Anti-human M-caderin (CDH15) antibody, conjugate, and its use for delivery of gene payloads to muscle cells

Abstract

Antibodies, parts, and drug conjugates thereof against cadherin 15 are provided herein. Also provided are nucleic acid sequences encoding it, viral particles for retargeting the viral particles to muscle cells, compositions for treating subjects in need of it, such as subjects suffering from skeletal muscle-related disorders (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, dystroglycan disorders, etc.), and methods of using it.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority from U.S. Provisional Applications No. 63 / 499,531 and No. 63 / 499,527, both filed on 2 May 2023, the contents of which are incorporated herein by reference.

[0002] This application generally relates to human antibodies and antigen-binding fragments of human antibodies that bind to human CDH15 (hCDH15), and methods of using them in, for example, in patients requiring treatment of a disorder. This application also relates to antigen-binding molecules comprising at least an antigen-binding fragment of an anti-hCDH15 antibody whose complex formation with CDH15 mediates the internalization of the antigen-binding molecule / CDH15 complex and / or inhibits the activity of CDH15. This application further 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, which may be useful for the treatment of diseases. This disclosure further relates to methods for producing and using recombinant viral particles, such as recombinant AAV particles, which contain a capsid protein retargeted to cadherin 15 (CDH15), which may be useful for the modification of muscle cells (such as muscle stem cells) in vitro or in vivo.

[0003] Sequence List The sequence listing in XML format, titled "11486 International Publication No. 01 Sequence Listing XML," was created on April 30, 2024, is 787KB in size, and is incorporated herein by reference in its entirety. [Background technology]

[0004] The delivery of genes to specific target cells has become one of the most important techniques in modern medicine for the potential treatment of various chronic and genetic diseases. Ideally, gene delivery vehicles should be able to stably introduce genetic material into the desired cells while avoiding the introduction of genetic material into non-target cells.

[0005] Recombinant AAV particles, particularly those based on adeno-associated virus (AAV), have been the focus of much research as gene delivery vehicles, due to AAV's ability to transduce a wide range of primates and tissues in vivo. (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Furthermore, AAV is safely transduced into postmittal tissues. While rare, viruses may integrate into host chromosomes, very rarely occurring at the safe harbor locus on human chromosome 19, but only if the replication (Rep) protein is supplied in trans. The AAV genome rapidly circularizes and chains within infected cells, residing in a stable episomal state within the cells, providing long-term, stable expression of the payload.

[0006] Furthermore, in recent years, it has been achieved to manipulate and retarget AAV infection to specific cells. Many of the advances in targeted gene therapy using recombinant AAV particles can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of the viral vector, resulting in pseudotyping, expansion, and / or retargeting of the recombinant AAV particles' inherent targeting capabilities. (As validated in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93 and Verheiji and Rottier (2012) Advances Virol 2012:1-15).

[0007] In direct recombinant targeting methods, the targeting ligand is either directly inserted into or ligated to the viral capsid; that is, the gene of the viral capsid protein is modified to express a capsid protein containing the heterologous targeting ligand. The targeting ligand is then redirected to, for example, a receptor or marker that is preferentially or exclusively expressed on the target cell, and binds to it. (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).

[0008] In indirect recombination methods, the viral capsid is modified using a heterologous "scaffold" and then ligated to an adapter containing a targeted ligand. The adapter then binds to the scaffold and target cells. (See also Arnold et al. (2006) Mol. Ther. 5:125-132, Ponazhagen et al. (2002) J. Virol. 76:12900-907, International Publication No. 97 / 05266). Scaffolds, such as (1) Fc-binding molecules that bind to the Fc of the antibody adapter (e.g., Fc receptors, protein A, etc.), (2) (strept)avidin that binds to the biotinylation adapter, (3) biotin that binds to the adapter fused with (strept)avidin, (4) detectable labels useful for the detection and / or isolation of recombinant AAV particles, which are bound to a bispecific adapter that has the ability to non-covalently bind to the detectable label and target molecule, and more recently, (5) protein:protein binding pairs that form isopeptide bonds have been reported for various recombinant AAV particles. (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, and Klimstra et al. (2005) Virology 338:9-21). Skeletal muscle is the largest organ in the body, accounting for approximately 40% of total body weight, and is one of the three major muscle tissues in the human body. When skeletal muscle is damaged, muscle stem cells (MuSCs) activate, proliferate, and differentiate functional muscle fibers to repair 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 useful for therapies, such as stimulating muscle repair, particularly in aging subjects, and / or for the treatment of muscle-related cancers. Furthermore, the anti-human antibodies described herein may be used in conjunction with recombinant viruses, such as AAV particles, for targeted introduction of target nucleic acids into cells expressing muscle stem cell-specific markers. In addition, certain muscle-related cancers, including rhabdomyosarcoma, may also benefit from therapeutic agents, such as the antibody-drug conjugates described herein that thereby target muscle stem cell-specific markers, retargeted recombinant AAV particles, and the like. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 97 / 05266 [Non-patent literature]

[0010] [Non-Patent Document 1] Muzyczka, et al. (1992)Current Topics in Microbiology and Immunology,158:97-129 [Non-Patent Document 2] Nicklin and Baker(2002)Curr.Gene Ther.2:273-93 [Non-Patent Document 3] Verheiji and Rottier(2012)Advances Virol 2012:1-15 [Non-Patent Document 4] Stachler et al.(2006)Gene Ther.13:926-931 [Non-Patent Document 5] White et al. (2004)Circulation 109:513-519 [Non-Patent Document 6] Park et al.,(2007)Frontiers in Bioscience 13:2653-59

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Summary of the Invention

Means for Solving the Problems

[0011] Antigen-binding proteins that bind to human cadherin 15 (CDH15) are described herein. 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 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, 127-129-131-135-137-13 9, 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, 58 2-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-6 Includes a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 selected from the group consisting of 64-666-670-672-674, 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.

[0012] Table 1 lists the HCVRs and LCVRs of each exemplary anti-hCDH15 antibody of this disclosure, as well as the amino acid and nucleic acid sequence identifiers of HCDR1, HCDR2, and HCDR3 within each HCVR, and LCDR1, LCDR2, and LCDR3 within each LCVR.

[0013] [Table 1-1] [Table 1-2] [Table 1-3]

[0014] In some embodiments, the antigen-binding protein includes a heavy chain variable region (HCVR or VH). In some embodiments, the HCVR includes a set of HCDR1-HCDR2-HCDR3 amino acid sequences selected from Table 1. In some embodiments, the HCVR includes an amino acid sequence selected from any of the HCVR 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 homology.

[0015] In some embodiments, the antigen-binding protein includes a light chain variable region (LCVR or VL). In some embodiments, the LCVR includes a set of LCDR1-LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the LCVR includes an amino acid sequence selected from any of the LCVR 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 homology.

[0016] In some embodiments, the antigen-binding protein comprises an anti-hCDH15 antibody or its antigen-binding fragment. In some embodiments, the anti-hCDH15 antibody or its antigen-binding fragment is selected from the group consisting of human or humanized antibodies or their antigen-binding fragments, mouse antibodies or their antigen-binding fragments, monovalent Fab', bivalent Fab2, F(ab)'3 fragments, single-chain variable fragments (scFv), bis-scFv, (scFv)2, diabodies, minibodies, nanobodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), single-domain antibodies (sdAb), Ig NAR, bispecific antibodies or their binding fragments, bispecific T-cell engagers (BiTE), triplicate antibodies, and chemically modified derivatives thereof. In some embodiments, the scFv comprises a variable region arranged from the N-terminus to the C-terminus in the following orientation: a domain arranged in HCVR-LCVR, as described in claim 5, the recombinant AAV particle. In some embodiments, the scFv includes a variable region arranged from the N-terminus to the C-terminus in the following orientation: a recombinant AAV particle according to claim 5, comprising a domain arranged in LCVR-HCVR. In some embodiments, the scFv variable region is linked 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.

[0017] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) is approximately 1 × 10⁻⁶ -7 It binds to hCDH15 with an affinity of or greater than the dissociation constant (KD) of M. In some embodiments, the antigen-binding protein is approximately 10 × 10 -8 From approximately 1 x 10 -10 It binds to hCDH15 with a dissociation constant (KD) of . In some embodiments, the antigen-binding protein binds to hCDH15 at approximately 5 × 10⁶. -9 From approximately 1 x 10 -10 They are joined by the dissociation constant (KD).

[0018] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) combining any HCVR amino acid sequence listed in Table 1 with any LCVR amino acid sequence listed in Table 1. In some embodiments, the antigen-binding protein described herein, e.g., an antibody or its antigen-binding fragment, comprises an HCVR / LCVR amino acid sequence pair contained in any of the anti-hCDH15 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is the SEQ ID NO: 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 The group is selected from 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.

[0019] Furthermore, this specification also describes nucleic acid molecules, i.e., polynucleotides, that encode antigen-binding proteins, such as anti-hCDH15 antibodies or their antigen-binding fragments. In some embodiments, the nucleic acid molecules described herein include nucleic acid sequences that encode the set of HCDR1-HCDR2-HCDR3 amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecules described herein include nucleic acid sequences that encode any of the HCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule includes a polynucleotide sequence selected from any of the HCVR 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 homology.

[0020] In some embodiments, the nucleic acid molecules described herein include nucleic acid sequences encoding the set of LCDR1-LCDR2-LCDR3 amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecules described herein include nucleic acid sequences encoding any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecules include a polynucleotide sequence selected from any of the LCVR 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 homology.

[0021] Furthermore, this specification also describes nucleic acid molecules encoding HCVR, where HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), the amino acid sequence set of HCDR1-HCDR2-HCDR3 being defined by one of the examples of anti-hCDH15 antibodies listed in Table 1.

[0022] Furthermore, this specification also describes nucleic acid molecules encoding LCVR, where LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), the amino acid sequence set of LCDR1-LCDR2-LCDR3 being defined by one of the examples of anti-hCDH15 antibodies listed in Table 1.

[0023] Furthermore, nucleic acid molecules encoding both HCVR and LCVR are also provided herein, in which case the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR 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 homology thereto, and a polynucleotide sequence selected from any of the LCVR 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 homology thereto. In certain embodiments, the nucleic acid molecule encodes both HCVR and LCVR, and both HCVR and LCVR are derived from the same anti-hCDH15 antibody listed in Table 1.

[0024] Pharmaceutical compositions comprising antigen-binding proteins, such as recombinant human antibodies or fragments thereof that bind to human CDH15, and pharmaceutically acceptable carriers, are also described herein. In some embodiments, the pharmaceutical compositions described herein include a combination of an anti-hCDH15 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-hCDH15 antibody. Additional combination therapies and co-formulations involving the anti-hCDH15 antibody are described herein.

[0025] Methods for inhibiting the activity of cells, for example, CDH15 in vivo, in vitro, or ex vivo, comprising contacting cells expressing CDH15 with an antigen-binding protein or pharmaceutical composition thereof that binds to human CDH15 as described herein. In some embodiments, the cells expressing CDH15 are muscle stem cells, myoblasts, or myocytes.

[0026] Also described herein are methods for accelerating the transition of muscle stem cells from quiescence to activation, such as in vivo, in vitro, or ex vivo, which include contacting muscle stem cells with an antigen-binding protein that binds to human CDH15 or a pharmaceutical composition thereof.

[0027] Also described herein are methods for treating a condition in a subject requiring such treatment, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antigen-binding protein that binds to human CDH15 as described herein. In some embodiments, the condition is muscle injury. 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 includes a cytotoxic chemotherapeutic agent. In some embodiments, the therapeutic agent is aflibercept, amsacrin, azacitidine, azathioprine, belantamab mafodotin, bendamustine, bleomycin, bortezomib, brentuximab vedotin, busulfan, cabazitaxel, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, cisplatin, cladribine, chloropharabine, cyclophosphamide, cytarabine, cytarabine liposome, dacarbazine, dactinomycin (dactinomycin D), daunorubicin, docetaxel. Doxorubicin, Doxorubicin liposome, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab 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 These include thioguanine, topotecan, trabectedin, trastuzumab deruxtecan, trastuzumab emtansine, trifluridine / tipiracil, valganciclovir, vinblastone, vincristine, vindesine, vinflunin, vinorelbine, or bismodegib. In some embodiments, the antigen-binding protein is conjugated to the therapeutic agent via valine-citrulline (VC).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 to the subject subcutaneously or intravenously.

[0028] Furthermore, methods for restoring the muscle regeneration capacity of a subject are described herein, the methods comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an antigen-binding protein that binds to human CDH15 as described herein to the subject. In some embodiments, the subject is an elderly subject. In some embodiments, the muscle regeneration capacity of the elderly subject is restored to the muscle regeneration capacity of a control subject or a functional state near that of the control subject. In some embodiments, the pharmaceutical composition is administered to the subject subcutaneously or intravenously.

[0029] Furthermore, a method for imaging muscle cells in a subject requiring such imaging, as described herein, includes administering a pharmaceutical composition containing an antigen-binding protein that binds to human CDH15 to the subject, wherein the antigen-binding protein is conjugated to a detectable portion. In some embodiments, the muscle cells include one or more selected from the group consisting of muscle stem cells, myoblasts, and muscle cells. In some embodiments, the detectable portion includes a radionuclide. In some embodiments, the pharmaceutical composition is administered to the subject subcutaneously or intravenously.

[0030] Furthermore, the use of antigen-binding proteins that bind to human CDH15 or a pharmaceutical composition thereof in the manufacture of pharmaceuticals, for example, for the treatment of the conditions described herein, is also described herein. For example, antigen-binding proteins that bind to human CDH15 or a pharmaceutical composition thereof for use in the treatment of the conditions described herein and / or for use in the treatment of the subject, such as for restoring the muscle regeneration capacity of the subject, are further described herein.

[0031] Furthermore, antibody or antigen-binding fragments that compete for binding to human CDH15 with reference antibodies containing the HCVR / LCVR amino acid sequence pairs listed in Table 1 are also described herein. In some embodiments, the antibody or antigen-binding fragments described herein are SEQ ID NOs. Compounds according to any one of Embodiments 1 to 11, including the sequences 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, 4 It antagonizes the binding of a reference antibody containing an HCVR / LCVR amino acid sequence pair selected from the group consisting of 36 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 to human CDH15.

[0032] Furthermore, this specification also describes antibodies or antigen-binding fragments that bind to homologous epitopes on human CDH15, as well as reference antibodies containing the HCVR / LCVR amino acid sequence pairs listed in Table 1. In some embodiments, the antibodies or antigen-binding fragments described herein are 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 46 Similar to a reference antibody containing an HCVR / LCVR amino acid sequence pair selected from the group consisting of 0, 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 binds to homologous epitopes on human CDH15.

[0033] Furthermore, isolated antibodies or antigen-binding fragments that bind to human CDH15 are described herein, and such antibodies or antigen-binding fragments include a complementation-determining region (CDR) of a heavy chain variable region (HCVR) having the amino acid sequence described in Table 1, and a CDR of a light chain variable region (LCVR) having the amino acid sequence described in Table 1. In some embodiments, the isolated antibody or antigen-binding fragment is SEQ ID NO: 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 44 The HCVR / LCVR amino acid sequence pair comprises heavy and light chain CDRs selected from the group consisting of 4, 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 yet another embodiment, the isolated antibody or antigen-binding fragment comprises, respectively, 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, 360-3 62-364-368-370-372, 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, 51 8-520-522-526-528-530, 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, 662-664-666-670-6 The domains include HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains selected from the group consisting of 72-674, 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. An isolated antibody or antigen-binding fragment that binds to human CDH15, wherein (a) SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145,A heavy chain variable region (HCVR) and / or (b) Sequence ID number having an amino acid sequence selected from the group consisting of 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. Isolated antibodies or antigen-binding fragments thereof, comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of 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, are also described herein. In some embodiments, the isolated antibody or antigen-binding fragment is SEQ ID NO: 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,It also includes HCVR / LCVR amino acid sequence pairs selected from the group consisting of 764 and 772.

[0034] Furthermore, this specification describes viral particles suitable for introducing a target nucleotide specifically targeted to muscle cells (e.g., muscle stem cells). This is because the viral capsids or viral capsid proteins described herein contain a targeting ligand that binds to a muscle cell-specific surface protein (e.g., the antigen-binding protein described herein that binds to human CDH15). In some embodiments, the viral capsid or viral capsid protein(s) described herein include direct insertion of a targeting ligand(s) (e.g., the targeting ligand is optionally directly bound to and fused to the viral capsid or viral capsid protein via a linker), for example, the viral capsid gene is modified to express a capsid protein containing the targeting ligand. In some embodiments, a viral capsid or viral capsid protein may include a targeting ligand via a scaffold or adapter, e.g., a first member of a protein:protein binding pair, and may be associated with a second member of the same gene of the protein:protein binding pair, in which case 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 described herein that binds to human CDH15). In some embodiments, the targeting ligand is operably linked to the second member and, for example, optionally fused to the second member via a linker. In some embodiments, the targeting ligand may be a binding moiety, e.g., a native ligand, an antibody, a multispecific binding molecule, etc. 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 that binds to a surface protein on a non-terminally differentiated muscle cell (e.g., a muscle stem cell, myoblast, muscle cell, or any combination thereof) (e.g., the variable domain of an antigen-binding protein described herein that binds to human CDH15), and a heavy chain constant domain.In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a non-terminally differentiated myocyte surface protein (e.g., the variable domain of the antigen-binding protein described herein that binds to human CDH15) on a target cell (e.g., myocyte stem cell, myoblast, myocyte, or any combination thereof), and optionally an IgG heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable region that binds to a target cell (e.g., myocyte stem cell, myoblast, myocyte, or any combination thereof) and an IgG heavy chain constant region, the IgG heavy chain constant region being functionally linked to a capsid protein, for example, directly or via a linker. In some embodiments, the targeted ligand has (i) a variable region that binds to a non-terminally differentiated muscle cell surface protein (e.g., the variable region of the antigen-binding protein described herein that binds to human CDH15), and (ii) an IgG heavy chain constant region, the IgG heavy chain constant region being functionally linked (optionally via a linker) to a protein (e.g., a second member of a protein:protein binding pair), the second member forming an isopeptide covalent bond with the corresponding first member of the protein:protein binding pair. In some embodiments, the capsid protein described herein comprises a first member of a protein:protein binding pair, including, for example, SpyTag (SEQ ID NO: 815) or its bioequivalent variant, operably linked to the viral capsid protein, wherein SpyTag or its bioequivalent variant is covalently linked (e.g., via an isopeptide bond) to SpyTag and to its second homogeneous protein:protein binding member, for example, SpyCatcher (SEQ ID NO: 816) or its bioequivalent variant, which may then be linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein 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 non-terminal differentiated muscle cell surface protein comprises CDH15. In some embodiments, the targeting ligand binds to CDH15, for example, human CDH15.In some embodiments, the targeting ligand includes a variable domain of an antigen-binding protein described herein that binds to human CDH15. In some embodiments, the targeting ligand includes an antibody variable domain comprising CDRs of HCVR sequences and / or LCVR sequences listed in Table 1, e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3. In some embodiments, the targeting ligand includes an antibody variable domain comprising CDRs, e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3, as listed in Table 1. In some embodiments, the targeting ligand includes an antibody variable domain comprising three sets of CDRs, e.g., HCDR1, HCDR2, and HCDR3, and / or LCDR1, LCDR2, and LCDR3, as listed in Table 1. In some embodiments, the targeted ligand includes an antibody variable domain comprising three CDRs, e.g., HCDR1, HCDR2, and HCDR3, and / or a set of LCDR1, LCDR2, and LCDR3, as shown in Table 1. In some embodiments, the targeted ligand includes an antibody variable domain comprising HCVR and / or LCVR as shown in Table 1.

[0035] This patent file or application file includes at least one drawing drawn in color. A copy of this patent or publication of this patent application with the color drawing will be provided by the United States Patent and Trademark Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0036] [Figure 1]Figure 1 shows a schematic diagram of myogenesis in non-terminally differentiated muscle cells. Muscle stem cells (MuSCs, also known as satellite cells) are mitotically quiescent and non-proliferative under steady-state conditions in adult muscle tissue. However, in response to injury, MuSCs actively begin to divide, generating daughter cells. Some daughter cells become quiescent again to replenish the MuSC pool, while others continue to proliferate as myoblasts, which align and fuse during differentiation into mature myotubes / muscle fibers that constitute muscle fibers. Quiescent muscle stem cells can be characterized by Pax7 expression, proliferative myoblasts by Pax7 and myogenic differentiation factor 1 (MyoD) expression, myoblasts involved in terminal differentiation (i.e., muscle cells) by MyoD and myogenin expression, and myotubes by myosin heavy chain (MyHC) expression.

[0037] [Figure 2]Figures 2A–2D show the promotion of muscle regeneration in mice with lost CDH15 expression. Figure 2A shows a schematic diagram of an exemplary experiment. Wild-type (WT) or homozygous CDH15 knockout mice (CDH15- / -) were intramuscularly injected with cardiotoxin (CTX) on day 0 to induce muscle injury. Muscle samples (dpi) collected 5, 15, and 25 days after injury were then histologically examined. Figure 2B shows examples of immunohistochemical sections of WT and CDH15- / - muscle samples stained with DAPI (blue, light grayscale), laminin (white), and embryonic myosin heavy chain (eMyHC, green, dark grayscale), which are transiently upregulated in immature muscle fibers but downregulated as muscle fibers mature (Rodgers, Growth Hormone & IGF Research, 2005, 15(6): 377-383). Sections were visualized by fluorescence microscopy. Figure 2C shows muscle fiber cross-sectional area (BMS) of WT and CDH15- / - mice at indicated time points after injury, quantified from histological images. See, for example, Figure 2B. Figure 2D shows the percentage of centrally nucleating muscle fibers in WT and CDH15- / - mice 15 days after injury, quantified from histological images. See, for example, Figure 2B. Data are reported as mean + / - SEM, *p<0.05, **p<0.01. [Figure 3] Figures 3A-3B show improved functional recovery from injury in mice with lost CDH15 expression. Figure 3A shows a schematic diagram of an exemplary experiment. WT or CDH15- / - mice were injected with CTX into the extensor digitorum longus (EDL) muscle on day 0. Contractile force of the EDL muscle was measured ex vivo at 15 dpi. Figure 3B shows the loss of maximal tetanic force in WT or CDH15- / - muscles compared to muscle isolated from an uninjured EDL. Data are reported as mean ± standard error (SEM), and * indicates p<0.05. [Figure 4]Figures 4A–4B show accelerated exit from quiescence in ex vivo in MuSCs with lost CDH15 expression. Figure 4A shows an example of immunohistochemical images of WT and CDH15- / - MuSCs after 48 hours of culture in a single muscle fiber. Arrows in the Pax7 and merge columns indicate individual cells in multicellular clusters. Figure 4B shows the percentage of Pax7+ single cells and multicellular clusters (top) of WT and CDH15- / - MuSCs, as well as the number of Pax7+ cells per cluster (bottom), quantified from histological images. See Figure 4A, for example. Data are reported mean ± standard error (SEM), and ** indicates p<0.01. [Figure 5] Figures 5A-5B show the upregulation of early response genes containing serum response factor (SRF) motifs. Figure 5A shows a Volcano plot of genes that are downregulated or upregulated in FACS-isolated CDH15- / - MuSCs compared to WT MuSCs. Figure 5B shows transcription factor motif analysis, with several relevant transcription factors highlighted in red. [Figure 6]Figures 6A–6D demonstrate that loss of CDH15 expression in aged mice rescues age-related decline in muscle regeneration. Figure 6A shows a schematic diagram of an exemplary experiment. Aged (23-month-old) wild-type (WT) or homozygous CDH15 knockout mice (CDH15- / -) were injected with cardiotoxin (CTX) on day 0 to induce muscle injury in the tibialis anterior muscle. Muscle samples were then collected 15 days after injury (dpi) and subjected to histological examination. Figure 6B shows examples of immunohistochemical sections of aged WT and CDH15- / - muscle samples, stained for nuclei (DAPI; blue; grayscale) and laminin (white) and visualized by fluorescence microscopy. Figure 6C shows the muscle fiber cross-sectional area (BMS) of aged WT and CDH15- / - mice at 15 dpi, quantified from histological images. See, for example, Figure 6B. The dotted line indicates the mean xxx of young mice at 15 dpi. Figure 6D shows the percentage of centronucleating muscle fibers containing three or more central nuclei in aged WT mice and CDH15- / - mice at 15 dpi, quantified from histological images. See, for example, Figure 6B. Data are reported as mean ± standard error (SEM), and * indicates p<0.05. [Figure 7]Figures 7A–7C show that the CDH15 protein is localized to the apical surface of most wild-type (WT) muscle stem cells (MuSCs) and is undetectable in CDH15- / -MuSCs. Figure 7A shows an immunohistochemical section of a WT single muscle fiber with the relevant WT MuSCs, where the CDH15 protein is present on the apical surface of the MuSCs. Anti-CDH15 staining is shown in red and indicated by arrows and dotted outlines, while DAPI staining is shown in blue and indicated by arrows and dashed outlines. Figure 7B shows an immunohistochemical section of WT mouse muscle, where Pax7-positive MuSCs are observed around the muscle fibers, and CDH15 is again present on the apical surface of the MuSCs. Anti-CDH15 is shown in red and indicated by arrows and dotted outlines in the enlarged inset, DAPI-stained nuclei are shown in blue, Pax7 is shown in green, and laminin is shown in white. The overlap between DAPI and Pax7 is indicated by arrows and dashed outlines in the enlarged inset image. Figure 7C shows the percentage of WT and CDH15-positive Pax7-positive MuSCs quantified from histological images. See, for example, Figures 7A and 7B. CDH15 The CDH15 protein is detected in the majority (approximately 95%) of WT Pax7-positive muscle cells, but CDH15 is, as expected, undetectable in CDH15- / Pax7-positive cells. [Figure 8] Figure 8 shows the specific binding of anti-hCDH15 antibodies to human rhabdomyosarcoma cells, but not to gliablastoma cells. Alveolar, embryonic, and glioblastoma cells were stained for survival for 30 minutes with the indicated anti-hCDH15 antibodies (REGN8787 and REGN9295), washed, stained with anti-human IgG Alexa Fluor™ 647 conjugate secondary antibody (red), washed again, fixed, and then stained with myogenin (green) and nuclei (DAPI; blue), after which they were visualized by fluorescence microscopy. Cells incubated with mouse IgG2a and human IgG4 were also used as controls. The last row shows the merge of the three markers. [Figure 9]Figure 9 shows the specific binding of anti-hCDH15 antibody to human rhabdomyosarcoma cells grown in 3D culture. Embryonic rhabdomyosarcoma tumor cells (i.e., rhabdomyosarcoma cells) were incubated with anti-hCDH15 or human IgG4 as a negative control for 30 minutes, washed, stained with anti-human IgG Alexa Fluor™ 647 conjugate secondary antibody (white), washed again, fixed, and stained for myogenin (red) and nucleus (DAPI; blue). The last column shows the merge of the three markers. [Figure 10] Figure 10 shows the retargeting of AAV9 to C2C12 mouse myoblasts via the anti-CDH15 antigen-binding domain. The figure shows representative immunofluorescence images, indicated by GFP fluorescence, of the transducing efficiency in C2C12 myoblasts transduced with eGFP-expressing AAV9 (2.5 × 10⁵ vg / cell). This includes the presence or absence of plasmids encoding the anti-CDH15 antigen-binding domain or a control antigen-binding domain (anti-ASGR1), and various mosaic capsid ratios. DAPI-stained nuclei are shown in blue (top panel), and eGFP is shown in green (bottom panel). The ratios provided represent the ratio of the amount of transfected plasmid encoding the SpyTag-conjugated AAV9 capsid to the amount of unconjugated N272A detargeted AAV9 capsid. When the antigen-binding domain is represented, for example, by an mAb or Fab, the AAV9 capsid contains a SpyTag inserted at position 453 and linked via a 10-amino acid linker, and the antigen-binding domain contains a SpyCatcher fused to the C-terminus of the heavy chain construct. [Figure 11]Figure 11 shows the retargeting of human skeletal myoblasts by the anti-CDH15 antigen-binding domain of AAV9. Representative immunofluorescence images are shown showing the transduction efficiency via GFP fluorescence of human skeletal myoblasts transduced with 2.5 × 10⁵ vg / cell AAV9-expressing eGFP + / - plasmids encoding either the anti-CDH15 antigen-binding domain or a control antigen-binding domain (anti-ASGR1) and various mosaic capsid ratios. DAPI-stained nuclei are shown in blue (top panel), and eGFP is shown in green (bottom panel). The ratios provided represent the ratio of the amount of transfected plasmid encoding the SpyTag-conjugated AAV9 capsid to the amount of unconjugated N272A detargeted AAV9 capsid. When the antigen-binding domain is represented, for example, by an mAb or Fab, the AAV9 capsid contains a SpyTag inserted at position 453 and linked via a 10-amino acid linker, and the antigen-binding domain contains a SpyCatcher fused to the C-terminus of the heavy chain construct. [Modes for carrying out the invention]

[0038] Each skeletal muscle is composed of thousands of muscle fibers encased together in a connective tissue sheath. Individual bundles of skeletal muscle fibers are known as fasciculi. The outermost connective tissue sheath surrounding the entire muscle is known as the epimysium. The connective tissue sheath covering each fascia is known as the perinodal sheath, and the innermost sheath surrounding individual muscle fibers is known as the endomysium. Each muscle fiber is composed of myofibrils, which contain multiple myofilaments.

[0039] When bundled together, all myofibrils are arranged in a unique striated pattern that forms the sarcomere, the basic contractile unit of skeletal muscle. The two most important myofiflals are actin and myosin filaments, which are characteristically arranged to form various bands on skeletal muscle.

[0040] The primary function of skeletal muscle is achieved through its inherent excitation-contraction coupling process. Because muscles are attached to tendons on bones, muscle contraction causes bones to move, enabling specific movements. Skeletal muscle also provides structural support and helps maintain body posture. Skeletal muscle also acts as a storage source of amino acids that can be used by different organs of the body to synthesize organ-specific proteins. Skeletal muscle also acts as a site for glucose processing in the form of muscle glycogen. Skeletal muscle also plays a central role in maintaining thermostasis and acts as an energy source during starvation. Thus, skeletal muscle plays a vital role in exercise, thermoregulation, and the control of systemic metabolism.

[0041] In many muscle diseases, as well as in normal aging, the size and function of skeletal muscle tissue decline, impairing functional mobility, and in severe muscle diseases, leading to long-term disability and premature death.

[0042] Treatment for muscle wasting and hereditary muscle diseases typically consists of broad-acting therapies, such as testosterone therapy for muscle wasting, glucocorticoids for muscular dystrophy, and systemic AAV delivery for the treatment of muscle diseases (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, and dystroglycanopathy). Detargeted delivery of these therapies reduces the efficiency of specific muscle uptake while also causing significant harmful off-target effects in other organs.

[0043] Muscle stem cells (MuSCs) are crucial for skeletal muscle regeneration. While normally quiescent in adult muscle, MuSCs become activated, proliferate, and differentiate into functional muscle fibers upon injury. However, MuSCs in aged subjects exhibit delayed activation and reduced motility in vitro, leading to impaired MuSC-mediated repair in vivo.

[0044] Mitotic, quiescent muscle stem cells (MuSCs, also called satellite cells) and proliferative myoblasts are embryonic precursors of muscle cells (also called myocytes) that have not yet fused to form myotubes or muscle fibers. Muscle stem cells and myoblasts differentiate into muscle cells through a process called myogenesis, schematically (not to exact scale) shown in Figure 1.

[0045] Generally, when exposed to signals from a damaged environment, muscle stem cells leave their quiescent state, re-enter the cell cycle, and begin to proliferate as myoblasts. Some daughter cells continue to differentiate, while others return to quiescence to replenish the reserve population of muscle stem cells. During the differentiation phase, specific genes (e.g., the striated alpha-actin gene) are expressed, causing the myoblasts to align with one another. The myoblasts then fuse to form muscle fibers, involving the recruitment of actin to the cell membrane.

[0046] Muscle stem cells can be characterized by a combination of several genetic markers, including Pax7 and muscle regulatory proteins. Pax7 is a pair of homeobox transcription factors that specify the myogenic properties of progenitor muscle cells. Therefore, muscle stem cells can be characterized as Pax7+, regardless of whether they are in the quiescent or proliferative phase. See, for example, Figure 1. Furthermore, myogenesis depends on the precise and dynamic integration of several muscle regulatory factors, such as myogenesis factor 5 (MYF5), myogenic differentiation factor 1 (MYOD), myogenin (MYOG), and embryonic myosin heavy chain (MyHC), making it possible to characterize myogenic cell lineages. For example, MYOD is expressed in myogenic cells but not in quiescent muscle stem cells, and can therefore 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. See, for example, Figure 1. Additional markers that may be used to identify the stage of myogenesis a cell is undergoing include, but are not limited to, embryonic myosin heavy chains (eMyHC). These are transiently upregulated in immature muscle fibers but downregulated as the muscle fibers mature. See, for example, Figure 1.

[0047] 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, muscle cells, and undifferentiated myotubes or muscle fibers can all be considered non-terminal differentiated muscle cells.

[0048] Many pathological conditions, such as muscular dystrophy (MD) or muscle wasting, may not provide sufficient signaling to muscle stem cells, or satellite cells may have inherent defects in these conditions, which can impair their regenerative potential. Therefore, the ability to specifically target non-terminally differentiated muscle cells, such as muscle stem cells, myoblasts, and myotubes, may be useful in the treatment of skeletal muscle disorders.

[0049] An example of a cell surface protein found on non-terminal differentiated muscle cells: cadherin 15 (CDH15). Cadherins are a class of calcium-dependent transmembrane proteins involved in cell-cell adhesion. Classical cadherins consist of an immunoglobulin-like cadherin domain, a single transmembrane region, and an extracellular domain containing five repeats of the cytoplasmic domain. Cadherin 15 (also known as M-cadherin) is expressed on the apical surface of muscle stem cells and is thought to regulate the adhesion of muscle stem cells to muscle fibers. 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 base pairs long. An exemplary sequence of the human CDH15 gene has been assigned NCBI accession number NM_004933.3 (sequence number 787). The exemplary human CDH15 protein is assigned NCBI accession number NP_004924.1 and / or UniProt accession number P55291 (sequence number 788).

[0050] Genetically modified animal models can prove particularly useful for studying the function of CDH15 in muscle. For example, deletion of Cdh15 in genetically modified animals does not alter the quiescence, proliferation, myogenic lineage progression, or differentiation ability of MuSCs isolated from them, but Cdh15 deletion in these mice leads to enhanced post-injury regeneration (Figures 2A-2D, 3A-3B). This may be at least partially due to accelerated exit from quiescence upon deletion of CDH15 expression (Figures 4A-4B), which is associated with the induction of early response genes for SRF regulation (Figures 5A-5B), which is then associated with a decrease in Rho / Rac signaling. Interestingly, Cdh15 - / - MuSCs were slightly larger than wild-type control MuSCs (data not shown), which is an indicator that these cells are more primed for activation. Similar to the effect observed in young mice, Cdh15 deletion also rescues age-related deficits in muscle regeneration (Figure 6A-6D). Therefore, antagonist antibodies, or CDH15 blocking antibodies, may be useful for enhancing muscle regeneration in both young subjects and older adults after injury stimuli (e.g., arthroplasty).

[0051] Furthermore, certain muscle-related cancers, including rhabdomyosarcoma, can benefit from therapies that target muscle-specific markers. Rhabdomyosarcoma (RMS) is characterized by the expression of myogenic genes, but several subtypes exist. For example, embryonic RMS is the most common subtype and typically has a more favorable prognosis, thought to be driven by the loss of tumor suppressor genes or the 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 of the Pax3 or Pax7 genes, or alternative gene fusions. Additional, rarer subtypes include spindle cell / sclerosing RMS and pleomorphic RMS. Rhabdomyosarcoma accounts for about 3% of all childhood cancers, with approximately 400-500 new cases annually in the United States (American Cancer Society, 2021). The prognosis is generally good in children (e.g., about 70% survival rate), but depends on the risk category. For example, one-third of patients with localized RMS and two-thirds of patients with metastatic RMS experience recurrent disease. When RMS recurs, the majority of patients have an estimated 5-year survival rate of about 10%. Furthermore, the prognosis is generally much worse in adults (overall survival is 20-50%).

[0052] Therefore, since high CDH15 expression is observed in all rhabdomyosarcomas (RMS) and may be associated with chemotherapy resistance and poor prognosis, anti-CDH15 antibodies may be useful for delivering therapeutic agents (e.g., cytotoxic agents) to RMS tumors.

[0053] Antigen-binding proteins that bind to human CDH15, such as antibodies and their antigen-binding fragments, are described herein. The antibodies described herein may be particularly useful for blocking the activity of CDH15 and / or specifically stimulating muscle regeneration, in order to specifically direct the internal delivery of drugs, such as drug conjugates, to muscle cells and / or muscle-associated cancer cells. Accordingly, antibodies or their antigen-binding fragments that bind to human CDH15, including antibody-protein fusion constructs comprising antibodies or their antigen-binding fragments that bind to human CDH15, and antibody-drug conjugates comprising antibodies or their antigen-binding fragments that bind to human CDH15, are also described herein. Furthermore, recombinant AAV particles, such as AAV recombinant AAV particles, that can target non-terminal differentiated muscle cell surface proteins such as mammalian CDH15, in order to enhance the muscle delivery of therapeutic payloads and reduce off-target effects are described herein.

[0054] The descriptions herein are not limited to the specific embodiments, compositions, methods, and experimental conditions described, as such embodiments, compositions, methods, and conditions may vary. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0055] Any methods and materials similar to or equivalent to those described herein may be used in the practices or tests described herein, but some preferred methods and materials are described herein. All publications cited herein are incorporated herein by reference for the purpose of illustrating their whole. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those universally understood by those skilled in the art.

[0056] The singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly indicated by the context. Therefore, for example, a reference to “one method” includes one or more methods and / or processes of the kind described herein and / or which would be apparent to those skilled in the art by reading this disclosure.

[0057] The term "approximately," when used in reference to a specific enumerated number, means that the value may vary by no more than 1% from the enumerated value. For example, the expression "approximately 100" includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0058] "Homologie percentages (%)" can be readily determined for amino acid or nucleotide sequences spanning the entire length or a portion of a protein. These portions may each be at least approximately 5 amino acids or 24 nucleotides long, or up to approximately 700 amino acids or 2100 nucleotides long. Generally, when referring to "homology," "similarity," or "similarity" between two different adeno-associated viruses, "homology," "similarity," or "similarity" is determined by referring to "aligned" sequences. "Aligned" sequences or "alignment" refer to multiple nucleic acid sequences or protein (amino acid) sequences that often include corrections for missing or added bases or amino acids compared to a reference sequence.

[0059] Alignment may be performed using one of the various publicly available or commercially available multiple sequence alignment programs. For amino acid sequences, available sequence alignment programs include, for example, "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box." Generally, one of these programs is used with its default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art may use another algorithm or computer program that provides at least the same level of homology or alignment as provided by the referenced algorithm and program. See, for example, JDThomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690 (1999).

[0060] For nucleic acid sequences, several 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 also known to those skilled in the art. Alternatively, the Vector NTI utility can be used. There are also numerous algorithms known in the art that can be used to measure the homology of nucleotide sequences, including the programs mentioned above. As another example, polynucleotide sequences can be compared using FASTA®, a program from GCG version 6.1. FASTA® provides alignment of best overlapping regions and sequence homology percentages between query sequences and search sequences. For example, the sequence homology percentage between nucleic acid sequences can be determined by using FASTA® with its default parameters (word size of 6 and NOPAM factor for the scoring matrix), as provided in GCG version 6.1, which is incorporated herein by reference.

[0061] "Significant homology" includes alignments of amino acid sequences or nucleic acid sequences that are homologous by at least 90%, e.g., at least 93%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, or e.g., at least 100%.

[0062] The term "chimera" encompasses functional genes or polypeptides that contain nucleic acid sequences or amino acid sequences (corresponding to each other) derived from at least two different AAV serotypes. For example, a gene or polypeptide that is part of at least a first and second AAV, where at least the first and second parts are functionally linked to form a functional chimeric AAV nucleic acid encoding a functional amino acid. Unless designated as a chimera, nucleotide sequences, genes, polypeptides, and amino acids are considered non-chimers in that each contains a nucleic acid sequence or amino acid sequence that has significant homology to a single AAV serotype nucleic acid sequence or amino acid sequence.

[0063] When used herein, the phrase “operably linked” includes the physical juxtaposition (e.g., in three-dimensional space) of members or elements that interact with each other directly or indirectly, or are positioned relative to each other in relation to a biological event, such juxtaposition that achieves or enables such interaction and / or positioning. For example, a regulatory element in a nucleic acid (e.g., an expression control sequence) 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, “operably linked” includes covalent bonding of members or elements that are related to each other. Those skilled in the art will readily understand that in some embodiments, covalent bonding is not necessary to achieve effective operably linked. For example, proteins that are operably linked together may associate with each other, for example, via covalent or non-covalent bonding. As a non-limiting example, the capsid proteins described herein may be operably ligated to a targeting ligand, and the capsid proteins may optionally have or may not have a scaffold and / or adapter between the capsid protein and the targeting ligand, and may be non-covalently or covalently ligated to the targeting ligand. As another example, in some embodiments, nucleic acid regulatory elements operably ligated to a coding sequence to be controlled are contiguous with the nucleotide of interest. Alternatively or in addition, in some embodiments, one or more such regulatory elements control the coding sequence of interest in trans or slightly apart. In some embodiments, the term “regulatory element,” as used herein, refers to the polynucleotide sequence necessary and / or sufficient to influence the expression and processing of the coding sequence to which they are ligated.In some embodiments, regulatory elements may be, or include, appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (e.g., Kozak consensus sequences); sequences that improve protein stability; and / or, in some embodiments, sequences that improve protein secretion. In some embodiments, one or more regulatory elements are preferentially or exclusively active in specific host cells or organisms, or types thereof. For example, in prokaryotes, regulatory elements typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, in many embodiments, regulatory elements typically include promoters, enhancers, and / or transcription termination sequences. Those skilled in the art will understand that in many embodiments, the term “regulatory element” refers to a member essential for expression and processing, and in some embodiments, includes a member whose presence is advantageous for expression (e.g., including leader sequences, targeting sequences, and / or fusion partner sequences).

[0064] Expressions such as "antibody that binds to CDH15" and "anti-hCDH15 antibody" include antibodies that specifically recognize a single CDH15 molecule and their antigen-binding fragments. The antibodies and their antigen-binding fragments described herein may bind to soluble CDH15 and / or cell surface-expressed CDH15. Soluble CDH15 includes the native CDH15 protein as well as recombinant CDH15 protein variants that lack a transmembrane domain or are otherwise not bound to the cell membrane.

[0065] The expression "cell surface-expressed CDH15" refers to one or more CDH15 proteins expressed on the surface of a cell in vitro or in vivo, in which at least a portion of the CDH15 protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface-expressed CDH15" may include or constitute CDH15 proteins expressed on the surface of cells that normally express the CDH15 protein. Alternatively, "cell surface-expressed CDH15" may include or constitute CDH15 proteins expressed on the surface of cells that do not normally express human CDH15 on their surface but have been artificially modified to express CDH15 on their surface.

[0066] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies.

[0067] The term "antibody" refers to any antigen-binding molecule or molecular complex that contains 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" refers to an immunoglobulin molecule that contains four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy-chain variable region (abbreviated herein as HCVR or VH) and a heavy-chain constant region. The heavy-chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light-chain variable region (abbreviated herein as LCVR or VL) and a light-chain constant region. The light-chain constant region contains 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 the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy-chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light-chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high-affinity" antibodies refers to antibodies whose binding affinity to their target is at least 10 -9 M, at least 10 -10 M, at least 10 -11 M, or at least 10 -12 M. The term "antibody" can encompass any type of antibody, e.g., monoclonal or polyclonal. Further, the antibody can be of any origin, e.g., mammalian or non-mammalian. In one embodiment, the antibody can be mammalian or avian. In a further embodiment, the antibody can be of human origin and even a human monoclonal antibody.

[0068] The term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. Terms such as “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be obtained from complete antibody molecules using any suitable standard technique, such as protein digestion or recombinant genetic engineering techniques, which involve 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 commercially available sources, DNA libraries (e.g., including phage antibody libraries), or can be synthesized. DNA may be sequenced and manipulated chemically or by using molecular biological techniques, for example, by arranging one or more variable domains and / or constant domains in a suitable configuration, or by introducing codons, creating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0069] 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., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included in the expression “antigen-binding fragment.”

[0070] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In an antigen-binding fragment having a domain, V H Domain and V L The domains can be positioned relative to each other in any preferred arrangement. For example, the variable region is a dimer, V H -V H , V H -V L or V L -V L It may contain dimers. Alternatively, the antigen-binding fragment of the antibody may contain a monomer V. H or V L You may include a domain name.

[0071] In certain embodiments, the antigen-binding fragment of an antibody may include at least one variable domain covalently bound to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within the antigen-binding fragments of antibodies described 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 -CH 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 This includes: In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a mobile or semi-mobile linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies described herein may be linked to each other and / or one or more monomers V H or V L Non-covalent associations with domains (e.g., via disulfide bonds) may include homodimers or heterodimers (or other polymers) of any of the variable domain and constant domain configurations listed above.

[0072] Similar to complete antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody form, including the exemplary bispecific antibody forms described herein, can be adapted for use in conjunction with the antibody antigen-binding fragment of the present invention using routine techniques available in the art.

[0073] In certain embodiments, the anti-hCDH15 antibodies described herein are human antibodies. The term “human antibody” refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences, for example, in the CDR, particularly in CDR3 (mutations introduced, for example, by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted onto a human framework sequence.

[0074] The antibodies described herein may, in some embodiments, be recombinant human antibodies. The term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (further described below), antibodies isolated from recombinant combinatorial human antibody libraries (further described below), antibodies isolated from animals transgenic for human immunoglobulin genes (e.g., mice) (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if transgenic animals are used for the human Ig sequence, in vivo mutagenesis of somatic cells), so the V of the recombinant antibody H Region and V L The amino acid sequence of the region is human germline V H Array and V LWhile it is derived from and related to the sequence, it cannot naturally exist in the in vivo human antibody germline repertoire.

[0075] Human antibodies can exist in two common forms related to hinge heterogeneity. In one common form, the immunoglobulin molecule contains a stable quad-chain construct of approximately 150–160 kDa, with the dimer held together by interchain heavy-chain disulfide bonds. In the second common form, the dimer is not linked via interchain disulfide bonds, and the molecule of approximately 75–80 kDa consists of covalently bonded light and heavy chains (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

[0076] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but is not limited to, structural differences related to 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 occurrence of the second form to the level typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The antibodies described herein are hinge, C H 2 regions or C H The antibody may have one or more mutations in the three regions, and these mutations may be desirable, for example, to improve the yield of the desired antibody morphology during production.

[0077] The antibodies described herein may be isolated antibodies. “Isolated antibody” means an antibody identified, isolated, and / or recovered from at least one component of its natural environment. For example, an antibody isolated or removed from at least one component of an organism, or from a tissue or cell in which antibodies naturally exist or are naturally produced, may be considered an “isolated antibody.” Isolated antibodies also include antibodies of insights within recombinant cells. An isolated antibody is an antibody subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may substantially contain no other cellular material and / or chemicals.

[0078] One-arm antibodies that bind to CDH15 are also described herein. The term "one-arm antibody" refers to an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-arm antibodies described herein may contain either the HCVR / LCVR or CDR amino acid sequence listed in Table 1.

[0079] The anti-hCDH15 antibodies discussed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. This specification also describes antibodies derived from any of the amino acid sequences described herein, and their antigen-binding fragments, where one or more amino acids in one or more framework regions and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibodies are derived, or to the corresponding residues of another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing 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 framework and / or CDR residues within the VH and / or VL domains are mutated back to residues found in the original germline sequence from which the antibody originates. In other embodiments, only certain residues, for example, only mutant residues found within the first eight amino acids of FR1, or within the last eight amino acids of FR4, or only mutant residues found within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more framework residues and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated). Furthermore, the antibodies described herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, certain individual residues may mutate to corresponding residues in a particular germline sequence, while certain other residues, different from the original germline sequence, are maintained or mutate 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 (in some cases) biological properties of the antagonist or agonist, or decreased immunogenicity. In some embodiments, the antibodies or antigen-binding fragments described herein are obtained in this general manner.

[0080] This specification also describes anti-hCDH15 antibodies containing variants of any 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 containing HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions to any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences listed in Table 1 of this specification.

[0081] The term "bispecific antibody" refers to an antibody that can selectively bind to two or more epitopes. A bispecific antibody typically contains two distinct heavy chains, each specifically binding to a different epitope—either on two different molecules (e.g., antigens) or on a homologous molecule (e.g., a homologous antigen). When a bispecific antibody can selectively bind to two different epitopes (a primary and a secondary epitope), the affinity of the primary heavy chain to the primary epitope is generally at least one to two orders of magnitude, or even three or four orders of magnitude, lower than the affinity of the primary heavy chain to the secondary epitope, and vice versa. The epitopes recognized by a bispecific antibody can be on homologous targets or different targets (e.g., on homologous proteins or different proteins). A bispecific antibody can be constructed, for example, by combining heavy chains that recognize different epitopes of a homologous antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of homologous antigens can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed (from N-terminus to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and the immunoglobulin light chain can bind to each heavy chain but not confer antigen-binding specificity, or can bind to each heavy chain and can bind to one or more epitopes to which the heavy chain antigen-binding region binds, or can bind to each heavy chain and can be involved in the process by which one or both heavy chains bind to one or both epitopes.

[0082] The terms “heavy chain” or “immunoglobulin heavy chain” include the constant region sequence of an immunoglobulin heavy chain of any biological origin, and unless otherwise specified, include the heavy chain variable domain. Unless otherwise specified, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. Heavy chain fragments include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a variable domain followed (from N-terminus to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Functional heavy chain fragments include fragments that are capable of specifically recognizing an antigen (e.g., recognizing an antigen at KD levels ranging from micromolar, nanomolar, or picomolar concentrations), are expressible and secretible from cells, and contain at least one CDR.

[0083] Terms such as "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single-domain antigen-binding protein," and "single-domain binding protein" refer to monomeric or homodimeric immunoglobulin molecules containing an immunoglobulin-like chain that includes a variable domain operably linked to a heavy chain constant region, where the heavy chain constant region is typically functional C H Because it lacks one domain, it cannot associate with the light chain. Therefore, terms such as "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single-domain antigen-binding protein," and "single-domain binding protein" are (i) functional C H (ii) A monomeric single-domain antigen-binding protein comprising one immunoglobulin-like chain containing a heavy chain constant region lacking one domain and a variable domain operably linked thereto, or (ii) comprising two immunoglobulin-like chains, each functionally C H This includes both homodimeric single-domain antigen-binding proteins, which contain a heavy chain constant region lacking one domain and a variable domain that is operably linked. In various embodiments, homodimeric single-domain antigen-binding proteins contain two homologous immunoglobulin-like chains, each of which is functionally C HIt contains a homologous heavy chain constant region lacking one domain and a homologous variable domain operably linked to it. Furthermore, each immunoglobulin-like chain of a single-domain antigen-binding protein contains a variable region, which has a variant region derived from a heavy chain variable region gene segment (e.g., VH, DH, JH), a light chain gene segment (e.g., VL, JL), or a combination thereof, and is linked to a heavy chain constant region (CH) gene sequence. This heavy chain constant region gene sequence contains deletion or inactivation mutations in the CH1 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 containing a variable domain derived from a heavy chain gene segment is "V H "Single-domain antibody" or "V H These may be referred to as "single-domain antibody-binding proteins," and for example, see U.S. Patent No. 8,754,287, U.S. Patent Publication Nos. 2014 / 0289876, 2015 / 0197553, 2015 / 0197554, 2015 / 0197555, 2015 / 0196015, 2015 / 0197556, and 2015 / 0197557, each of which is incorporated by reference as a whole. Single-domain antigen-binding proteins containing a variable domain derived from a light chain gene segment are referred to as "V L These may be referred to as "single-domain antigen-binding proteins," and the entire text is incorporated by reference, for example, to U.S. Publication No. 2015 / 0289489.

[0084] The term "light chain" includes the constant region sequence of an immunoglobulin light chain of any biological origin, and unless otherwise specified, includes the human kappa light chain and the human lambda light chain. The variable (VL) domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes a VL domain containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus, and a constant light chain domain. Potentially useful light chains include, for example, light chains that do not selectively bind to either a primary or secondary antigen selectively bound by an antigen-binding protein. Preferred light chains can be identified by screening for the light chains most commonly employed in existing antibody libraries (wet libraries or in silico), which do 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 to which the antigen-binding domain of an antigen-binding protein binds.

[0085] The term "variable domain" includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) containing the following amino acid regions in N-terminus to C-terminus (unless otherwise specified): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" contains an amino acid sequence that can fold into a standard domain (VH or VL) having a double beta-sheet structure, where the residues of the first beta-sheet and the residues of the second beta-sheet are connected by disulfide bonds.

[0086] The term “complementarity-determining region” or “CDR” refers to an amino acid sequence encoded by the nucleic acid sequence of an immunoglobulin gene in an organism, which is typically found between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor) in wild-type animals. CDRs can be encoded by, for example, germline sequences or reconfigured or non-reconfigured sequences, and by, for example, naive B cells or mature B cells, or T cells. Under certain circumstances (e.g., with respect to CDR3), a CDR may be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in non-reconfigured nucleic acid sequences) but are contiguous in B cell nucleic acid sequences, for example, as a result of sequence splicing or concatenation (e.g., VDJ reconfiguration to form heavy chain CDR3).

[0087] Methods and techniques for identifying CDRs within HCVR amino acid sequences and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within identified HCVR amino acid sequences and / or LCVR amino acid sequences disclosed herein. Examples of rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, 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 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0088] The term "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments included in the term "antibody fragment" include: (i) Fab fragments, which are monovalent fragments consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide crosslink at the hinge region; (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al. (1989) Nature 241:544-546); (vi) isolated CDRs; and (vii) scFv, which consists of the two domains VL and VH of the Fv fragment, linked by a synthetic linker to form a single protein chain in which the VL and VH regions are paired to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also included under the term "antibody" (see, for example, Holliger et al. (1993) PNAS USA 90:6444-6448 and Poljak et al. (1994) Structure 2:1121-1123).

[0089] The term "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least the functional portions of the CH2 and CH3 regions of immunoglobulins. "Functional portion" refers to the CH2 and CH3 regions capable of binding to an Fc receptor (e.g., FcyR or FcRn, i.e., fetal Fc receptor) and / or participating in complement activation. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions or other modifications that render them unable to bind any Fc receptor and also unable to activate complement, then the CH2 and CH3 regions are not functional.

[0090] Fc-containing proteins may include modifications to their immunoglobulin domains, such modifications including those that affect one or more effector functions of the binding protein (e.g., modifications affecting FcyR binding, FcRn binding, and therefore half-life and / or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to the EU numbering of the immunoglobulin constant region: 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.

[0091] For example, though not intended to be limiting, the binding protein is an Fc-containing protein that exhibits improved serum half-life (compared to homologous Fc-containing proteins without the described modifications) and has modifications 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 modifications at position 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at position 250 and / or 428; or modifications at position 307 or 308 (e.g., 308F, V308F) and 434. In another example, the modifications may include modifications of 428L (e.g., M428L) and 434S (e.g., N434S); modifications of 428L, 2591 (e.g., V259I), and 308F (e.g., V308F); modifications of 433K (e.g., H433K) and 434 (e.g., 434Y); modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E); modifications of 250Q and 428L (e.g., T250Q and M428L); and modifications of 307 and / or 308 (e.g., 308F or 308P).

[0092] As used herein, the term “antigen-binding protein” refers to a polypeptide or protein (one or more polypeptides complexed as functional units) that specifically recognizes an epitope on an antigen, such as a cell-specific antigen and / or a target antigen of the present invention as described herein. Antigen-binding proteins may be multispecific. The term “multispecific” in relation to an antigen-binding protein means that the protein recognizes different epitopes on either homologous or different antigens. Multispecific antigen-binding proteins described herein may be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides linked to each other by covalent or noncovalent bonds. The term “antigen-binding protein” includes antibodies or fragments thereof described herein that may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, gene fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as a protein or fragment thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity.

[0093] A "protein" refers to an amino acid polymer in which multiple amino acids are covalently linked via amide bonds. Proteins generally contain one or more amino acid polymer chains known in the art as "polypeptides." Therefore, polypeptides can be proteins, and proteins can contain multiple polypeptides to form a single functional biomolecule. Disulfide crosslinks (i.e., between cysteine ​​residues that form cysteine) may be present in some proteins. These covalent links may be within a single polypeptide chain or between two individual polypeptide chains. For example, disulfide crosslinks are essential for the proper structure and function of insulin, immunoglobulins, and protamines. 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).

[0094] As used herein, “protein” includes biotherapeutic proteins, recombinant proteins used in research or therapeutics, 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 be produced using recombinant cell-based production systems, e.g., insect baculovirus systems, yeast systems (e.g., Pichiasp.), mammalian systems (e.g., CHO cells, and CHO derivatives such as CHO-K1 cells). For a recent review discussing 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).

[0095] As used herein, the term “epitope” refers to a portion of an antigen recognized by a multispecific antigen-binding polypeptide. A single antigen (such as an antigen polypeptide) may have two or more epitopes. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and are defined as residues that directly contribute to the affinity of the interaction between the antigen-binding polypeptide and the antigen. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants, which are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, they may have specific three-dimensional structural properties and / or specific charge properties. Epitopes formed from continuous amino acids are typically retained even when exposed to denaturing solvents, whereas epitopes formed by three-dimensional folding are typically lost upon treatment with denaturing solvents.

[0096] The term “domain” refers to any portion of a protein or polypeptide having a specific function or structure. Preferably, the domains described herein bind to cell-specific antigens or target antigens. As used herein, cell-specific antigen domains or target antigen-binding domains include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen.

[0097] The terms “half-body” and “half-antibody” are used interchangeably and refer to half of an antibody that essentially contains one heavy chain and one light chain. The antibody heavy chain can form dimers, and therefore the heavy chain of one half-antibody can associate with a heavy chain that is associated with a different molecule (e.g., another half-antibody) or another Fc-containing polypeptide. The two slightly different Fc domains can “heterodimerize,” as is the case in the formation of bispecific antibodies, or other heterodimers, heterotrimers, and heterotetramers. 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 (2012), and Shimamoto et al., “Peptibodies: A flexible alternative format to antibodies,” 4(5)MAbs 586-91 (2012).

[0098] The terms “single-chain variable fragment” or “scFv” refer to 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 linked by a linker sequence of 10 to 25 amino acids. ScFv polypeptides may also contain other amino acid sequences, such as a CL region or a CH1 region. ScFv molecules may be produced by phage display or by directly subcloning the heavy and light chains from hybridomas or B cells. Ahmad et al., Clinical and Developmental Immunology, volume 2012, article ID 98025, is incorporated herein by reference to methods for producing scFv fragments by phage display and antibody domain cloning.

[0099] As used herein, the term muscle-related cancer refers to any cancer cell defined by the expression of myogenic genes, such as CDH15. Muscle-related cancers include, but are not limited to, rhabdomyosarcomas, such as embryonic, alveolar, spindle cell / scleral, and pleomorphic cancers.

[0100] The term “target cell” includes any cell on which the expression of the nucleotide of interest is desired. Preferably, target cells exhibit receptors on their surface that enable targeting by a targeting ligand, as described below. In certain embodiments, the target cell is a muscle cell. As used herein, a muscle cell means any cell that expresses a myogenic marker and / or is involved in skeletal muscle regeneration, e.g., a muscle stem cell (MuSC, also known as a satellite cell) that expresses Pax7 and therefore Pax7 + Cells, for example, at rest, Pax7 + / MyoD - , and during proliferation, Pax7 + / MyoD + ), myoblasts (e.g., MyoD + Myogenin +Cells), muscle cells, myotubes (also known as muscle fibers, e.g., MyHC + They are considered to be cells, etc.

[0101] "Retargeting" or "redirection" may include scenarios in which wild-type particles target several cells within a tissue and / or several organs within an organism, and the general targeting of the tissue or organ is reduced or neutralized by the insertion of heterologous amino acids, and retargeting of more specific cells within the tissue or more specific organs within the organism is achieved with a targeting ligand that binds to a marker expressed by the specific cell (e.g., via the targeting ligand). Such retargeting or redirection may also include scenarios in which wild-type particles target a tissue, and the targeting of the tissue is reduced or neutralized by the insertion of heterologous amino acids, and retargeting to a completely different tissue is achieved with the targeting ligand.

[0102] "Specific binding pairs," "binding pairs," and "protein:protein binding pairs" include two members (e.g., a first member (e.g., a first polypeptide) and a second congeneral member (e.g., a second polypeptide)) that interact to form a binding (e.g., a non-covalent binding between an epitope of the first member and the antigen-binding portion of the second member of an antibody that recognizes that epitope; e.g., a covalent binding between proteins that can form an isopeptide bond; or split inteins that recognize each other and mediate the ligation of adjacent proteins and their own removal through the process of protein trans-splicing). In some embodiments, the term "congeneral" refers to members that function together. Epitopes and their congeneral antibodies, particularly epitopes that can also function as detectable labels (e.g., c-myc), are known in the art. Certain protein:protein binding pairs capable of interacting to form covalent isopeptide bonds are outlined in Veggiani et al. (2014) Trends Biotechnol. 32:506, and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, isopeptag:pyrin C, SnoopTag:SnoopCatcher, and their biologically equivalent variants, such as SpyTag003:SpyCatcher003. Generally, the first member of a protein:protein binding pair refers to a member of the pair that is generally shorter than 30 amino acids and forms a spontaneous covalent isopeptide bond with a second congener protein, which is generally larger but can also be shorter than 30 amino acids, such as the SpyTag:KTag system.

[0103] The term "isopeptide bond" refers to an amide bond between a carboxyl group or carboxamide group and an amino group, at least one of which is not derived from the protein backbone or, alternatively, is not considered to be part of the protein backbone. Isopeptide bonds can be formed within a single protein or between two peptides or between a peptide and a protein. Thus, isopeptide bonds can be formed within molecules of a single protein, or between molecules, i.e., between two peptide / protein molecules, for example, between two peptide linkers. Typically, isopeptide bonds can be formed between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or between the terminal carboxyl group of a protein or peptide chain, or between the alpha-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid residue. Each residue in a pair involved in an isopeptide bond is referred to herein as a reactive residue. In preferred embodiments of the present invention, isopeptide bonds may be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, isopeptide bonds may occur between the side-chain amine of lysine and the carboxamide group of asparagine or the carboxyl group of aspartic acid.

[0104] The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zaveri et al. (2012) PNAS 109:E690-E697, each of which is incorporated herein by reference as a whole, and is derived from the CnaB2 domain of the fibronectin-binding protein FbaB of Streptococcus pyogenes. By splitting the domain, Zaveri et al. obtained the “SpyTag” peptide having the AHIVMVDAYKPTK sequence (SEQ ID NO: 815), which forms an amide bond with its congener protein “SpyCatcher,” a 112-amino acid polypeptide having the amino acid sequence described in SEQ ID NO: 816 (Zaveri (2012), above). A further specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase. (Fierer (2014) PNAS 111:E1176-1181) SpyLigase is manipulated by cleaving the β-chain from a reactive lysine-containing SpyCatcher, thereby obtaining a KTag, the first member of a 10-residue 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, which is incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, described as SEQ ID NO: 821, and binds to SpyCatcher002. SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK, described as SEQ ID NO: 822, and binds to SpyCatcher003.

[0105] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113:1202-07. The D4 Ig-like domain of RrgA, an adhesive derived from Streptococcus pneumoniae, was split to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher induces specific spontaneous isopeptide bonds between complementary proteins. (Veggiani (2016)), see above.

[0106] The isopeptag:pyrin-C specific binding pair was derived from Spy0128, a major pyrin protein from Streptococcus pyogenes (Zakeir and Howarth (2010) J.Am.Chem.Soc.132:4526-27). Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, described as SEQ ID NO: 820, and binds to pyrin-C (residues 18-299 of Spy0128). Incubation of isopeptag and pyrin-C results in a specific spontaneous isopeptide bond between complementary proteins. Zakeir and Howarth (2010), see above.

[0107] Terms such as "transduction" or "infection" refer to the introduction of nucleic acids into the nucleus of target cells (e.g., muscle stem cells, myoblasts, muscle cells, or any combination thereof) by viral particles. The efficiency related to transduction, such as the term "transduction efficiency," refers to the fraction (e.g., percentage) of cells expressing the target nucleotide after incubation with a set of recombinant AAV particles containing the target nucleotide. Known methods for determining transduction efficiency include flow cytometry of transduced cells using a fluorescent reporter gene and RT-PCR for the expression of the target nucleotide.

[0108] Generally, a “reference” viral capsid protein / capsid / particle is homologous to the test viral capsid protein / capsid / particle, except for modifications made to test the effect. For example, to determine the effect of inserting the first member of a specific binding pair into the test recombinant AAV particle, e.g., the effect on transduction efficiency, the transduction efficiency of the test recombinant AAV particle (in the presence or absence of a suitable targeting ligand) can be compared to the transduction efficiency of a reference recombinant AAV particle homologous to the test recombinant AAV particle in any example (e.g., additional point mutations, nucleotides of interest, number of recombinant AAV particles, and target cells), except for the presence of the first member of the specific binding pair (in the presence or absence of a suitable targeting ligand, as needed). In some embodiments, a reference viral capsid protein can form a capsid with a second viral capsid protein modified to include at least the first member of a protein:protein binding pair, while the reference viral capsid protein does not include the first member of a 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.

[0109] Antigen-binding protein Antigen-binding proteins, such as LCV1, LCVR2, antibodies, or antigen-binding fragments thereof, comprising amino acids of HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCV1, LCVR2, and / or LCVR3 as listed in Table 1, are described herein.

[0110] In certain embodiments, the antigen-binding proteins described herein, such as antibodies, or antigen-binding fragments thereof, have a heavy chain CDR1 (HCDR1) consisting of an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, or at least 97% homology, wherein the HCDR1 comprises an amino acid sequence selected from any 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 homology.

[0111] In certain embodiments, the antigen-binding proteins described herein, such as antibodies, or antigen-binding fragments thereof, have a heavy chain CDR2 (HCDR2) consisting of an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, or at least 97% homology, wherein the HCDR2 includes an amino acid sequence selected from any 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 homology.

[0112] In certain embodiments, the antigen-binding proteins described herein, such as antibodies, or antigen-binding fragments thereof, comprise a heavy chain CDR3 (HCDR3) or substantially similar sequence containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, wherein the sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology.

[0113] In certain embodiments, an antigen-binding protein described herein, such as an antibody, or an antigen-binding fragment thereof, has a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or substantially similar sequences having at least 90% and at least 95% homology, wherein the light chain CDR1 comprises an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence homology.

[0114] In certain embodiments, the antigen-binding protein described herein, for example, an antibody, or its antigen-binding fragment comprises a light chain CDR2 (LCDR2) or a substantially similar sequence, which includes an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, and which has sequence homology of at least 90%, at least 95%, at least 98%, or at least 99%.

[0115] In certain embodiments, the antigen-binding proteins described herein, such as antibodies, or antigen-binding fragments thereof, comprise a light chain CDR3 (LCDR3) or substantially similar sequence containing an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, wherein the sequence has at least 90%, at least 95%, at least 98%, or at least 99% sequence homology.

[0116] In some embodiments, the antibodies described herein, or their antigen-binding fragments, include an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) which is a combination of one of the HCDR3 amino acid sequences listed in Table 1 and one of the LCDR3 amino acid sequences listed in Table 1. In some embodiments, the antibodies described herein, or their antigen-binding fragments, include an HCDR3 / LCDR3 amino acid sequence pair which is contained in one of the exemplary anti-hCDH15 antibodies listed in Table 1. In some embodiments, the HCDR3 / LCDR3 amino acid sequence pair is SEQ ID NO: 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, 42 The group is selected from the group consisting of 6 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 778.

[0117] This specification describes antigen-binding proteins, such as antibodies, or antigen-binding fragments thereof, that have a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the anti-hCDH15 antibodies listed in Table 1. In some embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is the 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 The group is selected from -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.

[0118] Furthermore, this specification also describes antibodies or antigen-binding fragments thereof that include a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-hCDH15 antibodies listed in Table 1. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are 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 47 The set includes the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of 6, 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.

[0119] In some embodiments, the antigen-binding protein includes a heavy chain variable region (HCVR or VH). In some embodiments, the HCVR includes a set of HCDR1-HCDR2-HCDR3 amino acid sequences selected from Table 1. In some embodiments, the HCVR includes an amino acid sequence selected from any of the HCVR 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 homology.

[0120] In some embodiments, the antigen-binding protein includes a light chain variable region (LCVR or VL). In some embodiments, the LCVR includes a set of LCDR1-LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the LCVR includes an amino acid sequence selected from any of the LCVR 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 homology.

[0121] In some embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) combining any HCVR amino acid sequence listed in Table 1 with any LCVR amino acid sequence listed in Table 1. In some embodiments, the antigen-binding protein described herein, e.g., an antibody or its antigen-binding fragment, comprises an HCVR / LCVR amino acid sequence pair contained in any of the anti-hCDH15 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is the SEQ ID NO: 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 The group is selected from 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.

[0122] nucleic acid molecule Antigen-binding proteins described herein, such as antibodies, or nucleic acid molecules encoding antigen-binding fragments thereof, such as polynucleotides, are also described herein.

[0123] Furthermore, this specification also describes nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any 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 homology.

[0124] Furthermore, this specification also describes nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any 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 homology.

[0125] Furthermore, this specification also describes nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any 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 homology.

[0126] Furthermore, nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1 are also described. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any 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 homology.

[0127] This specification also describes nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any 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 homology.

[0128] This specification also describes nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any 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 homology. This specification also describes nucleic acid molecules encoding both HCVR and LCVR, in which case the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR 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 homology, and a polynucleotide sequence selected from any of the LCVR 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 homology. In some embodiments, the nucleic acid molecule encodes HCVR and LCVR, where both HCVR and LCVR are derived from homologous anti-hCDH15 antibodies listed in Table 1.

[0129] Furthermore, recombinant expression vectors capable of expressing polypeptides containing the heavy and / or light chain variable regions of an anti-hCDH15 antibody are also described. In some embodiments, the recombinant expression vector includes a nucleic acid molecule encoding any of the nucleic acid molecules mentioned above, namely any of the HCVR, LCVR, and / or CDR sequences listed in Table 1. Also described are host cells into which such vectors have been introduced, as well as methods for producing antibodies or antibody fragments by culturing the host cells under conditions capable of producing antibodies or antibody fragments, and recovering the produced antibodies or antibody fragments.

[0130] Multispecific antigen binding molecules: The anti-hCDH15 antibodies and their antigen-binding fragments described herein may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific to different epitopes of one target polypeptide, or they may contain antigen-binding domains specific to two or more target polypeptides. See, for example, Tutt et al., 1991, J.Immunol. 147:60-69 and Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-hCDH15 antibodies and their antigen-binding fragments described herein may be ligated to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment may be functionally ligated to one or more other molecular entities, such as another antibody or fragment (e.g., by chemical linkage, gene fusion, or non-covalent association, or otherwise) to produce a bispecific or multispecific antigen with a second or further binding specificity.

[0131] The use of the term “anti-hCDH15 antibody” herein is intended to include both monospecific anti-hCDH15 antibodies and bispecific antibodies comprising a CDH15-binding arm and a “target”-binding arm. Therefore, described herein are bispecific antibodies in which one arm of the immunoglobulin binds to human CDH15 and the other arm of the immunoglobulin is specific to another target molecule. The CDH15-binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences listed in Table 1 herein.

[0132] In certain embodiments, the CDH15-binding arm binds to human CDH15 and induces the internalization of CDH15 and the antibody bound to it. In certain embodiments, the CDH15-binding arm weakly binds to human CDH15 and induces the internalization of CDH15 and the antibody bound to it. In certain embodiments, the CDH15-binding arm binds to human CDH15 and blocks the activity of CDH15.

[0133] In certain embodiments, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody includes 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) containing a first antigen-binding domain and a second antigen-binding domain, the CDR of the first antigen-binding domain may be designated with the prefix "A1", and the CDR of 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.

[0134] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked to each other to form the bispecific antigen-binding molecule described herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate multimerizing domain. The association of one multimerizing domain with another facilitates the association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. A "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or configuration. For example, the multimerizing domain may be immunoglobulin C H The polypeptide may also contain three domains. Non-limiting examples of polymerizing components include (C H 2~C H The Fc portion of immunoglobulins (including 3 domains), for example, the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0135] The bispecific antigen-binding molecules described herein typically contain two multimerizing domains, for example, two Fc domains, each being a distinct portion of an antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype, for example, IgG1 / IgG1, IgG2 / IgG2, or IgG4 / IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes, for example, IgG1 / IgG2, IgG1 / IgG4, or IgG2 / IgG4.

[0136] In certain embodiments, the multimerizing 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 multimerizing domain is a cysteine ​​residue or a short-chain cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides containing or comprising a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0137] Any bispecific antibody form or technique may be used to produce the bispecific antigen-binding molecules described herein. For example, an antigen-binding molecule or fragment having a first antigen-binding specificity can be functionally linked (e.g., by chemical linkage, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second binding specificity, to produce a bispecific antigen-binding molecule. Specific examples of bispecificity formats include, for example, scFv-based or dibody bispecificity formats, IgG-scFv fusions, dual variable region (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), crossumabs, crossfabs, (SEED) bodies, leucine zippers, duobodies, IgG1 / IgG2, dual-acting fab (DAF)-IgG, and Mab2 bispecificity formats (for a review of the aforementioned formats, see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and the references cited therein).

[0138] In the context of the bispecific antigen-binding molecules described herein, the multimerizing domain, for example, the Fc domain, may include one or more amino acid modifications (e.g., insertions, deletions, or substitutions) compared to the naturally occurring version of the wild-type Fc domain. For example, a bispecific antigen-binding molecule includes a bispecific antigen-binding molecule that includes one or more modifications in the Fc domain, resulting in a modified Fc domain having a modified binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule is C H 2 regions or C H The modification involves three regions, and this modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F and / or 308P).

[0139] Also described herein is the first C H 3 domains and second Ig C HA bispecific antigen-binding molecule comprising three domains, wherein the first and second Ig C H The three domains are different from each other by at least one amino acid, and at least one amino acid difference reduces the binding of the bispecific antibody to protein A as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H The three domains bind to protein A, and the second Ig C H The three domains contain mutations that reduce or abolish protein A binding, such as, for example, the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second C H The three may further include the Y96F modification (according to IMGT; Y436F according to EU). See, for example, U.S. Patent No. 8,586,713. Further modifications that may be found in the second C H The three include the following: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT, D356E, L358M, N384S, K392N, V397M, and V422I in EU), for IgG2 antibodies, N44S, K52N, and V82I (IMGT, N384S, K392N, and V422I in EU), and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU).

[0140] In certain embodiments, the Fc domain may be a chimeric combination of Fc sequences derived from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be from human IgG1, human IgG2, or human IgG4 C H The C derived from the 2 domain H Part or all of the 2 sequence, and the C derived from human IgG1, human IgG2, or human IgG4 HIt may include some or all of the 3 - arrays. The chimeric Fc domain may also include a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived 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 described herein include, from the N - terminus to the C - terminus, [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 described herein includes, from the N - terminus to the C - terminus, [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen - binding molecules described herein are described in U.S. Patent Application Publication No. 2014 / 0243504, issued on August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements may have altered Fc receptor binding and, as a result, affect Fc effector functions.

[0141] In certain embodiments, the antibody heavy chains described herein include a heavy - chain constant (CH) region having an amino - acid sequence that is at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, at least 98%, at least 99%, 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 region (CH) region includes 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.

[0142] In some embodiments, the antibody heavy chain described herein includes 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% homologous 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.

[0143] Germline mutation The anti-hCDH15 antibodies discussed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework region and / or CDR region of the heavy chain variable domain compared to the corresponding germline sequence from which the antibody is derived.

[0144] The anti-hCDH15 antibodies and their antigen-binding fragments disclosed herein may be derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework regions and / or CDR regions are mutated to corresponding residues in the germline sequence from which the antibody is derived, or to corresponding residues in another human germline sequence, or to conserved amino acid substitutions in the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”), and may have weak or no-detectable binding to the CDH15 antigen. Some such exemplary antibodies that recognize CDH15 are listed in Table 1 herein.

[0145] Furthermore, the anti-hCDH15 antibodies and antigen-binding fragments disclosed herein may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, in which certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antibodies or antigen-binding fragments containing one or more germline mutations can be tested for one or more desired properties, such as improved binding specificity, weaker or reduced binding affinity, improved or enhanced pharmacokinetic properties, or reduced immunogenicity. In some embodiments, the antibodies or antigen-binding fragments described herein are obtained in this general manner.

[0146] This specification also describes anti-hCDH15 antibodies and their antigen-binding fragments, which include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, having one or more conservative substitutions. For example, the anti-hCDH15 antibodies or their antigen-binding fragments described herein may include HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions to any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences listed in Table 1 herein. The antibodies and their antigen-binding molecules described herein may include one or more amino acid substitutions, insertions, and / or deletions in the framework of the heavy and light chain variable domains and / or the CDR region compared to the corresponding germline sequence from which the individual antigen-binding domains are derived, while maintaining or improving binding from, for example, a desired weak binding to CDH15 to undetectable binding. A "conservative amino acid substitution" is a substitution of an amino acid residue with another amino acid residue that has a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein; that is, in the case of anti-hCDH15 binding molecules, the amino acid substitution maintains or improves the binding affinity from the desired weak to undetectable. Examples of amino acid groups with side chains having similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferably, the conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.Alternatively, a conservative permutation is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" permutation is any change with a non-negative value in the PAM250 log-likelihood matrix.

[0147] This specification also describes anti-hCDH15 antibodies and their antigen-binding fragments, which include an antigen-binding domain having an HCVR amino acid sequence and / or CDR amino acid sequence that is substantially homologous to either of the HCVR amino acid sequences and / or CDR amino acid sequences disclosed herein, while maintaining or improving a desired weak affinity for the CDH15 antigen. With respect to amino acid sequences, the terms “substantially homologous” or “substantially homologous” mean that two amino acid sequences share at least 95% sequence homology, and more preferably at least 98% or 99% sequence homology, when optimally aligned by programmed GAP or BESTFIT, etc., using default gap weights. Preferably, non-homologous residue positions differ only by conserved amino acid substitutions. If two or more amino acid sequences differ from each other by conserved substitutions, the percentage of sequence homology or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. For example, see Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference.

[0148] Sequence similarity to polypeptides, also referred to as sequence homology, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved 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 homology between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of best overlap regions and sequence homology percentages between query sequences and search sequences (Pearson (2000) above). Another preferred algorithm for comparing the sequences described herein with databases containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. For example, see Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402.

[0149] Once obtained, antigen-binding domains containing one or more germline mutations were tested for decreased binding affinity using one or more in vitro assays. Generally, antibodies that recognize a specific antigen are typically screened for that purpose by testing for high (i.e., strong) binding affinity to the antigen.

[0150] Further modification of the antibodies described herein by the methods described herein may result in unexpected benefits, such as improved pharmacokinetic properties and reduced toxicity to patients.

[0151] Antibody binding properties In the context of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein binding to any given antigen, such as a cell surface protein or a fragment thereof, the term "binding" typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0152] For example, when the binding affinity is determined by surface plasmon resonance (SPR) technology, for example in a BIAcore 3000 instrument, with the antigen as the ligand and the antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or anti-ligand), it is typically about 10. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 K below M D This corresponds to the value. Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used to provide data for analyzing binding characteristics of 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).

[0153] Therefore, the anti-hCDH15 antibody and its antigen-binding fragment described herein bind to a predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to a KD value at least 10 times lower than the affinity for binding to a nonspecific antigen (e.g., BSA, casein). According to the present invention, the KD value is 10 times or less than that of a nonspecific antigen. D The antibody affinity corresponding to the value may be considered undetectable binding, but such an antibody may pair with a second antigen-binding arm for the production of the bispecific antibodies described herein.

[0154] "K" in mole (M) unitsD The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen. D There is an inverse relationship between and binding affinity, therefore, K D The smaller the value, the higher the affinity, i.e., the stronger it is. Therefore, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and thus a smaller K. D Regarding the values, conversely, the terms "lower affinity" or "weaker affinity" indicate a lower ability to form an interaction, and therefore a higher K. D Regarding the value. In some situations, the binding affinity (or K) of a particular molecule (e.g., antibody) to its interaction partner molecule (e.g., antigen X) is considered. D If the binding affinity of that molecule (e.g., antibody) is higher compared to the binding affinity of another interaction partner molecule (e.g., antigen Y), then a larger K D The value (lower or weaker affinity) is smaller K D It can be expressed as a binding ratio determined by dividing by a value (higher or stronger affinity), and in some cases, it can be expressed as a binding affinity that is 5 or 10 times higher.

[0155] "k d The term (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. This value is k off It is also called a value.

[0156] "k a The term (M-1 × sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0157] "K A The term (M-1 or 1 / M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is k ato k d It is obtained by dividing by [a certain factor].

[0158] "EC50" or "EC 50 The term "semi-maximal effective concentration" refers to the concentration of the antibody that elicits an intermediate response between baseline and maximum after a specified exposure time. 50 This basically represents the antibody concentration at which 50% of its maximum effect is observed. In a particular embodiment, EC 50 The value is equal to the concentration of the antibody described herein that gives half of the maximum binding to cells expressing CDH15, as determined, for example, by a FACS binding assay or an androgen receptor-activated luciferase assay. Therefore, EC 50 Alternatively, a decrease or weakening of binding may be observed as the half-maximal effective concentration increases.

[0159] In one embodiment, reduced binding affinity may result in binding to the 50% maximum amount of target cells. 50 This can be defined as an increase in antibody concentration.

[0160] Also described herein are antigen-binding proteins, such as antibodies or their antigen-binding fragments, that bind to CDH15-expressing cells at single-digit nM or three-digit pM KD, as measured by surface plasmon resonance or an equivalent assay. Also described herein are antigen-binding proteins, such as antibodies or their antigen-binding fragments, that bind to CDH15-expressing cells at an EC50 greater than 100 nM, as measured by FACS analysis. Antigen-binding proteins, such as antibodies or their antigen-binding fragments, that bind to and internally migrate into CDH15-expressing cells are also described herein. Antigen-binding proteins, such as antibodies or their antigen-binding fragments, that bind to CDH15 and block its activity are also described herein.

[0161] Sequence variants The anti-hCACNG1 antibodies and antigen-binding fragments described herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains compared to the corresponding germline sequence from which each antigen-binding domain originates. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The antigen-binding molecules described herein may contain an antigen-binding domain derived from any of the exemplary amino acid sequences disclosed herein, where one or more amino acids in the framework and / or CDR region are mutated to the corresponding residues of the germline sequence from which the antibody originates, or to the corresponding residues of another human germline sequence, or to a conserved amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H Domain and / or V LAll framework residues and / or CDR residues within the domain are mutated again to residues found in the original germline sequence from which the antigen-binding domain originally originated. In other embodiments, only certain residues, for example, only mutant residues found in the first 8 amino acids of FR1, or only mutant residues found in the last 8 amino acids of FR4, or only mutant residues found in CDR1, CDR2, or CDR3, are mutated again to the original germline sequence. In other embodiments, one or more framework residues and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline variant sequence different from the germline sequence from which the antigen-binding domain originally originated). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, certain individual residues may mutate to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutate to corresponding residues in a different germline sequence. Once obtained, antigen-binding domains 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 (in some cases) biological properties of the antagonist or agonist, or decreased immunogenicity. This specification describes bispecific antigen-binding molecules containing one or more antigen-binding domains obtained by this general method.

[0162] This specification also describes antigen-binding molecules in which one or both antigen-binding domains have one or more conserved substitutions in any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. For example, the antigen-binding molecules described herein may include antigen-binding domains having HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conserved amino acid substitutions in any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences disclosed herein. A “conservative amino acid substitution” is a substitution 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, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of amino acid groups 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: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferably, the conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0163] This disclosure also includes antigen-binding molecules having antigen-binding domains having an HCVR amino acid sequence, an LCVR amino acid sequence, and / or a CDR amino acid sequence that is substantially homologous to any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences disclosed herein. With respect to amino acid sequences, the term “substantially homologous” means that two amino acid sequences share at least 95% sequence homology, and more preferably at least 98% or 99% sequence homology, when optimally aligned by programmed GAP or BESTFIT, etc., using default gap weights. Preferably, non-homologous residue positions differ only by conserved amino acid substitutions. If two or more amino acid sequences differ from each other by conserved substitutions, the percentage of sequence homology or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference.

[0164] Sequence similarity to polypeptides, also called sequence homology, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved 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 homology between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of best overlap regions and sequence homology percentages between the query sequence and the search sequence (Pearson (2000) above). Another preferred algorithm when comparing sequences to databases containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. For example, see Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402.

[0165] pH dependent binding Anti-hCDH15 antibodies and their antigen-binding fragments having pH-dependent binding properties are also described herein. For example, the anti-hCDH15 described herein may exhibit reduced binding to CDH15 at acidic pH compared to neutral pH. Alternatively, the anti-hCDH15 described herein may exhibit enhanced binding to CDH15 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than approximately 6.2, such as approximately 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 approximately 7.0 to approximately 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0166] In a particular case, "reduced binding at acidic pH compared to neutral pH" is due to the K of the antibody that binds to that antigen at acidic pH. D The value and neutral pH of the antibody that binds to the antigen D It is expressed as a ratio to (or vice versa) the value. For example, an antibody or its antigen-binding fragment is expressed as an acid / neutral potassium ratio of approximately 3.0 or higher. D Where a ratio is given, for the purposes of the explanation herein, it may be considered to indicate "reduced binding to CACNG1 at acidic pH compared to neutral pH." In certain embodiments, the acidic / neutral K for the antibody or antigen-binding fragment described herein D The ratio can be approximately 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, and 100.0 or higher.

[0167] Antibodies exhibiting pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for decreased (or enhanced) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modifications to the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues can yield antibodies with decreased antigen binding at acidic pH compared to neutral pH.

[0168] Antibodies containing Fc variants In some embodiments, an anti-hCDH15 antibody and its antigen-binding fragment (including a multispecific antigen-binding molecule and a multi-domain therapeutic protein containing the anti-hCDH15 antibody or its antigen-binding fragment) are provided, which include an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the antibody described herein, the C of the Fc domain H 2 or C HThe antibody contains mutations in three regions, which increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may result in an extension of the serum half-life of the antibody when administered to animals. Non-restrictive examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, the modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F and / or 308P).

[0169] For example, the anti-hCDH15 antibodies and antigen-binding fragments described herein may contain 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 assumed to be within the scope of the description herein.

[0170] Biological characteristics of antibodies and bispecific antigen-binding molecules Furthermore, this specification also describes antibodies and their antigen-binding fragments that bind to human CDH15 with high, medium, or low affinity, depending on the treatment situation and desired specific targeting characteristics. For example, in the case of a bispecific antigen-binding molecule in which one arm binds to CDH15 and the other arm binds to a target antigen (e.g., a tumor-associated antigen), it may be desirable for the target antigen-binding arm to bind to the target antigen with high affinity, while the anti-hCDH15 arm binds to CDH15 only with medium or low affinity. In this way, preferential targeting of antigen-binding molecules to cells expressing the target antigen can be achieved while avoiding general / untargeted CDH15 binding and the associated adverse side effects.

[0171] Furthermore, antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to human CDH15 with weak (i.e., low) affinity or even undetectable affinity are described herein. In some embodiments, the antibodies and antigen-binding fragments described herein have a K+ of about 100 nM as measured by surface plasmon resonance. D Then, it binds to human CDH15 (for example, at 37°C). In some embodiments, the antibody or antigen-binding fragment described herein is measured by surface plasmon resonance (e.g., mAb capture or antigen capture form) or a substantially similar assay, with a K content 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. D It binds to CHD15 either by or without detectable affinity.

[0172] Epitope mapping and related technologies The epitopes on CDH15 to which the anti-hCDH15 antibodies and their antigen-binding fragments described herein bind may consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) amino acids of the CDH15 protein. Alternatively, the epitope may consist of multiple discontinuous amino acids (or amino acid sequences) of CDH15. The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have two or more epitopes. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be either conformal or linear. Conformal epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, an epitope may contain a sugar, phosphoryl group, or sulfonyl group portion on an antigen.

[0173] 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. Illustrative techniques include, for example, the standard cross-blocking assay described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Furthermore, methods such as antigen epitope excision, epitope extraction, and chemical modification can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that may be used to identify the amino acids within a polypeptide with which the antigen-binding domain of an antigen-binding molecule interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange involves deuterizing the protein of interest and then conjugating the antibody to this deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and hydrogen-deuterium exchange is induced at all residues except those protected by the antibody (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259 and 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.

[0174] This specification also describes anti-hCDH15 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (for example, antibodies containing any of the amino acid sequences listed in Table 1 of this specification). Similarly, this specification also describes anti-hCDH15 antibodies that compete for binding to CDH15 with any of the specific exemplary antibodies described herein (for example, antibodies containing any of the amino acid sequences listed in Table 1 of this specification).

[0175] Whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as the reference antigen-binding molecule described herein, or competes for binding, can be readily determined using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope on CDH15 as the reference bispecific antigen-binding molecule described herein, the reference bispecific molecule is first bound to the CDH15 protein. The ability of the test antibody to bind to the CDH15 molecule is then evaluated. If the test antibody can bind to CDH15 after saturated binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope on CDH15 than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CDH15 molecule after saturated binding with the reference bispecific antigen-binding molecule, the test antibody may bind to the same epitope on CDH15 as the epitope bound by the reference bispecific antigen-binding molecule described herein. Additional conventional experiments (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether stereoblocking (or another phenomenon) is the cause of the observed lack of binding. This type of experiment 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, if a competitive binding assay measures, for example, that a 1, 5, 10, 20, or 100-fold excess of one antigen-binding protein inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even up to 99%, then the two antigen-binding proteins bind to homologous (or overlapping) epitopes (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other, the two antigen-binding proteins are considered to bind to a homologous epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other, the two antigen-binding proteins are considered to have a “duplicate epitope.”

[0176] 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, the reference antigen-binding molecule is bound to the CDH15 protein under saturated conditions, and then the binding of the test antibody to the CDH15 molecule is evaluated. In the second direction, the test antibody is bound to the CDH15 molecule under saturated conditions, and then the binding of the reference antigen-binding molecule to the CDH15 molecule is evaluated. In both directions, if only the first (saturated) antigen-binding molecule can 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 understood by those skilled in the art, an antibody that competes for binding to a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antigen-binding molecule, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0177] Preparation of antigen-binding domains and preparation of bispecificity constructs Antigen-binding domains specific to a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, two different antigen-binding domains specific to two different antigens (e.g., CDH15 and a target antigen) can be appropriately arranged opposite each other to produce a bispecific antigen-binding molecule as described herein using conventional methods. (A consideration of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules described herein is provided elsewhere in this specification.) In certain embodiments, one or more of the individual members (e.g., heavy and light chains) of the antigen-binding molecules described herein are derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the antigen-binding molecules described herein can be prepared using VELOCIMMUNE® technology. Using VELOCIMMUNE® technology (or any other human antibody production technique), a high-affinity chimeric antigen-binding molecule for a specific antigen (e.g., CDH15) having a human variable region and a mouse constant region is first isolated. Antibodies are characterized and selected for desirable features, including affinity, selectivity, and epitopes. The mouse constant region is substituted with a desired human constant region to produce a complete human heavy and / or light chain that can be incorporated into the antigen-binding molecules described herein.

[0178] Human bispecific antigen-binding molecules may be produced using genetically modified animals. For example, a genetically modified mouse in which the endogenous mouse immunoglobulin light chain variable sequence cannot be rearranged and expressed may be used, and this mouse expresses only one or two human light chain variable domains encoded by a human immunoglobulin sequence operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Using such a genetically modified mouse, the heavy chain variable region and the light chain variable region can be isolated to produce a fully human bispecific antigen-binding molecule. Thus, a fully human bispecific antigen-binding molecule contains two different heavy chains that associate with the same light chain. (See, for example, U.S. Patent Application Publication 2011 / 0195454). Fully human means an antibody or its antigen-binding fragment or its immunoglobulin domain, which contains an amino acid sequence encoded by DNA derived from a human sequence throughout the entire length of each polypeptide of the antibody or its antigen-binding fragment or its immunoglobulin domain. In some cases, the fully human sequence is derived from a protein endogenous to humans. In other cases, a complete human protein or protein sequence contains a chimeric sequence in which each member sequence is derived from a human sequence. While not bound by any theory, chimeric proteins or sequences are generally designed to minimize the generation of immunogenic epitopes at member sequence junctions compared to, for example, any wild-type human immunoglobulin region or domain.

[0179] The bispecific antigen-binding molecule may be constructed with a single heavy chain having a modified Fc domain that disrupts its binding to protein A, thus enabling a purification method for producing heterodimeric proteins. See, for example, U.S. Patent No. 8,586,713. Thus, the bispecific antigen-binding molecule is first C H 3 domains and second Ig C H It contains three domains, including the first and second IgC HThe three domains differ from each other by at least one amino acid, and this difference by at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking amino acid differences. In one embodiment, the first Ig C H The 3 domains bind to protein A, and the second Ig C H The 3 domains include mutations / modifications that reduce or eliminate protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). Second C H 3 may further include the Y96F modification (according to IMGT; Y436F according to the EU).

[0180] biological equivalent This specification provides antigen-binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein but retain the ability to bind to CDH15. Such variant molecules may involve the addition, deletion, or substitution of one or more amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the bispecific antigen-binding molecules described herein.

[0181] Antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules described herein are also described. Two antigen-binding proteins or antibodies are considered bioequivalents or pharmaceutically equivalents if, for example, when administered in the same molar dose in a single or multiple dose under similar experimental conditions, their absorption rates and extents do not show significant differences. Some antigen-binding proteins are considered equivalents or pharmaceutically equivalents if their absorption rates are equivalent but not equivalent, and furthermore, such differences in absorption rates are intentional and reflected in the labeling, and are considered not medically important to the particular drug studied, for example, because they are not essential for achieving effective drug concentrations in the body in chronic use.

[0182] In one embodiment, the two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

[0183] In one embodiment, the patient may be switched between a reference product and a biological product one or more times, and the two antigen-binding proteins are bioequivalent if there is no expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or a decrease in efficacy, compared to continuous therapy without such switching.

[0184] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by one or more common mechanisms for one or more usage conditions, to a known degree of such mechanisms.

[0185] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence assays include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.

[0186] Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein may be constructed, for example, by various substitutions of residues or sequences, or by deletions of terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or replaced with other amino acids to prevent the formation of unnecessary or inaccurate intramolecular disulfide crosslinks during regeneration. In other contexts, bioequivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules described herein that involve amino acid changes that modify the glycosylation properties of the molecule, such as mutations that eliminate or remove glycosylation.

[0187] Antigen-binding proteins, such as antibodies or antigen-binding fragments, including modified glycosylation patterns, including anti-hCDH15 antibodies, are also described herein. In some embodiments, modifications to remove undesirable glycosylation sites may be useful. Alternatively, for example, antibodies lacking fucose residues present on the oligosaccharide chain can be used to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shields et al. (2002) JBC 277:26733). In this case, cytotoxicity is desirable. In other applications, galactosylation modifications can be performed to modify complement-dependent cell-mediated cytotoxicity (CDC) activity.

[0188] Species selectivity and species cross-reactivity In some embodiments, the antigen-binding molecules described herein bind to human CDH15 but not to CDH15 from other species. Antigen-binding molecules that bind to both human CDH15 and CDH15 from one or more non-human species are also described herein.

[0189] In some embodiments, the antigen-binding molecules described herein that bind to human CDH15 may or may not bind to one or more of the following: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, crab-eating macaque, marmoset, rhesus macaque, or chimpanzee CDH15.

[0190] Antibody-drug conjugates (ADCs) Also described herein are antibody-drug conjugates (ADCs) comprising an anti-hCDH15 antibody or its antigen-binding fragment conjugated to a payload, e.g., a drug or molecular unload (e.g., a small molecule and / or therapeutic portion). Anti-hCDH15 antibodies conjugated to a therapeutic portion, their antigen-binding fragments, and / or their multiselective antigen-binding molecules are also provided. Generally speaking, ADCs are A-[LP] y And the formula In this equation, A is an antigen-binding molecule, such as an anti-hCDH15 antibody or a fragment thereof (for example, a fragment containing at least HCDR3 selected from any of the HCDR3 amino acid sequences listed in Table 1), L is a linker, P is the payload or molecular load, and y is an integer from 1 to 30.

[0191] In various embodiments, the ADC is one of the sequence numbers listed in Table 1 (for example, sequence numbers 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, 1 Anti-hCDH15 antibodies or their antigen-binding fragments containing CDRs of HCVR or LCVR having the amino acid sequences 53, 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 HCVR / LCVR pairs (e.g., SEQ ID NO: 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, 4 (including 20+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 may be used in the sequence numbers listed in Table 1 (for example, sequence numbers 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、The CDR contains amino acid sequences of 750-752-754-758-760-762 and 766-768-770-774-776-778). In some cases, the anti-hCDH15 antibody or fragment may be used as the sequence number listed in Table 1 (e.g., sequence number: 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 The amino acid sequence of 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 a specific amino acid sequence 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, 404+412, 4 (including 20+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).

[0192] In some embodiments, the payload or molecular load may include small molecules as therapeutic agents, for example, therapeutic agents that may be useful in treating muscle wasting or hereditary muscle diseases and / or muscle-related cancers. Small molecules (SMs) can easily enter cells because they have a low molecular weight (typically up to about 1 kDa). Once inside cells, small molecules can affect other molecules, such as proteins, and may, for example, kill cancer cells. This is different from many high molecular weight molecules, such as antibodies. An example of a small molecule may be conjugated to an anti-CDH15 antigen-binding protein to form an anti-CDH15:SM conjugate.

[0193] Potential therapeutic agents that may be useful in treating muscle wasting or hereditary muscle diseases include testes and their biologically active variants (e.g., dihydrostrosterone (DHT)), β2-adrenergic receptor agonists (e.g., clenbuterol), rapamycin or its analogues, MAPK inhibitors, or histone deacetylase inhibitors.

[0194] In some embodiments, the therapeutic payload is testosterone, or a bioactive derivative and / or part thereof, such as dihydrosterone. In some embodiments, the therapeutic payload is rapamycin or an analogue, a MAPK inhibitor, a histone deacetylase inhibitor, or a Notch ligand. In some embodiments, the payload is a chemotherapeutic agent, such as a cytotoxic agent.

[0195] Potential therapeutic agents that may be useful in treating muscle-related cancers include any chemotherapeutic agent known to delay, stop, and / or destroy cancer cells. Examples of therapeutic agents that may be useful in treating muscle-related cancers include aflibercept, amsacrin, azacitidine, azathioprine, belantamab mafodotin, bendamustine, bleomycin, bortezomib, brentuximab vedotin, busulfan, cabazitaxel, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, cisplatin, cladribine, chlorofarabine, cyclophosphamide, cytarabine, cytarabine liposome, dacarbazine, dactinomycin (dactinomycin D), daunorubicin, and docetaxel. Doxorubicin, Doxorubicin liposome, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab 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 Examples include, but are not limited to, thioguanine, topotecan, trabectedin, trastuzumab deruxtecan, trastuzumab emtansine, trifluridine / tipiracil, valganciclovir, vinblastone, vincristine, vindesine, vinflunin, vinorelbine, or bismodegib.

[0196] Furthermore, this specification provides 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 the embodiments of this disclosure include, but are not limited to, the following: 225 Ac, 212 Bi, 213 Bi,131 I, 186 Re, 227 Th, 222 Rn, 223 Ra, 224 Ra, and 90 Y is one example.

[0197] In certain embodiments described herein, for example, an ADC is provided comprising an anti-hCDH15 antigen-binding protein conjugated to a therapeutic agent (e.g., any of the therapeutic agents disclosed above) via a linker molecule. A linker is any group or part that links, connects, or binds the antibody or antigen-binding protein described herein to 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*; Ducrry, L., Ed.; Humana Press, 2013; and *Antibody-Drug Conjugates*; Wang, J., Shen, W.-C., and Zaro, JL, Eds.; Springer International Publishing, 2015. The contents of these documents are incorporated herein by reference in their entirety. In general, a suitable linker for the antibody conjugate described herein is one that is stable enough to take advantage of the antibody's circulating half-life while simultaneously being able to release the payload after the antigen-mediated internalization of the conjugate. The linker may be cleavable or incleavable. Cleavable linkers include those that are cleaved by intracellular metabolism after internalization, such as hydrolysis, reduction, or enzymatic cleavage. Incleavable linkers include those that release the attached payload via lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-unstable linkers, hydrolysis-unstable linkers, enzymatically cleavable linkers, reductively unstable linkers, self-immolative linkers, and incleavable linkers.Appropriate linkers include, but are not limited to, peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units or linkers containing them.

[0198] The ADCs of this disclosure can be fabricated or constructed using any linker molecule or linker technology known in the art. In certain embodiments, the linker is a cleavable linker. According to other embodiments, the linker is an incleavable linker. Examples of linkers that may be used in the context of this disclosure include, for example, MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), val-gly (valine-glycine), dipeptide moieties in protease-cleavable linkers, ala-phe (alanine-phenylalanine), dipeptide moieties in protease-cleavable linkers, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate), SIAB (N-succinimidyl (4-iodoacetyl)aminobenzoate), and linkers comprising or consisting of these, as well as their variants and combinations. Additional exemplary linkers that may be used in the context of this disclosure are provided, for example, in U.S. Patent No. 7,754,681, whose contents are incorporated herein in their entirety by reference, and in Ducry, Bioconjugate Chem., 2010, 21:5-13, and in the references cited herein.

[0199] In certain embodiments, the linker is stable under physiological conditions. In certain embodiments, the linker is cleavable and can release at least the payload portion, for example, in the presence of an enzyme or at a specific pH range or pH value. In some embodiments, the linker includes an enzymatically cleavable portion. Exemplary enzymatically cleavable portions include, but are not limited to, peptide bonds, ester bonds, hydrazones, and disulfide bonds. In some embodiments, the linker includes a cathepsin-cleavable linker.

[0200] In some embodiments, the linker includes an inseparable portion.

[0201] Suitable linkers also include, but are not limited to, linkers that are chemically bonded to two cysteine ​​residues of a single binder, such as an antibody. Such linkers can mimic the disulfide bonds of the antibody that are disrupted as a result of the conjugation process.

[0202] In some embodiments, the linker comprises one or more amino acids. Preferred amino acids include natural, unnatural, standard, non-standard, protein-constituting, non-protein-constituting, 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, their derivatives, or combinations thereof. In certain embodiments, one or more amino acid side chains are linked to the group of side chains 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 embodiments, the linker comprises valine and alanine.

[0203] In some embodiments, the linker comprises a self-immolative group. The self-immolative group can 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 is capable of undergoing a chemical reaction that releases the linker's remaining atoms from the payload.

[0204] In some embodiments, the linker is as follows:

[0205]

Chemical formula

[0206] Wherein,

[0207]

Chemical formula

[0208]

Chemical formula

[0212] [Chemistry] is a binding to a therapeutic payload (e.g., testosterone or a biologically equivalent variant thereof). In certain embodiments, the linker is:

[0213] [Chemistry]

[0214] In certain embodiments, the linker is:

[0215] [Chemistry]

[0216] In some embodiments, the linker is derived from maleimidylmethyl-4-trans-cyclohexanecarboxylate:

[0217] [Chemistry]

[0218] In some embodiments, the linker is:

[0219] [Chemistry]

[0220] wherein

[0221] [Chemistry] is a binding to an antibody or antigen-binding protein (e.g., via a lysine residue),

[0222] [Chemistry] This involves binding with a therapeutic payload (e.g., steroidone or its bioequivalent variant).

[0223] In some embodiments, the linker is as follows:

[0224] [ka]

[0225] During the ceremony,

[0226] [ka] This is binding to an antibody or antigen-binding protein (for example, via a lysine residue),

[0227] [ka] This involves binding with a therapeutic payload (e.g., testosterone or its biologically equivalent variant).

[0228] This disclosure includes an ADC in which a linker links the anti-hCDH15 antigen-binding protein described herein to a therapeutic agent via an addition site at a specific amino acid within an antibody or antigen-binding molecule. An exemplary amino acid addition that may be used in the context of this embodiment is, for example, lysine (e.g., U.S. Patent No. 5,208,020, U.S. Patent Application Publication No. 2010 / 0129314, Holander et al., Bioconjugate). See Chem., 2008, 19:358-361, International Publication No. 2005 / 089808, U.S. Patent No. 5,714,586, and U.S. Patent Application Publication No. 2013 / 0101546), cysteine ​​(see, for example, U.S. Patent Application Publication No. 2007 / 0258987, International Publication No. 2013 / 055993, International Publication No. 2013 / 055990, International Publication No. 2013 / 053873, International Publication No. 2013 / 053872, International Publication No. 2011 / 130598, U.S. Patent Application Publication No. 2013 / 0101546, and U.S. Patent No. 7,750,116), selenocysteine ​​(see, for example, International Publication No. 2008 / 122039; and Hofer et al.) See, for example, Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456; and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067; unnatural amino acids (see, for example, publication numbers 2013 / 068874 and 2012 / 166559); and acidic amino acids (see, for example, publication number 2012 / 05982).The linker may also be conjugated to an antigen-binding protein via attachment to a carbohydrate (see, e.g., U.S. Patent Application Publication No. 2008 / 0305497, International Publication No. 2014 / 065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and a disulfide linker (see, e.g., International Publication No. 2013 / 085925, International Publication No. 2010 / 010324, International Publication No. 2011 / 018611, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313). Alternatively, site-specific conjugation techniques may be employed to directly conjugate to specific residues of the antibody or antigen-binding protein (see, for example, Schumacher et al. J Clin Immunol (2016) 36(Suppl 1):100). Site-specific conjugation techniques include, but are not limited to, transglutaminase-mediated glutamine conjugation (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995). In some embodiments, residues of the antibody described herein, for example, residues in the constant region of the antibody's heavy chain, may be substituted with glutamine to further promote transglutaminase-mediated glutamine conjugation. As an unspecified example, the human heavy chain constant region may be modified with the N180Q substitution found in the sequence of the human IgG1 heavy chain constant region. Such substitutions provide a total of four glutamines for transglutaminase-mediated conjugation.

[0229] The antibody-drug conjugates described herein can be prepared using conjugation conditions known to those skilled in the art (see, for example, Doronina et al. Nature Biotechnology 2003, 21, 7, 778, which is incorporated herein in its entirety by reference). In some embodiments, an anti-hCDH15 antigen-binding protein-drug conjugate is prepared by contacting the anti-hCDH15 antigen-binding protein described herein with a compound comprising a desired linker and a therapeutic agent, wherein the linker has a portion that is reactive with the antibody or antigen-binding protein, for example, a portion that is reactive at a desired residue of the antibody or antigen-binding protein.

[0230] 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 features two terminal inversion sequences (ITRs) and two open reading frames (ORFs), rep and cap. The reading frame of wild-type rep 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 play a role in regulating 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 83–85 kD (VP1), 72–73 kD (VP2), and 61–62 kD (VP3). Over 80% of the total protein in AAV virions (capsids) is composed of VP3, and in mature virions, VP1, VP2, and VP3 are present in a relative ratio of approximately 1:1:10, although a ratio of 1:1:8 has also been reported. Padron et al. (2005) J. Virology 79:5047-58.

[0231] The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal inversion (ITR), Rep protein, and capsid subunit, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. For example, see GenBank acceptance numbers 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 with respect to teaching the nucleic acid and amino acid sequences of AAV. For example, Srivistava et al. (1983) J. Virology 45:555, Chiorini et al. (1998) J. Virology 71:6823, Chiorini et al. (1999) J. Virology 73: 1309, Bantel-Schaal et al. (1999) J. Virology 73:939, Xiao et al. 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, Morris et al. al.(2004) Virology See also 33:375-383, U.S. Patent Publication No. 2017 / 0130245, International Patent Publication Nos. 00 / 28061, 99 / 61601, 98 / 11244, and U.S. Patent No. 6,156,303, each of which is incorporated in whole by reference. Table 2 of this specification provides a sequence of various non-primate AAVs.

[0232] "AAV" encompasses all subtypes known in the art, as well as both naturally occurring and modified forms. AAVs include primate AAVs (for example, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, AAV rh10, AAV h10, AAV hu11, AAV rh32, 33 (AAV This includes rh32.33), AAV retro (AAV retro), AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAV2 / 8, etc.; non-primate AAVs (e.g., bird AAV (AAAV)), and other non-primate AAVs, such as mammal AAVs (e.g., bat AAV, sea lion AAV, cattle AAV, dog AAV, horse AAV, goat AAV, and sheep AAV, etc.), and squamate AAVs (e.g., snake AAV, bearded dragon AAV, etc.). "Primate AAV" generally refers to AAVs isolated from primates. Similarly, "non-primate AAV" refers to AAVs isolated from non-primate animals.

[0233] As used herein, 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 the capsid protein of a designated AAV (e.g., PLA2 region, VP1-u region, VP1 / VP2 common region, VP3 region), a nucleotide sequence (e.g., ITR sequence), and a cap gene or capsid protein such as AAV, "[of the designated] AAV" includes, in addition to the gene or polypeptide containing the nucleic acid sequence or amino acid sequence described herein with respect to each designated AAV, variants containing the minimum number of nucleotides or amino acids required to retain one or more biological functions. As used herein, a mutant gene or mutant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence described herein with respect to a specified AAV gene or polypeptide, the difference generally does not alter at least one biological function of the gene or polypeptide and / or alter the phylogenetic characteristics of the gene or polypeptide, for example, the difference may be due to degeneracy of the gene code, variation in isolation, sequence length, etc. For example, as used herein, rep genes and cap genes may include rep genes and cap genes that differ from the wild-type gene in that the gene may 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 selective start codons, or sequences between one or more selective start codons, are removed, thereby encoding only a single Cap protein. For example, the start codons for VP2 and / or VP3 are removed or substituted, so that the cap gene encodes a functional VP1 capsid protein but not the VP2 or VP3 capsid proteins.Therefore, as used herein, the rep gene encompasses any sequence encoding a functional Rep protein. The cap gene encompasses any sequence encoding at least one functional cap gene.

[0234] The wild-type cap gene is known to express all three VP1, VP2, and VP3 capsid proteins from a single open reading frame of the cap gene under the control of the p40 promoter present in the rep ORF. Terms such as “capsid protein” and “Cap protein” refer to proteins that are part of the viral capsid. For adeno-associated viruses, the capsid proteins are commonly referred to as VP1, VP2, and / or VP3, and they can be encoded by a single cap gene. In the case of AAV, the three AAV capsid proteins are naturally produced in a redundant manner utilizing the selective translation start codons of the cap ORF, although all three proteins use a common stop codon. The ORF of the wild-type cap gene encodes three selective start codons from 5' to 3', namely the “VP1 start codon,” the “VP2 start codon,” and the “VP3 start codon,” as well as one “common stop codon.” VP1, the largest viral protein, is commonly encoded from the VP1 start codon to the “common stop codon.” VP2 is generally encoded from the VP2 start codon to the common stop codon. VP3 is generally encoded from the VP3 start codon to the common stop codon. Therefore, VP1 contains an N-terminal sequence that is not shared with VP2 or VP3, and this is referred to as the VP1-specific region (VP1-u). The VP1-u region is generally encoded by the wild-type cap gene sequence starting from the VP1 start codon to the "VP2 start codon". VP1-u contains the phospholipase A2 domain (PLA2), which may be important for infection and nuclear localization signaling, and the signaling may assist the virus in targeting the nucleus for uncoating and genome release. The VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that constitutes the entire VP3, which may also be referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the common stop codon. VP2 has approximately 60 additional amino acids that it shares with VP1. This region is called the VP1 / VP2 common region.

[0235] In some embodiments, one or more of the Cap proteins of the present invention may be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the present invention may be expressed from two or more ORFs containing nucleotide sequences encoding any combination of VP1, VP2, and / or VP3, by using separate nucleotide sequences operably linked to at least one expression regulatory sequence for expression in packaging cells, each producing one or more of the VP1, VP2, and / or VP3 capsid proteins of the present invention. In some embodiments, the VP capsid proteins of the present invention may be individually expressed from ORFs containing nucleotide sequences encoding any one of VP1, VP2, or VP3, by using separate nucleotide sequences operably linked to one expression regulatory sequence for expression in viral replication cells, each producing only one of the VP1, VP2, or VP3 capsid proteins. In another embodiment, the VP protein may be expressed from a single ORF containing nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins, which are operably linked to at least one expression control sequence for expression in viral replication cells, each producing the VP1, VP2, and VP3 capsid proteins. Thus, the amino acid positions provided herein may be provided in relation to the VP1 capsid protein of a reference AAV, and those skilled in the art will be able to readily determine the positions of the same amino acids in the VP2 and / or VP3 capsid proteins of an AAV, and the corresponding amino acid positions between different AAVs, respectively.

[0236] The term "terminal inversion sequence" or "ITR" refers to a symmetrical nucleic acid sequence in the adeno-associated virus genome required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs function as the origin of replication for viral DNA synthesis and are essential cis-members for AAV particle production, including packaging into AAV particles.

[0237] The AAV ITR contains the recognition site for the replication protein Rep78 or Rep68. The "D" region of the ITR contains a DNA nicking site where DNA replication is initiated, providing direction to the nucleic acid replication process. AAV replication in mammalian cells typically involves two ITR sequences.

[0238] A single ITR may be manipulated using Rep-binding sites on both strands of the "A" region and the two symmetrical D regions of the ITR palindrome. On a double-stranded circular DNA template, a construct subjected to such manipulation can allow nucleic acid replication initiated by Rep78 or Rep68 to proceed in both directions. A single ITR is sufficient for AAV replication of circular particles. In the method for producing AAV recombinant AAV particles of the present invention, the rep coding sequence codes for a Rep protein or a Rep protein equivalent, and these proteins can bind to an ITR contained on a transfer plasmid.

[0239] The Cap protein of the present invention, when expressed by packaging cells together with an appropriate Rep protein, can capsidize a transfer plasmid containing the target nucleotide and an even number of two or more ITR sequences. In some embodiments, the transfer plasmid contains one ITR sequence. In some embodiments, the transfer plasmid contains two ITR sequences.

[0240] Either Rep78 and / or Rep68 bind to a unique and known site on the ITR hairpin sequence, functioning to disrupt and unwind the hairpin structure at the end of the AAV genome, thereby providing access to the replication mechanism of viral replicating cells. As is known, Rep proteins may be expressed from two or more ORFs containing nucleotide sequences encoding any combination of Rep78, Rep68, Rep52 and / or Rep40 by using separate nucleotide sequences operably linked to at least one expression regulatory sequence for expression in viral replicating cells, each producing one or more of the Rep78, Rep68, Rep52 and / or Rep40 Rep proteins. Alternatively, the Rep proteins may be individually expressed from an ORF containing a nucleotide sequence encoding any one of Rep78, Rep68, Rep52, or Rep40 by using a separate nucleotide sequence operably linked to a single expression regulatory sequence for expression in packaging cells, each producing only one Rep78, Rep68, Rep52, or Rep40 Rep protein. In another embodiment, the Rep proteins may be expressed from a single ORF containing nucleotide sequences encoding Rep78 and Rep52 Rep proteins operably linked to at least one expression regulatory sequence for expression in viral replication cells, each producing Rep78 and Rep52 Rep proteins.

[0241] For example, in a method for producing AAV virions such as recombinant AAV particles according to 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 such a condition is not essential. Such recombinant AAV particles may or may not contain a genome.

[0242] A "chimeric AAV capsid protein" includes an AAV capsid protein that contains, for example, a portion of an amino acid sequence from two or more different AAVs, and has and / or forms the ability to form an AAV viral capsid / recombinant AAV particle. A chimeric AAV capsid protein may also be encoded by a chimeric AAV capsid gene, such as a plurality of chimeric nucleotides, each of which is homologous to a portion of a capsid gene encoding a capsid protein of a distinct AAV, and together they encode a functional chimeric AAV capsid protein. The association of a chimeric capsid protein with a particular AAV indicates that the capsid protein contains one or more portions derived from the capsid protein of an AAV and one or more portions derived from the capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein comprises a capsid protein that includes one or more portions of the VP1, VP2, and / or VP3 capsid proteins of AAV2, as well as one or more portions of the VP1, VP2, and / or VP3 capsid proteins of different AAVs.

[0243] The term “part” refers to at least five amino acids or at least fifteen nucleotides, but smaller than a full-length polypeptide or nucleic acid molecule, and having 100% homology to the sequence from which the part is derived (Penzes (2015) J. General Virol. 2769). “Part” encompasses any consecutive segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule from which the part is derived is “of the [designated] AAV,” such as a non-primate AAV or a distantly related AAV, or has “significant homology” to a particular AAV. In some embodiments, a part comprises at least five amino acids or fifteen nucleotides having 100% homology to a sequence associated with the designated AAV. In some embodiments, a part comprises at least ten amino acids or thirty nucleotides having 100% homology to a sequence associated with the designated AAV. In some embodiments, a part comprises at least fifteen amino acids or forty-five nucleotides having 100% homology to a sequence associated with the designated AAV. In some embodiments, a portion contains at least 20 amino acids or 60 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 25 amino acids or 75 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 30 amino acids or 90 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 35 amino acids or 105 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 40 amino acids or 120 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 45 amino acids or 135 nucleotides having 100% homology to the sequence associated with the specified AAV.In some embodiments, a portion contains at least 50 amino acids or 150 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 60 amino acids or 180 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 70 amino acids or 210 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 80 amino acids or 240 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 90 amino acids or 270 nucleotides having 100% homology to the sequence associated with the specified AAV. In some embodiments, a portion contains at least 100 amino acids or 300 nucleotides having 100% homology to the sequence associated with the specified AAV.

[0244] Modified viral capsid proteins, recombinant AAV particles, nucleic acids In some embodiments, the Cap protein, for example, the VP1 capsid protein described herein, the VP2 capsid protein described herein, and / or the VP3 capsid protein described herein, is modified to include, for example, insertion of a targeted ligand, chemical modification, first member of a binding pair, detectable labeling, point mutation, etc., one or a combination thereof.

[0245] In general, modifications to the gene or polypeptide of a designated AAV or its variants result in a nucleic acid sequence or amino acid sequence different from the nucleic acid sequence or amino acid sequence described herein with respect to the designated AAV, in which case the modification alters, confers, or removes one or more biological functions, but does not alter the phylogenetic characteristics of the gene or polypeptide as an AAV gene or AAV polypeptide. The modification may include one or a combination of the following: substituting the sequence of a first AAV serotype with the sequence of a second AAV serotype to create chimerism, chemical modification, insertion of the first member of a binding pair, and / or point mutation, thereby reducing or inactivating the innate directivity of the capsid protein, making the directivity of the capsid protein more easily redirected, and / or thereby including a detectable label for the capsid protein. The modifications described herein generally include modifications that do not alter the recognition of capsids modified with existing antibodies present in the general population, produced during infection by another AAV, such as serotypes like AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or combinations thereof, and preferably reduce low recognition to no recognition.

[0246] Targeted ligand The modifications described herein may relate to the association (e.g., presentation, operable linkage, binding) of the targeted ligand to the modified capsid protein and / or the capsid containing the modified capsid protein. Generally, the targeted ligands described herein bind to surface proteins expressed by mammalian muscle cells, e.g., proteins expressed on the surface of mammalian muscle cells, e.g., mammalian non-terminally differentiated muscle cell-specific surface proteins. In some embodiments, the modified capsid protein and / or the modified capsid contains a targeted ligand that binds to mammalian CDH15, e.g., human CDH15.

[0247] Table 1 outlines the sequence numbers of non-limiting and exemplary anti-human CDH15 monoclonal antibodies (mAb IDs) that can be used to redirect AAV capsids by the methods described herein, for each binding site (e.g., heavy chain variable region (HCVR), light chain variable region (LCVR), CDR1, CDR2, CDR3). In some embodiments, the AAV capsid described herein comprises a targeted ligand that binds to human CDH15, the targeted ligand comprising a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, and the amino acid sequences of the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, each at least 90% homologous to the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 described in any one of SEQ ID NOs: 1 to 786. In some embodiments, the AAV capsid described herein comprises a target ligand that binds to human CDH15, wherein the target ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, and the amino acid sequences of the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, each at least 95% homologous to the set of heavy chain variable domains, light chain variable domains, heavy chain variable domain / light chain variable domain pairs, and the amino acid sequences of the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively.In some embodiments, the AAV capsid described herein comprises a target ligand that binds to human CDH15, the target ligand comprising a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, and the amino acid sequence of the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, which is at least 97% homologous to the set of heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, and the amino acid sequence of the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, as described in any one of SEQ ID NOs: 1 to 786. In some embodiments, the AAV capsid described herein comprises a target ligand that binds to human CDH15, the target ligand comprising a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, and the amino acid sequence of the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, which is at least 98% homologous to the set of heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, and the amino acid sequence of the set of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, as described in any one of SEQ ID NOs: 1 to 786.In some embodiments, the AAV capsid described herein comprises a target ligand that binds to human CDH15, the target ligand comprising a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, and amino acid sequences of the set HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, which are 99% homologous to the heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, and amino acid sequences of the set HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, as described in any one of SEQ ID NOs: 1 to 786. Furthermore, this specification also describes antibodies or antigen-binding fragments thereof that include a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by one of the representative anti-hCDH15 antibodies listed in Table 1. In some embodiments, the targeted ligand described herein is SEQ ID NO: 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, 436+444, 452+460, 468+476, 484+49 Includes the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within HCVR / LCVR amino acid sequence pairs selected from 2, 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.

[0248] Non-limiting examples of target ligands that bind to CDH15 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., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapies (SMIPs), and other manipulated molecules such as shark variable IgNAR domains are also included in the expression “targeting ligand” as used herein. In non-limiting embodiments, the anti-CDH15 targeting ligand that binds to CDH15 and is useful for retargeting the viral capsid described herein includes scFv. In non-limiting embodiments, V is useful for retargeting the viral capsid described herein. L -(Gly4Ser)3-V H The scFv sequence in this format may include a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 that is 90%, 95%, 97%, 98%, 99%, or 100% homologous to any one of the amino acid sequences of 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 set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 described in any one of sequence numbers 1 to 786.

[0249] Target ligands that bind to mammalian non-terminally differentiated muscle cell surface proteins can be associated (e.g., presented, functionally linked, bound) with modified AAV capsid proteins and the resulting AAV capsids according to known methods (e.g., direct approaches involving direct insertion of the target ligand, such as recombination). See, for example, Stachler et al. (2006), White et al. (2004), Girod et al. (1999), Grifman et al. (2001), Shi et al. (2001), and Shi and Bartlett (2003). Targeted ligands that bind to mammalian non-terminally differentiated muscle cell-specific surface proteins can be linked to modified AAV capsid proteins and the resulting AAV capsids using known chemical linkers. For example, AAV capsid proteins can be chemically modified to include dibenzothicotinyl or azide groups. Furthermore, if necessary, the targeted ligands described herein may be attached to a dibenzocyclootin group or an azide group (see, for example, U.S. Patent Application Publication 2022 / 028234, which is incorporated herein by reference in its entirety). Here, the targeted ligand is covalently linked to the primary amino acid group of the AAV capsid protein, for example, via a -CSNH- linkage. In some embodiments, the modified capsid described herein includes a targeted ligand, such as an anti-CDH15 antibody or its binding moiety, which is directly inserted or bound thereto according to a well-known direct recombination method.

[0250] bond pair In some embodiments, a target ligand that binds to a mammalian non-terminally differentiated muscle cell surface protein can be associated (e.g., presented, functionally linked, bound) with a modified AAV capsid protein and the resulting AAV capsid, following an indirect recombination approach, where the AAV capsid protein is modified to include a first member of the binding pair (e.g., a heterologous scaffold), and optionally, the first member of the binding pair is linked (e.g., covalently or non-covalently) to a second corresponding member of the binding pair (e.g., an adapter), and optionally, the second corresponding member of the binding pair is fused to the target ligand. Non-limiting exemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12:248-265.

[0251] Therefore, in some embodiments, the capsid protein modifications described herein include those that generally result from modifications at the gene level, such as modifications to the cap gene, including modifications to the first member of a binding pair (e.g., protein:protein binding pair, protein:nucleic acid binding pair), modifications to insert a detectable label presented by the Cap protein.

[0252] In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member is a metal ion, for example, Ni 2+ Co 2+ Cu 2+ Zn 2+ Fe 3+ It forms binding pairs with such as the above. In some embodiments, the first member is selected from the group consisting of streptavidin, StrepII, HA, L14, 4C-RGD, LH, and protein A.

[0253] 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, for example, U.S. Patent Application No. 2021 / 0180082, which is incorporated herein by reference in its entirety.

[0254] In some embodiments, the capsid protein of the present invention comprises at least a first member of a peptide:peptide bond pair.

[0255] In some embodiments, each of the first and second members of a peptide:peptide bond pair contains an intein. See, for example, Wagner et al., (2021) Adv. Sci. 8:2004018(1 / 22) and Muik et al. (2017) Biomaterials 144: 84. Each of these is incorporated herein by reference in its entirety.

[0256] In some embodiments, the first member is a B cell epitope, for example, approximately 1 to 35 amino acids in length, and forms a binding pair with an antibody paratope, such as an immunoglobulin variable domain. In some embodiments, the capsid protein of the present invention is modified to include a detectable label as the first member of the binding pair. Many detectable labels are known in the art. (See, for example, 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).Detectable labels include, but are not limited to, polyhistidine detectable labels that bind to immobilized divalent cations (e.g., Ni2+) (e.g., His-6, His-8, or His-10), biotin moieties that bind to immobilized avidin (e.g., on biotinylated polypeptide sequences in vivo), GST (glutathione S-transferase) sequences that bind to immobilized glutathione, S tags that bind to immobilized S proteins, and antigens that bind to immobilized antibodies or domains or fragments thereof (e.g., T7, myc, FLAG, and that bind to corresponding antibodies). Examples of detectable labels include B tags, FLASH tags (highly detectable labels that link to specific arsenic moieties), receptors or receptor domains that bind to immobilized ligands (or vice versa), protein A or its derivatives (e.g., Z) that bind to immobilized IgG, maltose-binding proteins (MBPs) that bind to immobilized amylose, albumin-binding proteins that bind to immobilized albumin, chitin-binding domains that bind to immobilized chitin, calmodulin-binding peptides that bind to immobilized calmodulin, and cellulose-binding domains that bind to immobilized cellulose. Another example of a detectable label is the SNAP-tag, commercially available from Covalys (www.covalys.com). In some embodiments, the detectable labels disclosed herein include detectable labels recognized by an antibody paratope, where the detectable label and the antibody paratope form a protein:protein binding pair.

[0257] In some embodiments, the capsid protein of the present invention comprises a first member of a protein:protein binding pair containing a detectable label, which may also be used for the detection and / or isolation of the Cap protein and / or as a first member of the protein:protein binding pair. In some embodiments, the detectable label plays a role as the first member of the protein:protein binding pair for the binding of a targeting ligand, which comprises a multispecific binding protein capable of binding to both the detectable label and the target expressed by the cell of interest. In some embodiments, the Cap protein of the present invention comprises a first member of a protein:protein binding pair containing c-myc (SEQ ID NO: 818). The use of the detectable label as the first member of a protein:protein binding pair is described, for example, in International Publication No. 2019 / 006043, which is incorporated herein by reference in its entirety.

[0258] In some embodiments, the first member comprises the B1 epitope (SEQ ID NO: 819). In some embodiments, the capsid protein is modified to include 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).

[0259] In some embodiments, the capsid protein comprises a first member of a protein:protein binding pair, which forms a covalent isopeptide bond. In some embodiments, the first member of the protein:protein binding pair is covalently bonded to a second member of the same gene of the protein:protein binding pair via an isopeptide bond, and optionally, the second member of the same gene of the protein:protein binding pair is fused with a targeting ligand, which binds to a target expressed by the cell of interest. In some embodiments, the protein:protein binding pair may be selected from the group consisting of SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag003:SpyCatcher003, SpyTag:KTag, Isopeptag:Pyrin-C, and SnoopTag:SnoopCatcher. In some embodiments, the first member is SpyTag (or its biologically active moiety or variant), and the protein (second congener member) is SpyCatcher (or its biologically active moiety or variant). In some embodiments, the first member is SpyTag (or its biologically active moiety or variant), and the protein (second congener member) is SpyCatcher (or its biologically active moiety or variant). In some embodiments, the first member is KTag (or its biologically active moiety or variant), and the protein (second congener member) is SpyTag (or its biologically active moiety or variant). In some embodiments, the first member is SnoopTag (or its biologically active moiety or variant), and the protein (second congener member) is SnoopCatcher (or its biologically active moiety or variant). In some embodiments, the first member is Isopeptag (or its biologically active moiety or variant), and the protein (second congener member) is pyrin-C (or its biologically active moiety or variant).In some embodiments, the first member is SpyTag002 (or its biologically active moiety or variant), and the protein (second congener member) is SpyCatcher002 (or its biologically active moiety or variant). In some embodiments, the first member is SpyTag003 (or its biologically active moiety or variant), and the protein (second congener member) is SpyCatcher003 (or its biologically active moiety or variant). In some embodiments, the Cap protein of the present invention comprises SpyTag, or its biologically active moiety or variant. The use of the first member of the protein-protein binding pair is described in International Publication No. 2019 / 006046, which is incorporated herein by reference in whole.

[0260] In some embodiments, the first member of a protein:protein binding pair, and / or a detectable label, is operably linked to the Cap protein of the present invention via first and / or second linkers, such as amino acid spacers that are at least one amino acid length (translated in frame using the Cap protein of the present invention, chemically added to the Cap protein of the present invention, and / or presented by the Cap protein of the present invention). In some embodiments, the first member of a protein:protein binding pair is adjacent to first and / or second linkers, such as first and / or second amino acid spacers, each of which is at least one amino acid length.

[0261] In some embodiments, the first and / or second linkers are not homologous. In some embodiments, the first and / or second linkers are each independently the length of one or two amino acids. In some embodiments, the first and / or second linkers are each independently the length of one, two, or three amino acids. In some embodiments, the first and / or second linkers are each independently the length of one, two, three, or four amino acids. In some embodiments, the first and / or second linkers are each independently the length of one, two, three, four, or five amino acids. In some embodiments, the first and / or second linkers are each independently the length of one, two, three, four, or five amino acids. In some embodiments, the first and / or second linkers are each independently the length of one, two, three, four, five, or six amino acids. In some embodiments, the first and / or second linkers are each independently the length of one, two, three, four, five, six, or seven amino acids. In some embodiments, the first and / or second linker is independently the length of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids. In some embodiments, the first and / or second linker is independently the length of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids. In some embodiments, the first and / or second linker is independently the length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the first and / or second linker is independently the length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0262] In some embodiments, the first and second linkers have homologous sequences and / or lengths, each being the length of one amino acid. In some embodiments, the first and second linkers have homologous lengths, each being the length of one amino acid. In some embodiments, the first and second linkers have homologous lengths, each being the length of two amino acids. In some embodiments, the first and second linkers have homologous lengths, each being the length of three amino acids. In some embodiments, the first and second linkers have homologous lengths, each being the length of four amino acids; for example, the linker is GLSG (SEQ ID NO: 823). In some embodiments, the first and second linkers have homologous lengths, each being the length of five amino acids. In some embodiments, the first and second linkers are homologous in length, each being the length of six amino acids. For example, the first and second linkers each contain the sequences GLSGSG (SEQ ID NO: 824) or GSGESG (SEQ ID NO: 828). In some embodiments, the first and second linkers are homologous in length, each having a length of 7 amino acids. In some embodiments, the first and second linkers are homologous in length, each having a length of 8 amino acids, for example, the first and second linkers each contain the sequence GLSGLSGS (SEQ ID NO: 825). In some embodiments, the first and second linkers are homologous in length, each having a length of 9 amino acids. In some embodiments, the first and second linkers are homologous in length, each having a length of 10 amino acids, for example, the first and second linkers each contain the sequence GLSGLSGLSG (SEQ ID NO: 826) or GLSGGSGLSG (SEQ ID NO: 827). In some embodiments, the first and second linkers are homologous in length, each having a length of more than 10 amino acids.

[0263] Generally, the amino acid sequences of the first member of the protein:protein binding pairs described herein, for example, include the first member of the specific binding pair alone or in combination with one or more linkers, and are approximately 5 to 50 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is at least 5 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 6 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 7 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 8 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 9 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 10 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 11 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 12 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 13 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 14 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 15 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 16 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 17 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 18 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 19 amino acids long.In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 20 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 21 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 22 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 23 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 24 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 25 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 26 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 27 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 28 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 29 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 30 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 31 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 32 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 33 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 34 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 35 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 36 amino acids long.In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 37 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 38 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 39 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 40 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 41 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 42 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 43 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 44 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 45 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 46 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 47 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 48 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 49 amino acids long. In some embodiments, the amino acid sequence of the first member of the protein:protein bond pair is 50 amino acids long.

[0264] In some embodiments of the present invention, which include 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, which can be conjugated to the surface of beads (e.g., for purification) or expressed by target cells (e.g., muscle stem cells, myoblasts, myocytes, or any combination thereof). Thus, the multispecific binding molecule comprises (i) an antibody paratope that specifically binds to the detectable label, and (ii) a second binding domain that specifically binds to a receptor that targets recombinant AAV particles. Such “targeting” or “direction” may include a scenario in which wild-type recombinant AAV particles target certain cells in tissues and / or organs within an organism, and the insertion of the detectable label reduces or neutralizes the broad targeting of the tissue or organ, and retargeting to more specific cells in tissues or more specific organs within the organism is achieved by the multispecific binding molecule. Such retargeting or redirection may also include a scenario in which wild-type recombinant AAV particles target a tissue, and this tissue targeting is reduced and rendered ineffective by the insertion of a detectable label, and retargeting to a completely different tissue is achieved with a multispecific binding molecule. The antibody paratopes described herein generally include at least a complementarity-determining region (CDR) that specifically recognizes the detectable label, e.g., a CDR3 region of the heavy chain and / or light chain variable domains. In some embodiments, the multispecific binding molecule includes an antibody (or a portion thereof) containing an antibody paratope that specifically binds to the detectable label. For example, the multispecific binding molecule may include a single-domain heavy chain variable region or a single-domain light chain variable region, in which case the single-domain heavy chain variable region or single-domain light chain variable region contains an antibody paratope that specifically binds to the detectable label. In some embodiments, the multispecific binding molecule may include an Fv region, e.g., the multispecific binding molecule may include an scFv containing an antibody paratope that specifically binds to the detectable label.In some embodiments, the multispecific binding molecules described herein include an antibody paratope (SEQ ID NO: 818) that specifically binds to c-myc.

[0265] Modified capsid containing modified capsid protein In some embodiments, the viral capsid containing the modified viral capsid proteins described herein is a mosaic capsid, for example, comprising at least two sets of VP1, VP2, and / or VP3 proteins, each set encoded by a different cap gene. The mosaic capsid as used herein generally refers to a mosaic of a first viral capsid protein modified to include the first member of a binding pair and a second corresponding viral capsid protein lacking the first member of the binding pair. In relation to mosaic capsids, the second viral capsid protein lacking the first member of the binding pair may also be referred to as the reference capsid protein and is encoded by a reference cap gene. In some embodiments of mosaic capsids, preferably, if the VP1, VP2, and / or VP3 capsid proteins modified with the first member of the protein:protein pair are not chimeric capsid proteins, the VP1, VP2, and / or VP3 reference capsid proteins may contain an amino acid sequence homologous to the amino acid sequence of the viral VP1, VP2, and / or VP3 capsid proteins modified with the first member of the binding pair, but the reference capsid protein lacks the first member of the binding pair. In some embodiments of mosaic capsids, the VP1, VP2, and / or VP3 reference capsid proteins correspond to the viral VP1, VP2, and / or VP3 capsid proteins modified with the first member of the binding pair, but the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP1 reference capsid protein corresponds to the viral VP1 capsid protein modified with the first member of the binding pair, but 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 the binding pair, while 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 the binding pair, while the reference capsid protein lacks the first member of the binding pair.In some embodiments of mosaic capsids, which include chimeric VP1, VP2, and / or VP3 capsid proteins further modified to include the first member of the binding pair, the reference protein may be a corresponding capsid protein, a portion of which forms a portion of the chimeric capsid protein. In some embodiments, as a non-limiting example, a mosaic capsid containing a chimeric AAV2 / AAAV VP1 capsid protein modified to include the first member of the binding pair may further include an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, and a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member as reference capsid proteins. Similarly, in some embodiments, a mosaic capsid containing a chimeric AAV2 / AAAV VP2 capsid protein modified to include the first member of the binding pair may further include, as reference capsid proteins, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, and a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid containing a chimeric AAV2 / AAAV VP3 capsid protein modified to include the first member of the binding pair may further include, as reference capsid proteins, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, and a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member. In some embodiments of mosaic capsids, the reference capsid protein may be any capsid protein, as long as it can form a capsid with a first capsid protein that lacks the first member of the binding pair and is modified with the first member of the binding pair.

[0266] In general, mosaic particles may be produced by transfecting a production cell with a mixture of modified and reference Cap genes in a specified ratio. The protein subunit ratio within the particle (e.g., modified VP protein:unmodified VP protein ratio) may, but not necessarily, stoichiometrically reflect the ratio of at least two of the cap genes (e.g., modified cap gene:reference cap gene transfected into the packaging cell) between the cap gene encoding the first capsid protein modified by 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 ratio of modified cap gene:reference cap gene transfected into the packaging cell.

[0267] In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is in the range of approximately 1:59 to approximately 59:1. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least approximately 1:1 (for example, the mosaic recombinant AAV particles contain approximately 30 modified capsid proteins and approximately 30 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least approximately 1:2 (for example, the mosaic recombinant AAV particles contain approximately 20 modified capsid proteins and approximately 40 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least approximately 3:5. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least approximately 1:3 (for example, the mosaic recombinant AAV particles contain approximately 15 modified capsid proteins and approximately 45 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:4 (for example, the mosaic recombinant AAV particles contain about 12 modified capsid proteins and 48 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:5 (for example, the mosaic recombinant AAV particles contain 10 modified capsid proteins and 50 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:6. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:7. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:8. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:9 (for example, the mosaic recombinant AAV particles contain about 6 modified capsid proteins and about 54 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:10.In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:11 (for example, a mosaic recombinant AAV particle contains about 5 modified capsid proteins and about 55 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:12. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:13. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:14 (for example, a mosaic recombinant AAV particle contains about 4 modified capsid proteins and about 56 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:15. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:19 (for example, a mosaic recombinant AAV particle contains about 3 modified capsid proteins and about 57 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:29 (for example, a mosaic recombinant AAV particle contains about 2 modified capsid proteins and about 58 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 1:59. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 2:1 (for example, a mosaic recombinant AAV particle contains about 40 modified capsid proteins and about 20 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 5:3. In some embodiments of mosaic recombinant AAV particles, the ratio of protein subunits is at least about 3:1 (for example, a mosaic recombinant AAV particle contains about 45 modified capsid proteins and about 15 reference capsid proteins).In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 4:1 (for example, the mosaic recombinant AAV particles contain about 48 modified capsid proteins and 12 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 5:1 (for example, the mosaic recombinant AAV particles contain 50 modified capsid proteins and 10 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 6:1. In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 7:1. In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 8:1. In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 9:1 (for example, the mosaic recombinant AAV particles contain about 54 modified capsid proteins and about 6 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 10:1. In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 11:1 (for example, the mosaic recombinant AAV particles contain about 55 modified capsid proteins and about 5 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 12:1. In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 13:1. In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 14:1 (for example, the mosaic recombinant AAV particles contain about 56 modified capsid proteins and about 4 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 15:1.In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 19:1 (for example, a mosaic recombinant AAV particle contains about 57 modified capsid proteins and about 3 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 29:1 (for example, a mosaic recombinant AAV particle contains about 58 modified capsid proteins and about 2 reference capsid proteins). In some embodiments of mosaic recombinant AAV particles, the protein subunit ratio is at least about 59:1.

[0268] In some embodiments of non-mosaic recombinant AAV particles, the ratio of protein subunits may be 1:0, and each capsid protein in the non-mosaic recombinant AAV particle is modified with the first member of the binding pair. In some embodiments of non-mosaic recombinant AAV particles, the ratio of protein subunits may be 0:1, and each capsid protein in the non-mosaic recombinant AAV particle is not modified with the first member of the binding pair.

[0269] Furthermore, nucleic acids encoding the VP3 capsid protein of the present invention are also provided herein. The AAV capsid protein may, but not necessarily, be encoded by overlapping the reading frames of homologous genes with shifted start codons. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the present invention also does not encode the VP2 capsid protein or the VP1 capsid protein of the present invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the present invention may also encode the VP2 capsid protein of the present invention, but not the VP1 capsid of the present invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the present invention may also encode the VP2 capsid protein and the VP1 capsid of the present invention.

[0270] One embodiment of the present invention is a multimeric structure comprising the modified viral capsid proteins of the present invention. The multimeric structure comprises at least five, preferably at least ten, more preferably at least 30, and most preferably at least 60 modified viral capsid proteins, each comprising the first member of the specific binding pair described herein. They can form ordinary viral capsids (empty recombinant AAV particles) or recombinant AAV particles (capsids capsidizing the nucleotide of interest). The formation of recombinant AAV particles containing the viral genome is a highly preferred characteristic for the use of the modified viral capsids described herein.

[0271] Further embodiments of the present invention relate to the manufacture and use of at least one modified viral capsid protein and / or nucleic acid encoding it, preferably at least one multimeric structure (e.g., a viral particle), for transporting a nucleotide of interest to a target cell (e.g., muscle stem cells, myoblasts, muscle cells, or any combination thereof).

[0272] Insertion site Due to the high conservation of at least a wide range of closely related family members, the corresponding insertion sites of AAVs other than those listed can be identified by performing amino acid alignment or capsid structure comparisons. For example, see 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) provide overlays of ribbons from different dependoparvoviruses in Figure 7, depicting the variable regions VR I–VR IX. Using structural and sequence analyses as described in the relevant literature, a person skilled in the art can determine which amino acids within the variable region correspond to the amino acid sequence of AAV that can accept, for example, the insertion of a target ligand, the first member of a binding pair, and / or a detectable label as described herein.

[0273] Generally, the targeting ligand, the first member of the binding pair, and / or the detectable label may be inserted into the variable region or variable loop of the AAV capsid protein, the GH loop of the AAV capsid protein, etc. In some embodiments, the target ligand, the first member of the binding pair, and / or the detectable label may be inserted into the variable region or variable loop VRI of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or the detectable label may be inserted into the variable region or variable loop VRII of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or the detectable label may be inserted into the variable region or variable loop VRIII of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or the detectable label may be inserted into the variable region or variable loop VRIV of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or the detectable label may be inserted into the variable region or variable loop VRV of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or a detectable label may be inserted into the variable region or variable loop (VRV) of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or a detectable label may be inserted into the variable region or variable loop VRVI of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or a detectable label may be inserted into the variable region or variable loop VRVII of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or a detectable label may be inserted into the variable region or variable loop VRIII of the AAV capsid protein. In some embodiments, the target ligand, the first member of the binding pair, and / or a detectable label may be inserted into the variable region or variable loop VRIX of the AAV capsid protein.

[0274] Therefore, in some embodiments, the first member of the binding pair and / or the detectable label is inserted in the VP1 capsid protein of non-primate AAVs after the amino acid position corresponding to the amino acid position selected from the group consisting of G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the first member of the binding pair and / or the detectable label is inserted in the VP1 capsid protein of non-primate AAVs between the amino acids corresponding to N587 and R588 of AAV2 VP1 capsid.

[0275] In this specification, nomenclature such as I-### and I# refers to an insertion site (I) that names the amino acid number of the VP1 protein of the AAV capsid protein as ###. Such insertions may be located directly at the N-terminus or C-terminus, preferably at the C-terminus of one amino acid in the N-terminus or C-terminus sequence of five amino acids of a given amino acid, preferably at three amino acids of a given amino acid, more preferably at two amino acids, and particularly at the C-terminus of one amino acid in the N-terminus or C-terminus sequence of one amino acid. Furthermore, the positions referred to herein are in comparison to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and their point mutations) can be easily identified by performing sequence alignment of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid gene to identify the VP2 and VP3 capsid proteins encoded by the capsid gene.

[0276] Suitable insertion sites for additional non-primate VP1 capsid protein 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 AAV2 VP1 capsid protein (Wu et al. (2000) J. Virol. 74:8635-8647). The modified viral capsid proteins described herein may be non-primate animal capsid proteins containing a first member of a binding pair and / or a detectable label inserted at a position corresponding to the 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 preferred insertion sites for non-primate AAVs include sites 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 proteins described herein may be non-primate capsid proteins comprising a target ligand, a first member of a binding pair, and / or a detectable label, inserted at 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.

[0277] In some embodiments, the first member of the binding pair, and / or the detectable label, is I444 of the VP1 capsid protein of non-primate AAVs, I580 of the avian AAV capsid protein VP1, I573 of the bearded dragon AAV capsid protein VP1, I436 of the bearded dragon AAV capsid protein VP1, I429 of the sea lion AAV capsid protein VP1, and I4 30 is inserted after the amino acid position corresponding to the position of an amino acid selected from the group consisting of I431, I432, I433, I434, I436, I437, and I565 of the sea lion AAV capsid protein VP1.

[0278] Because the capsid protein is encoded by overlapping reading frames of homologous genes with shifted start codons, an insertion of the coding nucleic acid at a corresponding position in one of these sites of the cap gene also results in insertions in VP1, VP2, and / or VP3. Therefore, for example, in AAV2, according to this nomenclature, an insertion of amino acids 1-138 is inserted only in VP1, an insertion of 138-203 is inserted in both VP1 and VP2, and an insertion of 203-C-terminus is inserted in VP1, VP2, and VP3, and this naturally also applies to insertion site I-587. Accordingly, the present invention encompasses structural genes of AAV having corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0279] Transduction efficiency In some embodiments, a viral capsid containing a modified viral capsid protein including the first and second members of a binding pair (e.g., the second member is operably linked to a targeting ligand and includes a multispecific binding protein) can infect specific cells and, for example, has an enhanced ability to target and bind to specific cells compared to a control viral capsid (the control viral capsid is homologous to the modified viral capsid protein except that it lacks one or both of the first and second members of the binding pair and, for example, includes a control capsid protein). In some embodiments, a viral capsid containing the modified viral capsid protein described herein, linked to the first and second members of a binding pair linked to a targeting ligand, exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a 10% higher transduction efficiency than a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a 20% higher transduction efficiency than a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a 30% higher transduction efficiency than a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a 40% higher transduction efficiency than a control viral capsid.In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 50% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 60% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 70% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 75% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 80% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 85% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 90% higher than that of a control capsid.In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 95% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 99% higher than that of a control viral capsid.

[0280] In some embodiments, a viral capsid containing a modified viral capsid protein including the first and second members of a binding pair (e.g., the second member is operably linked to a targeting ligand and includes a multispecific binding protein) can infect specific cells and, for example, has an enhanced ability to target and bind to specific cells compared to a control viral capsid (the control viral capsid is homologous to the modified viral capsid protein except that it lacks one or both of the first and second members of the binding pair and, for example, includes a control capsid protein). In some embodiments, a viral capsid containing the modified viral capsid protein described herein, linked to the first and second members of a binding pair linked to a targeting ligand, exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a 10% higher transduction efficiency than a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a 20% higher transduction efficiency than a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a 30% higher transduction efficiency than a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a 40% higher transduction efficiency than a control viral capsid.In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 50% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 60% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 70% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 75% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 80% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 85% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 90% higher than that of a control capsid.In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to the appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 95% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency 99% higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 1.5 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least twice as high as that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least three times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least four times higher than that of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 5 times higher than that of a control viral capsid.In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 6 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 7 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 8 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 9 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 10 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 20 times higher than that of a control capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, conjugated to appropriate first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 30 times higher than that of a control viral capsid.In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 40 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 50 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 60 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 70 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 80 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 90 times higher than that of a control viral capsid. In some embodiments, a viral capsid containing the modified viral capsid protein described herein, bound to the first and second members of a binding pair linked to a targeted ligand, exhibits a transduction efficiency at least 100 times higher than that of a control viral capsid.In some embodiments, recombinant AAV particles of the present invention include a viral capsid protein containing the amino acid sequence of a capsid protein of a non-primate AAV, a distantly related AAV, or a combination thereof, and optionally include the first and second members of the binding pair (e.g., the second member is operably linked to a targeted ligand and includes a multispecific binding protein). Compared to suitable control recombinant AAV particles (e.g., including a viral capsid of an AAV serotype that contains a portion of the viral capsid of the present invention as part of a viral capsid protein containing the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof), the recombinant AAV particles of the present invention can better avoid neutralization by existing antibodies in serum isolated from human patients (control recombinant AAV particles also optionally include the first and second members of the binding pair (e.g., the second member is operably linked to a targeted ligand and includes a multispecific binding protein)). In some embodiments, recombinant AAV particles of the present invention, comprising a viral capsid protein containing the amino acid sequence of a capsid protein of a non-primate AAV, a distantly related AAV, or a combination thereof, require at least twice as much total IVIG or IgG to neutralize (e.g., 50% or more inhibition of infection) compared to suitable control recombinant AAV particles (e.g., recombinant AAV particles of the present invention). The offspring has an IC50 value at least twice that of the control recombinant AAV particle.

[0281] Administration of therapeutic formulations and antigen-binding molecules Pharmaceutical compositions containing antigen-binding molecules described herein are also described herein. In some embodiments, the pharmaceutical compositions of the present invention may be formulated with suitable carriers, excipients, and other agents that provide improvements in transport, delivery, and tolerability. Numerous suitable formulations can be found in the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (e.g., LIPOFECTIN®, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0282] The dose of antigen-binding molecules administered to a patient may vary depending on the patient's age and physique, the target disease, condition, and route of administration. Preferred doses are typically calculated according to body weight or body surface area. When the antigen-binding molecules of the present invention are used for therapeutic purposes in adult patients, it may be advantageous to administer the antigen-binding molecules described in the present invention intravenously as a single dose at approximately 0.01 to 20 mg per kg of body weight, more preferably approximately 0.02 to 7 mg, 0.03 to 5 mg, or 0.05 to 3 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and schedules for administering bispecific antigen-binding molecules can be determined empirically, but the patient's progress can be monitored by periodic assessments, for example, and the dose can be adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0283] Various delivery systems are known and may be used to administer the pharmaceutical compositions described herein, such as liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-dependent endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of delivery are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example, by injection or bolus injection, by absorption through the epithelium or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other biologically active agents. Administration may be systemic or topical.

[0284] The pharmaceutical compositions described herein can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable to the delivery of the pharmaceutical compositions described herein. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices come pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0285] Numerous reusable pen-type and automated injector delivery devices find applications in the subcutaneous delivery of the pharmaceutical compositions described herein. To give just a few examples, though not limited to them, are AUTOPEN (trademark) (Owen Mumford, Inc., International Publication No. odstock, UK), DISETRONIC (trademark) pens (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 (trademark) pens, HUMALOG (trademark) pens, HUMALIN 70 / 30 (trademark) pens (Eli Lilly and Co., Indianapolis, IN), NOVOPEN (trademark) I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (trademark) (Novo Nordisk, Copenhagen, Denmark), BD (trademark) pens (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN (trademark), OPTIPEN PRO (trademark), and OPTIPEN Examples include STARLET® and OPTICLIK® (sanofi-aventis, Frankfurt, Germany). Some examples of disposable pen delivery devices for use in subcutaneous delivery of the pharmaceutical compositions described herein include, but are not limited to, SOLOSTAR® pen (sanofi-aventis), FLEXPEN® (Novo Nordisk), KWIKPEN® (Eli Lilly), SURECLICK® Autoinjector (Amgen, Thousand Oaks, CA), PENLET® (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA® Pen (Abbott Labs, Abbott Park IL).

[0286] In certain circumstances, pharmaceutical compositions may be delivered by a controlled-release system. In one embodiment, a pump may be used (see Langer; Sefton, 1987, CRC Crit Ref Biomed.Eng.14:201 above). In another embodiment, a polymer material may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system may be positioned near the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, Medical Applications of Controlled Release, above, vol.2, pp.115-138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0287] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibodies or salts thereof described above in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with suitable solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers, such as benzyl benzoate and benzyl alcohol. The injection solutions thus prepared are preferably filled into suitable ampoules.

[0288] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in unit doses suitable for adapting the dose of the active ingredient. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antigen-binding molecule contained is generally about 5 to about 500 mg per dosage form in a unit dose, and in particular in the form of injection, the antibody is preferably contained in about 5 to about 100 mg, and for other dosage forms, it is preferably contained in about 10 to about 250 mg.

[0289] Pharmaceutical compositions, dosage forms, and administration methods for virus particles. Further embodiments provide a pharmaceutical product comprising at least one modified viral capsid protein, a suitable targeting ligand according to the present invention, and / or a nucleic acid according to the present invention. Preferably, such a pharmaceutical product is useful as a gene transfer particle.

[0290] Pharmaceutical compositions comprising recombinant AAV particles described herein and pharmaceutically acceptable carriers and / or excipients are also disclosed herein. In addition, pharmaceutical dosage forms comprising recombinant AAV particles described herein are disclosed herein.

[0291] As discussed herein, the recombinant AAV particles described herein can be used for a variety of therapeutic applications (in vivo and ex vivo) and as research tools.

[0292] Pharmaceutical compositions based on recombinant AAV particles disclosed herein may be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. Recombinant AAV particles may be prescribed for administration, for example, by injection, inhalation, or insulation (through either the mouth or nose), or by oral, intraoral, parenteral, or rectal administration, or by direct administration to a tumor.

[0293] Pharmaceutical compositions can be formulated for a variety of administration modes, 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, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections. For injection, pharmaceutical compositions can preferably be formulated in a liquid solution with a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, pharmaceutical compositions can be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms of pharmaceutical compositions are also preferred.

[0294] For oral administration, pharmaceutical compositions are prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate), and may take the form of tablets or capsules. Tablets may also be coated by methods well known in the art. Liquid preparations for oral administration may take the form of solutions, syrups, or suspensions, or may be presented as dried products using a composition containing water or other suitable medium before use. Such liquid preparations may be prepared by conventional means using 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 media (e.g., oils, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoic acid or sorbic acid). The preparations may also contain buffer salts, flavorings, colorants, and sweeteners, as needed.

[0295] Pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may optionally contain preservatives and be presented in unit dosage forms, for example, in ampoules or multi-dose containers. Pharmaceutical compositions can be further formulated as suspensions, solutions, or emulsions in oily or aqueous media and may contain other agents, including suspending agents, stabilizers, and / or dispersants.

[0296] In addition, pharmaceutical compositions can also be formulated as depot preparations. These long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. For example, compounds can be formulated with suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion-exchange resins, or as poorly soluble derivatives, such as poorly soluble salts. Another suitable delivery system is microspheres, which offer the possibility of long-term, local, non-invasive delivery of drugs. This technique may include microspheres having a pre-capillary size that can be injected into any selected portion of an organ via a coronary catheter without causing inflammation or ischemia. The administered therapeutic agent is gradually released from the microspheres and absorbed by surrounding cells present in the selected tissue.

[0297] Systemic administration is also possible by transmucosal or transdermal means. For transmucosal or transdermal administration, a suitable penetrating agent for the penetration barrier is used in the formulation. Such penetrating agents are generally known in the art, and examples of bile salts and fusidic acid derivatives for transmucosal administration include. In addition, detergents may be used to promote penetration. Transmucosal administration may be performed using intranasal sprays or suppositories. For topical administration, the recombinant AAV particles described herein can be formulated into ointments, plasters, gels, or creams, which are generally known in the art. To accelerate healing, a cleansing solution may also be used topically to treat the injury or inflammation.

[0298] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical form must be sterile and fluid. It must also be stable under manufacturing conditions and certain storage parameters (e.g., cooling and freezing) and must be protected from contamination by microorganisms such as bacteria and fungi.

[0299] When the formulations disclosed herein are used as therapeutic agents to promote an immune response in a subject, the therapeutic agents may be formulated into neutral or salt-form compositions. 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 organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0300] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents known in the art. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride. Long-term absorption of the injectable composition can be achieved by the use of absorption-delaying agent compositions, such as aluminum monostearate and gelatin.

[0301] Sterile, injectable solutions can be prepared by incorporating the active compound or construct in the required amount into a suitable solvent, using various other components listed above as needed, and then sterilizing by filtration.

[0302] When prescribed, the solution may be administered in a form compatible with the administered formulation and in a therapeutically effective amount. The formulation can be readily administered in a variety of dosage forms, such as the injectable solution types described above, but sustained-release capsules or microparticles and microspheres may also be used.

[0303] For parenteral administration in aqueous solutions, the solution should be buffered appropriately as needed, for example, and the liquid diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration. In this context, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid, or injected into the proposed injection site.

[0304] The person responsible for administration will, in any case, determine the appropriate dose for each individual subject. For example, a subject may be administered the recombinant AAV particles described herein daily, weekly, monthly, every two years, or annually for a period of time, depending on the subject's need or exposure to pathogenic microorganisms or to the subject's condition (e.g., cancer).

[0305] In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, subcutaneous, 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.

[0306] In addition, intranasal or inhalable solutions or sprays, aeros...

Claims

1. An antigen-binding protein that binds to human cadherin 15 (CDH15), wherein the antigen-binding protein is sequence number 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, 187-189-191-52-3 4-196, 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,An antigen-binding protein comprising an aggregate of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from 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.

2. The antigen-binding protein according to claim 1, wherein the antigen-binding protein includes a heavy chain variable region (HCVR or VH).

3. The antigen-binding protein according to claim 1, wherein the antigen-binding protein includes a light chain variable region (LCVR or VL).

4. The 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.

5. The antigen-binding protein according to claim 4, wherein the anti-hCDH15 antibody or its antigen-binding fragment is selected from the group consisting of human or humanized antibodies or their antigen-binding fragments, monovalent Fab', bivalent Fab2, F(ab)'3 fragments, single-chain variable region fragments (scFv), bis-scFv, (scFv)2, dibodies, minibodies, nanobodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), single-domain antibodies (sdAb), Ig NAR, bispecific antibodies or their binding fragments, bispecific T cell engagers (BiTE), trispecific antibodies, and chemically modified derivatives thereof.

6. The antigen-binding protein according to claim 5, wherein the anti-hCDH15 antibody or its antigen-binding fragment comprises a fragment antigen-binding region (Fab).

7. The antigen-binding protein according to claim 5, wherein the anti-hCDH15 antibody or its antigen-binding fragment comprises a single-chain variable region fragment (scFv).

8. The antigen-binding protein according to claim 7, wherein the scFv has a variant region arranged in the sequence HCVR-LCVR from the N-terminus to the C-terminus.

9. The antigen-binding protein according to claim 7, wherein the scFv has a variant region arranged in the sequence LCVR-HCVR from the N-terminus to the C-terminus.

10. The antigen-binding protein according to any one of claims 7 to 8, wherein the scFv variable region is connected by a linker.

11. The antigen-binding protein according to claim 10, wherein the linker is a peptide linker.

12. The antigen-binding protein according to claim 11, wherein the peptide linker is -(GGGGS)n-(SEQ ID NO: 789), and n is 1 to 10.

13. The aforementioned antigen-binding protein is approximately 1 × 10⁻ ７ An antigen-binding protein according to any one of claims 1 to 12, which binds to hCDH15 with M KD or a stronger affinity.

14. The aforementioned antigen-binding protein is approximately 10 × 10 －８ ~Approx. 1×10 －１０ An antigen-binding protein according to any one of claims 1 to 13, which binds to hCDH15 in KD.

15. The aforementioned antigen-binding protein is approximately 5 × 10 －９ From approximately 1 x 10 －１０ An antigen-binding protein according to any one of claims 1 to 14, which binds to hCDH15 with a dissociation constant (KD) in the range of .

16. The antigen-binding protein is sequence numbers 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 An antigen-binding protein according to any one of claims 1 to 15, comprising an HCVR / LCVR amino acid sequence pair having at least 90% sequence homology with an HCVR / LCVR amino acid sequence pair selected from the group consisting of 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, 764 and 772.

17. A polynucleotide comprising a sequence encoding an antigen-binding protein according to any one of claims 1 to 16.

18. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

19. A method for inhibiting the activity of CDH15 in cells, comprising contacting the cells expressing CDH15 with an antigen-binding protein according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 18.

20. The method according to claim 19, wherein the cells expressing CDH15 are muscle stem cells, myoblasts, or muscle cells.

21. A method for accelerating the transition of muscle stem cells from quiescence to activation, comprising contacting the muscle stem cells with an antigen-binding protein according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 18.

22. A method for treating a disease in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in claim 18.

23. The method according to claim 22, wherein the disease is muscle injury.

24. A method for improving muscle regeneration after muscle injury in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 18 to the subject.

25. A method for restoring the muscle regeneration capacity of a target, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 18 to the target.

26. The method according to claim 25, wherein the muscle regeneration capacity of the target is restored to a functional state equivalent to or near the muscle regeneration capacity of the control.

27. A method for treating a condition in a subject requiring such treatment, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition according to claim 18 to the subject, wherein the antigen-binding protein is conjugated to the therapeutic agent.

28. The method according to claim 27, wherein the disease is cancer.

29. The method according to claim 28, wherein the cancer is rhabdomyosarcoma.

30. The method according to any one of claims 27 to 29, wherein the therapeutic agent comprises a cytotoxic chemotherapeutic agent.

31. The aforementioned therapeutic agent includes aflibercept, amsacrin, azacitidine, azathioprine, belantamab mafodotin, bendamustine, bleomycin, bortezomib, brentuximab vedotin, busulfan, cabazitaxel, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, cisplatin, cladribine, chloropharabine, cyclophosphamide, cytarabine, and cytarabine liposomes. Dacarbazine, Dactinomycin (Dactinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin Liposome, Epirubicin, Eribulin, Etoposide, Etoposide Phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab 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 The method according to any one of claims 27 to 30, comprising romidepsin, sacituzumab govitecan, temozolomide, teniposide, thiotepa thioguanine, topotecan, trabectedin, trastuzumab deruxtecan, trastuzumab emtansine, trifluridine / tipiracil, valganciclovir, vinblastone, vincristine, vindesine, vinflunin, vinorelbine, or bismodegib.

32. The 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.

33. A method for imaging muscle cells in a subject requiring such imaging, wherein the method comprises administering the pharmaceutical composition according to claim 18 to the subject, the antigen-binding protein being conjugated to a detectable portion.

34. The method according to claim 33, wherein the muscle cells include one or more cells selected from the group consisting of muscle stem cells, myoblasts, and muscle cells.

35. The method according to claim 33 or 34, wherein the detectable portion includes a radionuclide.

36. The method according to any one of claims 22 to 35, wherein the administration includes administering the pharmaceutical composition to the subject intravenously or subcutaneously.

37. Recombinant adeno-associated virus (AAV) particles, (i) AAV capsid containing modified AAV capsid protein, (ii) A targeted ligand that binds to the surface protein of non-terminal differentiated muscle cells, Recombinant AAV particles in which the modified AAV capsid is operably linked to the targeted ligand.

38. (a) The modified AAV capsid protein comprises a first member and a second member of a protein:protein binding pair, The first member of the protein:protein bond pair is operably connected to the second member of the protein:protein bond pair, (b) The protein: The second member of the protein binding pair comprises a target ligand that binds to the surface protein of the non-terminally differentiated muscle cell, The recombinant AAV particle according to claim 37, wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair associate in such a way that the affinity of the recombinant AAV particle is directed toward the non-terminally differentiated muscle cell.

39. The recombinant AAV particle according to claim 37 or claim 38, wherein the non-terminal differentiated muscle cells include one or more cells selected from the group consisting of muscle stem cells, myoblasts, muscle cells, myotubes, and combinations thereof.

40. Recombinant AAV particles according to any one of claims 37 to 39, comprising an AAV capsid bound to the surface protein of the non-terminally differentiated muscle cell, wherein the surface protein is expressed on the surface of a mammalian cell.

41. It contains an AAV capsid that is expressed on the surface of mammalian muscle cells and is bound to the surface protein of non-terminally differentiated muscle cells. The surface protein of the non-terminal differentiated muscle cell is the human surface protein of the non-terminal differentiated muscle cell. Recombinant AAV particles according to any one of claims 37 to 40, wherein the mammalian muscle cells are non-human animal muscle cells genetically modified to express the human muscle cell-specific surface protein.

42. The AAV capsid is bound to a surface protein of non-terminally differentiated muscle cells, and the surface protein is expressed on the surface of mammalian muscle cells. The surface protein of the non-terminally differentiated muscle cells is a human surface protein of the non-terminally differentiated muscle cells. The recombinant AAV particle according to any one of claims 37 to 41, wherein the mammalian muscle cell is a rodent muscle cell genetically modified to express the human surface protein of the non-terminally differentiated muscle cell.

43. The AAV capsid is bound to the surface protein of the non-terminally differentiated muscle cell, and the surface protein of the non-terminally differentiated muscle cell is expressed on the surface of the mammalian muscle cell. The surface protein of the non-terminal differentiated muscle cell is the human surface protein of the non-terminal differentiated muscle cell. Recombinant AAV particles according to any one of claims 37 to 42, wherein the mammalian muscle cells are rat or mouse muscle cells genetically modified to express human surface proteins of non-terminally differentiated muscle cells.

44. The AAV capsid is bound to the surface protein of the non-terminally differentiated muscle cell, and the surface protein is expressed on the surface of the mammalian muscle cell. The surface protein of the non-terminal differentiated muscle cells is a human cell-specific surface protein of the non-terminal differentiated muscle cells. The recombinant AAV particle according to any one of claims 37 to 43, wherein the mammalian muscle cells are mouse muscle cells genetically engineered to express human cell-specific surface proteins of non-terminally differentiated muscle cells.

45. The AAV capsid is bound to the surface protein of the non-terminally differentiated muscle cell, and the surface protein is expressed by mammalian muscle cells. The surface protein of the non-terminal differentiated muscle cell is the human surface protein of the non-terminal differentiated muscle cell. Recombinant AAV particles according to any one of claims 37 to 41, wherein the mammalian muscle cells are human muscle cells.

46. Recombinant AAV particles according to any one of claims 37 to 45, wherein the AAV is in vitro.

47. Recombinant AAV particles according to any one of claims 37 to 45, wherein the AAV is in vivo.

48. Recombinant AAV particles according to any one of claims 37 to 42, wherein the surface protein of the non-terminal differentiated muscle cells is mammalian cadherin 15 (CDH15).

49. Recombinant AAV particles according to any one of claims 37 to 48, wherein the surface protein of the non-terminal differentiated muscle cells is human CDH15.

50. (a) The protein: The first member of the protein binding pair includes SpyTag, IsopTag, SnoopTag, or SpyTag002, (b) The protein: The second member of the protein binding pair is (i) SpyCatcher, KTag, pilin-C, SnoopCatcher, or SpyCatcher002, and (ii) comprising the targeting ligand that binds to mammalian muscle cell-specific surface proteins, (c) The recombinant AAV particle according to any one of claims 38 to 49, wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are associated by an isopeptide bond.

51. (a) The protein: The first member of the protein binding pair comprises SpyTag, (b) The recombinant AAV particle according to any one of claims 38 to 50, wherein the second member of the protein-binding pair comprises a SpyCatcher fused to a targeted ligand that binds to a mammalian muscle-specific surface protein.

52. (a) The protein: The first member of the protein binding pair comprises the c-myc amino acid sequence described in Sequence ID No. 818, (b) The recombinant AAV particle according to any one of claims 38 to 49, wherein the second member of the protein-binding pair comprises a bispecific binding protein comprising an anti-c-myc antibody and a target ligand that binds to a mammalian muscle-specific surface protein.

53. The recombinant AAV particle according to any one of claims 38 to 52, comprising a first and / or second linker functionally linking the first member of a protein-binding pair to the capsid protein of the recombinant AAV particle.

54. The recombinant AAV particle according to claim 53, wherein the first and second linkers are not homologous.

55. The recombinant AAV particle according to claim 53, wherein the first and second linkers are homologous.

56. The recombinant AAV particle according to any one of claims 53 to 55, wherein the length of the first linker is 10 amino acids and / or the length of the second linker is 10 amino acids, and optionally the amino acid sequence of the first linker and / or the amino acid sequence of the second linker includes the amino acid sequence described in SEQ ID NO: 826 or SEQ ID NO:

827.

57. (a) The AAV capsid comprises a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein, which are encoded by a mutant cap gene. (b) The modified cap gene or a portion thereof comprises a sequence that is at least 90% homologous to the cap gene or a portion thereof of the AAV, and is genetically modified to include an insertion of a nucleotide sequence encoding the first member of a protein:protein binding pair, thereby the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises the first member of a protein:protein binding pair, according to any one of claims 38 to 56.

58. The modified cap gene is genetically modified such that the mutant VP1 capsid protein, mutant VP2 capsid protein, and / or mutant VP3 capsid protein contain one or more of the following mutations in addition to the first member of the protein:protein binding pair: (i) Substitution, insertion, or deletion of amino acids, (ii) Chimeric amino acid sequence, or (iii) Recombinant AAV particles according to claim 57, comprising both a point mutation and a chimeric amino acid sequence.

59. The recombinant AAV particle according to claim 58, wherein the substitution, insertion, or deletion of an amino acid reduces the intrinsic directivity of the recombinant AAV particle and / or produces a detectable label.

60. (a) The AAV capsid comprises a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein, which are encoded by a mutant cap gene. (b) The modified cap gene or a portion thereof contains a sequence that is at least 90% homologous to the AAV cap gene or a portion thereof, and is genetically modified to include an insertion of a nucleotide sequence encoding the first member of a protein:protein binding pair, thereby the modified VP1 capsid protein, modified VP2 capsid protein, and / or modified VP3 capsid protein contain the first member of a protein:protein binding pair, (c) Recombinant AAV particles according to any one of claims 38 to 59, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, non-primate-derived AAVs listed in Table 2, and combinations thereof.

61. Recombinant AAV particle according to any one of claims 57 to 60, wherein the AAV is AAV2.

62. The recombinant AAV particle according to any one of claims 57 to 61, wherein the AAV capsid comprises a modified AAV2VP1 capsid protein in which the first member of a protein:protein binding pair is linked to amino acids at positions I453 and / or I587, optionally via a linker.

63. The AAV capsid comprises a modified AAV2VP1 capsid protein containing the first member of the protein-protein binding pair, which is indicated at position G453 via a linker. Optionally, the modified AAV2 VP1 capsid protein further comprises R585A modification, R588A modification, or both R585A modification and R588A modification. Recombinant AAV particles according to any one of claims 57 to 62, wherein the modified AAV2 VP1 capsid protein optionally further comprises R484A modification, R487A modification, R585A modification, R588A modification, and K532A modification, or any combination of R484A modification, R487A modification, R585A modification, R588A modification, and K532A modification.

64. The AAV capsid is a mosaic AAV capsid comprising a second set of AAV2VP1 capsid proteins lacking the first member of the protein binding pair, and comprising a second group of AAV2VP1 capsid proteins lacking the first member of the protein binding pair. Recombinant AAV particles according to claim 62 or 63, wherein the second set of AAV2 VP1 capsid proteins optionally comprises R585A modification, R588A modification, or both R585A modification and R588A modification.

65. Recombinant AAV particle according to any one of claims 57 to 60, wherein the AAV is AAV9.

66. Recombinant AAV particles according to any one of claims 57 to 60 and 65, comprising a modified AAV9 VP1 capsid protein, wherein the AAV capsid optionally links to an amino acid at position I453 or I589 via a linker, and the first member of the protein:protein binding pair.

67. The AAV capsid comprises a modified AAV9 VP1 capsid protein, which includes the first member of the protein:protein binding pair presented at G453 via a linker. The recombinant viral capsid protein according to any one of claims 57-60 and 65-66, wherein the modified AAV9 VP1 capsid protein optionally further comprises N272A modification, W503A modification, or both N272A modification and W503A modification.

68. 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, The recombinant AAV particle according to claim 66 or 67, wherein the modified AAV2 VP1 capsid protein optionally further comprises N272A modification, W503A modification, or both N272A modification and W503A modification.

69. The recombinant AAV particle according to claim 60, wherein the non-primate animal AAV is avian AAV (AAAV), non-human mammal AAV, or squamate AAV.

70. Recombinant AAV particles according to claim 60 or claim 69, wherein the non-primate AAV is AAAV.

71. Recombinant AAV particles according to claim 60, 69, or 70, wherein the AAV capsid comprises a modified AAAV VP1 capsid protein in which the first member of the protein binding pair is optionally linked via a linker to an amino acid at position I444 or I580.

72. The recombinant AAV particle according to claim 60 or claim 69, wherein the non-primate AAV is a lepidosaurian AAV.

73. Recombinant AAV particles according to claim 60, 69, or 72, wherein the non-primate AAV is bearded dragon AAV.

74. Recombinant AAV particles according to any one of claims 60, 69, 72, or 73, comprising a modified bearded dragon VP1 capsid protein, wherein the AAV capsid optionally links to an amino acid at position I573 or I436 via a linker, and the first member of the protein:protein binding pair.

75. The recombinant AAV particle according to claim 60 or 69, wherein the non-primate AAV is a non-human mammalian AAV.

76. Recombinant AAV particles according to any one of claims 60, 69, or 75, wherein the non-primate AAV is sea lion AAV.

77. Recombinant AAV particles according to any one of claims 60, 69, 75, or 76, comprising a modified AAAV VP1 capsid, wherein the AAV capsid comprises the first member of the protein:protein binding pair, which optionally links to an amino acid via a linker at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and I565.

78. Recombinant AAV particle according to any one of claims 37 to 77, wherein the AAV capsid is a mosaic AAV capsid, and optionally the mosaic AAV capsid comprises a plurality of first reference capsid proteins that do not include the first member of a protein:protein binding pair, and a plurality of second capsid proteins, each of which contains the first member of a protein:protein binding pair, and optionally the mosaic AAV capsid does not include the first plurality of reference capsid proteins and the first plurality of second capsid proteins, each of which contains the first component of a protein:protein binding pair, and optionally the mosaic AAV capsid comprises the plurality of first reference capsid proteins and the plurality of second capsid proteins in a ratio of 1:

7.

79. Recombinant AAV particles according to any one of claims 37 to 78, wherein the targeted ligand is an antibody or a part thereof.

80. The recombinant AAV particle according to any one of claims 37 to 79, further comprising the target nucleotide encapsulated within the AAV capsid.

81. The recombinant AAV particle according to claim 80, wherein the target nucleotide is a reporter gene.

82. Recombinant AAV particles according to claim 80 or 81, wherein the target nucleotide encodes β-galactosidase, green fluorescent protein (GFP), highly sensitive green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), highly sensitive blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), highly sensitive yellow fluorescent protein (eYFP), Emerald, CyPet, cyanide fluorescent protein (CFP), Cerulea, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

83. The recombinant AAV particle according to claim 80, wherein the target nucleotide encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

84. The recombinant AAV particle according to any one of claims 37 to 83, wherein the targeted ligand comprises an antigen-binding protein according to any one of claims 1 to 16, and optionally the targeted 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.

85. A pharmaceutical composition comprising (a) recombinant AAV particles according to any one of claims 37 to 84, and (b) a pharmaceutically acceptable carrier or excipient.

86. A method for delivering a target nucleotide to a mammalian muscle cell, comprising contacting the mammalian muscle cell with (a) recombinant AAV particles according to any one of claims 37 to 84, or (b) the pharmaceutical composition according to claim 85. A method for which the aforementioned mammalian muscle cells express surface proteins specific to mammalian muscle.

87. The method according to claim 86, wherein the contact is performed exvivo.

88. The method according to claim 86, wherein the contact is performed on a subject, and optionally the subject is modified to express a targeted ligand, for example, from a safe harbor locus.

89. The method according to claim 88, wherein the subject is a primate, preferably a human.

90. The method according to any one of claims 86 to 89, wherein the mammalian muscle cell is a mammalian skeletal muscle cell, and optionally, the mammalian skeletal muscle cell is not terminally differentiated, and optionally, the mammalian skeletal muscle cell comprises one or more cells selected from the group consisting of muscle stem cells, myoblasts, and muscle cells.

91. The method according to any one of claims 86 to 90, wherein the mammalian muscle cell-specific surface protein is CDH15.

92. The method according to any one of claims 86 to 91, wherein the target nucleotide encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

93. A method for doing so in patients who require treatment for muscle wasting or hereditary muscle disease, The patient is given either (a) recombinant AAV particles according to any one of claims 37 to 84, or (b) the pharmaceutical composition according to claim 85. The recombinant AAV particle contains the target nucleotide capsidized within the viral capsid, A method for encoding a target nucleotide that encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

94. Use of recombinant AAV particles or compositions according to any one of claims 37 to 84, or the pharmaceutical composition according to claim 85, for the manufacture of a pharmaceutical for the treatment of muscle wasting or hereditary muscle disease.

95. The method according to claim 93 or the use according to claim 94, wherein the muscle wasting or hereditary muscle disease is selected from the group consisting of 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, and dystroglycanopathy.

96. A method for performing treatment in patients who require treatment for rhabdomyosarcoma, The patient is given either (a) recombinant AAV particles according to any one of claims 37 to 84, or (b) the pharmaceutical composition according to claim 85. The recombinant AAV particles contain the target nucleotide capsidized within the AAV capsid. A method for encoding a target nucleotide that encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

97. Use of recombinant AAV particles according to any one of claims 37 to 84, or the pharmaceutical composition according to claim 85, in the manufacture of a pharmaceutical product for the treatment of rhabdomyosarcoma.

98. The method according to claim 96 or the use according to claim 97, wherein the rhabdomyosarcoma is selected from the group consisting of embryonic, alveolar, pleomorphic, botryoidal, and spindle / tonic rhabdomyosarcoma.

Images