 3.05E-08 2 8794 280 ± 2.4 3 NB NB NB NB 8795 405 ± 2.3 59 1.34E+05 1.70E-03 1.26E-08 7 8796 607 ± 3.4 23 NB NB NB NB 9271 NT NT NT NT NT NT 9272 NT NT NT NT NT NT 9273 NT NT NT NT NT NT 9274 NT NT NT NT NT NT 9275 NT NT NT NT NT NT 9276 NT NT NT NT NT NT 9277 NT NT NT NT NT NT 9290 NT NT NT NT NT NT 9291 NT NT NT NT NT NT 9292 NT NT NT NT NT NT 9293 NT NT NT NT NT NT 9294 NT NT NT NT NT NT 9295 NT NT NT NT NT NT 9296 NT NT NT NT NT NT 9297 NT NT NT NT NT NT 9298 NT NT NT NT NT NT 9299 NT NT NT NT NT NT 9300 NT NT NT NT NT NT 9316 NT NT NT NT NT NT 9317 NT NT NT NT NT NT 9318 NT NT NT NT NT NT 9319 NT NT NT NT NT NT 9320 NT NT NT NT NT NT 9399 NT NT NT NT NT NT 9400 NT NT NT NT NT NT 9402 NT NT NT NT NT NT Isotype control group 1 NT NT NT NT NT NT Isotype control group 2 350 ± 0.5 -2 NB NB NB NB NB = No binding; IC = Not determined; NT = Not tested

As shown in Table 8 below, at 25°C, anti-CDH15 monoclonal antibodies bound to mfCDH15-MMH with KD values ranging from 86.7 pM to 1.33 μM.

Kinetic binding parameters for the interaction of mfCDH15-MMH and anti-CDH15 monoclonal antibodies at 25°C. Antibody name mAb capture level (RU) Combined 100 nM Ag (RU) k _a k _d K _D t½ (1/Ms) (1/s) (M) (minute) 8674 375 ± 0.8 39 5.91E+04 1.69E-03 2.86E-08 7 8675 278 ± 0.4 67 1.68E+05 1.18E-03 7.02E-09 10 8676 303 ± 0.7 113 1.43E+05 1.49E-05 1.05E-10 775 8677 388 ± 2.3 91 4.86E+05 3.63E-04 7.48E-10 32 8678 212 ± 0.6 79 1.50E+05 1.86E-04 1.24E-09 62 8679 342 ± 1.6 69 8.47E+04 3.07E-04 3.63E-09 38 8680 132 ± 1.7 13 3.37E+04 9.41E-04 2.79E-08 12 8681 323 ± 0.3 45 6.98E+04 2.63E-04 3.76E-09 44 8682 353 ± 0.5 -2 NB NB NB NB 8683 283 ± 0.1 3 NB NB NB NB 8684 197 ± 0.9 12 IC IC IC IC 8685 233 ± 0.2 0 NB NB NB NB 8686 436 ± 1 3 NB NB NB NB 8687 512 ± 1.3 5 IC IC IC IC 8784 405 ± 0.5 9 IC IC IC IC 8785 262 ± 0.5 1 NB NB NB NB 8786 257 ± 0.4 15 4.38E+04 5.84E-02 1.33E-06 0.20 8787 582 ± 0.7 6 IC IC IC IC 8788 221 ± 2.1 95 3.07E+05 8.69E-03 2.83E-08 1 8789 337 ± 0.9 84 3.64E+05 3.66E-04 1.01E-09 32 8790 277 ± 0.3 43 1.74E+05 9.14E-03 5.27E-08 1 8791 462 ± 0.6 13 1.45E+05 1.66E-02 1.14E-07 1 8792 296 ± 1.1 74 1.41E+05 1.90E-03 1.34E-08 6 8793 343 ± 1.4 82 2.73E+05 5.10E-04 1.87E-09 23 8794 264 ± 1.7 2 NB NB NB NB 8795 362 ± 2.1 58 1.96E+05 5.63E-04 2.88E-09 21 8796 540 ± 0.7 15 1.11E+05 5.02E-04 4.52E-09 23 9271 485 ± 0.5 31 2.72E+04 1.40E-03 5.15E-08 8 9272 476 ± 0.7 34 1.66E+04 1.16E-03 6.96E-08 10 9273 493 ± 2.5 176 8.11E+04 2.74E-04 3.37E-09 42 9274 497 ± 1.2 96 1.11E+05 1.24E-02 1.12E-07 1 9275 497 ± 0.2 65 3.71E+04 4.29E-04 1.16E-08 27 9276 490 ± 0.9 22 5.25E+04 6.44E-03 1.23E-07 2 9277 493 ± 0.9 48 4.34E+04 3.09E-04 7.12E-09 37 9290 478 ± 2.1 132 6.24E+04 5.34E-04 8.57E-09 22 9291 513 ± 0.7 49 3.26E+04 3.83E-04 1.17E-08 30 9292 475 ± 0.6 110 3.86E+04 2.06E-04 5.33E-09 56 9293 490 ± 2.7 46 2.99E+04 2.02E-03 6.75E-08 6 9294 493 ± 1.1 115 4.28E+04 1.12E-04 2.61E-09 104 9295 496 ± 0.8 292 3.55E+05 7.98E-05 2.25E-10 145 9296 508 ± 3.2 97 4.94E+04 4.61E-04 9.32E-09 25 9297 473 ± 1 194 2.64E+05 2.60E-03 9.82E-09 4 9298 479 ± 0.8 65 5.78E+04 1.28E-04 2.21E-09 90 9299 486 ± 1.4 31 2.47E+04 1.65E-04 6.67E-09 70 9300 479 ± 0.5 38 1.76E+04 4.32E-04 2.45E-08 27 9316 553 ± 0.3 81 3.13E+04 8.84E-05 2.82E-09 131 9317 481 ± 2.8 151 6.73E+04 3.86E-04 5.74E-09 30 9318 476 ± 1.7 206 1.30E+05 1.37E-04 1.05E-09 84 9319 499 ± 0.9 56 1.02E+05 9.56E-03 9.36E-08 1 9320 463 ± 1.6 137 1.15E+05 1.00E-05 * 8.67E-11 1155 * 9399 491 ± 3.4 26 3.56E+04 8.32E-04 2.33E-08 14 9400 462 ± 0.8 12 6.58E+03 9.90E-04 1.50E-07 12 9402 485 ± 2.3 69 1.24E+05 2.81E-03 2.27E-08 4 Isotype control group 1 474 ± 2.1 -1 NB NB NB NB Isotype control group 2 339 ± 0.5 -3 NB NB NB NB * = No dissociation was observed under the current experimental conditions, and the k _a value was determined by manually fixing the k _d value to 1.00E-05 s ^-1 while fitting the real-time binding sensorgram; NB = No binding; IC = Not determined

As shown in Table 9 below, at 37°C, anti-CDH15 monoclonal antibodies bound to mfCDH15-MMH with KD values ranging from 151 pM to 539 nM.

Kinetic binding parameters for the interaction of mfCDH15-MMH and anti-CDH15 monoclonal antibodies at 37°C. Antibody name mAb capture level (RU) Combined 100 nM Ag (RU) k _a k _d K _D t½ (1/Ms) (1/s) (M) (minute) 8674 412 ± 1.9 36 1.19E+05 8.44E-03 7.12E-08 1 8675 316 ± 0.5 81 2.40E+05 2.59E-03 1.08E-08 4 8676 337 ± 1.3 129 3.55E+05 5.36E-05 1.51E-10 215 8677 370 ± 0.6 93 4.74E+05 1.84E-03 3.89E-09 6 8678 233 ± 0.7 85 2.09E+05 1.32E-03 6.32E-09 9 8679 366 ± 2.1 99 1.97E+05 1.51E-03 7.69E-09 8 8680 163 ± 1.2 21 IC IC IC IC 8681 400 ± 0.8 70 1.13E+05 8.98E-04 7.99E-09 13 8682 372 ± 0.7 -1 NB NB NB NB 8683 337 ± 0.6 9 4.47E+04 1.80E-03 4.04E-08 6 8684 214 ± 0.4 16 IC IC IC IC 8685 262 ± 0.4 4 NB NB NB NB 8686 488 ± 1.3 5 NB NB NB NB 8687 563 ± 0.6 13 6.40E+04 2.24E-04 3.50E-09 52 8784 469 ± 0.8 19 IC IC IC IC 8785 310 ± 1.5 4 NB NB NB NB 8786 270 ± 0.4 8 3.35E+05 1.81E-01 5.39E-07 0.06 8787 642 ± 1.4 12 1.70E+04 8.37E-05 4.92E-09 138 8788 242 ± 3 55 4.22E+05 3.63E-02 8.62E-08 032 8789 347 ± 2.8 92 4.14E+05 6.99E-04 1.69E-09 17 8790 310 ± 0.3 43 2.48E+05 2.03E-02 8.17E-08 1 8791 504 ± 0.2 17 1.98E+05 4.13E-03 2.08E-08 3 8792 347 ± 0.5 87 2.61E+05 5.80E-03 2.22E-08 2 8793 402 ± 1.1 103 3.26E+05 1.99E-03 6.10E-09 6 8794 274 ± 2 3 NB NB NB NB 8795 398 ± 2.3 74 2.74E+05 1.98E-03 7.23E-09 6 8796 589 ± 3.6 17 IC IC IC IC 9271 NT NT NT NT NT NT 9272 NT NT NT NT NT NT 9273 NT NT NT NT NT NT 9274 NT NT NT NT NT NT 9275 NT NT NT NT NT NT 9276 NT NT NT NT NT NT 9277 NT NT NT NT NT NT 9290 NT NT NT NT NT NT 9291 NT NT NT NT NT NT 9292 NT NT NT NT NT NT 9293 NT NT NT NT NT NT 9294 NT NT NT NT NT NT 9295 NT NT NT NT NT NT 9296 NT NT NT NT NT NT 9297 NT NT NT NT NT NT 9298 NT NT NT NT NT NT 9299 NT NT NT NT NT NT 9300 NT NT NT NT NT NT 9316 NT NT NT NT NT NT 9317 NT NT NT NT NT NT 9318 NT NT NT NT NT NT 9319 NT NT NT NT NT NT 9320 NT NT NT NT NT NT 9399 NT NT NT NT NT NT 9400 NT NT NT NT NT NT 9402 NT NT NT NT NT NT Isotype control group 1 NT NT NT NT NT NT Isotype control group 2 348 ± 0.8 -2 NB NB NB NB NB = No binding; IC = Not determined; NT = Not tested

As shown in Table 10 below, at 25°C, anti-CDH15 monoclonal antibodies bound to mCDH15-MMH with KD values ranging from 129 pM to 323 nM.

Kinetic binding parameters for the interaction of mCDH15-MMH and anti-CDH15 monoclonal antibodies at 25°C. Antibody name mAb capture level (RU) 100 nM Ag (RU) bound k _a k _d K _D t½ (1/Ms) (1/s) (M) (minute) 8674 374 ± 0.4 11 1.26E+05 2.33E-02 1.86E-07 0.50 8675 278 ± 0.1 55 2.09E+05 1.59E-03 7.63E-09 7 8676 303 ± 0.6 2 IC IC IC IC 8677 389 ± 0.3 65 2.64E+05 1.60E-03 6.06E-09 7 8678 212 ± 0.7 -1 NB NB NB NB 8679 339 ± 0.1 -1 NB NB NB NB 8680 133 ± 0.4 14 2.21E+05 8.32E-04 3.77E-09 14 8681 322 ± 0.1 47 1.19E+05 2.80E-04 2.36E-09 41 8682 352 ± 0.2 -2 NB NB NB NB 8683 283 ± 0.1 33 5.86E+04 1.52E-04 2.60E-09 76 8684 197 ± 1.2 9 IC IC IC IC 8685 233 ± 0.3 18 4.86E+04 3.09E-04 6.35E-09 37 8686 434 ± 0.2 14 3.89E+04 1.00E-05 * 2.57E-10 1155 8687 512 ± 0.2 14 3.62E+04 1.00E-05 * 2.76E-10 1155 8784 404 ± 0.4 15 5.20E+04 1.00E-05 * 1.92E-10 1155 8785 261 ± 0.5 23 2.43E+05 1.85E-02 7.61E-08 1 8786 257 ± 0.1 -1 NB NB NB NB 8787 581 ± 0.2 32 5.76E+04 1.00E-05 * 1.74E-10 1155 8788 217 ± 5.6 68 5.70E+05 6.15E-03 1.08E-08 2 8789 335 ± 0.1 64 2.64E+05 1.28E-03 4.83E-09 9 8790 277 ± 0.2 0 NB NB NB NB 8791 463 ± 0.3 100 2.58E+05 4.18E-04 1.62E-09 28 8792 295 ± 1.7 3 NB NB NB NB 8793 343 ± 0.4 61 2.08E+05 7.25E-04 3.49E-09 16 8794 261 ± 0.5 -2 NB NB NB NB 8795 358 ± 0.6 54 2.01E+05 9.22E-04 4.59E-09 13 8796 537 ± 1 41 7.73E+04 1.00E-05 * 1.29E-10 1155 9271 484 ± 1.3 14 4.01E+04 4.31E-03 1.07E-07 3 9272 475 ± 0.7 15 2.63E+04 3.71E-03 1.41E-07 3 9273 491 ± 4.5 132 1.53E+05 7.00E-04 4.58E-09 17 9274 497 ± 2.2 0 NB NB NB NB 9275 496 ± 1.3 45 1.47E+05 1.20E-02 8.16E-08 1 9276 490 ± 1.1 45 6.23E+04 2.16E-03 3.47E-08 5 9277 490 ± 1.4 16 1.11E+04 5.50E-04 4.96E-08 21 9290 478 ± 1.2 101 9.94E+04 6.19E-04 6.23E-09 19 9291 513 ± 0.6 35 4.82E+04 5.39E-04 1.12E-08 21 9292 474 ± 1 1 NB NB NB NB 9293 488 ± 1.5 16 1.67E+04 3.88E-03 2.32E-07 3 9294 492 ± 1.4 1 NB NB NB NB 9295 496 ± 1.2 189 4.37E+05 8.94E-05 2.04E-10 129 9296 507 ± 2.9 2 NB NB NB NB 9297 471 ± 1 63 4.07E+05 2.75E-02 6.75E-08 0.42 9298 478 ± 0.3 27 6.59E+04 6.19E-03 9.39E-08 2 9299 487 ± 0.3 23 3.86E+04 3.24E-04 8.39E-09 36 9300 477 ± 0.7 26 3.25E+04 6.81E-04 2.10E-08 17 9316 553 ± 1.9 101 7.07E+04 3.49E-04 4.93E-09 33 9317 478 ± 2 134 1.63E+05 4.15E-04 2.54E-09 28 9318 477 ± 2.3 128 1.28E+05 1.02E-03 7.95E-09 11 9319 498 ± 4.5 10 5.62E+04 1.82E-02 3.23E-07 1 9320 463 ± 1 -2 NB NB NB NB 9399 490 ± 2.5 20 3.68E+04 1.26E-03 3.41E-08 9 9400 461 ± 0.6 6 NB NB NB NB 9402 484 ± 1.9 40 9.26E+04 4.38E-03 4.74E-08 3 Isotype control group 1 474 ± 0.6 6 NB NB NB NB Isotype control group 2 338 ± 0.3 -3 NB NB NB NB NB = No binding; IC = Not determined; NT = Not tested

As shown in Table 11 below, at 37°C, anti-CDH15 monoclonal antibodies bound to mCDH15-MMH with KD values ranging from 637 nM to 391 nM.

Kinetic binding parameters for the interaction of mCDH15-MMH and anti-CDH15 monoclonal antibodies at 37°C. Antibody name mAb capture level (RU) Combined 100 nM Ag (RU) k _a k _d K _D t½ (1/Ms) (1/s) (M) (minute) 8674 408 ± 1.5 4 NB NB NB NB 8675 316 ± 0.1 62 3.02E+05 3.12E-03 1.03E-08 4 8676 335 ± 0.3 1 NB NB NB NB 8677 370 ± 0.5 49 4.65E+05 1.06E-02 2.29E-08 1 8678 233 ± 0.6 -1 NB NB NB NB 8679 362 ± 0.9 -1 NB NB NB NB 8680 161 ± 0.8 17 IC IC IC IC 8681 399 ± 0.7 68 1.77E+05 5.78E-04 3.27E-09 20 8682 370 ± 0.2 -2 NB NB NB NB 8683 336 ± 0 47 9.86E+04 9.74E-04 9.87E-09 12 8684 213 ± 2.4 5 IC IC IC IC 8685 262 ± 0.1 25 7.81E+04 3.57E-03 4.57E-08 3 8686 484 ± 0.4 22 4.16E+04 2.03E-04 4.87E-09 57 8687 562 ± 0.1 23 4.56E+04 1.98E-04 4.34E-09 58 8784 467 ± 0.2 31 7.66E+04 1.56E-04 2.04E-09 74 8785 307 ± 0.9 11 3.65E+05 1.43E-01 3.91E-07 0.08 8786 269 ± 0.3 -1 NB NB NB NB 8787 639 ± 0.2 48 7.87E+04 5.02E-05 6.37E-10 230 8788 240 ± 1.8 44 7.08E+05 6.70E-02 9.47E-08 0.17 8789 347 ± 0.8 55 3.84E+05 3.60E-03 9.38E-09 3 8790 309 ± 0.4 -1 NB NB NB NB 8791 503 ± 0.6 110 3.16E+05 1.75E-03 5.55E-09 7 8792 346 ± 0.7 11 IC IC IC IC 8793 401 ± 0.6 72 2.39E+05 3.27E-03 1.37E-08 4 8794 270 ± 0.6 -1 NB NB NB NB 8795 393 ± 0.9 63 2.53E+05 4.13E-03 1.63E-08 3 8796 589 ± 0.2 60 8.25E+04 2.41E-04 2.92E-09 48 9271 NT NT NT NT NT NT 9272 NT NT NT NT NT NT 9273 NT NT NT NT NT NT 9274 NT NT NT NT NT NT 9275 NT NT NT NT NT NT 9276 NT NT NT NT NT NT 9277 NT NT NT NT NT NT 9290 NT NT NT NT NT NT 9291 NT NT NT NT NT NT 9292 NT NT NT NT NT NT 9293 NT NT NT NT NT NT 9294 NT NT NT NT NT NT 9295 NT NT NT NT NT NT 9296 NT NT NT NT NT NT 9297 NT NT NT NT NT NT 9298 NT NT NT NT NT NT 9299 NT NT NT NT NT NT 9300 NT NT NT NT NT NT 9316 NT NT NT NT NT NT 9317 NT NT NT NT NT NT 9318 NT NT NT NT NT NT 9319 NT NT NT NT NT NT 9320 NT NT NT NT NT NT 9399 NT NT NT NT NT NT 9400 NT NT NT NT NT NT 9402 NT NT NT NT NT NT Isotype control group 1 NT NT NT NT NT NT Isotype control group 2 347 ± 1 -1 NB NB NB NB NB = No binding; IC = Not determined; NT = Not tested

Example 5: Cross-competition between exemplary CDH15 antibodies

Binding competition between anti-CDH15 monoclonal antibodies (mAbs) was determined using a real-time, label-free bio-layer interferometry (BLI) assay on an Octet HTX biosensor platform (Sartorius ForteBio Corp.). The entire experiment was performed at 25°C in 10 mM HEPES (HBS-P) buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM MgCl ₂ , 1 mM CaCl ₂ , 1 mg/mL BSA, 0.02% NaN ₃ , and 0.05% v/v surfactant Tween-20, with plate shaking at 1000 rpm. To assess the ability of one antibody to compete with another for binding to CDH15, 0.3 nM of recombinant human CDH15 extracellular domain expressed with a C-terminal myc-myc-hexahistidine (hCDH15-MMH) was first captured on the tip by immersing an Octet biosensor tip (Fortebio Inc, # 18-5122) coated with an anti-Penta-His antibody into a well containing a 20 μg/mL hCDH15-MMH solution for 180 s. The captured biosensor tip was then saturated with mAb-1 by immersing the well containing a 50 μg/mL anti-CDH15 monoclonal antibody (hereinafter referred to as mAb-1) solution for 4 min. The biosensor tip was then immersed in a well containing a 50 μg/mL solution of the second anti-CDH15 monoclonal antibody (hereinafter referred to as mAb-2) for 3 minutes. Between each step, the biosensor tip was washed in HBS-P buffer. The real-time binding reaction was monitored, and the binding reaction was recorded after each step. The binding reaction of mAb-2 to hCDH15-MMH pre-complexed with mAb-1 was compared with the binding reaction of hCDH15-MMH alone (mAb-1 = isotype control), and if the pre-complexed mAb-1 reduced the binding of mAb-2 by more than 60%, mAb-1 was classified as a competitor of mAb-2. The competitors of each antibody tested are summarized in Table 12 below.

[Table 12]

Cross-competition between different anti-CDH15 monoclonal antibodies for binding to hCDH15-MMH.

Example 6: Binding of purified CDH15 antibody to mouse muscle stem cells

As described above, CDH15 mRNA is highly expressed in MuSCs. As shown in Figure 7A , the exemplary anti-hCDH15 antibody, 8787, was able to bind to mouse MuSCs associated with individual myofibers. Therefore, this demonstrates that the anti-hCDH15 antibody of the present disclosure can specifically bind to mCDH15 located within its unique niche on muscle stem cells (i.e., attached to single myofibers).

Furthermore, as shown in Figure 7b , an exemplary commercial anti-mCDH15 antibody was also able to bind to MuSCs adjacent to myofibers in muscle cross-sections from WT mice. Furthermore, Figure 7c shows that in wild-type mice, nearly all Pax7+ MuSCs (approximately 95%) exhibited some level of CDH15 protein expressed on the cell surface, whereas CDH15 protein was absent from Pax7+ MuSCs from CDH15-/- mice. Thus, this demonstrates that the commercial anti-mouse CDH15 antibody binds to mCDH15 in a similar pattern as the anti-hCDH15 antibody of the present disclosure, whereas, as expected, no binding was detected in CDH15-/- mice.

Example 7: Binding of purified CDH15 antibody to rhabdomyosarcoma cells.

CDH15 is highly expressed in RMS and can specifically differentiate RMS from other sarcomas. Furthermore, high CDH15 expression is associated with chemoresistance and a poorer prognosis in soft tissue sarcomas. The ability of the anti-hCDH15 antibodies of the present disclosure to bind to RMS cells was investigated. As shown in Figure 8 , exemplary anti-hCDH15 antibodies 8787 and 9295 specifically bound to alveolar and embryonal RMS cells, but not to glioblastoma cells used as a control. Furthermore, 9295 was able to specifically bind to RMS tumor cells (see Figure 9 ). Therefore, this demonstrates that the anti-hCDH15 antibodies of the present disclosure can specifically bind to hCDH15 on RMS cells and tumor cells.

method

In vivo muscle damage and histological analysis

To assess muscle regenerative capacity in vivo ( Figures 2a–c, 6a–d ), WT and CDH15−/− mice were anesthetized with isoflurane, their legs shaved, and injected intramuscularly with 50 μL of 10 μM cardiotoxin (CTX, Sigma #217503, lot #3123340 diluted in 0.9% saline) into the tibialis anterior (TA) muscle. At 5, 15, or 25 days post-injury (dpi), mice were sacrificed, and TA muscles were dissected and cryopreserved in OCT compound (Tissue-Tek catalog # 4583) by freezing in isopentane cooled with liquid nitrogen. Tissues were stored at -80°C and then sectioned at 12 μm thickness onto charged SuperFrost Plus glass slides (ThermoFisher Scientific catalog # 12-550-15). Sections were fixed with 4% PFA for 15 minutes. The slides were then washed three times with PBS for 5 minutes each. The tissue sections were covered with blocking buffer (20% goat serum, 0.3% Triton in PBS) for 1 hour at room temperature. The primary antibody was added (for laminin, diluted 1:100 in blocking buffer; Sigma L9393) and incubated overnight. The slides were then washed with PBS and incubated with the secondary antibody (diluted 1:250 in blocking buffer; Invitrogen A32733) for 1 hour. The slides were washed with PBS, counterstained with Hoechst 33342 (ThermoFisher, Cat. #00-4958-02, 1:1000) for 5 minutes, and mounted in Fluoromount G (ThermoFisher Cat. #00-4958-02). Slides were dried overnight and then imaged using a Zeiss Axioscan microscope. Cross-sectional area and the number of nuclei per myofiber were quantified using HALO imaging software (Indica Labs) and Python.

In vitro muscle function analysis

To assess functional regeneration ( Figures 3a–b ), WT and CDH15−/− mice were anesthetized, and 50 μL of 10 μM cardiotoxin (Sigma #217503, lot #3123340 diluted in 0.9% saline) was injected into the tibialis anterior and extensor digitorum longus (EDL) muscles. Fifteen days post-injury, mice were sacrificed, and the EDL muscles were carefully dissected and assessed for force production using the Muscle Strip System (840MD) from MyoDynamics. Sutures were tied around the EDL tendon, and the EDL was then placed and secured within the 840MD system. The medium used in the 840MD system was Tyrode's solution (modified II) BSS-375 from Boston BioProducts. The EDL was stretched to an appropriate length and then stimulated with various tonic stimuli (20 V for 2 ms, pulse frequency starting at 25 Hz and increasing to 150 Hz, with a 2-minute interval between each stimulus). Before removing the EDL from the 840MD system, the EDL length was recorded. The EDL was then removed and weighed, and these data were used to calculate the specific force. The maximum tonic force generated by the EDL at 15 days post-injury was then plotted against the maximum tonic force of uninjured mice of the same genotype.

Activation of muscle stem cells in isolated single myofibers

To assess the quiescent-to-activated state transition of MuSCs within the intrinsic myofibrillar niche ex vivo ( Fig. 4a–b ), gastrocnemius muscles from WT and CDH15−/− mice were removed, digested with collagenase, and individual myofibers were isolated, washed, and incubated in DMEM + 20% FBS at 37°C in 5% CO2 for 48 h. After 48 h, myofibers were fixed with 4% paraformaldehyde and stained for Pax7 (Developmental Studies Hybridoma Bank: PAX7), Hoechst (Invitrogen: H3570), and actin (Invitrogen: A22287). Myofibers were then transferred to microscope slides, coverslipped, and imaged using a Zeiss LSM 710 confocal microscope. Using Zen software (Zeiss), the total number of Pax7+ MuSCs as well as the number of Pax7+ MuSCs within each clonal cluster were manually quantified (n = 3 from WT and 3 from CDH15-/- mice), and the percentage of clones containing only one Pax7+ MuSC or containing two or more MuSCs as well as the total number of MuSCs per cluster were plotted.

RNAseq of FACS-isolated MuSCs

To determine whether CDH15 deletion in quiescent MuSCs affects their transcriptional profile, MuSCs from healthy, intact WT and CDH15−/− mice were isolated by FACS ( Figures 5a–b ). Mice were anesthetized and sacrificed, and gastrocnemius, soleus, quadriceps femoris, tibialis anterior, and extensor digitorum longus muscles were isolated and minced. Minced muscles were incubated in 700 U/mL collagenase, type II (Gibco, 16050-122) in Ham's F10 buffer (Life Technologies) on a shaker at 37°C for 1.5 h and then switched to digestion medium containing 100 U/mL collagenase type II and 2 U/mL dispase II (Roche). After enzymatic digestion, the samples were pipetted up and down 10 times with a 10 mL syringe fitted with a 20G needle and then passed through a 70 μm cell strainer. The samples were purified using satellite cell isolation beads (Miltenyi Biotec.), and then labeled with fluorophore-conjugated antibodies against α7-integrin (R&D Systems, FAB3518G) and CD34 (BD Biosciences, 560230) to stain MuSCs on ice for 20 min. The samples were washed, suspended in FACS buffer, and α7-integrin+/CD34+ MuSCs were sorted using a FACSAria Fision. The sorted MuSCs were dissolved in Trizol, and RNA was isolated using the miRNeasy Micro Kit (Qiagen).

Strand-specific RNA-seq libraries were prepared using KAPA mRNA HyperPrep (Roche Sequencing Solutions). Libraries were amplified by 12 cycles of PCR. Sequencing was performed on an Illumina HiSeq 2500 (Illumina) using multiplexed single-read sequences performed for 33 cycles. Raw BCL sequence data were converted to FASTQ format using Illumina Casava v1.8.2. Reads were decoded based on read barcodes, and read quality was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Array Studio (QIAGEN OmicSoft) was used to map reads to the mouse transcriptome (NCBI GRCm38), allowing two mismatches. Reads mapped to exons of genes were aggregated at the gene level.

Transcriptome analysis was performed using custom R code. The DESeq2 package was used for differential gene expression analysis, and significantly perturbed genes were defined as those with a fold change ≥1.5 in either the up or down direction and a Benjamini and Hochberg multiple comparison-adjusted P ≤.05. The genome browser PWM analysis within the Enrichr application was used to identify the abundance of genes harboring specific transcription factor binding motifs that were differentially regulated in WT compared to CDH15−/− MuSCs.

CDH15 antibody binds to single myofibrillar-associated MuSCs

For isolation of individual muscle fibers ( Figure 7a ), gastrocnemius muscles were removed, digested with collagenase, and individual muscle fibers were isolated, washed, and incubated overnight in DMEM + 10% horse serum at 37°C under 5% CO2. After overnight incubation, single muscle fibers were incubated with 100 nM anti-hCDH15 antibody (REGN8787) or isotype control antibody for 30 min. Muscle fibers were then washed twice in DMEM + 10% horse serum, incubated with 10 μg/mL fluorescence-conjugated secondary antibody for 30 min, washed twice in DMEM + 10% horse serum, and then fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. Individual fibers were then washed twice with PBS, stained with Hoechst for 5 min, washed once more with PBS, transferred to microscope slides, coverslipped, and imaged using a Zeiss LSM 710 confocal microscope.

Quantification of the percentage of MuSCs expressing CDH15 protein on the cell surface

To detect CDH15 protein expression and localization in tissue sections ( Figures 7b–c ), gastrocnemius muscles from wild-type or CDH15−/− mice were removed and cryopreserved in OCT compound (Tissue-Tek catalog # 4583) by freezing in isopentane cooled with liquid nitrogen. Tissues were stored at -80°C and then sectioned at 12 μm thickness onto charged SuperFrost Plus glass slides (ThermoFisher Scientific catalog # 12-550-15). Sections were fixed in 4% PFA for 15 min. Slides were then washed three times with phosphate-buffered saline (PBS) for 5 min each. Tissue sections were covered with blocking buffer (20% goat serum, 0.3% Triton in PBS) for 1 h at room temperature. Primary antibodies were added (1:100 diluted in blocking buffer for laminin: Sigma L9393, CDH15: BD 611101, and Pax7 DSHB: Pax7) and incubated overnight. The slides were then washed with PBS and incubated with secondary antibodies (1:250 diluted in blocking buffer; Invitrogen) for 1 hour. The slides were washed with PBS, counterstained with Hoechst 33342 (ThermoFisher, Cat. #00-4958-02, 1:1000) for 5 minutes, and mounted in Fluoromount G (ThermoFisher Cat. #00-4958-02). The slides were dried overnight and then imaged using a Zeiss Axioscan microscope. The total number of Pax7+ cells, as well as the number of Pax7+ cells positive for CDH15 staining, were manually counted and quantified.

Binding of CDH15 antibodies to rhabdomyosarcoma cells

To determine whether the CDH15 antibody could specifically bind to rhabdomyosarcoma cells but not to other tumor cells (e.g., glioblastoma), in vitro binding assays were performed ( Figures 8 and 9 ). The rhabdomyosarcoma cell lines tested were RH30 (ATCC) and RD (ATCC), and the glioblastoma cell line U87 (ATCC) was used as a negative control. For adherent cell culture media, RPMI-1640 containing 10% FBS was used for the RH30 cell line, and DMEM (11995-065) containing 10% FBS was used for RD and U87.

For live-stained adherent cells, cell lines were seeded in collagen-coated 96-well plates and stained 72 hours after plating. Cells were incubated with 100 nM CDH15 antibody or isotype control antibody for 30 minutes, washed twice with medium, and then incubated with 10 μg/mL of fluorescently conjugated secondary (Invitrogen A-21445 for human antibodies, Invitrogen A-21235 for mouse antibodies) for 30 minutes. Cells were washed twice more with medium and fixed with 4% PFA for 15 minutes. Cells were then washed with PBS, stained with Hoescht (1:1000) for 5 minutes, and washed twice more with PBS. Cells were then incubated for 1 hour in blocking buffer (20% goat serum, 0.3% Triton in PBS) at room temperature. The primary antibody was then added (diluted 1:100 in blocking buffer containing myogenin; Abcam AB124800) and incubated overnight. Cells were washed twice with PBS, and the secondary antibody was added for 1 hour (diluted 1:200 in blocking buffer; Invitrogen A11035). Cells were washed twice with PBS and imaged using a Zeiss Axio observation microscope.

For tumorsphere culture, RD cells were seeded in low attachment 6-well plates (Corning 3471) and cultured in tumorsphere medium (1:1 DMEM (11995-065) & Ham's F12 (11765-054) + 2X B27 supplement (17504004) + 10 ng/mL human bFGF (Thermo, RP-8628) + 10 ng/mL human EGF (Thermo, PHG0311) + 1% penicillin streptomycin (Catalog # 15140148)) until tumorspheres formed. After tumorspheres were formed, cells were transferred into 15 mL conical tubes and allowed to settle by gravity for 10 minutes (cells were allowed to settle by gravity for 10 minutes after each wash step). Cells were incubated with 100 nM CDH15 or isotype antibodies for 30 minutes, washed twice with medium, and incubated with 10 μg/mL fluorescently conjugated secondary antibody (Invitrogen A-21445) for 30 minutes. Cells were washed twice more with medium and fixed with 4% PFA for 15 minutes. Cells were then washed with PBS, stained with Hoescht (1:1000) for 5 minutes, and washed twice more with PBS. Cells were then incubated for 1 hour in blocking buffer (20% goat serum, 0.3% Triton in PBS) at room temperature. Primary antibodies were then added (diluted 1:100 in blocking buffer containing myogenin: Abcam AB124800) and incubated overnight. Cells were washed twice with PBS, and secondary antibodies (diluted 1:200 in blocking buffer; Invitrogen A11035) were added for 1 h. Cells were washed twice with PBS, mounted on slides in Fluoromount G (Invitrogen 00-4958-02), covered with coverslips, and imaged using a Zeiss LSM 710 confocal microscope.

Example 8: Retargeting AAV with an anti-cadherin 15 antigen binding domain

Figure 10 shows AAV9-based retargeting viral transduction of C2C12 mouse myoblasts 3 days after transduction with 2.5 x 10 ⁵ vg/cell of AAV9 expressing eGFP under the control of the CAG promoter. WT AAV9 particles, N272A detargeted AAV9 particles, N272A detargeted AAV9 particles conjugated to anti-ASGR1 mAb with 1:8 mosaic decoration, N272A detargeted AAV9 particles conjugated to anti-CDH15 mAb with 1:8 mosaic decoration (9295 mAb; HC = SEQ ID NOs: 832 [DNA] and 833 [protein]; LC = SEQ ID NOs: 834 [DNA] and 835 [protein]), N272A detargeted AAV9 particles conjugated to anti-ASGR1 Fab with 1:8 mosaic decoration, anti-CDH15 Fab with 1:8 mosaic decoration (9295 Fab; HC = SEQ ID NOs: 836 [DNA] and 837 [protein]; LC = SEQ ID NOs: 834 [DNA] and 835 Representative immunofluorescence images are provided showing the transduction efficiency via GFP fluorescence of C2C12 myoblasts transduced with N272A detargeted AAV9 particles conjugated to [protein]), N272A detargeted AAV9 particles conjugated to anti-ASGR1 Fab in 1:2 mosaic decoration, and N272A detargeted AAV9 particles conjugated to 9295 Fab in 1:2 mosaic decoration. As shown in Figure 10 , conjugation of REGN9295-based anti-CDH15 mAb and Fab, when conjugated in 1:8 mosaic, improves transduction of C2C12 myoblasts compared to WT AAV9.

Figure 11 shows AAV9-based retargeting viral transduction of human skeletal myoblasts 3 days after transduction with 2.5 x 10 ⁵ vg/cell of AAV9 expressing eGFP under the control of the CAG promoter. Representative immunofluorescence images are provided showing the transduction efficiency via GFP fluorescence of human myoblasts transduced with WT AAV9 particles, N272A detargeted AAV9 particles, N272A detargeted AAV9 particles conjugated to anti-ASGR1 mAb with 1:8 mosaic decoration, N272A detargeted AAV9 particles conjugated to 9295 mAb with 1:8 mosaic decoration, N272A detargeted AAV9 particles conjugated to anti-ASGR1 Fab with 1:8 mosaic decoration, N272A detargeted AAV9 particles conjugated to 9295 Fab with 1:8 mosaic decoration, N272A detargeted AAV9 particles conjugated to anti-ASGR1 Fab with 1:2 mosaic decoration, and N272A detargeted AAV9 particles conjugated to 9295 Fab with 1:2 mosaic decoration. As shown in Figure 4 , conjugation of 9295-based anti-CDH15 mAb and Fab, when conjugated in a 1:8 mosaic, improves transduction of human myoblasts compared to WT AAV9.

The heavy chain (HC) for the aforementioned 9295 mAb comprises the heavy chain variable region (HCVR) of 9295, three heavy chain constant regions (CH1 to CH3), and a SpyCatcher, e.g., SEQ ID NO: 833 = SEQ ID NO: 202 + CH1 to CH3 + SEQ ID NO: 816. The HC for the aforementioned 9295 Fab comprises the HCVR, CH1, and SpyCatcher of 9295, e.g., SEQ ID NO: 837 = SEQ ID NO: 202 + CH1 + SEQ ID NO: 816. The light chain (LC) for the aforementioned 9295 mAb and Fab comprises the light chain variable region and the light chain constant region (CL) of 9295, e.g., SEQ ID NO: 835 = SEQ ID NO: 210 + CL.

Materials and Methods

AAV capsid protein constructs

GeneBlocks encoding the desired SpyTag inserts, flanking linker amino acids, and additional mutations were purchased from IDT and cloned into BsiWI and XcmI-digested pAAVR2C9 (AAV9 wild-type RepCap plasmid) using Gibson Assembly according to the manufacturer's protocol (NEB).

Cloning SpyCatcher into an antibody

GeneBlocks encoding antibody heavy chain variables were purchased from IDT and cloned into appropriate SpyCatcher-containing backbone plasmids using Gibson assembly. The heavy chain was cloned as the entire Fc and Fab. The light chain was cloned into a standard human light chain expression plasmid.

Production of AAV viral vectors

Viruses were produced by transfecting 293T packaging cells using PEI Pro with the following plasmids: pAd helper, an AAV2 ITR-containing genomic plasmid encoding eGFP under the control of CAG, and pAAV-CAP plasmid encoding AAV Rep and Cap genes (with or without a 10 amino acid linker and a sequence encoding SpyTag inserted at position 453 of the encoded capsid protein). Under predetermined conditions, 293T cells were further transfected with an additional plasmid encoding an antigen-binding domain, e.g., a complete monoclonal antibody (mAb) or a Fab fragment thereof. The antibody heavy chain construct is fused to SpyCatcher at its C-terminus, as described in WO2019006046, which is incorporated herein by reference in its entirety, so that upon contact with the AAV9 capsid protein containing SpyTag, a covalent isopeptide bond is formed between the capsid protein and the antigen-binding domain. Transfection complexes were prepared in incomplete DMEM (no additional supplements) and incubated at room temperature for 10 minutes.

Each virus was generated by transfecting 15 cm plates of 293T packaging cells with the following plasmids and the following amounts:

AAV9 N272A with WT AAV9/eGFP

pAd helper 16 μg

pAAV-CAG eGFP 8 μg

8 μg of pAAV9-CAP WT or pAAV9-CAP N272A

AAV9 anti-human ASGR1 or anti-CDH15 (REGN9295) mAb and Fab with eGFP (1:8 ratio)

pAd helper 16 μg

pAAV-CAG eGFP 8 μg

pAAV9-CAP G453 Linker10 SpyTag 1 μg

pAAV9-CAP N272A 7 μg

:

pAnti-human ASGR1 or anti-human CDH15 hIgG4US SpyCatcher Vh 1.5 μg

ULC1-39 or p-anti-human CDH15 Vk 3 μg

or

p-anti-human ASGR1 or p-anti-human CDH15 Fab SpyCatcher Vh 1.5 μg

ULC1-39 or p-anti-human CDH15 Vk 3 μg

AAV9 anti-human ASGR1 or anti-CDH15 (REGN9295) Fab with eGFP (1:2 ratio)

pAd helper 16 μg

pAAV-CAG eGFP 8 μg

pAAV9-CAP G453 Linker10 SpyTag 4 μg

pAAV9-CAP N272A 4 μg

:

p-anti-human ASGR1 or p-anti-human CDH15 Fab SpyCatcher Vh 1.5 μg

ULC1-39 or p-anti-human CDH15 Vk 3 μg

After incubation, the complexes were added to DMEM supplemented with 10% FBS, 1XNEAA, 1% Pen/Strep, and 1% L-glutamine.

Transfected packaging cells were incubated at 37°C for 3 days and then harvested from the cell lysate using a standard freeze-thaw protocol. Packaging cells were scraped, lifted, and pelleted. The supernatant was removed, and the cells were resuspended in a solution of 50 mM Tris-HCl, 150 mM NaCl, and 2 mM MgCl ₂ , pH 8.0. Cell lysis was induced to release intracellular viral particles through three consecutive freeze-thaw cycles, which consisted of vigorously vortexing the cell suspension between a dry ice/ethanol bath and a 37°C water bath. The lysate was treated with EMD Millipore Benzonase (50 U/ml cell lysate) for 60 minutes at 37°C with intermittent mixing to reduce viscosity. The debris was then pelleted by centrifugation, and the resulting supernatant was filtered through a 0.22 μm PVDF Millex-GV filter. For crude virus to be tested in vitro, the filtered lysate was added directly to an Amicon Ultra-15 centrifugal filter unit equipped with an Ultracel-100 membrane (100 KDa MWCO) filter cartridge. The filter unit was centrifuged at 5-10 minute intervals until the desired volume was reached in the upper chamber, and the concentrated crude virus was then pipetted into a low-protein binding tube and stored at 4°C.

Titers (viral genomes per milliliter, vg/mL) were determined by qPCR using a standard curve of known virus concentrations.

cell

C2C12 myoblast cell lines were maintained in DMEM supplemented with 10% FBS, 1% Pen/Strep, and 1% L-glutamine and grown in a 37°C incubator containing 5% CO ₂ . The C2C12 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Human skeletal myoblasts were purchased from Cook Myosite (Pittsburgh, PA; SkMDC; lot # P01059-14M), maintained in MyoTonic basal medium (MB-2222) supplemented with MyoTonic growth supplement (MS-3333), and grown in a 37°C incubator with 5% CO ₂ .

Cell transduction and analysis

C2C12 myoblasts and human myoblasts were seeded at a density of 8,000 cells/well in collagen-coated 96-well plates with a clear bottom and black walls. To infect cells, virus particles were added directly to the culture medium at 2.5 x 10 ⁵ vg/cell, and the mixture was incubated at 37°C. Three days after infection, cells were fixed with 4% PFA for 15 min, washed, and stored in PBS until analysis.

After washing, cells were stained with Hoescht (1:1,000) and imaged on an Axio Observer microscope (Zeiss, Oberkochen, Germany) for GFP detection.

Immunostaining of single muscle fibers and muscle cross-sections

For single-fiber immunohistochemistry, wild-type C57BL/6 mice were sacrificed, and the gastrocnemius muscles were carefully dissected and digested in 700 units/mL collagenase II (Gibco, 17101-015) in DMEM (high glucose, + glutamine, + 110 mg/mL sodium pyruvate, Fisher 11995-065) at 37°C for 90 min. Muscles were placed on plates coated with horse serum, and intact single fibers were picked up and transferred to plates containing washing medium (DMEM, 10% horse serum, 1% pen/strep). Single fibers were washed three times in fresh washing medium from the horse serum-coated plates, then transferred to plates containing DMEM alone and incubated for 1 h. Single fibers were transferred to low-adhesion 6-well plates (Corning, 3474) and incubated overnight in DMEM containing 2% horse serum. Single fibers were live-stained with 100 nM anti-CDH15 Ab for 30 min, washed twice, and stained with 1:200 APC-conjugated human secondary (Jackson Cat# 109-136-170). Single fibers were then washed twice, fixed with 4% PFA, and incubated overnight in Pax7 (1:100, DSHB). Single fibers were then incubated with Hoescht (1:1000) for 5 min, washed with PBS, mounted on coverslips, and imaged using a Zeiss LSM 710 confocal microscope.

For immunohistochemical staining of muscle sections, wild-type C57BL/6 mice were sacrificed, and striated muscles were embedded in optical coherence tomography (OCT) compound, frozen in liquid nitrogen-cooled isopentane, and cryosectioned at 10 μm onto microscope slides. Tissue sections were fixed in 4% PFA, permeabilized by blocking with 0.1% Triton-X100 in 20% goat serum, stained overnight with anti-CDH15 Ab, anti-Pax7 Ab, and anti-laminin Ab, and then detected with Alexa-Fluor conjugated secondary antibodies (1:250). Tissues were then washed, counterstained with Hoescht (1:1000), coverslipped, and imaged with an Axio Observer microscope.

While the present invention has been particularly illustrated and described with reference to numerous embodiments, it will be understood by those skilled in the art that changes in form and detail may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention, and that the various embodiments disclosed herein are not intended to limit the scope of the claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some preferred methods and materials are now described. All publications cited herein are incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as broadly understood by one of ordinary skill in the art to which this invention belongs.

Attach an electronic file of the sequence list

Claims (98)

An antigen binding protein that binds to human cadherin 15 (CDH15), wherein the antigen binding protein has a sequence number of 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311-313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392-396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490-494-496-498, 502-504-506-510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730-734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and An antigen binding protein comprising a set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 selected from the group consisting of 766-768-770-774-776-778. In claim 1, the antigen binding protein comprises a heavy chain variable region (HCVR or VH). In claim 1, the antigen binding protein comprises a light chain variable region (LCVR or VL). An antigen binding protein according to any one of claims 1 to 3, wherein the antigen binding protein comprises an anti-hCDH15 antibody or an antigen binding fragment thereof. In claim 4, the anti-hCDH15 antibody or antigen-binding fragment thereof is an antigen-binding protein selected from the group consisting of a human or humanized antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a trispecific antibody, and a chemically modified derivative thereof. In claim 5, the anti-hCDH15 antibody or antigen-binding fragment thereof is an antigen-binding protein comprising a fragment antigen-binding region (Fab). In claim 5, the anti-hCDH15 antibody or antigen binding protein thereof comprises a single-chain variable fragment (scFv). In claim 7, the scFv is an antigen binding protein comprising variable regions arranged in the following orientation from the N-terminus to the C-terminus: HCVR-LCVR. In claim 7, the scFv is an antigen binding protein comprising variable regions arranged in the following orientation from the N-terminus to the C-terminus: LCVR-HCVR. An antigen binding protein according to any one of claims 7 to 8, wherein the scFv variable region is connected by a linker. An antigen binding protein according to claim 10, wherein the linker is a peptide linker. An antigen binding protein in claim 11, wherein the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), and n in the sequence is 1 to 10. An antigen binding protein according to any one of claims 1 to 12, wherein the antigen binding protein binds to hCDH15 with a KD of about ^1X10-7 M or stronger affinity. An antigen binding protein according to any one of claims 1 to 13, wherein the antigen binding protein binds to hCDH15 with a KD of about 10X10 ^-8 to about 1X10 ^-10 . An antigen binding protein according to any one of claims 1 to 14, wherein the antigen binding protein binds to hCDH15 with a KD of about 5X10 ^-9 to about 1X10 ^-10 . In any one of claims 1 to 15, the antigen binding protein is selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and An antigen binding protein comprising an HCVR/LCVR amino acid sequence pair having at least 90% sequence identity to an HCVR/LCVR amino acid sequence pair selected from the group consisting of 772. A polynucleotide comprising a sequence encoding an antigen binding protein of any one of claims 1 to 16. A pharmaceutical composition comprising an antigen binding protein according to any one of claims 1 to 16, and a pharmaceutically acceptable carrier. A method for inhibiting the activity of CDH15 in a cell, comprising the step of contacting a cell expressing CDH15 with an antigen binding protein according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 18. A method according to claim 19, wherein the cell expressing CDH15 is a muscle stem cell, a myoblast, or a muscle cell. A method for accelerating the transition from quiescence to activation of muscle stem cells, comprising the step of contacting said muscle stem cells with an antigen binding protein of any one of claims 1 to 16 or a pharmaceutical composition of claim 18. A method for treating a disease in a subject requiring treatment, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 18. A method according to claim 22, wherein the condition is muscle damage. A method for improving muscle regeneration after muscle damage in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 18. A method for restoring muscle regeneration ability of a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 18. A method according to claim 25, wherein the muscle regeneration ability of the subject is restored to or close to the functional state of the control subject. A method for treating a condition in a subject in need of treatment of the condition, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 18, wherein the antigen binding protein is conjugated to a therapeutic agent. A method according to claim 27, wherein the condition is cancer. A method according to claim 28, wherein the cancer is rhabdomyosarcoma. A method according to any one of claims 27 to 29, wherein the therapeutic agent comprises a cytotoxic chemotherapeutic agent. In any one of claims 27 to 30, the therapeutic agent is selected from the group consisting of Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Cytarabine liposomal, Dacarbazine, Dactinomycin (Actinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab Gemtuzumab ozogamicin, Hydroxyurea, Idarubicin, Ifosfamide, Inotuzumab ozogamicin, Irinotecan, Ixazomib, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitozantrone, Nab-paclitaxel, Oxaliplatin, Paclitaxel, Pemetrexed, Pegaspargase, Polatuzumab Polatuzumab vedotin, Pralatrexate, Procarbazine, Raltitrexed, Romidepsin, Sacituzumab govitecan, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Trabectedin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trifluridine/tipiracil, Valganciclovir, Vinblastine, A method comprising vincristine, vindesine, vinflunine, vinorelbine, or vismodegib. A method according to any one of claims 27 to 31, wherein the antigen binding protein is conjugated to the therapeutic agent via a valine-citrulline (VC) and/or para-aminobenzyl (PAB) linker. A method for imaging muscle cells in a subject requiring imaging of the same, the method comprising administering to the subject the pharmaceutical composition of claim 18, wherein the antigen binding protein is conjugated to a detectable moiety. A method according to claim 33, wherein the muscle cells comprise one or more cells selected from the group consisting of muscle stem cells, myoblasts, and muscle cells. A method according to claim 33 or 34, wherein the detectable moiety comprises a radioactive nuclide. A method according to any one of claims 22 to 35, wherein the administering step comprises administering the pharmaceutical composition intravenously or subcutaneously to the subject. As a recombinant adeno-associated virus (AAV) particle,
(i) an AAV capsid comprising a modified AAV capsid protein, and
(ii) comprising a targeting ligand that binds to a surface protein of terminally differentiated muscle cells;
A recombinant AAV particle wherein the AAV capsid is operably linked to the targeting ligand. In Article 37,
(a) the modified AAV capsid protein comprises a first member of a protein:protein binding pair,
wherein the first member of the protein:protein binding pair is operably linked to the second member of the protein:protein binding pair; and
(b) the second member of the protein:protein binding pair comprises a targeting ligand that binds to a surface protein of terminally differentiated muscle cells;
A recombinant AAV particle wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are combined to direct the tropism of the recombinant AAV particle to terminally undifferentiated muscle cells. A recombinant AAV particle according to claim 37 or 38, wherein the terminally differentiated muscle cells comprise one or more cells selected from the group consisting of muscle stem cells, myoblasts, myocytes, myotubes, and combinations thereof. A recombinant AAV particle comprising an AAV capsid bound to a surface protein of a terminally differentiated muscle cell expressed on the surface of a mammalian muscle cell, according to any one of claims 37 to 39. In any one of claims 37 to 40, comprising an AAV capsid bound to a surface protein of a non-terminally differentiated muscle cell expressed on the surface of a mammalian muscle cell,
The surface protein of the above terminally undifferentiated muscle cells is a human surface protein of the above terminally undifferentiated muscle cells, and
The recombinant AAV particle, wherein the mammalian muscle cell is a non-human animal muscle cell genetically modified to express the human surface protein of the terminally undifferentiated muscle cell. In any one of claims 37 to 41, comprising an AAV capsid bound to a surface protein of a non-terminally differentiated muscle cell expressed on the surface of a mammalian muscle cell,
The surface protein of the above terminally undifferentiated muscle cells is a human surface protein of the above terminally undifferentiated muscle cells, and
The recombinant AAV particle, wherein the mammalian muscle cell is a rodent muscle cell genetically modified to express the human surface protein of the terminally undifferentiated muscle cell. In any one of claims 37 to 42, comprising an AAV capsid bound to a surface protein of said non-terminally differentiated muscle cell expressed on the surface of said mammalian muscle cell,
The surface protein of the above terminally undifferentiated muscle cells is a human surface protein of the above terminally undifferentiated muscle cells, and
A recombinant AAV particle wherein the mammalian muscle cell is a rat or mouse muscle cell genetically modified to express the human surface protein of the terminally undifferentiated muscle cell. In any one of claims 37 to 43, comprising an AAV capsid bound to a surface protein of a non-terminally differentiated muscle cell expressed on the surface of a mammalian muscle cell,
The surface protein of the above terminally undifferentiated muscle cells is a human cell-specific surface protein of the above terminally undifferentiated muscle cells, and
A recombinant AAV particle wherein the mammalian muscle cell is a mouse muscle cell genetically modified to express the human cell-specific surface protein of the terminally undifferentiated muscle cell. An AAV capsid bound to a surface protein of a non-terminally differentiated muscle cell expressed by a mammalian muscle cell according to any one of claims 37 to 41, wherein:
The surface protein of the above terminally undifferentiated muscle cells is a human surface protein of the above terminally undifferentiated muscle cells, and
The mammalian muscle cells above are human muscle cells, recombinant AAV particles. A recombinant AAV particle according to any one of claims 37 to 45, wherein the recombinant AAV particle is in a test tube. A recombinant AAV particle according to any one of claims 37 to 45, wherein the recombinant AAV particle is in vivo. A recombinant AAV particle according to any one of claims 37 to 42, wherein the surface protein of the terminally undifferentiated muscle cell is mammalian cadherin 15 (CDH15). A recombinant AAV particle according to any one of claims 37 to 48, wherein the surface protein of the terminally undifferentiated muscle cell is human CDH15. In any one of Articles 38 to 49,
(a) the first member of the protein:protein binding pair comprises SpyTag, Isopeptag, SnoopTag, or SpyTag002,
(b) the second member of the protein:protein binding pair
(i) SpyCatcher, KTag, pilin-C, SnoopCatcher, or SpyCatcher002, and
(ii) comprising a targeting ligand that binds to a mammalian muscle-specific surface protein, and
(c) A recombinant AAV particle, wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are linked by an isopeptide bond. In any one of Articles 38 to 50,
(a) the first member of the protein:protein binding pair comprises a SpyTag, and
(b) a recombinant AAV particle comprising a SpyCatcher fused to a targeting ligand that binds to a mammalian muscle-specific surface protein, wherein the second member of the protein:protein binding pair is a mammalian muscle-specific surface protein. In any one of Articles 38 to 49,
(a) the first member of the protein:protein binding pair comprises the c-myc amino acid sequence set forth as SEQ ID NO: 818, and
(b) a recombinant AAV particle comprising a bispecific binding protein comprising an anti-c-myc antibody and a targeting ligand that binds to a mammalian muscle-specific surface protein, wherein the second member of the protein:protein binding pair comprises a bispecific binding protein. A recombinant AAV particle according to any one of claims 38 to 52, comprising a first and/or second linker operably linking the first member of the protein:protein binding pair to a capsid protein of the recombinant AAV particle. A recombinant AAV particle in claim 53, wherein the first and second linkers are not identical. In claim 53, the first and second linkers are the same, a recombinant AAV particle. A recombinant AAV particle according to any one of claims 53 to 55, wherein the first linker is 10 amino acids in length and/or the second linker is 10 amino acids in length, and optionally, the amino acid sequence of the first linker and/or the amino acid sequence of the second linker comprises an amino acid sequence set forth as SEQ ID NO: 826 or SEQ ID NO: 827. In any one of Articles 38 to 56,
(a) the AAV capsid comprises a modified VP1 capsid protein, a modified VP2 capsid protein, and/or a modified VP3 capsid protein encoded by a mutated cap gene, and
(b) a recombinant AAV particle, wherein said mutated cap gene or a portion thereof comprises a nucleotide sequence that is at least 90% identical to a cap gene or a portion thereof of AAV, and wherein said mutated cap gene or a portion thereof is genetically modified to include an insertion of a nucleotide sequence encoding a first member of said protein:protein binding pair, such that said modified VP1 capsid protein, said modified VP2 capsid protein, and/or said modified VP3 capsid protein comprises the first member of said protein:protein binding pair. In claim 57, the mutated cap gene is genetically modified to include one or more additional mutations such that the modified VP1 capsid protein, the modified VP2 capsid protein, and/or the modified VP3 capsid protein comprises, in addition to the first member of the protein:protein binding pair, the recombinant AAV particle:
(i) substitution, insertion, or deletion of amino acids;
(ii) a chimeric amino acid sequence, or
(iii) Both point mutations and chimeric amino acid sequences. A recombinant AAV particle in claim 58, wherein the substitution, insertion, or deletion of an amino acid reduces the natural tropism of the AAV capsid and/or produces a detectable label. In any one of Articles 38 to 59,
(a) the AAV capsid comprises a modified VP1 capsid protein, a modified VP2 capsid protein, and/or a modified VP3 capsid protein encoded by a mutated cap gene, and
(b) the mutated cap gene or a portion thereof comprises a nucleotide sequence that is at least 90% identical to a cap gene or a portion thereof of AAV, and the mutated cap gene or a portion thereof is genetically modified to include an insertion of a nucleotide sequence encoding a first member of the protein:protein binding pair, such that the modified VP1 capsid protein, the modified VP2 capsid protein, and/or the modified VP3 capsid protein comprises the first member of the protein:protein binding pair, and
(c) a recombinant AAV particle selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, non-primate animal AAVs listed in Table 2, and combinations thereof. A recombinant AAV particle according to any one of claims 57 to 60, wherein the AAV is AAV2. A recombinant AAV particle according to any one of claims 57 to 61, wherein the AAV capsid comprises a modified AAV2 VP1 capsid protein comprising a first member of the protein:protein binding pair, optionally linked via a linker, to amino acids at positions I453 and/or I587. In any one of claims 57 to 62, the AAV capsid comprises a modified AAV2 VP1 capsid protein comprising a first member of the protein:protein binding pair indicated via a linker at position G453,
Optionally, the modified AAV2 VP1 capsid protein further comprises an R585A variant, an R588A variant, or both an R585A variant and an R588A variant, and
Optionally, the modified AAV2 VP1 capsid protein further comprises an R484A variant, an R487A variant, an R585A variant, an R588A variant, and a K532A variant, or any combination of an R484A variant, an R487A variant, an R585A variant, an R588A variant, and a K532A variant. In claim 62 or 63, the AAV capsid is a mosaic virus capsid comprising a second set of AAV2 VP1 capsid proteins lacking the first member of the protein:protein binding pair,
Optionally, a recombinant AAV particle wherein the second set of AAV2 VP1 capsid proteins further comprises an R585A variant, an R588A variant, or both an R585A variant and an R588A variant. A recombinant AAV particle according to any one of claims 57 to 60, wherein the AAV is AAV9. A recombinant AAV particle according to any one of claims 57 to 60 and 65, wherein the AAV capsid comprises a modified AAV9 VP1 capsid protein comprising a first member of the protein:protein binding pair, optionally linked via a linker, to an amino acid at position I453 or I589. In any one of claims 57 to 60 and 65 to 66, the AAV capsid comprises a modified AAV9 VP1 capsid protein comprising a first member of the protein:protein binding pair displayed via a linker at position G453,
Optionally, the modified AAV9 VP1 capsid protein further comprises an N272A modification, a W503A modification, or both an N272A modification and a W503A modification. In claim 66 or 67, the AAV capsid is a mosaic virus capsid comprising a second set of AAV9 VP1 capsid proteins lacking the first member of the protein:protein binding pair,
Optionally, a recombinant AAV particle wherein the second set of AAV2 VP1 capsid proteins comprises an N272A variant, a W503A variant, or both an N272A variant and a W503A variant. A recombinant AAV particle in claim 60, wherein the non-primate animal AAV is avian AAV (AAAV), non-human mammalian AAV, or squamate AAV. A recombinant AAV particle according to claim 60 or 69, wherein the non-primate animal AAV is AAAV. A recombinant AAV particle according to claim 60, 69, or 70, wherein the AAV capsid comprises a modified AAAV VP1 capsid protein comprising a first member of the protein:protein binding pair, optionally linked via a linker, to an amino acid at position I444 or I580. A recombinant AAV particle according to claim 60 or 69, wherein the non-primate animal AAV is a primate AAV. A recombinant AAV particle according to claim 60, claim 69, or claim 72, wherein the non-primate animal AAV is a bearded dragon AAV. A recombinant AAV particle according to claim 60, 69, 72, or 73, wherein the AAV capsid comprises a modified bearded dragon VP1 capsid protein comprising a first member of the protein:protein binding pair, optionally linked via a linker, to an amino acid at position I573 or I436. A recombinant AAV particle according to claim 60 or 69, wherein the non-primate animal AAV is a non-human mammalian AAV. A recombinant AAV particle according to claim 60, claim 69, or claim 75, wherein the non-primate animal AAV is a sea lion AAV. A recombinant AAV particle according to claim 60, claim 69, claim 75, or claim 76, wherein the AAV capsid comprises a modified AAAV VP1 capsid protein comprising a first member of the protein:protein binding pair, optionally linked via a linker, to an amino acid at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and I565. A recombinant AAV particle according to any one of claims 37 to 77, wherein the AAV capsid is a mosaic AAV capsid, and optionally wherein the mosaic AAV capsid comprises a first plurality of reference capsid proteins, each of which does not comprise the first member of the protein:protein binding pair, and a second plurality of capsid proteins, each of which comprises the first member of the protein:protein binding pair, and optionally wherein the mosaic AAV capsid comprises the first plurality of reference capsid proteins and the second plurality of capsid proteins in a ratio of 1:7. A recombinant AAV particle according to any one of claims 37 to 78, wherein the retargeting ligand is an antibody or a portion thereof. A recombinant AAV particle according to any one of claims 37 to 79, further comprising a nucleotide of interest encapsulated within the AAV capsid. A recombinant AAV particle in claim 80, wherein the nucleotide of interest is a reporter gene. A recombinant AAV particle according to claim 80 or 81, wherein the nucleotide of interest encodes β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, cyan fluorescent protein (BFP), enhanced cyan fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof. In claim 80, a recombinant AAV particle wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or part(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. A recombinant AAV particle according to any one of claims 37 to 83, wherein the targeting ligand comprises an antigen binding protein of any one of claims 1 to 16, and optionally, the targeting ligand comprises a sequence selected from the group consisting of SEQ ID NO: 833, SEQ ID NO: 835, SEQ ID NO: 837, and any combination thereof. A pharmaceutical composition comprising (a) a recombinant AAV particle of any one of claims 37 to 84 and (b) a pharmaceutically acceptable carrier or excipient. A method of delivering a nucleotide of interest to a mammalian muscle cell, comprising the step of contacting the mammalian muscle cell with (a) a recombinant AAV particle of any one of claims 37 to 84 or (b) a pharmaceutical composition of claim 85, wherein:
A method wherein the mammalian muscle cells express the mammalian muscle-specific surface protein. A method according to claim 86, wherein the contacting step is performed in vitro. A method according to claim 86, wherein the contacting step is performed within a subject, and optionally, the subject is modified to express the targeting ligand, for example, from a safe harbor locus. A method according to claim 88, wherein the subject is a primate, preferably a human. A method according to any one of claims 86 to 89, wherein the mammalian muscle cell is a mammalian skeletal muscle cell, optionally wherein the mammalian skeletal muscle cell is not terminally differentiated, and optionally wherein the mammalian skeletal muscle cell comprises one or more cells selected from the group consisting of muscle stem cells, myoblasts, and myocytes. A method according to any one of claims 86 to 90, wherein the mammalian muscle-specific surface protein is CDH15. A method according to any one of claims 86 to 91, wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or part(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. As a method of treating a patient requiring treatment for a muscle wasting disease or a genetic muscle disease,
Comprising the step of administering to the subject (a) a recombinant AAV particle of any one of claims 37 to 84 or (b) a pharmaceutical composition of claim 85,
The recombinant AAV particle comprises a nucleotide of interest encapsulated within the AAV capsid,
A method wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or part(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. Use of a recombinant AAV particle according to any one of claims 37 to 84 or a pharmaceutical composition according to claim 85 for the manufacture of a medicament for treating a muscle wasting disease or a genetic muscle disease. A method or use according to claim 93 or 94, wherein the muscle wasting disease or genetic muscle disease is selected from the group consisting of X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), myosohumeral muscular dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, and dystroglycanopathy. As a method of treating rhabdomyosarcoma in patients requiring treatment,
Comprising the step of administering to the subject (a) a recombinant AAV particle of any one of claims 37 to 84 or (b) a pharmaceutical composition of claim 85,
The recombinant AAV particle comprises a nucleotide of interest encapsulated within the AAV capsid,
A method wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or part(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. Use of a recombinant AAV particle according to any one of claims 37 to 84 or a pharmaceutical composition according to claim 85 for the manufacture of a medicament for treating rhabdomyosarcoma. A method or use according to claim 96 or 97, wherein the rhabdomyosarcoma is selected from the group consisting of embryonal, alveolar, pleomorphic, staphylococcal, and spindle/sclerosing rhabdomyosarcoma.