Treatment of muscle-related disorders with anti-human CACNG1 antibodies

Abstract

Adeno-associated virus (AAV) particles are provided that are redirected with antibodies against CACNG1 and carry a therapeutic nucleotide of interest. Also provided are methods of making and using AAV particles, e.g., for treating patients in need of treatment or for manufacturing a medicament therefor. Described herein are viral vectors (e.g., adeno-associated virus (AAV) vectors) that are retargeted with antibodies and antigen-binding fragments thereof that bind to human CACNG1. The retargeted AAV vectors described herein may be particularly useful for specifically directing the internalization of nucleotides, e.g., nucleotides encoding therapeutic proteins, to skeletal muscle cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 484,675, filed February 13, 2023, U.S. Provisional Patent Application No. 63 / 494,119, filed April 4, 2023, and U.S. Provisional Patent Application No. 63 / 583,724, filed September 19, 2023, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Reference to sequence listing submitted as an XML file The sequence listing in xml format, entitled "11459WO01 Sequence Listing XML," was created on February 8, 2024, is 280 Kb, and is incorporated herein by reference in its entirety.

[0003] The present application is generally directed to human antibodies and antigen-binding fragments of human antibodies that bind to human CACNG1 (hCACNG1), and methods of using them, for example, in methods of treating disorders in patients in need thereof. The application also relates to antigen-binding molecules comprising at least an antigen-binding fragment of an anti-hCACNG1 antibody, wherein complex formation of the antigen-binding molecule with CACNG1 mediates internalization of the antigen-binding molecule / CACNG1 complex. The application further relates to anti-hCACNG1 antibody (or antigen-binding molecule comprising an antigen-binding fragment of an anti-hCACNG1 antibody)-conjugated viral vectors comprising a therapeutic nucleotide of interest, which conjugates may be useful for treating muscle-related disorders. [Background technology]

[0004] Skeletal muscle is the largest organ in the body, accounting for approximately 40% of total body weight. Skeletal muscle is one of the three major muscle tissues in the human body. Each skeletal muscle is composed of thousands of muscle fibers wrapped together by connective tissue sheaths. The individual bundles of muscle fibers in a skeletal muscle are known as fasciculi. The outermost connective tissue sheath that surrounds the entire muscle is known as the epimysium. The connective tissue sheath that covers each fascia is known as the perimysium, and the innermost sheath that surrounds individual muscle fibers is known as the endomysium. Each muscle fiber is composed of many myofibrils, which contain multiple myofilaments.

[0005] When bundled together, all myofibrils are arranged in a unique cross-striated pattern to form the sarcomere, the basic contractile unit of skeletal muscle. The two most important myofilaments are actin and myosin filaments, which are characteristically arranged to form various bands on skeletal muscle.

[0006] The primary function of skeletal muscle is carried out through its intrinsic excitation-contraction coupling process. Because muscles are attached to bone tendons, muscle contraction causes bone movement, enabling specific movements to be performed. 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 serves as a site for glucose disposal 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 an important role in locomotion, thermoregulation, and the control of whole-body metabolism.

[0007] Many muscle diseases, as well as normal aging, result in a decline in the size and function of skeletal muscle tissue, impairing functional mobility and, in severe muscle diseases, leading to long-term disability and premature death.

[0008] Treatments for muscle wasting and genetic muscle diseases typically consist of broad-spectrum therapies, such as corticosteroid therapy for muscle wasting and glucocorticoids for muscular dystrophies. Non-targeted delivery of these therapies reduces the efficiency of specific muscle uptake while also causing significant adverse off-target effects in other organs.

[0009] There is a need in the art for new anti-human antibodies that can bind to muscle-specific markers and result in internalization of therapeutic payloads by muscle cells. Summary of the Invention [Means for solving the problem]

[0010] Described herein are viral vectors (e.g., adeno-associated viral (AAV) vectors) retargeted with antibodies and antigen-binding fragments thereof that bind to human CACNG1. The retargeted AAV vectors described herein can be particularly useful for specifically directing the internalization of nucleotides, such as nucleotides encoding therapeutic proteins, to skeletal muscle cells.

[0011] The viral particles described herein are particularly suitable for targeted introduction of nucleotides into muscle cells because the viral capsids or viral capsid proteins described herein comprise a targeting ligand that binds to a muscle cell-specific surface protein. In some embodiments, the viral capsid or viral capsid protein comprises a first member of a binding pair associated with its cognate second member of the binding pair, where the second member is linked (e.g., fused) to a targeting ligand that binds to the muscle cell-specific surface protein. In some embodiments, the targeting ligand is operably linked to the second member, e.g., fused to the second member, optionally via a linker. In some embodiments, the targeting ligand may be a binding moiety, such as a natural ligand, an antibody, a multispecific binding molecule, or the like. In some embodiments, the targeting ligand is an antibody or portion thereof. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a muscle-specific surface protein on a muscle cell and a heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a muscle-specific surface protein on a target cell and an IgG heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a muscle-specific surface protein on a target cell and an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked, e.g., via a linker, to a protein (e.g., a second member of a protein:protein binding pair) that forms an isopeptide covalent bond with the first member. In some embodiments, the capsid protein described herein comprises a first member that comprises a SpyTag operably linked to a viral capsid protein and is covalently linked to the SpyTag, and a second member that comprises a SpyCatcher linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein the SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, such as, for example, GSGESG (SEQ ID NO: 253). In some embodiments, the muscle-specific surface protein comprises CACNG1.In some embodiments, the targeting ligand binds to CACNG1, e.g., human CACNG1. In some embodiments, the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising 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 set forth in any one of SEQ ID NOs: 1-240.

[0012] In some embodiments, the viral particles described herein, e.g., AAV particles retargeted with an anti-CACNG1 antibody or fragment thereof described herein, comprise a nucleotide, e.g., a nucleotide of interest. In some embodiments, the nucleotide of interest is a reporter gene. In some embodiments, the nucleotide of interest encodes microdystrophin, e.g., human microdystrophin, e.g., for use in methods of treating Duchenne muscular dystrophy or a model thereof and / or for use in the manufacture of a medicament for treating Duchenne muscular dystrophy or a model thereof. In some embodiments, the nucleotide of interest encodes fukutin-related protein (FKRP), e.g., human FKRP, e.g., for use in methods of treating limb-girdle muscular dystrophy or a model thereof and / or for use in the manufacture of a medicament for treating limb-girdle muscular dystrophy or a model thereof. In some embodiments, the nucleotide of interest encodes, for example, myotubularin (MTM1), e.g., human MTM1, for use in a method of treating a myotubular myopathy or a model thereof, and / or for use in the manufacture of a medicament for treating a myotubular myopathy or a model thereof.

[0013] An exemplary nucleotide molecule of interest described herein may comprise, for example, the sequence set forth as SEQ ID NO: 270 for use in a method of treating Duchenne muscular dystrophy or a model thereof and / or for use in the manufacture of a medicament for treating Duchenne muscular dystrophy or a model thereof. An exemplary nucleotide molecule of interest described herein may comprise, for example, the sequence set forth as SEQ ID NO: 271 for use in a method of treating limb-girdle muscular dystrophy or a model thereof and / or for use in the manufacture of a medicament for treating limb-girdle muscular dystrophy or a model thereof. An exemplary nucleotide molecule of interest described herein may comprise, for example, the sequence set forth as SEQ ID NO: 272 for use in a method of treating myotubular myopathy or a model thereof and / or for use in the manufacture of a medicament for treating myotubular myopathy or a model thereof.

[0014] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0015] [Figure 1A]In vitro and ex vivo evaluation of CACNG1 antibody properties is shown. Mouse and human myotubes were used as an in vitro muscle model to assess cell binding (Figures 1A-1B) and internalization (Figure 1C) of CACNG1 antibodies. Live myotubes were incubated with anti-CACNG1 antibodies followed by fluorophore-conjugated secondary detection to assess antibody binding (Figures 1A-1B). Myotubes were incubated with anti-CACNG1 antibodies described herein, such as REGN7854, and other anti-CACNG1 antibodies, followed by duocarmycin-conjugated secondary (2°Ab-cytotoxic drug) detection to assess antibody internalization via a cell killing assay (Figure 1C). Immunostaining with anti-CACNG1 antibody in single myofibers from live CACNG1Hu / Hu mice (Fig. 1D, left panel) and in cross sections of unfixed muscle tissue (Fig. 1D, right panel) shows the localization of CACNG1 on the myofiber cell surface. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 2] Figure 1 provides data on human myotube acetylcholine-induced calcium flux (relative light units, y-axis) after incubation with different concentrations (0.01 μM, 0.1 μM, 1 μM, and 10 μM, x-axis) of anti-hCACNG1 antibodies (REGN5972, REGN10728, or H2aM31944N), isotype control antibodies (REGN3892, REGN1945, or REGN1097), or 20 μM nicardipine as a positive control for calcium blockade. The anti-hCACNG1 antibodies tested here do not inhibit acetylcholine-induced calcium flux in human myotubes at these concentrations. [Figure 3]Fluorescence immunohistochemistry images taken at 20x magnification of single ex vivo myofibers isolated from wild-type ("WT") mice, mice homozygous for deletion of CACNG1 ("KO"), or mice expressing only human CACNG1 ("CACNG1Hu / Hu") were incubated with anti-human CACNG1 antibody (H1M31941N or REGN5972) or isotype control antibody (REGN653 or REGN1945), and labeled with a fluorescently conjugated secondary antibody. The CACNG1 antibody bound to CACNG1Hu / Hu myofibers, but the isotype control antibody did not. [Figure 4] Figure 1 provides single-plane confocal fluorescence immunohistochemistry images taken at 20x magnification of single ex vivo myofibers isolated from wild-type ("WT") mice, mice homozygous for deletion of CACNG1 ("KO"), or mice expressing only human CACNG1 ("CACNG1Hu / Hu"), after 30 minutes, 4 hours, or 8 hours of incubation with an anti-human CACNG1 antibody (REGN10728) or an isotype control antibody (REGN4439) conjugated with the Alexa647 (A647) fluorophore. Confocal imaging revealed that the fluorophore-conjugated CACNG1 antibody bound to the surface of CACNG1Hu / Hu myofibers after 30 minutes of incubation, and that a portion of the CACNG1 antibody was internalized and detected within the myofibers by 4 and 8 hours of incubation. No binding or internalization into CACNG1Hu / Hu muscle fibers was detected with a fluorophore-conjugated isotype control antibody. [Figure 5]The level of androgen receptor (AR) activation is shown in relative light units (RLU, y-axis) after 24 hours of incubation of an LNCaP cell line modified to express luciferase upon androgen receptor activation (AR.Luc) with dihydrotestosterone (DHT) alone (M608; unconjugated DHT), an anti-hCACNG1 antibody (REGN14570, REGN14571, REGN14572, REGN14573, REGN14574, or REGN14647) conjugated to DHT (M3004) via a VC-PAB linker, or an anti-FelD isotype control antibody (REGN3892) conjugated to DHT (M3004) via a VC-PAB linker at various concentrations (Log[concentration (M)], x-axis). This assay showed that only unconjugated DHT activated the androgen receptor, whereas none of the CACNG1 antibodies conjugated to DHT showed any appreciable activation of the androgen receptor in this cell line, which does not express hCACNG1. [Figure 6A]The hCACNG1-expressing LNCaP cell line (hCACNG1.AR.Luc), which was modified to also express luciferase upon androgen receptor activation, was incubated with dihydrotestosterone (DHT) alone (M608; unconjugated DHT), anti-hCACNG1 antibodies conjugated to DHT via a VC-PAB linker (M3004) (REGN14570, REGN14571, REGN14572, REGN14573, REGN14574). Figures 6A-6C, 6D-6F, and 6G-6I show the levels of androgen receptor (AR) activation in relative light units (RLU, y-axis) after incubation with various concentrations (Log[concentration (M)]; x-axis) of either CACNG1 antibody-DHT (REGN14574 or REGN14647) or an anti-FelD isotype control antibody (REGN3892) conjugated to DHT (M3004) via a VC-PAB linker for 24 hours (Figures 6A-6C), 48 hours (Figures 6D-6F), and 72 hours (Figures 6G-6I). Several CACNG1 antibody-DHT conjugates activated the androgen receptor in this hCACNG1-expressing cell line. Although the efficacy and potency of androgen receptor activation were lower than those of unconjugated DHT at 24 hours posttreatment, androgen receptor activation persisted for 48 and 72 hours compared to unconjugated DHT. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above. [Figure 6H] Same as above. [Figure 6I] Same as above. [Figure 7] 1 provides cryofluorescence tomography images of mice 6 days after systemic injection of 10 mg / kg of Alexa647-conjugated anti-hCACNG1 antibody (REGN10728 or REGN5972) or Alexa647-conjugated isotype control antibody (REGN4439). [Figure 8A]These figures show tile fluorescence immunohistochemistry images taken at 20x magnification of sections of the gastrocnemius / plantaris / plantaris / soleus (Figure 8A), tibialis anterior (Figure 8B), diaphragm (Figure 8C), tongue (Figure 8D), triceps (Figure 8E), trapezius (Figure 8F), or pelvic floor muscle (Figure 8G) from mice expressing only human CACNG1 ("CACNG1Hu / Hu") that were sacrificed 6 days after tail vein injection of 10 mg / kg of anti-human CACNG1 antibody (REGN5972 or REGN10728) or isotype control antibody (REGN4439) conjugated with Alexa647 (A647) fluorophore. Fluorophore-conjugated CACNG1 antibodies were detected in all of these skeletal muscles, with REGN10728 exhibiting a stronger signal in muscle compared to REGN5972. Only low levels of fluorescence were detected in muscles from isotype control and saline-injected mice. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above. [Figure 9A] Figure 9 shows tiled fluorescence immunohistochemistry images taken at 20x magnification of liver (Figure 9A), kidney (Figure 9B), spleen (Figure 9C), or brown adipose tissue (Figure 9D) sections from mice expressing only human CACNG1 (CACNG1Hu / Hu) after tail vein injection of 10 mg / kg of anti-human CACNG1 antibodies (REGN5972 or REGN10728) conjugated with Alexa647 (A647) fluorophore or an isotype control antibody (REGN4439). The mice were sacrificed 6 days after injection. The fluorophore-conjugated CACNG1 antibody did not show significant signal in these organs, and Alexa647 levels were similar to those of isotype and saline-injected controls. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10] A schematic diagram showing an exemplary experimental timeline (top panel) and photomicrographs (bottom panel) showing CACNG1 antibody distribution to the soleus muscle under sedentary and exercise conditions at either 10 mg / kg or 50 mg / kg (high dose) are provided. CACNG1 distribution varies with exercise and dose. [Figure 11] Cryofluorescence tomography images are provided of mouse models of Duchenne muscular dystrophy (D2-mdx), limb-girdle muscular dystrophy (FkrpP448L), or myotubular myopathy (MTM1 KO) that were sacrificed 2 weeks after systemic injection of 5 × 10 12 viral genomes / kg of wild-type AAV9 particles expressing eGFP under the control of the CAG promoter, or AAV9 particles containing the W503A mutation (REGN10717) retargeted with an anti-hCACNG1 antibody. [Figure 12A] FIG. 1 provides a schematic diagram of treatment of D2-mdx mice with an AAV expressing a nucleotide of interest encoding microdystrophin (μDys) under the control of the CK8 promoter. [Figure 12B] Levels of μDys mRNA expressed in the quadriceps, gastrocnemius, diaphragm, and liver of D2-mdx mice injected with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing the nucleotide of interest encoding μDys, or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing the nucleotide of interest encoding μDys are provided (y-axis, compared to levels in mice injected with WT AAV9). [Figure 12C]The left panel shows (i) Western blots detecting μDys or β-actin from the quadriceps muscles of D2-mdx mice injected with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing a μDys-encoding nucleotide of interest, or AAV9 particles with the N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) containing a μDys-encoding nucleotide of interest, and (ii) graphs presenting protein abundance levels. The right panel of Figure 12C shows immunohistochemistry images of the gastrocnemius muscles of untreated wild-type (WT) or D2-mdx mice, or after injection with wild-type (WT) AAV9 particles containing a μDys-encoding nucleotide of interest, or AAV9 particles with the N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) containing a μDys-encoding nucleotide of interest, taken after staining for dystrophin. [Figure 12D] The left panel provides the percent change in serum creatine kinase (CK) (compared to baseline levels before injection) 4 weeks after injection of D2-mdx mice with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing a nucleotide of interest encoding μDys, or AAV9 particles with the N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding μDys. The right panel of Figure 12D provides the maximum grip strength (grams, y-axis) 12 weeks after injection of D2-mdx mice with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing a nucleotide of interest encoding μDys, or AAV9 particles with the N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding μDys. [Figure 13A] FIG. 1 provides a schematic diagram of treatment of FkrpP448L mice with an AAV expressing a nucleotide of interest encoding human FKRP (hFKRP) under the control of the CK7 promoter. [Figure 13B]Levels of hFKRP mRNA expressed in the quadriceps, gastrocnemius, diaphragm, and liver of FkrpP448L mice injected with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing a nucleotide of interest encoding hFKRP, or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding hFKRP are provided (y-axis, compared to levels in mice injected with WT AAV9). [Figure 13C] The left panel shows immunohistochemical images of the diaphragm of untreated wild-type (WT) or FkrpP448L mice, or after injection of phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing a nucleotide of interest encoding hFKRP, or AAV9 particles with the N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding hFKRP, after incubation with IIH6 (which stains glycosylated α-dystroglycan), laminin, and DAPI. The right panel of Figure 13C shows the intensity of IIH6 (y-axis) in these animals in arbitrary units (upper graph) or as a percentage of the area within the laminin region (lower graph). [Figure 13D] The maximum treadmill distance (meters, y-axis) run by FkrpP448L mice 7 weeks after injection with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing a nucleotide of interest encoding hFKRP, or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding hFKRP is provided. [Figure 14A] FIG. 1 provides a schematic diagram of treatment of MTM1 knockout (KO) mice with an AAV expressing a nucleotide of interest encoding human MTM1 (hMTM1) under the control of the desmin promoter. [Figure 14B]Levels of hMTM1 mRNA expressed in the quadriceps, gastrocnemius, diaphragm, and liver of MTM1 KO mice injected with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing a nucleotide of interest encoding hMTM1, or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding hMTM1 are provided (y-axis, compared to levels from mice injected with WT AAV9). [Figure 14C] The left panel provides immunohistochemistry images of soleus muscles from untreated wild-type (WT) or MTM1 KO mice, or after injection of wild-type (WT) AAV9 particles containing a nucleotide of interest encoding hMTM1, or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding hMTM1, after incubation with laminin and DAPI. The right panel of Figure 14C provides the percentage of surviving MTM1 KO mice (up to 60 days) injected at day 32 with PBS, wild-type (WT) AAV9 particles containing a nucleotide of interest encoding hMTM1, or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding hMTM1. [Figure 15] Images of cardiac GFP expression in FkrpP488L mice after injection of wild-type AAV9 particles or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) expressing a nucleotide of interest encoding hFKRP under the control of the CK7 promoter (left panel) are provided; as well as the levels of hFKRP mRNA in the hearts of FkrpP488L mice after injection of PBS, wild-type AAV9 particles containing a nucleotide of interest encoding hFKRP under the control of the CK7 promoter, or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing a nucleotide of interest encoding hFKRP (right panel). [Figure 16]We provide the rationale and study protocol to determine whether cardiac transduction by AAV9 particles can be preserved with robust skeletal muscle retargeting by conjugating CACNG1 antibodies to non-detargeted AAV9 capsids. [Figure 17A] Figures 17A-17C show images of the liver, quadriceps, or heart of C57BL / 6 healthy mice (Figure 17A) or D2-mdx mice (Figures 17B-17C) after injection of wild-type AAV9 particles encapsulating a nucleotide of interest encoding eGFP under the control of a CAG promoter, AAV9 particles retargeted with an anti-hCACNG1 antibody (REGN10717) and carrying a detargeting mutation (e.g., W503A) encapsulating a nucleotide of interest encoding eGFP under the control of a CAG promoter, or WT AAV9 particles (without a detargeting mutation) retargeted with an anti-hCACNG1 antibody (REGN10717) and encapsulating a nucleotide of interest encoding eGFP under the control of a CAG promoter at different doses of 2 x 10 vg / mouse (high), 4 x 10 vg / mouse (medium), or 8 x 10 vg / mouse (low). Figure 17C shows the same tissue as Figure 17B, but at a higher magnification. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 18A]Wild-type AAV9 particles (AAV WT) encapsulating a nucleotide of interest encoding eGFP under the control of the CAG promoter, WT AAV9 particles (without detargeting mutations) retargeted with anti-hCACNG1 antibody (REGN10717) encapsulating a nucleotide of interest encoding eGFP under the control of the CAG promoter (AAV WT + anti-CACNG1), and AAV9 particles containing a detargeting mutation (e.g., W503A) and retargeted with hCACNG1 antibody (REGN10717) encapsulating a nucleotide of interest encoding eGFP under the control of the CAG promoter (AAV Expression levels of GFP mRNA (y-axis) relative to the housekeeping gene Rplp0 are shown in the liver, heart, or quadriceps muscle of C57BL / 6 healthy mice (FIG. 18A) or D2-mdx mice (FIG. 18B) after injection of 2×10 vg / mouse (high), 4×10 vg / mouse (medium), or 8×10 vg / mouse (low) with either W503A+anti-CACNG1 or phosphate-buffered saline (PBS) (x-axis). [Figure 18B] Same as above. [Figure 19A] Wild-type AAV9 particles (AAV WT) encapsulating a nucleotide of interest encoding eGFP under the control of the CAG promoter, WT AAV9 particles (without detargeting mutations) retargeted with anti-hCACNG1 antibody (REGN10717) encapsulating a nucleotide of interest encoding eGFP under the control of the CAG promoter (AAV WT + anti-CACNG1), and AAV9 particles containing a detargeting mutation (e.g., W503A) and retargeted with anti-hCACNG1 antibody (REGN10717) encapsulating a nucleotide of interest encoding eGFP under the control of the CAG promoter (AAV Figure 19A shows the expression levels of GFP mRNA (y-axis) relative to the housekeeping gene Rplp0 in the gastrocnemius, quadriceps, diaphragm, soleus, tibialis anterior, or tongue of C57BL / 6 healthy mice (Figure 19A) or D2-mdx mice (Figure 19B) after injection of 2 x 10 vg / mouse (high), 4 x 10 vg / mouse (medium), or 8 x 10 vg / mouse (low) with either W503A + anti-CACNG1 or phosphate-buffered saline (PBS) (x-axis). [Figure 19B] Same as above. [Figure 20-1] We provide an exemplary schematic for refining the detargeting and retargeting of AAV9 viral particles by engineering the AAV capsid, retargeting antibodies, or both. This modular design provides flexibility for adjusting the degree of detargeting, and the addition of antibodies directs viral particles to new tissues and cell types that can be fine-tuned for the treatment of specific diseases. [Figure 20-2] Same as above. [Figure 21A] An exemplary schematic diagram (not to scale) of the single-stranded (ss) viral genome is provided, which includes, in the 5' to 3' direction, a 141 base pair inverted terminal repeat (ITR), a CAGG promoter, a sequence encoding enhanced green fluorescent protein (GFP), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a 32 base pair barcode, a human (h) growth hormone (GH) polyA tail, and the 141 base pair ITR. [Figure 21B]Figure 21A provides a bar graph showing enhanced transduction of various muscles in vivo in non-human primates (cynomolgus monkeys) after administration of AAV9 viral particles (e.g., containing capsid mutations such as N272A, W503A, etc.) containing the viral genome shown in Figure 21A, each bearing a unique barcode, and retargeted with an anti-CACNG1 antibody, compared to wild-type AAV9 viral particles (AAV9) containing the viral genome shown in Figure 21A. Each candidate AAV was packaged with a unique barcoded genome as described in Figure 21A. After IV administration of the 12 candidate barcoded pools, the indicated tissues were collected, and the relative abundance of each barcode in total RNA purified from each tissue was assessed using next-generation sequencing (NGS). The percentage of NGS reads (y-axis) that mapped to each barcode and associated capsid in several tissues (x-axis) is shown (normalized to the injected virus pool): liver (lateral left lobe) and a subset of skeletal muscles (diaphragm, biceps brachii, biceps femoris, extensor digitorum longus (EDL), gastrocnemius, intercostal muscles, soleus, tibialis anterior, transverse abdominus, triceps, vastus lateralis, psoas, and tongue). Data shown here are the average of two animals in this study. [Figure 21C] Figure 21B shows that systemically delivered detargeted AAV9 conjugated to anti-CACNG1 exhibits antibody-dependent transduction of skeletal muscle in non-human primates, where the data in Figure 21B are plotted as relative mRNA expression compared to wild-type AAV9 expression (y-axis) and show enhanced transduction of the diaphragm, psoas, triceps, and interfibular muscles with anti-CACNG1 antibodies #3 and #5. [Figure 22A]Serum levels of liver enzymes (ALT; FIG. 22A) and complement pathway biomarkers (sC5b-9; FIG. 22B), as well as markers of thrombotic microangiopathy (platelet count; FIG. 22C), are shown at the indicated time points (x-axis) following injection of 2×10 vg / kg of wild-type AAV9 or AAV9 W503A expressing eGFP under the control of the CAG promoter in non-human primates (cynomolgus monkeys) that were either seropositive (serum(+)) or seronegative (serum(-)) for AAV9. Administration of wild-type AAV9 particles resulted in an increase in ALT (FIG. 22A), an increase in sC5b-9, a marker of the complement terminal membrane attack complex (FIG. 22B), and a decrease in platelet count (FIG. 22C), as expected, whereas administration of AAV9 Administration of W503A particles resulted in ALT levels (Figure 22A), sC5b-9 levels (Figure 22B), and platelet counts (Figure 22C) similar to those of cynomolgus monkeys receiving saline alone (negative control). These data suggest that liver-detargeted AAV9 W503A particles offer a safety advantage over liver-tropic wild-type AAV serotypes. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 23A] Figure 23A shows the degree of thrombocytopenia (Fig. 23A, platelet count), hemolytic anemia (Fig. 23B, red blood cell distribution width), and renal filtration impairment (Fig. 23C, serum creatinine) as markers of the thrombotic microangiopathy (TMA) triad at the indicated time points (x-axis) after injection of wild-type AAV9 or AAV9 W503A expressing eGFP under the control of the CAG promoter in non-human primates (cynomolgus monkeys) that were either seropositive (serum (+)) or seronegative (serum (-)) for AAV9. A decrease in platelet count suggests transient thrombocytopenia, an increase in red blood cell distribution width, a marker of schistocytes, suggests mild, transient hemolytic anemia, and an increase in serum creatinine level, a marker of renal filtration impairment, suggests transient acute kidney injury. Monkeys administered wild-type AAV9, but not AAV9 W503A, exhibit some symptoms of the TMA triad. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 24A] 24A and 24B are line graphs (Figure 24A and 24B) showing serum creatine kinase levels in wild-type mice treated with PBS (50500 vehicle) and mice treated with various doses (4E12vg / kg, 1E13vg / kg, and 5E13vg / kg) of AAV9 particles containing N272A, a P448L point mutation in fukutin-associated protein (FKRP) as a model of limb-girdle muscular dystrophy type 2I (FKRPP448L / P448L), humanized laminin subunit alpha 2 (LAMA2; Lama2HU / HU), and humanized dystroglycan 1 (DAG1; DAG1HU / HU), retargeted with anti-hCACNG1 antibody (REGN10717), and encapsulating a nucleotide of interest encoding human FKRP (hFKRP) under the control of the CK7 promoter. [Figure 24B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0016] Provided herein are novel anti-human CACNG1 antibodies and monovalent antigen-binding fragments thereof useful for mediating the internalization of CACNG1. The anti-human CACNG1 antibodies and monovalent antigen-binding fragments thereof may be useful, for example, in the treatment of disease, as part of multispecific antigen-binding proteins and / or multidomain therapeutic proteins, and / or as antibody-drug conjugates.

[0017] The description herein is not limited to the specific embodiments, compositions, methods, and experimental conditions described, as such embodiments, compositions, methods, and conditions may vary, and the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0018] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing described herein, some preferred methods and materials are described here. All publications cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0019] The term "about," when used in reference to a specific recited numerical value, means that the value may vary by no more than 1% from the recited value. For example, the phrase "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0020] Voltage-gated calcium channels generally consist of five subunits. The protein encoded by the CACNG1 gene represents one of these subunits. The term "CACNG1" includes the protein encoded by the CACNG1 gene, one of two known gamma-subunit proteins. CACNG1 is part of the skeletal muscle 1,4-dihydropyridine-sensitive calcium channel and is an integral membrane protein that plays a role in excitation-contraction coupling. CACNG1 is part of the functionally diverse eight-member protein subfamily of the PMP-22 / EMP / MP20 family, clustered with two family members that function as transmembrane AMPA receptor regulatory proteins (TARPs). CACNG1 is highly and specifically expressed in skeletal muscle. The gene encoding human CACNG1 (CACNG1) is located on the long arm of chromosome 17. CACNG1 contains four exons and is approximately 12,244 bases long. An exemplary sequence of the human CACNG1 gene has been assigned NCBI accession number NM_0007582.2 (SEQ ID NO: 241). An exemplary human CACNG1 protein has been assigned UniProt accession number O70578 (SEQ ID NO: 242).

[0021] The phrase "antibody that binds to CACNG1" or "anti-hCACNG1 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize a single CACNG1 molecule. The antibodies and antigen-binding fragments thereof described herein can bind to soluble CACNG1 and / or cell surface-expressed CACNG1. Soluble CACNG1 includes native CACNG1 protein as well as recombinant CACNG1 protein variants that lack the transmembrane domain or are otherwise not associated with the cell membrane.

[0022] The phrase "cell surface-expressed CACNG1" refers to one or more CACNG1 proteins that are expressed on the surface of a cell in vitro or in vivo, and at least a portion of the CACNG1 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 CACNG1" may include or consist of a CACNG1 protein that is expressed on the surface of a cell that normally expresses CACNG1 protein. Alternatively, "cell surface-expressed CACNG1" may include or consist of a CACNG1 protein that is expressed on the surface of a cell that does not normally express human CACNG1 on its surface but has been artificially engineered to express CACNG1 on its surface.

[0023] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies.

[0024] The term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CACNG1). As used herein, the term "antibody" includes immunoglobulin molecules containing four polypeptide chains, 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 regions of hypervariability, 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 from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibodies refers to antibodies whose binding affinity to their target is at least 10 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -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. Furthermore, antibodies 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 can even be a human monoclonal antibody.

[0025] The term "antibody" also includes antigen-binding fragments of intact antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically produced, 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 intact antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0026] 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 mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment."

[0027] Antigen-binding fragments of antibodies typically contain at least one variable domain, which 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 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 suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may also include a domain.

[0028] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragments of antibodies described herein include: (i) a V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any 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 may be linked by a full or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies described herein may be linked to each other and / or to one or more monomeric V H or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association (e.g., via disulfide bonds) of the domains.

[0029] Like intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, can be adapted for use in connection with the antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0030] In certain embodiments, the anti-hCACNG1 antibodies described herein are human antibodies. The term "human antibody" refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0031] 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, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, 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 in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thereby increasing the V of the recombinant antibody. H Area and V L The amino acid sequence of the region is human germline V H Sequence and V LWhile derived from and related to sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0032] Human antibodies can exist in two general forms related to hinge heterogeneity. In one general form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, where the dimers are held together by interchain heavy chain disulfide bonds. In the second general form, the dimers are not linked via interchain disulfide bonds, and the approximately 75-80 kDa molecule is composed of covalently linked light and heavy chains (half-antibody). These forms have been very difficult to separate, even after affinity purification.

[0033] The frequency of occurrence of the second form in various intact IgG isotypes depends on, but is not limited to, structural differences associated with the antibody hinge region isotype. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The antibodies described herein contain a hinge, C H Area 2 or C H One or more mutations in the three regions may be present, which may be desirable, for example, to improve the yield of the desired antibody form in production.

[0034] An antibody described herein may be an isolated antibody. An "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, may be considered an "isolated antibody." An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0035] Also described herein are one-arm antibodies that bind to CACNG1. 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 comprise any of the HCVR / LCVR or CDR amino acid sequences listed in Table 1.

[0036] The anti-hCACNG1 antibodies discussed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. Also described herein are antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences described herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies described herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue 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 antagonist or agonist biological properties (in some cases), reduced immunogenicity, etc. In some embodiments, the antibodies or antigen-binding fragments described herein are obtained in this general manner.

[0037] Also described herein are anti-hCACNG1 antibodies containing variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, with one or more conservative substitutions. For example, some embodiments include anti-hCACNG1 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences listed in Table 1 herein.

[0038] "Biologically equivalent portions," "biologically equivalent variants," and the like, of a reference nucleic acid sequence or polypeptide sequence disclosed herein include sequences that exhibit similar biological activity as the reference nucleic acid sequence or reference polypeptide sequence. Biologically equivalent portions or variants of a reference nucleic acid sequence include shorter nucleic acids than the reference nucleic acid that encode either the same polypeptide as that encoded by the reference nucleic acid sequence or a polypeptide that exhibits the same biological activity as the polypeptide encoded by the reference nucleic acid. The term "portion" refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, that has at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the sequence from which it is derived. A "portion" encompasses any contiguous segment of amino acids or nucleotides sufficient to determine the reference polypeptide or nucleic acid molecule from which it is derived. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 10 amino acids or 30 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 15 amino acids or 45 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 20 amino acids or 60 nucleotides that have 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 25 amino acids or 75 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence.In some embodiments, the portion comprises at least 30 amino acids or 90 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 35 amino acids or 105 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 40 amino acids or 120 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 45 amino acids or 135 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 50 amino acids or 150 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 60 amino acids or 180 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 70 amino acids or 210 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 80 amino acids or 240 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the reference polypeptide or nucleic acid sequence. In some embodiments, the portion comprises at least 100 amino acids or 300 nucleotides that have at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to a reference polypeptide or nucleic acid sequence.

[0039] In a non-limiting example, biologically equivalent variants of the nucleic acid sequences disclosed herein can be developed through codon optimization of the nucleic acid sequence. "Codon optimization" takes advantage of codon degeneracy, as seen in the multiplicity of three-base pair codon combinations that define amino acids. This typically involves modifying a nucleic acid sequence to enhance expression in a particular host cell by replacing at least one codon in the native sequence with a codon that is more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. For example, a nucleic acid encoding a Cas9 protein can be modified to replace a codon with a more frequently used codon compared to the native nucleic acid sequence in a given prokaryotic or eukaryotic cell, including, for example, bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host cell. Codon usage tables are readily available, for example, from the "Codon Usage Database." These tables can be adapted in many ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, which is incorporated by reference in its entirety for all purposes. Computer algorithms are also available for codon optimization of particular sequences for expression in particular hosts (see, e.g., Gene Forge). One of skill in the art will understand that the nucleic acid sequences disclosed herein encompass variants thereof, including variants that differ due to degeneracy and / or codon optimization of the genetic code and encode the same or substantially similar amino acid sequences of biologically equivalent polypeptides.

[0040] The phrase "bispecific antibody" includes antibodies that can selectively bind two or more epitopes. Bispecific antibodies typically contain two different heavy chains, each of which specifically binds a different epitope—either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least one to two, or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same target or on different targets (e.g., on the same protein or on different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and these sequences can be expressed in cells that express immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs followed (N- to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen binding specificity but can associate with each heavy chain, or that can associate with each heavy chain and bind one or more of the epitopes bound by the heavy chain antigen-binding region, or that can associate with each heavy chain and enable one or both of the heavy chains to bind one or both epitopes.

[0041] The phrases "heavy chain" or "immunoglobulin heavy chain" include immunoglobulin heavy chain constant region sequences from any organism, and, unless otherwise specified, include a heavy chain variable domain. Unless otherwise specified, the heavy chain variable domain comprises three heavy chain CDRs and four FR regions. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain comprises (from N- to C-terminus) the variable domain followed by a CH1 domain, hinge, CH2 domain, and CH3 domain. Functional fragments of heavy chains include fragments that are capable of specifically recognizing an antigen (e.g., recognizing an antigen with a KD in the micromolar, nanomolar, or picomolar range), that are capable of being expressed and secreted from a cell, and that comprise at least one CDR.

[0042] The term "light chain" includes immunoglobulin light chain constant region sequences from any organism, including human kappa light chains and human lambda light chains, unless otherwise specified. A light chain variable (VL) domain typically contains three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain contains a VL domain, including, from the amino terminus to the carboxyl terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Potentially useful light chains include, for example, light chains that do not selectively bind to either the first antigen or the second antigen selectively bound by the antigen-binding protein. Suitable light chains include those that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or in silico), and 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 one or both epitopes bound by the antigen-binding region of the antigen-binding protein.

[0043] The phrase "variable domain" includes the amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) which comprises the following amino acid regions, from N-terminus to C-terminus (unless otherwise specified): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" comprises an amino acid sequence that can fold into a canonical domain (VH or VL) having a two-part beta-sheet structure, the beta-sheets being connected by disulfide bonds between residues of the first beta-sheet and the second beta-sheet.

[0044] The phrase "complementarity determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes, which sequences are normally (i.e., in wild-type animals) 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). CDRs can be encoded, for example, by germline sequences or rearranged or unrearranged sequences, e.g., by naive or mature B cells, or T cells. Under some circumstances (e.g., with respect to a CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B-cell nucleic acid sequence, e.g., as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form a heavy chain CDR3).

[0045] 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 encompassed within the term "antibody fragment" include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) an isolated CDR; and (vii) an scFv, which consists of the two domains, VL and VH, of an Fv fragment connected by a synthetic linker to form a single protein chain in which the VL and VH domains pair to form a monovalent molecule. Other forms of single chain antibodies, such as diabodies, are also encompassed under the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0046] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least a functional portion of the CH2 and CH3 regions of an immunoglobulin. "Functional portion" refers to CH2 and CH3 regions that can bind an Fc receptor (e.g., FcyR or FcRn, i.e., fetal Fc receptor) and / or participate in complement activation. CH2 and CH3 regions are not functional if they contain deletions, substitutions, and / or insertions, or other modifications that render them incapable of binding any Fc receptor and of activating complement.

[0047] An Fc-containing protein can contain modifications in an immunoglobulin domain, including those that affect one or more effector functions of the binding protein (e.g., modifications that affect 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 respect to the EU numbering of the immunoglobulin constant regions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309 , 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0048] For example, and not by way of limitation, the binding protein is an Fc-containing protein that exhibits improved serum half-life (compared to the same Fc-containing protein without the described modifications) and has modifications at positions 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 positions 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 positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In another example, modifications can include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); 307 and / or 308 modifications (e.g., 308F or 308P).

[0049] As used herein, the term "antigen-binding protein" refers to a polypeptide or protein (one or more polypeptides complexed as a functional unit) that specifically recognizes an epitope on an antigen, such as a cell-specific antigen described herein and / or a target antigen of the present invention. An antigen-binding protein may be multispecific. The term "multispecific" in reference to an antigen-binding protein means that the protein recognizes different epitopes, either on the same antigen or on different antigens. The multispecific antigen-binding proteins described herein may be a single multifunctional polypeptide or may be a multimeric complex of two or more polypeptides covalently or noncovalently linked to each other. The term "antigen-binding protein" includes antibodies or fragments thereof described herein that may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or other methods) to one or more other molecular entities, such as proteins or fragments thereof, to produce a bispecific or multispecific antigen-binding molecule with a second binding specificity.

[0050] The term "protein" refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. Proteins contain one or more amino acid polymer chains, commonly known in the art as "polypeptides." Thus, a polypeptide can be a protein, and a protein can contain multiple polypeptides to form a single functional biomolecule. Disulfide bridges (i.e., between cysteine ​​residues, such as those forming cysteines) can be present in some proteins. These covalent bonds can be within a single polypeptide chain or between two individual polypeptide chains. For example, disulfide bridges 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).

[0051] As used herein, "protein" includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins may be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), and 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).

[0052] 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 antigenic polypeptide) may have more than one epitope. Epitopes may 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 an antigen-binding polypeptide and an antigen. Epitopes may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural and / or specific charge characteristics. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by three-dimensional folding are typically lost upon treatment with denaturing solvents.

[0053] The term "domain" refers to any portion of a protein or polypeptide that has a specific function or structure. Preferably, the domains described herein bind to a cell-specific antigen or a target antigen. As used herein, a cell-specific antigen domain or a target antigen-binding domain, etc., includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen.

[0054] 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. Antibody heavy chains can form dimers, and thus the heavy chain of one half-antibody can associate with a heavy chain associated with a different molecule (e.g., another half-antibody) or another Fc-containing polypeptide. Two slightly different Fc domains can "heterodimerize," as 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).

[0055] The term "single-chain variable fragment" or "scFv" includes a single-chain fusion polypeptide containing an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL). In some embodiments, the VH and VL are connected by a linker sequence of 10-25 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 direct subcloning of heavy and light chains from hybridomas or B cells. Ahmad et al., Clinical and Developmental Immunology, volume 2012, article ID98025, is incorporated herein by reference for methods of generating scFv fragments by phage display and antibody domain cloning.

[0056] Adeno-associated virus (AAV) "AAV" is an abbreviation for adeno-associated virus and can refer to the virus itself or its derivatives. AAV is a small, non-enveloped, single-stranded DNA virus. Generally, the wild-type AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames (ORFs), rep and cap. The wild-type rep reading frame encodes four proteins with molecular weights of 78 kD ("Rep78"), 68 kD ("Rep68"), 52 kD ("Rep52"), and 40 kD ("Rep40"). Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. These proteins primarily function to regulate the transcription and replication of the AAV genome. The wild-type cap reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2), and 61-62 kD (VP3). VP3 accounts for over 80% of the total protein in the AAV virion (capsid). In mature virions, VP1, VP2, and VP3 are present in relative amounts of approximately 1:1:10, although ratios as high as 1:1:8 have also been reported. (Padron et al. (2005) J. Virology 79:5047-58)

[0057] The genomic sequences of various AAV serotypes, as well as the sequences of the natural inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases, such as GenBank. See, for example, GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8), the disclosures of which are incorporated herein by reference for their teaching of AAV nucleic acid and amino acid sequences. 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 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 by reference in its entirety. Table 2 herein provides sequences for various non-primate AAVs.

[0058] "AAV" includes all subtypes known in the art and both naturally occurring and modified forms. AAVs include primate AAVs (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV type rh10 (AAV rh10), AAV type h10 (AAV h10), AAV type hu11 (AAV hu11), AAV type rh32.33 (AAV rh32.33), and AAV type rh33. rh32.33), AAV retro (AAV retro), AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAV2 / 8, etc.; non-primate AAV (e.g., avian AAV (AAAV)), and other non-primate AAV, such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, and ovine AAV), squamate AAV (e.g., snake AAV, bearded dragon AAV, etc.). "Primate AAV" generally refers to AAV isolated from primates. Similarly, "non-primate AAV" refers to AAV isolated from non-primates.

[0059] 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 a capsid protein of a specified AAV (e.g., the PLA2 region, the VP1-u region, the VP1 / VP2 common region, the VP3 region), a nucleotide sequence (e.g., an ITR sequence), e.g., the cap gene or capsid protein of an AAV, "of a [specified] AAV" encompasses not only a gene or polypeptide comprising a nucleic acid sequence or amino acid sequence described herein for the specified AAV, respectively, but also variants of the gene or polypeptide, including variants that include the minimum number of nucleotides or amino acids required to retain one or more biological functions. As used herein, a variant gene or variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence described herein for the specified AAV gene or polypeptide, where the difference generally does not alter at least one biological function of the gene or polypeptide and / or does not alter the phylogenetic characteristics of the gene or polypeptide; for example, the difference may be due to degeneracy in the genetic code, isolated variation, sequence length, etc. For example, as used herein, rep and cap genes can encompass rep and cap genes that differ from the wild-type genes in that the genes may encode one or more Rep and Cap proteins, respectively. In some embodiments, the Rep gene encodes at least Rep78 and / or Rep68. In some embodiments, the cap gene may differ from the wild-type in that one or more alternative start codons, or the sequence between one or more alternative start codons, have been removed, such that the cap gene encodes only a single Cap protein, e.g., the VP2 and / or VP3 start codons have been removed or replaced, such that the cap gene encodes a functional VP1 capsid protein, but not a VP2 or VP3 capsid protein. Thus, as used herein, a rep gene encompasses any sequence that encodes a functional Rep protein.A cap gene includes any sequence that encodes at least one functional cap gene.

[0060] Wild-type cap genes are known to express all three VP1, VP2, and VP3 capsid proteins from a single open reading frame in the cap gene under the control of the p40 promoter present in the rep ORF. Terms such as "capsid protein" and "Cap protein" include 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 may be encoded by a single cap gene. For AAV, the three AAV capsid proteins are naturally produced in an overlapping manner using alternative translation initiation codons in the cap ORF, but all three proteins use a common stop codon. The wild-type cap gene ORF encodes three alternative initiation codons from 5' to 3': the "VP1 initiation codon," the "VP2 initiation codon," and the "VP3 initiation codon," as well as a single "consensus stop codon." VP1, the largest viral protein, is generally encoded from the VP1 initiation codon to the "consensus stop codon." VP2 is generally encoded from the VP2 start codon to the consensus stop codon. VP3 is generally encoded from the VP3 start codon to the consensus stop codon. Therefore, VP1 contains an N-terminal sequence not shared with VP2 or VP3, referred to as the VP1-unique region (VP1-u). The VP1-u region is generally encoded by the sequence of the wild-type cap gene, starting from the VP1 start codon to the "VP2 start codon." VP1-u contains a phospholipase A2 domain (PLA2), which may be important for infection and a nuclear localization signal that may assist in targeting the virus to 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 is sometimes referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the consensus stop codon. VP2 shares approximately 60 additional amino acids with VP1. This region is called the VP1 / VP2 common region.

[0061] 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, each producing one or more of the VP1, VP2, and / or VP3 capsid proteins of the present invention, by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in a packaging cell. In some embodiments, the VP capsid proteins of the present invention may be expressed individually from ORFs containing nucleotide sequences encoding any one of VP1, VP2, or VP3, by using separate nucleotide sequences operably linked to a single expression control sequence for expression in a viral replication cell, each producing only one of the VP1, VP2, or VP3 capsid proteins. In another embodiment, the VP proteins may be expressed from a single ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a viral replicating cell, each producing a VP1, VP2, and VP3 capsid protein. Thus, the amino acid positions provided herein may be provided relative to the VP1 capsid protein of the referenced AAV, and one of skill in the art would readily be able to determine the same amino acid position within the VP2 and / or VP3 capsid proteins of an AAV, and the corresponding amino acid positions among different AAVs, respectively.

[0062] The term "inverted terminal repeats" or "ITRs" refers to symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs function as origins of replication for viral DNA synthesis and are essential cis-elements for AAV particle production, including packaging into AAV particles.

[0063] The AAV ITRs contain recognition sites for the replication proteins Rep78 and Rep68. The "D" region of the ITR contains the DNA nick site where DNA replication is initiated, providing directionality for the nucleic acid replication process. AAV replication in mammalian cells typically involves two ITR sequences.

[0064] A single ITR may be engineered with Rep binding sites on both strands of the "A" region and the two symmetric D regions on each side of the ITR palindrome. On a double-stranded circular DNA template, such engineered constructs allow Rep78- or Rep68-initiated nucleic acid replication to proceed in both directions. A single ITR is sufficient for AAV replication in circular particles. In the methods of generating AAV viral particles of the invention, the rep coding sequence encodes a Rep protein or a Rep protein equivalent, which can bind to the ITRs contained on a transfer plasmid.

[0065] When expressed by a packaging cell together with an appropriate Rep protein, the Cap protein of the present invention can encapsidate a transfer plasmid containing a nucleotide of interest 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.

[0066] Either Rep78 and / or Rep68 binds to a unique and known site on the ITR hairpin sequence and functions to disrupt and unwind the hairpin structure at the end of the AAV genome, thereby providing access to the replication machinery of the viral replicating cell. 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, each producing one or more of the Rep78, Rep68, Rep52, and / or Rep40 Rep proteins, by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in the viral replicating cell. Alternatively, the Rep proteins may be individually expressed from ORFs containing nucleotide sequences encoding any one of Rep78, Rep68, Rep52, or Rep40 by using separate nucleotide sequences operably linked to a single expression control sequence for expression in a packaging cell, 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 the Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in a viral replication cell, each producing the Rep78 and Rep52 Rep proteins.

[0067] In the methods of producing AAV virions, e.g., viral particles, of the invention, the rep coding sequence and cap gene of the invention may be provided in a single packaging plasmid. However, one skilled in the art will recognize that this requirement is not required. Such viral particles may or may not contain a genome.

[0068] A "chimeric AAV capsid protein" includes an AAV capsid protein that contains amino acid sequences, e.g., portions, from two or more different AAVs and that has the ability to form and / or forms an AAV viral capsid / virion. A chimeric AAV capsid protein may be encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide sequence containing multiple, e.g., at least two, nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a separate AAV, and which together encode a functional chimeric AAV capsid protein. Association of a chimeric capsid protein with a particular AAV indicates that the capsid protein contains one or more portions derived from a capsid protein of an AAV and one or more portions derived from a capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein includes a capsid protein that includes one or more portions of the VP1, VP2, and / or VP3 capsid proteins of AAV2 and one or more portions of the VP1, VP2, and / or VP3 capsid proteins of a different AAV.

[0069] The term "portion" refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, having 100% identity to the sequence from which the portion is derived; see Penzes (2015) J. General Virol. 2769. A "portion" encompasses any contiguous segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule from which the portion is derived is "of a [designated] AAV," or has "significant identity" to a particular AAV, e.g., a non-primate AAV or a distantly related AAV. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides that are 100% identical to a sequence associated with the designated AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides that are 100% identical to a sequence associated with the designated AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides that are 100% identical to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 20 amino acids or 60 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 25 amino acids or 75 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 30 amino acids or 90 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 35 amino acids or 105 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 40 amino acids or 120 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 45 amino acids or 135 nucleotides that have 100% identity to a sequence associated with the designated AAV.In some embodiments, the portion comprises at least 50 amino acids or 150 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 60 amino acids or 180 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 70 amino acids or 210 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 80 amino acids or 240 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 90 amino acids or 270 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 100 amino acids or 300 nucleotides that have 100% identity to a sequence associated with a designated AAV.

[0070] Modified viral capsid proteins, viral particles, and viral nucleic acids In some embodiments, a Cap protein, such as a VP1 capsid protein described herein, a VP2 capsid protein described herein, and / or a VP3 capsid protein described herein, is modified to include, for example, any one or combination of an insertion of a targeting ligand, a chemical modification, a first member of a binding pair, a detectable label, a point mutation, etc.

[0071] Generally, modifications of a designated AAV gene or polypeptide or variant thereof result in a nucleic acid or amino acid sequence that differs from the nucleic acid or amino acid sequence described herein for the designated AAV, in which case the modification alters, imparts, or eliminates one or more biological functions, but does not change the phylogenetic character of the gene or polypeptide as an AAV gene or polypeptide. Modifications may include any one or combination of the following: replacing a sequence of a first AAV serotype with a sequence of a second AAV serotype to create a chimerization, chemical modification, insertion of a first member of a binding pair, and / or point mutations, etc., thereby reducing or abolishing the natural tropism of the capsid protein, making the tropism of the capsid protein more easily redirected, and / or causing the capsid protein to contain a detectable label. The modifications described herein generally do not alter the recognition of the modified capsid by pre-existing antibodies present in the general population that were produced during infection with another AAV, such as a serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, etc., virions based on such serotypes, virions from currently used AAV gene therapy modalities, or combinations thereof, and preferably include modifications that reduce low recognition to no recognition.

[0072] Targeting Ligands The modifications described herein may involve the association (e.g., presentation, operative linkage, binding) of a targeting ligand to the modified capsid protein and / or a capsid comprising the modified capsid protein. Generally, the targeting ligands described herein bind to a surface protein expressed by a mammalian muscle cell, e.g., a protein expressed on the surface of a mammalian muscle cell, e.g., a mammalian muscle cell-specific surface protein. In some embodiments, the modified capsid protein and / or modified capsid comprises a targeting ligand that binds to mammalian CACNG1, e.g., human CACNG1.

[0073] antigen binding molecule The anti-hCACNG1 antibodies and antigen-binding fragments thereof described herein may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-hCACNG1 antibodies and antigen-binding fragments thereof described herein may be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical conjugation, genetic fusion, noncovalent association, or other methods) to one or more other molecular entities, such as other antibodies or fragments thereof, to produce bispecific or multispecific antigens with second or additional binding specificities.

[0074] The use of the phrase "anti-hCACNG1 antibody" herein is intended to include both monospecific anti-hCACNG1 antibodies and bispecific antibodies comprising a CACNG1-binding arm and a "target"-binding arm. Thus, described herein are bispecific antibodies in which one immunoglobulin arm binds to human CACNG1 and the other immunoglobulin arm is specific for another target molecule. The CACNG1-binding arm may comprise any of the amino acid sequences of the HCVR / LCVR or CDRs listed in Table 1 herein.

[0075] In certain embodiments, the CACNG1-binding arm binds to human CACNG1 and induces internalization of CACNG1 and antibodies bound thereto. In certain embodiments, the CACNG1-binding arm binds weakly to human CACNG1 and induces internalization of CACNG1 and antibodies bound thereto.

[0076] In certain exemplary embodiments, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first antigen-binding domain and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be designated with the prefix "A1," and the CDRs of the second antigen-binding domain may be designated with the prefix "A2." Accordingly, 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.

[0077] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected 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 connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. A "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of the same or similar structure or configuration. For example, a multimerization domain may be connected to the second multimerization domain of immunoglobulin C. H The multimer-forming component may be a polypeptide comprising three domains. H 2~C H The Fc portion of an immunoglobulin (comprising three domains), for example, the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0078] The bispecific antigen-binding molecules described herein typically comprise two multimerization domains, e.g., two Fc domains, each of which is part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, or IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, or IgG2 / IgG4.

[0079] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0080] Any bispecific antibody format or technology may be used to generate the bispecific antigen-binding molecules described herein. For example, an antigen-binding molecule or fragment thereof having a first antigen-binding specificity can be operably linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody or antibody fragment having a second binding specificity, to generate the bispecific antigen-binding molecule. Specific exemplary bispecific formats include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2Bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for a discussion of the aforementioned formats) are included.

[0081] In the context of the bispecific antigen-binding molecules described herein, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid alterations (e.g., insertions, deletions, or substitutions) compared to a naturally occurring version of the wild-type Fc domain. For example, bispecific antigen-binding molecules include bispecific antigen-binding molecules containing one or more modifications in the Fc domain that result in a modified Fc domain with modified binding interactions (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H Area 2 or C H The FcRn-binding domain contains modifications in three regions that increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes with a pH in the range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include modifications at positions 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 positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P).

[0082] Also described herein is a first C H 3 domain and second Ig C H A bispecific antigen-binding molecule comprising three domains, a first and a second Ig C H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds to protein A and the second Ig C H The third domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). See, e.g., U.S. Patent No. 8,586,713. H Further modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT).

[0083] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be a human IgG1, human IgG2, or human IgG4 C H C derived from 2 regions H 2 sequences, and C derived from human IgG1, human IgG2, or human IgG4 HThe chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also comprise a chimeric hinge region. For example, the chimeric hinge may comprise 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. A specific example of a chimeric Fc domain that may be comprised in any of the antigen-binding molecules described herein is, from the N-terminus to the C-terminus, a chimeric Fc domain consisting of: [IgG4 C H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein comprises, 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, published August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains with these general structural arrangements, and variants thereof, may have altered Fc receptor binding, thereby affecting Fc effector function.

[0084] In certain embodiments, the antibody heavy chains described herein comprise a heavy chain constant region (CH) region comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, or SEQ ID NO:255. In some embodiments, the heavy chain constant region (CH) region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, or SEQ ID NO:255.

[0085] In some embodiments, an antibody heavy chain described herein comprises an Fc domain comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, or SEQ ID NO: 267. In some embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, or SEQ ID NO: 267.

[0086] Germline mutations The anti-hCACNG1 antibodies discussed herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domain compared to the corresponding germline sequence from which the antibody is derived.

[0087] The anti-hCACNG1 antibodies and antigen-binding fragments thereof disclosed herein can be derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"), and can have weak or no detectable binding to the CACNG1 antigen. Some such exemplary antibodies that recognize CACNG1 are listed in Table 1 herein.

[0088] Furthermore, the anti-hCACNG1 antibodies and antigen-binding fragments disclosed herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue 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, reduced immunogenicity, etc. In some embodiments, the antibodies or antigen-binding fragments described herein are obtained in this general manner.

[0089] Also described herein are anti-hCACNG1 antibodies and antigen-binding fragments thereof comprising 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-hCACNG1 antibodies or antigen-binding fragments thereof described herein may comprise an HCVR amino acid sequence, an LCVR amino acid sequence, and / or a CDR amino acid sequence with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative 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 antigen-binding molecules thereof described herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains are derived, while maintaining or improving, for example, desired weak to undetectable binding to CACNG1. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with 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 a protein; i.e., in the case of anti-hCACNG1 binding molecules, the amino acid substitution maintains or improves the desired binding affinity, from weak to undetectable. Examples of amino acid groups with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0090] Also described herein are anti-hCACNG1 antibodies and antigen-binding fragments thereof comprising an antigen-binding domain having an HCVR amino acid sequence and / or CDR amino acid sequence that is substantially identical to any of the HCVR amino acid sequences and / or CDR amino acid sequences disclosed herein, while maintaining or improving a desired weak affinity for the CACNG1 antigen. With respect to amino acid sequences, the term "substantial identity" or "substantially identical" means that two amino acid sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using default gap weights. Preferably, non-homologous residue positions differ only by conservative amino acid substitutions. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitutions. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference.

[0091] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences described herein to databases containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0092] Once obtained, antigen-binding domains containing one or more germline mutations were tested for reduced binding affinity using one or more in vitro assays. In general, antibodies that recognize a particular antigen are typically screened for this purpose by testing for high (i.e., strong) binding affinity to the antigen.

[0093] By further modifying the antibodies described herein by the methods described herein, unexpected benefits may be realized, such as improved pharmacokinetic properties and reduced toxicity to patients.

[0094] Antibody binding properties The term "binding" in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to any given antigen, such as, for example, a cell surface protein or fragment thereof, typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0095] For example, binding affinities are typically about 10 when determined by surface plasmon resonance (SPR) techniques, e.g., on a BIAcore3000 instrument, using an antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 K below M D These values ​​correspond to the values ​​of the quantification of binding sites. Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used to provide binding characterization data for cell surface-expressed proteins. FACS data correlate well with other methods, such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0096] Thus, the anti-hCACNG1 antibodies and antigen-binding fragments thereof described herein have a K that is at least 10-fold lower than the affinity for binding to a non-specific antigen (e.g., BSA, casein). D According to the present invention, the antibody binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K value that is 10 times or less than that of a non-specific antigen. D An antibody affinity corresponding to a value may be considered undetectable binding, but such an antibody may be paired with a second antigen-binding arm to produce a bispecific antibody as described herein.

[0097] Molar (M) unit "K DThe term "KD" or "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 the binding affinity and the K D The smaller the value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and therefore a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction and, therefore, a larger K D In some situations, the binding affinity (or K) of a particular molecule (e.g., an antibody) to an interaction partner molecule (e.g., antigen X) is used. D ) is higher compared to the binding affinity of the molecule (e.g., an antibody) to another interacting partner molecule (e.g., antigen Y), a larger K D The smaller the K value (lower, or weaker, affinity), the D This can be expressed as a binding ratio determined by dividing by a value (higher, or stronger, affinity), and in some cases can be expressed as, for example, 5-fold or 10-fold higher binding affinity.

[0098] "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 known as the k off Also called the value.

[0099] "k a The term "(M-1 x 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.

[0100] "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 a k dIt is obtained by dividing by

[0101] "EC50" or "EC 50 The term "half-maximal effective concentration" refers to the concentration of antibody that elicits a response halfway between baseline and maximum after a specified exposure time. 50 The EC essentially represents the concentration of an antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The EC value is equal to the concentration of an antibody described herein that confers half-maximal binding to cells expressing CACNG1, as determined, for example, by a FACS binding assay or an androgen receptor-activated luciferase assay. Thus, the EC 50 Alternatively, the greater the half-maximal effective concentration value, the lower or weaker binding is observed.

[0102] In one embodiment, the reduction in binding is determined by measuring the EC2 concentration that allows binding to half the maximum amount of target cells. 50 It can be defined as an increase in antibody concentration.

[0103] 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 regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules described herein may contain antigen-binding domains derived from any of the exemplary amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LAll of the framework and / or CDR residues within the domain are mutated back to the residue found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline variant sequence that differs from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue 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 antagonist or agonist biological properties (in some cases), reduced immunogenicity, etc. Described herein are bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained by this general method.

[0104] Also described herein are antigen-binding molecules in which one or both antigen-binding domains comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the antigen-binding molecules described herein may comprise antigen-binding domains having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with 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 a protein. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0105] The present disclosure also provides that the antigen-binding molecules described herein may comprise antigen-binding domains having HCVR, LCVR, and / or CDR amino acid sequences substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. With respect to amino acid sequences, the term "substantial identity" or "substantially identical" means that two amino acid sequences, when optimally aligned using default gap weights, such as with the programs GAP or BESTFIT, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, non-homologous residue positions differ by conservative amino acid substitutions. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitutions. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference.

[0106] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing sequences to databases containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0107] pH dependent binding Also described herein are anti-hCACNG1 antibodies and antigen-binding fragments thereof with pH-dependent binding properties. For example, the anti-hCACNG1 antibodies described herein may exhibit reduced binding to CACNG1 at acidic pH compared to neutral pH. Alternatively, the anti-hCACNG1 antibodies described herein may exhibit enhanced binding to CACNG1 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, e.g., about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or lower. The term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0108] In certain instances, "reduced binding at acidic pH compared to neutral pH" refers to the K of an antibody that binds to its antigen at acidic pH. D value and K of an antibody that binds to its antigen at neutral pH D For example, an antibody or antigen-binding fragment thereof may be used where the antibody or antigen-binding fragment thereof has an acidic / neutral K value of about 3.0 or greater. D When a ratio is provided, for purposes of the description herein, it may be considered to indicate "reduced binding to CACNG1 at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K D The ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0109] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for decreased (or increased) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antibody with decreased antigen binding at acidic pH compared to neutral pH can be obtained.

[0110] Antibodies containing Fc variants In some embodiments, anti-hCACNG1 antibodies and antigen-binding fragments thereof (including multispecific antigen-binding molecules and multidomain therapeutic proteins comprising anti-hCACNG1 antibodies or antigen-binding fragments thereof) are provided, which comprise an Fc domain containing one or more mutations that enhance or decrease binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the antibodies described herein, the C of the Fc domain, H 2 or C HThe antibodies include those containing mutations in the 3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations can result in an extended serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 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 positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P).

[0111] For example, the anti-hCACNG1 antibodies and antigen-binding fragments described herein can comprise an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated as being within the scope of the description herein.

[0112] Biological characteristics of antibodies and bispecific antigen-binding molecules Also described herein are antibodies and antigen-binding fragments thereof that bind to human CACNG1 with high, medium, or low affinity, depending on the therapeutic situation and the desired specific targeting properties. For example, in the context of a bispecific antigen-binding molecule in which one arm binds to CACNG1 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-hCACNG1 arm binds to CACNG1 with only medium or low affinity. In this way, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while avoiding general / non-targeted CACNG1 binding and the resulting adverse side effects associated therewith.

[0113] Also described herein are antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to human CACNG1 with weak (i.e., low) or even undetectable affinity. In some embodiments, the antibodies and antigen-binding fragments thereof described herein have a K of greater than about 100 nM as measured by surface plasmon resonance. D In some embodiments, the antibodies or antigen-binding fragments described herein bind to human CACNG1 (e.g., at 37° C.) at a K 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, as measured by surface plasmon resonance (e.g., mAb capture or antigen capture format), or a substantially similar assay. D It binds to CACNG1 with or without detectable affinity.

[0114] Epitope mapping and related techniques The epitope on CACNG1 to which the anti-hCACNG1 antibodies and antigen-binding fragments thereof described herein bind may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids of the CACNG1 protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of CACNG1. 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 the paratope. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational 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 in a polypeptide chain. In certain circumstances, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on an antigen.

[0115] 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" in a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. Furthermore, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in 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 deuterium-labeling a protein of interest and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of antigen / antibody complexes can also be used for epitope mapping purposes.

[0116] Also described herein are anti-hCACNG1 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Table 1 herein). Similarly, also described herein are anti-hCACNG1 antibodies that compete for binding to CACNG1 with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Table 1 herein).

[0117] Whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to or competes for binding with the same epitope as a reference antigen-binding molecule described herein can be easily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope on CACNG1 as a reference bispecific antigen-binding molecule described herein, the reference bispecific molecule is first bound to the CACNG1 protein. The ability of the test antibody to bind to the CACNG1 molecule is then evaluated. If the test antibody can bind to CACNG1 after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope on CACNG1 than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CACNG1 molecule after saturation binding with the reference bispecific antigen-binding molecule, the test antibody may bind to the same epitope on CACNG1 as the epitope bound by the reference bispecific antigen-binding molecule described herein. Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to some embodiments described herein, two antigen-binding proteins bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antigen-binding proteins are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein also reduce or eliminate binding of the other. Two antigen-binding proteins are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein also reduce or eliminate binding of the other.

[0118] To determine whether an antibody or its antigen-binding domain competes for binding with a reference antigen-binding molecule, the above-described binding methodology is carried out in two ways: In the first way, the reference antigen-binding molecule is bound to the CACNG1 protein under saturating conditions, and then the binding of the test antibody to the CACNG1 molecule is evaluated. In the second way, the test antibody is bound to the CACNG1 molecule under saturating conditions, and then the binding of the reference antigen-binding molecule to the CACNG1 molecule is evaluated. In both ways, if only the first (saturating) antigen-binding molecule can bind to the CACNG1 molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CACNG1. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antigen-binding molecule, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0119] Preparation of antigen-binding domains and construction of multispecific molecules Antigen-binding domains specific for a particular antigen can be prepared by any antibody generation technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CACNG1 and a target antigen) can be appropriately positioned relative to each other to produce a bispecific antigen-binding molecule as described herein using conventional methods. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules described herein is provided elsewhere herein.) In certain embodiments, one or more of the individual components (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 generation technique), high-affinity chimeric antigen-binding molecules for a particular antigen (e.g., CACNG1) with human variable regions and mouse constant regions are first isolated. Antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The murine constant regions are replaced with desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the antigen-binding molecules described herein.

[0120] Genetically engineered animals can also be used to produce human bispecific antigen-binding molecules. For example, genetically modified mice that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Using such genetically modified mice, heavy and light chain variable regions can be isolated to produce fully human bispecific antigen-binding molecules. Thus, fully human bispecific antigen-binding molecules contain two different heavy chains associated with the same light chain. (See, for example, US2011 / 0195454.) "Fully human" refers to an antibody or antigen-binding fragment thereof or immunoglobulin domain thereof that contains amino acid sequences encoded by DNA derived from human sequences across the entire length of each polypeptide of the antibody or antigen-binding fragment thereof or immunoglobulin domain thereof. In some cases, the fully human sequence is derived from a protein endogenous to humans. In other cases, a fully human protein or protein sequence includes a chimeric sequence in which each component sequence is derived from a human sequence. Without being bound by any theory, chimeric proteins or sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of the component sequences, for example, compared to any wild-type human immunoglobulin region or domain.

[0121] Bispecific antigen-binding molecules may be constructed with one heavy chain having a modified Fc domain that disrupts its binding to Protein A, thus enabling a purification method that produces a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. Thus, bispecific antigen-binding molecules comprise a first C H 3 domain and second Ig C H It contains three domains, the first and second Ig C H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference.H The 3 domain binds to protein A and the second Ig C H The third domain contains a mutation / modification that reduces or eliminates Protein A binding, for example, an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU).

[0122] biological equivalent Provided herein are antigen-binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein but that retain the ability to bind to CACNG1. Such variant molecules may contain one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen-binding molecules.

[0123] Also described are antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered to be bioequivalents if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption when administered at the same molar dose, either single or multiple doses, under similar experimental conditions, for example. Some antigen-binding proteins are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their absorption rate is not, and further, such differences in absorption rate are considered to be bioequivalents because they are intentional, reflected in labeling, and are not considered medically significant for the particular pharmaceutical product being studied, for example.

[0124] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0125] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or decreased efficacy, compared to continuous therapy without such switching.

[0126] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms and to the known extent of such mechanisms for one or more conditions of use.

[0127] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals in which the concentration of the 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 relevant acute pharmacological effect of the 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 the antigen binding protein.

[0128] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antigen-binding proteins can include variants of the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation characteristics of the molecule, for example, mutations that eliminate or remove glycosylation.

[0129] Species selectivity and species cross-reactivity In some embodiments, the antigen binding molecules described herein bind to human CACNG1 but do not bind to CACNG1 from other species. Also described herein are antigen binding molecules that bind to human CACNG1 and CACNG1 from one or more non-human species.

[0130] In some embodiments, antigen binding molecules that bind to human CACNG1 described herein may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee CACNG1, as the case may be.

[0131] Non-limiting examples of targeting ligands that bind to CACNG1 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 regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "targeting ligand" as used herein. In a non-limiting embodiment, the anti-CACNG1 targeting ligand that binds CACNG1 useful for retargeting viral capsids described herein comprises an scFv. As a non-limiting example, the V useful for retargeting viral capsids described herein comprises an scFv. L -(Gly4Ser)3-V HThe scFv sequence of this format may comprise 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 that is 90%, 95%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of the heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, respectively, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1 to 240.

[0132] A targeting ligand that binds to a mammalian muscle cell-specific surface protein can be associated with (e.g., presented by, operably linked to, or bound to) the modified AAV capsid protein and the resulting AAV capsid according to known methods, e.g., a direct approach in which the targeting ligand is directly inserted (e.g., using recombinant methods) according to known methods. See, e.g., Stachler et al. (2006), supra, White et al. (2004), supra, Girod et al. (1999), supra, Grifman et al. (2001), supra, Shi et al. (2001), supra, Shi and Bartlett (2003). A targeting ligand that binds to a mammalian muscle cell-specific surface protein may be attached to the modified AAV capsid protein and the resulting AAV capsid using known chemical linkers; for example, the AAV capsid protein may be chemically modified to include a dibenzocycootyne group or an azide group, and optionally, a targeting ligand described herein may be attached to the dibenzocycootyne group or azide group; see, for example, U.S. Patent Application Publication No. 2022 / 028234, the entire contents of which are incorporated herein by reference. Here, the targeting ligand is covalently attached to a primary amino acid group of the AAV capsid protein, e.g., via a -CSNH- bond. In some embodiments, the modified capsids described herein include a targeting ligand, e.g., an anti-CACNG1 antibody or binding portion thereof, that is directly inserted into or attached to the capsid according to well-known direct recombinant methods.

[0133] bond pair In some embodiments, a targeting ligand that binds to a mammalian muscle cell-specific surface protein can be associated with (e.g., presented by, operably linked to, or bound to) a modified AAV capsid protein and the resulting AAV capsid according to an indirect recombinant approach, where the AAV capsid protein is modified to include a first member of a binding pair (e.g., a heterologous scaffold), optionally linked (e.g., covalently or non-covalently) to a second cognate member of the binding pair (e.g., an adapter), and further optionally fused to the targeting ligand. Non-limiting exemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12:248-265.

[0134] Thus, in some embodiments, modifications of capsid proteins described herein include those that typically result from modifications at the genetic level, such as through modifications of the cap gene, such as modifications to insert a first member of a binding pair (e.g., a protein:protein binding pair, a protein:nucleic acid binding pair), a detectable label displayed by the Cap protein, and the like.

[0135] In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member binds a metal ion, e.g., Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ In some embodiments, the first member is selected from the group consisting of streptavidin, StrepII, HA, L14, 4C-RGD, LH, and Protein A.

[0136] In some embodiments, the binding pair comprises an enzyme:nucleic acid binding pair. In some embodiments, a first member comprises an HUH-endonuclease or an HUH tag, and a second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises an HUH tag. See, e.g., U.S. Patent Application No. 2021 / 0180082, which is incorporated herein by reference in its entirety.

[0137] In some embodiments, the capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

[0138] In some embodiments, each of the first and second members of the peptide:peptide binding pair comprises an intein. See, e.g., Wagner et al., (2021) Adv. Sci. 8:2004018(1 / 22); Muik et al. (2017) Biomaterials 144:84, each of which is incorporated herein by reference in its entirety.

[0139] In some embodiments, the first member is a B-cell epitope, e.g., about 1 amino acid to about 35 amino acids in length, that forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, the capsid proteins of the invention are modified to include a detectable label as the first member of the binding pair. Many detectable labels are known in the art. (See, e.g., Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of modified proteins" Protein Expression and Purification 11:1-16; Terpe et al. (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60:523-533, and references therein.) Detectable labels include, but are not limited to, immobilized divalent cations (e.g., Ni 2+), a biotin moiety that binds to immobilized avidin (e.g., on a biotinylated polypeptide sequence in vivo), a GST (glutathione S-transferase) sequence that binds to immobilized glutathione, an S tag that binds to immobilized S protein, an antigen that binds to an immobilized antibody or domain or fragment thereof (including, for example, T7, myc, FLAG, and B tags that bind to the corresponding antibody), a FLASH tag (specific Examples of detectable labels include a highly detectable label linked to an arsenic-based moiety (e.g., a highly detectable label linked to an arsenic-based moiety), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds to immobilized IgG, maltose-binding protein (MBP) that binds to immobilized amylose, albumin-binding protein that binds to immobilized albumin, a chitin-binding domain that binds to immobilized chitin, a calmodulin-binding peptide that binds to immobilized calmodulin, and a cellulose-binding domain that binds to immobilized cellulose. Another exemplary detectable label is a SNAP tag. In some embodiments, the detectable labels disclosed herein include a detectable label that is recognized by an antibody paratope, wherein the detectable label and the antibody paratope form a protein:protein binding pair.

[0140] In some embodiments, the capsid proteins of the invention comprise a first member of a protein:protein binding pair comprising a detectable label, which may also be used for detection and / or isolation of the Cap protein and / or may be used as the first member of a protein:protein binding pair. In some embodiments, the detectable label serves as the first member of a protein:protein binding pair for binding of a targeting ligand comprising a multispecific binding protein capable of binding both the detectable label and a target expressed by a cell of interest. In some embodiments, the Cap proteins of the invention comprise a first member of a protein:protein binding pair comprising c-myc (the use of a detectable label as the first member of a protein:protein binding pair is described, for example, in WO2019 / 006043, incorporated herein by reference in its entirety).

[0141] In some embodiments, the capsid protein comprises a first member of a protein:protein binding pair, wherein the protein:protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of the protein:protein binding pair is covalently linked to a cognate second member of the protein:protein binding pair via an isopeptide bond, and optionally, the cognate second member of the protein:protein binding pair is fused to a targeting ligand, wherein the targeting ligand binds to a target expressed by a 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 a biological equivalent or variant thereof) and the protein (second cognate member) is SpyCatcher (or a biological equivalent or variant thereof). In some embodiments, the first member is SpyTag (or a biological equivalent or variant thereof) and the protein (second cognate member) is KTag (or a biological equivalent or variant thereof). In some embodiments, the first member is KTag (or a biological equivalent or variant thereof) and the protein (second cognate member) is SpyTag (or a biological equivalent or variant thereof). In some embodiments, the first member is SnoopTag (or a biological equivalent or variant thereof) and the protein (second cognate member) is SnoopCatcher (or a biological equivalent or variant thereof). In some embodiments, the first member is Isopeptag (or a biologically equivalent or variant thereof) and the protein (the second cognate member) is Pirin-C (or a biologically equivalent or variant thereof).In some embodiments, the first member is SpyTag002 (or a biologically equivalent or variant thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically equivalent or variant thereof). In some embodiments, the first member is SpyTag003 (or a biologically equivalent or variant thereof) and the protein (second cognate member) is SpyCatcher003 (or a biologically equivalent or variant thereof). In some embodiments, the Cap protein of the invention comprises SpyTag, or a biologically equivalent or variant thereof. The use of the first member of a protein:protein binding pair is described in WO2019 / 006046, which is incorporated herein in its entirety.

[0142] The phrase "operably linked," as used herein, includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other or are positioned relative to each other so as to participate in a biological event, which juxtaposition achieves or enables such interaction and / or positioning. By way of example, a regulatory element (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when 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 a covalent bond between the components or elements involved. Those skilled in the art will readily appreciate that in some embodiments, a covalent bond is not required to achieve effective operable linkage. For example, proteins that are operably linked together can be associated with each other via, for example, covalent or non-covalent bonds. As a non-limiting example, the capsid proteins described herein may be operably linked to a targeting ligand, where the capsid protein is non-covalently bound to the targeting ligand, optionally with or without a scaffold and / or adapter between the capsid protein and the targeting ligand, or covalently bound to the targeting ligand. As another example, in some embodiments, nucleic acid regulatory elements operably linked to a coding sequence they control are contiguous with the nucleotide of interest. Alternatively or additionally, in some embodiments, one or more such regulatory elements act in trans or at a distance to control the coding sequence of interest. In some embodiments, the term "regulatory element," as used herein, refers to polynucleotide sequences necessary and / or sufficient to affect the expression and processing of the coding sequence to which they are linked.In some embodiments, regulatory elements may be or include appropriate transcription initiation, 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 only active in a particular host cell or organism, or type thereof. By way of example, in prokaryotes, regulatory elements typically include a promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, regulatory elements typically include a promoter, enhancer, and / or transcription termination sequence. Those skilled in the art will understand that in many embodiments, the term "regulatory elements" refers to components essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (e.g., including leader sequences, targeting sequences, and / or fusion partner sequences).

[0143] "Retargeting" or "redirection" can include scenarios in which wild-type particles target some cells within a tissue and / or some organs within an organism, where general targeting of the tissue or organ is reduced or abolished by the insertion of heterologous amino acids, and retargeting to more specific cells within the tissue or more specific organs within the organism is achieved with (e.g., via) a targeting ligand that binds to a marker expressed by the specific cells. Such retargeting or redirection can also include scenarios in which wild-type particles target a tissue, where tissue targeting is reduced or abolished by the insertion of heterologous amino acids, and retargeting to an entirely different tissue is achieved with a targeting ligand.

[0144] A "specific binding pair," "binding pair," "protein:protein binding pair," and the like, comprises two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non-covalent bond between 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 bond between proteins that can form an isopeptide bond; 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 "cognate" refers to components that function together. Epitopes and their cognate antibodies are known in the art, particularly epitopes that can also serve as detectable labels (e.g., c-myc). Specific protein:protein binding pairs that can interact to form covalent isopeptide bonds are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506 and include peptide:peptide binding pairs, e.g., SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, isopeptag:Pyrin C, SnoopTag:SnoopCatcher, and variants thereof, e.g., SpyTag003:SpyCatcher003. Generally, a first member of a protein:protein binding pair refers to a member of the protein:protein binding pair that is generally shorter than 30 amino acids in length and that forms a spontaneous covalent isopeptide bond with a second cognate protein, which is generally larger but may also be shorter than 30 amino acids in length, e.g., the SpyTag:KTag system.

[0145] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, at least one of which is not derived from the protein backbone or, alternatively, is not found to be part of the protein backbone. Isopeptide bonds can form within a single protein, or between two peptides or between a peptide and a protein. Thus, isopeptide bonds can form intramolecularly within a single protein, or between molecules, i.e., between two peptide / protein molecules, for example, between two peptide linkers. Typically, an isopeptide bond can occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or the terminal carboxyl group of a protein or peptide chain, or between the alpha-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid residue. Each residue of a pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, an isopeptide bond may be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, an isopeptide bond may occur between the side chain amine of a lysine and the carboxamide group of an asparagine or the carboxyl group of an aspartic acid.

[0146] 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 in its entirety, and is derived from the CnaB2 domain of the fibronectin-binding protein FbaB of Streptococcus pyogenes. By splitting the domain, Zakeri et al. obtained a peptide "SpyTag" with the sequence AHIVMVDAYKPTK (SEQ ID NO: 243), which forms an amide bond with its cognate protein, "SpyCatcher," a 112-amino acid polypeptide with the amino acid sequence set forth in SEQ ID NO: 244 (Zakeri (2012), supra). Another 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 has been engineered by excising a beta strand from SpyCatcher containing a reactive lysine, resulting in the 10-residue first member of the protein:protein binding pair, KTag, with the amino acid sequence ATHIKFSKRD (SEQ ID NO: 245). The SpyTag002:SpyCatcher002 system is described in Keeble et al (2017) Angew Chem Int Ed Engl 56:16521-25, incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO: 255, and binds to SpyCatcher002. SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK, set forth as SEQ ID NO: 259, and binds to SpyCatcher003.

[0147] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113:1202-07. The D4 Ig-like domain of RrgA, an adhesin from Streptococcus pneumoniae, was split to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher results in the formation of specific spontaneous isopeptide bonds between the complementary proteins (Veggiani (2016)), supra).

[0148] The isopeptag:pilin-C specific binding pair was derived from SpyO128, a major pilin protein from Streptococcus pyogenes (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). The isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, set forth as SEQ ID NO: 254, and binds to pilin-C (residues 18-299 of SpyO128). Incubation of the SnoopTag and SnoopCatcher results in the formation of a specific spontaneous isopeptide bond between the complementary proteins (Zakeir and Howarth (2010), supra).

[0149] The term "detectable label" includes, for example, a polypeptide sequence that is a member of a specific binding pair that specifically binds with high affinity to another polypeptide sequence, such as an antibody paratope, via a non-covalent bond. Exemplary and non-limiting detectable labels include a hexahistidine tag, a FLAG tag, a Strep II tag, a streptavidin-binding peptide (SBP) tag, a calmodulin-binding peptide (CBP), a glutathione S-transferase (GST), a maltose-binding protein (MBP), an S-tag, an HA tag, and a myc tag (SEQ ID NO: 246) from c-myc. (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8, incorporated herein by reference.) A common detectable label for primate AAV is the B1 epitope (SEQ ID NO: 247). Some AAV capsid proteins described herein do not naturally contain the B1 epitope, but can be modified herein to contain the B1 epitope. Generally, the AAV capsid proteins described herein can contain a sequence with substantial homology to the B1 epitope within the last 10 amino acids of the capsid protein. Thus, in some embodiments, the non-primate AAV capsid proteins of the present invention can be modified with one or more but less than five point mutations within the last 10 amino acids of the capsid protein, so that the AAV capsid protein contains the B1 epitope.

[0150] The term "target cell" includes any cell in which expression of a nucleotide of interest is desired. Preferably, target cells exhibit receptors on their surface that allow the cells to be targeted by targeting ligands, as described below.

[0151] Terms such as "transduction" or "infection" refer to the introduction of nucleic acid into the nucleus of a target cell by a viral particle. Efficiency related to transduction, e.g., the term "transduction efficiency," refers to the fraction (e.g., percentage) of cells that express a nucleotide of interest after incubation with a set of viral particles containing the nucleotide of interest. Known methods for determining transduction efficiency include flow cytometry of transduced cells using a fluorescent reporter gene, RT-PCR for expression of the nucleotide of interest, etc.

[0152] Typically, the "reference" viral capsid protein / capsid / particle is identical to the test viral capsid protein / capsid / particle, except for the modification to be tested. For example, to determine the effect of inserting a first member of a specific binding pair into a test viral particle, e.g., on transduction efficiency, the transduction efficiency of the test viral particle (in the presence or absence of an appropriate targeting ligand) can be compared to the transduction efficiency of a reference viral particle that is identical to the test viral particle in every instance (e.g., additional point mutations, nucleotides of interest, number of viral particles, target cells, etc.) except for the presence of the first member of the specific binding pair (in the absence or presence of an appropriate targeting ligand, as needed). In some embodiments, the reference viral capsid protein can form a capsid with a second viral capsid protein that has been modified to include at least a first member of a protein:protein binding pair, and the reference viral capsid protein does not include the first member of the protein:protein binding pair, and preferably the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.

[0153] In some embodiments, the first member of the protein:protein binding pair and / or the detectable label is operably linked to the Cap protein of the invention (translated in frame with the Cap protein of the invention, chemically attached to the Cap protein of the invention, and / or displayed by the Cap protein of the invention) via a first or second linker, e.g., an amino acid spacer that is at least one amino acid in length. In some embodiments, the first member of the protein:protein binding pair is flanked by first and / or second linkers, e.g., first and / or second amino acid spacers, each of which is at least one amino acid in length.

[0154] In some embodiments, the first and / or second linkers are not identical. In some embodiments, the first and / or second linkers are each independently 1 or 2 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, or 3 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, or 4 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, or 6 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, or 7 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids or more in length.

[0155] In some embodiments, the first and second linkers are the same sequence and / or length, each one amino acid long. In some embodiments, the first and second linkers are the same length, each one amino acid long. In some embodiments, the first and second linkers are the same length, each two amino acids long. In some embodiments, the first and second linkers are the same length, each three amino acids long. In some embodiments, the first and second linkers are the same length, each four amino acids long, e.g., the linkers are GLSG (SEQ ID NO: 248). In some embodiments, the first and second linkers are the same length, each five amino acids long. In some embodiments, the first and second linkers are the same length, each six amino acids long, e.g., the first and second linkers each comprise the sequence GLSGSG (SEQ ID NO: 249). In some embodiments, the first and second linkers are the same length, each seven amino acids long. In some embodiments, the first and second linkers are the same length, each 8 amino acids in length, e.g., the first and second linkers each comprise the sequence GLSGLSGS (SEQ ID NO: 250). In some embodiments, the first and second linkers are the same length, each 9 amino acids in length. In some embodiments, the first and second linkers are the same length, each 10 amino acids in length, e.g., the first and second linkers each comprise the sequence GLSGLSGLSG (SEQ ID NO: 251) or GLSGGSGLSG (SEQ ID NO: 252). In some embodiments, the first and second linkers are the same length, each more than 10 amino acids in length.

[0156] Generally, the amino acid sequence of the first member of a protein:protein binding pair described herein, e.g., comprising the first member of a specific binding pair alone or in combination with one or more linkers, is from about 5 amino acids to about 50 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is at least 5 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 6 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 7 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 8 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 9 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 10 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 11 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 12 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 13 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 14 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 15 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 16 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 17 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 18 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 19 amino acids in length.In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 20 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 21 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 22 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 23 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 24 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 25 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 26 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 27 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 28 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 29 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 30 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 31 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 32 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 33 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 34 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 35 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 36 amino acids in length.In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 37 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 38 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 39 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 40 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 41 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 42 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 43 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 44 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 45 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 46 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 47 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 48 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 49 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 50 amino acids in length.

[0157] Modified capsids containing modified capsid proteins

[0158] In some embodiments, a viral capsid comprising a modified viral capsid protein described herein is a mosaic capsid, e.g., comprising at least two pairs of VP1, VP2, and / or VP3 proteins, each pair encoded by a different cap gene. A mosaic capsid herein generally refers to a mosaic of a first viral capsid protein modified to include a first member of a binding pair and a second corresponding viral capsid protein lacking the first member of the binding pair. In the context of a mosaic capsid, the second viral capsid protein lacking the first member of the binding pair may also be referred to as a reference capsid protein and is encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP1, VP2, and / or VP3 capsid protein modified with a first member of a protein:protein pair is not a chimeric capsid protein, the VP1, VP2, and / or VP3 reference capsid protein may comprise an amino acid sequence identical to the amino acid sequence of a viral VP1, VP2, and / or VP3 capsid protein modified with a first member of a binding pair, but the reference capsid protein lacks the first member of the binding pair. In some mosaic capsid embodiments, the VP1, VP2, and / or VP3 reference capsid protein corresponds to a viral VP1, VP2, and / or VP3 capsid protein modified with a first member of a 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 a viral VP1 capsid protein modified with a first member of a 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 a first member of a binding pair, but the reference capsid protein lacks the first member of the binding pair, hi some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with a first member of a binding pair, but the reference capsid protein lacks the first member of the binding pair.In some mosaic capsid embodiments comprising chimeric VP1, VP2, and / or VP3 capsid proteins further modified to comprise a first member of a binding pair, the reference protein may be the corresponding capsid protein, a portion of which forms part of the chimeric capsid protein. As a non-limiting example, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP1 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member. Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP2 capsid protein modified to comprise a first member of a binding pair may further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP3 capsid protein modified to comprise a first member of a binding pair may further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member. In some mosaic capsid embodiments, the reference capsid protein may be any capsid protein so long as it lacks the first member of the binding pair and is capable of forming a capsid with a first capsid protein modified with the first member of the binding pair.

[0159] Generally, mosaic particles may be produced by transfecting a mixture of modified and reference cap genes into producer cells in a specified ratio. The ratio of protein subunits in the particle, e.g., the ratio of modified VP protein:unmodified VP protein, may, but does not necessarily, stoichiometrically reflect the ratio of at least two of the cap gene encoding a first capsid protein modified with a first member of a binding pair and one or more reference cap genes, e.g., the ratio of modified cap gene:reference cap gene transfected into packaging cells. In some embodiments, the ratio of protein subunits in the particle does not stoichiometrically reflect the ratio of modified cap gene:reference cap gene transfected into packaging cells.

[0160] In some mosaic virus particle embodiments, the ratio of protein subunits ranges from about 1:59 to about 59:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:1 (e.g., a mosaic virus particle includes about 30 modified capsid proteins and about 30 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:2 (e.g., a mosaic virus particle includes about 20 modified capsid proteins and about 40 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 3:5. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:3 (e.g., a mosaic virus particle includes about 15 modified capsid proteins and about 45 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:4 (e.g., the mosaic virus particle comprises about 12 modified capsid proteins and 48 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:5 (e.g., the mosaic virus particle comprises 10 modified capsid proteins and 50 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:6. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:7. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:8. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:9 (e.g., the mosaic virus particle comprises about 6 modified capsid proteins and about 54 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:10.In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:11 (e.g., the mosaic virus particle comprises about 5 modified capsid proteins and about 55 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:12. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:13. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:14 (e.g., the mosaic virus particle comprises about 4 modified capsid proteins and about 56 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:15. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:19 (e.g., the mosaic virus particle comprises about 3 modified capsid proteins and about 57 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:29 (e.g., the mosaic virus particle comprises about 2 modified capsid proteins and about 58 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:59. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 2:1 (e.g., the mosaic virus particle comprises about 40 modified capsid proteins and about 20 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 5:3. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 3:1 (e.g., the mosaic virus particle comprises about 45 modified capsid proteins and about 15 reference capsid proteins).In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 4:1 (e.g., the mosaic virus particle comprises about 48 modified capsid proteins and 12 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 5:1 (e.g., the mosaic virus particle comprises 50 modified capsid proteins and 10 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 6:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 7:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 8:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 9:1 (e.g., the mosaic virus particle comprises about 54 modified capsid proteins and about 6 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 10:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 11:1 (e.g., the mosaic virus particle comprises about 55 modified capsid proteins and about 5 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 12:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 13:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 14:1 (e.g., the mosaic virus particle comprises about 56 modified capsid proteins and about 4 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 15:1.In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 19:1 (e.g., the mosaic virus particle comprises about 57 modified capsid proteins and about 3 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 29:1 (e.g., the mosaic virus particle comprises about 58 modified capsid proteins and about 2 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 59:1.

[0161] In some non-mosaic viral particle embodiments, the ratio of protein subunits may be 1:0, and each capsid protein of the non-mosaic viral particle is modified with a first member of a binding pair. In some non-mosaic viral particle embodiments, the ratio of protein subunits may be 0:1, and each capsid protein of the non-mosaic viral particle is not modified with a first member of a binding pair.

[0162] Insertion site Due to the high degree of conservation, at least among widespread and numerous closely related family members, corresponding insertion sites in AAVs other than those listed can be identified by performing amino acid alignments or comparisons of capsid structures. For example, for exemplary alignments of different AAV capsid proteins, 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 an overlay of ribbons from different dependoparvoviruses in Figure 7, depicting variable regions VR I through VR IX. Using structural and sequence analysis as described in the literature, one skilled in the art can determine which amino acids within the variable regions correspond to amino acid sequences of AAV that are amenable to insertion of, for example, a targeting ligand, a first member of a binding pair, and / or a detectable label as described herein.

[0163] Generally, the targeting ligand, first member of the binding pair, and / or detectable label may be inserted into a variable region or loop of an AAV capsid protein, the GH loop of an AAV capsid protein, or the like.

[0164] Thus, in some embodiments, the first member of the binding pair and / or the detectable label is inserted in the VP1 capsid protein of the non-primate AAV after an amino acid position corresponding to an amino acid position selected from the group consisting of G453 of the AAV2 capsid protein VP1, N587 of the AAV2 capsid protein VP1, G453 of the AAV9 capsid protein VP1, and A589 of the AAV9 capsid protein VP1. In some embodiments, the first member of the binding pair and / or the detectable label is inserted in the VP1 capsid protein of the non-primate AAV between amino acids corresponding to N587 and R588 of the AAV2 VP1 capsid. Additional suitable insertion sites for non-primate VP1 capsid proteins include sites 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 protein described herein can be a non-primate capsid protein comprising a first member of a binding pair and / or a detectable label inserted at a position corresponding to a position in an AAV2 capsid protein selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. Additional suitable insertion sites in non-primate AAVs include sites corresponding to I-587 or I-590 in AAV1, I-589 in AAV1, I-585 in AAV3, I-584 or I-585 in AAV4, and I-575 or I-585 in AAV5.In some embodiments, the modified viral capsid protein described herein may be a non-primate capsid protein comprising a targeting ligand, a first member of a binding pair, and / or a detectable label inserted at a position corresponding to a position selected from the group consisting of I-587(AAV1), I-589(AAV1), I-585(AAV3), I-585(AAV4), I-585(AAV5), and combinations thereof.

[0165] In some embodiments, the first member of the binding pair and / or the detectable label is selected from the group consisting of I444 of avian AAV capsid protein VP1, I580 of avian AAV capsid protein VP1, I573 of bearded dragon AAV capsid protein VP1, I436 of bearded dragon AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, and I43 of sea lion AAV capsid protein VP1 in the VP1 capsid protein of non-primate AAV. and after an amino acid position corresponding to an amino acid position selected from the group consisting of: I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I433 of sea lion AAV capsid protein VP1, I434 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I437 of sea lion AAV capsid protein VP1, and I565 of sea lion AAV capsid protein VP1.

[0166] As used herein, the nomenclature I-###, I#, etc. refers to an insertion site (I) in the VP1 protein of the AAV capsid protein, where ### designates the amino acid numbering; however, such insertions may be located directly N- or C-terminally, preferably at the C-terminus of one amino acid in a sequence five amino acids N- or C-terminal to a given amino acid, preferably three amino acids, more preferably two amino acids, and particularly one amino acid C-terminal to a sequence N- or C-terminal to a given amino acid. Furthermore, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene; corresponding positions (and point mutations thereof) can be readily identified in the VP2 and VP3 capsid proteins encoded by the capsid genes by performing sequence alignments of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid genes.

[0167] Thus, because capsid proteins are encoded by overlapping reading frames of the same gene with offset start codons, insertion of the coding nucleic acid of one of these sites in the cap gene into the corresponding position also results in insertion of VP1, VP2, and / or VP3. Thus, for example, for AAV2, according to this nomenclature, an insertion of amino acids 1-138 is inserted only into VP1, an insertion of 138-203 is inserted into VP1 and VP2, and an insertion of 203 to the C-terminus is inserted into VP1, VP2, and VP3, and of course, this also applies to insertion site I-587. Thus, the present invention encompasses AAV structural genes with corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0168] Also provided herein are nucleic acids encoding the VP3 capsid proteins of the present invention. AAV capsid proteins may, but need not, be encoded by overlapping reading frames of the same gene with shifted start codons. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention does not also encode a VP2 capsid protein or a VP1 capsid protein of the present invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention may also encode a VP2 capsid protein of the present invention, but not a VP1 capsid of the present invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention may also encode a VP2 capsid protein of the present invention and a VP1 capsid of the present invention.

[0169] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a binding pair (e.g., the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) is capable of infecting a particular cell, e.g., has an enhanced ability to target and bind to a particular cell compared to a control viral capsid (the control viral capsid is identical to the modified viral capsid protein except that it lacks either or both of the first and second members of the binding pair, e.g., comprises a control capsid protein). In some embodiments, a viral capsid comprising a modified viral capsid protein described herein associated with a first and second member of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 10% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 20% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to an appropriate first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 30% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to an appropriate first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 40% greater than that of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 50% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 60% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 70% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 75% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 80% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 85% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid.In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.

[0170] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a binding pair (e.g., the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) is capable of infecting a particular cell, e.g., has an enhanced ability to target and bind to a particular cell compared to a control viral capsid (the control viral capsid is identical to the modified viral capsid protein except that it lacks either or both of the first and second members of the binding pair, e.g., comprises a control capsid protein). In some embodiments, a viral capsid comprising a modified viral capsid protein described herein associated with a first and second member of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 10% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 20% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to an appropriate first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 30% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to an appropriate first and second member of a binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 40% greater than that of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 50% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 60% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 70% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 75% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 80% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 85% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 1.5-fold greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 2-fold greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 3-fold greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least four times higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least five times higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least six times higher than that of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 7-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 8-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 9-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 10-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 20-fold higher than that of a control capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 30-fold higher than that of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 40-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 50-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 60-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 70-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 80-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 90-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 100-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral particles of the invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a distantly related AAV, or a combination thereof, and optionally comprising first and second members of a binding pair (e.g., the second member is operably linked to a targeting ligand and comprises a multispecific binding protein, etc.), are better able to avoid neutralization by pre-existing antibodies in serum isolated from a human patient compared to a suitable control viral particle (e.g., comprising a viral capsid of an AAV serotype that includes a portion of the viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof) (the control viral particle also optionally comprising the first and second members of a binding pair (e.g., the second member is operably linked to a targeting ligand and comprises a multispecific binding protein, etc.)). In some embodiments, viral particles of the invention comprising a viral capsid protein comprising 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 for neutralization (e.g., 50% or greater inhibition of infection) as appropriate control viral particles (e.g., viral particles of the invention have an IC50 value at least twice that of the control viral particles).

[0171] In some embodiments of the invention comprising a detectable label, the targeting ligand comprises a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor that can be bound to the surface of a bead (e.g., for purification) or expressed by a target cell. 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 a receptor that targets the viral particle. Such "targeting" or "directing" can include a scenario in which wild-type viral particles target tissues and / or some cells within some organs within an organism, and insertion of a detectable label reduces or abolishes broad targeting of tissues or organs, 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 viral particles target a tissue, tissue targeting is reduced or abolished by the insertion of a detectable label, and retargeting to an entirely different tissue is achieved with the multispecific binding molecule. The antibody paratopes described herein generally minimally comprise a complementarity-determining region (CDR), e.g., the CDR3 region of the heavy and / or light chain variable domain, that specifically recognizes the detectable label. In some embodiments, the multispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a detectable label. For example, the multispecific binding molecule may comprise 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 the single-domain light chain variable region comprises an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecule may comprise an Fv region, e.g., an scFv, comprising an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecules described herein comprise an antibody paratope that specifically binds to c-myc (SEQ ID NO: 246).

[0172] 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 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 modified viral capsid proteins comprising the first members of the specific binding pairs described herein. They can form normal viral capsids (empty viral particles) or viral particles (capsids encapsidating a nucleotide of interest). The formation of viral particles containing a viral genome is a highly desirable characteristic for the use of the modified viral capsids described herein.

[0173] A further embodiment of the present invention is the use of at least one modified viral capsid protein and / or nucleic acid encoding same, preferably at least one multimeric structure (e.g., viral particle) for the manufacture and for use in the introduction of a nucleotide of interest into a target cell.

[0174] Use and preparation method A further embodiment of the modified viral capsid proteins described herein is their use for delivering a nucleotide of interest, such as a reporter gene or a therapeutic gene, to a target cell. Generally, packaging a nucleotide of interest involves substituting the gene of interest between the AAV ITR sequences into the AAV genome to create a transfer plasmid, which is then packaged into an AAV capsid according to well-known methods. Thus, the modified viral capsids described herein can package a transfer plasmid and / or a nucleotide of interest, which generally may include 5' and 3' inverted repeat (ITR) sequences flanking a gene of interest, such as a reporter gene or a therapeutic gene, or a portion of a gene of interest (which may be under the control of a viral or non-viral promoter). According to well-known methods for packaging AAV viral particles, the modified viral capsid, 5' ITR, and 3' ITR do not need to be of the same AAV serotype. In one embodiment, the transfer plasmid and / or nucleotide of interest comprises, from 5' to 3', a 5' ITR, a promoter, a gene (eg, a reporter and / or therapeutic gene), and a 3' ITR.

[0175] Genes of interest disclosed herein include, but are not limited to, genes encoding microdystrophin, FKRP, and MTM1, such as genes encoding human microdystrophin, human FKRP, and human MTM1. A non-limiting sequence encoding microdystrophin is set forth as SEQ ID NO: 270. A non-limiting sequence encoding FKRP is set forth as SEQ ID NO: 271. A non-limiting sequence encoding MTM1 is set forth as SEQ ID NO: 272. Genes of interest described herein also include, but are not limited to, biologically equivalent portions or variants of the genes disclosed herein. For example, a gene of interest may comprise a biologically equivalent portion or variant of the sequence set forth as SEQ ID NO: 270. A gene of interest may comprise a biologically equivalent portion or variant of the sequence set forth as SEQ ID NO: 271. A gene of interest may comprise a biologically equivalent portion or variant of the sequence set forth as SEQ ID NO: 272.

[0176] A consideration in AAV transfer plasmid design is that the wild-type AAV genome is approximately 4.7 kb. Therefore, well-known strategies for providing packaging of a target nucleotide that exceeds the packaging capacity of individual AAVs are included herein. Such strategies include, but are not limited to, dual vector strategies that utilize ITR-mediated recombination to express a gene of interest that is larger than the wild-type AAV genome by transcriptional splicing across the intermolecularly recombined ITRs from two complementary vector genomes, homologous vector recombination, RNA trans-splicing, and / or protein "trans-splicing" via split intein design. See, for example, Nakai, H. et al. (2000) Nat. Biotechnol. 18:527-532, Sun, L. (2000) Nat. Med. 6:599-602 (2000), Ghosh, A., et al. (2008) Mol. Ther. 16:124-130 (2008), Lai, Y. (2005) Nat. Biotechnol. 23:1435-1439, Chew, W. Let al. (2016) Nat. Methods 13:868-874, Li, J. (2008) Hum. Gene Ther. 19:958-964, each of which is incorporated herein by reference in its entirety.

[0177] Dual AAV vector strategies for introducing large genes into target cells have been described, relying on various mechanisms, including but not limited to trans-splicing, overlapping regions in the dual vector, and hybrids of the two. See also Tornabene and Trapani (2020) Human Gene Ther. 31:47-56; U.S. Patent No. 8,236,557, each of which is incorporated herein by reference in its entirety.

[0178] The trans-splicing approach utilizes the ability of AAV ITR sequences to concatenate to reconstitute a full-length genome. Two or more viral capsids each contain one of two or more transfer plasmids, each containing a portion of a gene of interest. For example, in a dual vector approach, two transfer plasmids can be designed as follows: the 5'-transfer plasmid contains a promoter, the 5' portion of the coding sequence of the gene of interest, and a splicing donor (SD) signal; the 3'-transfer plasmid contains a splicing acceptor (SA) signal, the 3' portion of the gene of interest, and a poly(A) signal. Upon tail-to-head ITR-mediated concatenation of the two AAV genomes, the SD and SA signals allow splicing of the recombinant genome.

[0179] Large genes of interest can also be divided when using an overlapping region approach, in which the 5' and 3' portions (and thus the 5' transfer plasmid and 3' transfer plasmid) share recombinogenic sequences, e.g., homologous regions, e.g., each portion contains overlapping sequences. The gene of interest is generated in its entirety in the targeted cells via homologous recombination mediated by the recombinogenic sequences, e.g., the homologous / overlapping regions.

[0180] In the hybrid approach, the 5'-transfer plasmid and the 3'-transfer plasmid each contain highly recombinogenic sequences, which are located downstream of the SD signal in the 5' portion of the coding sequence of the gene of interest and upstream of the SA signal in the 3' portion of the coding sequence of the gene of interest. In this hybrid system, the gene of interest can be completed either through ITR-mediated concatenation and splicing and / or by homologous recombination.

[0181] Trans-splicing at the RNA or protein level can also be utilized. In the RNA trans-splicing approach, two transfer plasmids encode the 5' and 3' fragments, respectively, of the pre-mRNA of a large gene, and may share an intron hybridization domain that can favor trans-splicing to join the two half-transcripts into the complete full-length mRNA.

[0182] Protein trans-splicing occurs post-translationally and is catalyzed by an intervening protein called a split intein. Split inteins are expressed as two independent polypeptides (N-intein and C-intein) at the termini of two host proteins. The N-intein and C-intein polypeptides remain catalytically inactive until they encounter each other. Upon encountering each other, each intein precisely excises itself from the host protein, mediating the ligation of the N-host polypeptide and the C-host polypeptide via a peptide bond. The use of split inteins has been used for AAV-based delivery of therapeutic genes of interest in muscle, liver, and retinal diseases. For example, simultaneous delivery of two halves of a mini-dystrophin cDNA fused to the N- and C-intein coding sequences demonstrated efficient production of two polypeptides. Li et al. (2008) Hum Gene Ther 19:958-64. Similarly, AAV split inteins have been widely used for the expression and ligation of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases.

[0183] The above-mentioned dual vector approach is well known in the art. See, for example, Tornabene and Trapani (2020), U.S. Patent No. 8,236,557, supra. Thus, in some embodiments, the modified viral capsid described herein encapsulates a nucleotide of interest, wherein the nucleotide of interest comprises a portion of a gene of interest. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest further comprises a splicing donor signal or a splicing acceptor signal and / or a recombination-inducing sequence. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest comprises an intron hybridization domain-encoding sequence. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest comprises an N-intein or C-intein-encoding sequence.

[0184] The design of the transfer plasmid / nucleotide of interest includes the inclusion of one or more regulatory elements, such as promoters and / or enhancer elements, that control the expression of the gene of interest. Non-limiting examples of useful promoters include the cytomegalovirus (CMV) promoter, the spleen focus-forming virus (SFFV) promoter, the elongation factor 1 alpha (EF1a) promoter (1.2 kb EF1a promoter or 0.2 kb EF1a promoter), the chimeric EF1a / IF4 promoter, the phosphoglycerate kinase (PGK) promoter, and biologically equivalent portions or variants thereof. Internal enhancers can also be present in the viral construct to increase the expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, incorporated herein by reference in its entirety) can be used. In some embodiments, tissue-specific regulatory elements, such as muscle-specific promoters and / or regulatory elements, may be used to drive expression of a gene of interest, such as muscle-specific promoters based on the skeletal α-actin, muscle creatine kinase, and desmin genes, as well as other genes expressed in muscle. A non-limiting example of an actin gene predominant in adult muscle is the human skeletal α-actin gene (HSA). The HSA promoter, as well as biologically equivalent portions or variants thereof, and other regulatory regions of homologous chicken, rat, and bovine genes, have been used in vitro and in vivo in transgenic animal models and AAV-mediated gene transfer. Skopenkova et al. Acta Naturae 13:47-58. In some embodiments, an enhancer, such as a CMV enhancer, can be used in combination with an actin gene promoter, such as the chicken β-actin promoter, as well as biologically equivalent portions or variants thereof. For example, the use of muscle-specific regulatory elements based on the muscle creatine kinase gene (MCK) has been employed in muscle gene therapy treatments such as Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy (LGMD).See, for example, Salva, MZ et al. (2007) Mol. Ther. 15:320-329, which is incorporated herein by reference in its entirety. In some embodiments, the transfer plasmid and / or nucleotide of interest herein comprises an enhancer and / or promoter of MCK, or a biologically equivalent portion or variant thereof, which drives expression of a gene of interest. In some embodiments, the MCK enhancer and / or promoter, or a biologically equivalent portion or variant thereof, is selected from the group consisting of CK6, MHCK7, dMCK, tMCK, CK8, and CK8e. In some embodiments, the transfer plasmid and / or nucleotide of interest herein comprises an enhancer and / or promoter element that recruits RNA polymerase II, and the MCK enhancer and / or promoter (or a biologically equivalent portion or variant thereof) drives expression of a gene of interest. In some embodiments, the transfer plasmids and / or nucleotides of interest herein comprise an enhancer and / or promoter element that recruits RNA polymerase III, and the MCK enhancer and / or promoter (or a biologically equivalent portion or variant thereof) drives expression of the gene of interest. In some embodiments, the transfer plasmids and / or nucleotides of interest herein comprise a desmin promoter, or a biologically equivalent portion or variant thereof. In some embodiments, the transfer plasmids and / or nucleotides of interest herein comprise a human myosin heavy chain gene (αMHC) promoter, or a biologically equivalent portion or variant thereof. In some embodiments, the transfer plasmids and / or nucleotides of interest herein comprise a CMV-IE enhancer ligated to an MLC promoter, or a biologically equivalent portion or variant thereof, for example, the rat MLC promoter.In some embodiments, transfer plasmids and / or nucleotides of interest herein comprise the ΔUSEx3 promoter, or a biologically equivalent portion or variant thereof, based on the human troponin I (TNN1) gene. In some embodiments, transfer plasmids and / or nucleotides of interest herein comprise the unc45b promoter, or a biologically equivalent portion or variant thereof.

[0185] In some embodiments, bidirectional promoters and / or vectors are also employed to deliver dual therapeutic gene cassettes. An example of this is the bidirectional chicken β-actin ubiquitous promoter driving simultaneous expression of the hexosaminidase α and β subunits of the HexA enzyme, two respective genes involved in Tay-Sachs disease and Sandhoff disease. See Lahey, et al. (2020) Mol. Ther. 28:2150-2160, the entire contents of which are incorporated herein by reference. In some embodiments, transfer plasmids and / or nucleotides of interest herein comprise a bidirectional promoter, which drives expression of two different genes of interest.

[0186] A variety of reporter genes (or detectable moieties) can be encapsidated into the multimeric structures containing the modified viral capsid proteins described herein. Exemplary reporter genes include, for example, b-galactosidase (encoded by the lacZ gene), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof. Although the methods described herein demonstrate the construction of targeted particles that employ the use of a reporter gene encoding green fluorescent protein, one of skill in the art upon reading this disclosure will understand that the viral capsids described herein can be produced in the absence of a reporter gene or with any reporter gene known in the art.

[0187] A variety of therapeutic genes can also be encapsidated into multimeric structures comprising the modified viral capsid proteins described herein, e.g., as part of a transfer particle. Non-limiting examples of therapeutic genes include genes encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or portions thereof, antisense RNA, siRNA, shRNA, etc.

[0188] A further embodiment of the present invention is a process for the preparation of a modified capsid protein, the method comprising: a. expressing a nucleic acid encoding a modified capsid protein under suitable conditions; b. isolating the expressed capsid protein of step a).

[0189] In some embodiments, the viral particles described herein comprise a mosaic capsid, e.g., the capsid comprises a capsid protein that has been genetically modified as described herein (in the presence or absence of covalent attachment to a targeting ligand) in a specific ratio to a reference capsid protein. Methods for producing such mosaic viral particles include: a. expressing under suitable conditions a nucleic acid encoding the modified capsid protein and a nucleotide encoding a reference capsid protein in a ratio (wt / wt) of at least about 60:1 to 1:60, e.g., 2:1, 1:1, 3:5, 1:2, 1:3, etc.; b. isolating the expressed capsid protein of step a).

[0190] In some embodiments, the compositions described herein comprise, or the methods described herein combine, modified cap genes:reference cap genes (or combinations of reference cap genes) in a ratio ranging from at least about 1:60 to about 60:1, e.g., 2:1, 1:1, 3:5, 1:2, 1:3, etc. In some embodiments, the ratio is at least about 1:2. In some embodiments, the ratio is at least about 1:3. In some embodiments, the ratio is at least about 1:4. In some embodiments, the ratio is at least about 1:5. In some embodiments, the ratio is at least about 1:6. In some embodiments, the ratio is at least about 1:7. In some embodiments, the ratio is at least about 1:8. In some embodiments, the ratio is at least about 1:9. In some embodiments, the ratio is at least about 1:10. In some embodiments, the ratio is at least about 1:11. In some embodiments, the ratio is at least about 1:12. In some embodiments, the ratio is at least about 1:13. In some embodiments, the ratio is at least about 1:14. In some embodiments, the ratio is at least about 1:15. In some embodiments, the ratio is at least about 1:16. In some embodiments, the ratio is at least about 1:17. In some embodiments, the ratio is at least about 1:18. In some embodiments, the ratio is at least about 1:19. In some embodiments, the ratio is at least about 1:20. In some embodiments, the ratio is at least about 1:25. In some embodiments, the ratio is at least about 1:30. In some embodiments, the ratio is at least about 1:35. In some embodiments, the ratio is at least about 1:40. In some embodiments, the ratio is at least about 1:45. In some embodiments, the ratio is at least about 1:50. In some embodiments, the ratio is at least about 1:55. In some embodiments, the ratio is at least about 1:60. In some embodiments, the ratio is at least about 2:1. In some embodiments, the ratio is at least about 3:1. In some embodiments, the ratio is at least about 4: 1. In some embodiments, the ratio is at least about 5: 1.In some embodiments, the ratio is at least about 6:1. In some embodiments, the ratio is at least about 7:1. In some embodiments, the ratio is at least about 8:1. In some embodiments, the ratio is at least about 9:1. In some embodiments, the ratio is at least about 10:1. In some embodiments, the ratio is at least about 11:1. In some embodiments, the ratio is at least about 12:1. In some embodiments, the ratio is at least about 13:1. In some embodiments, the ratio is at least about 14:1. In some embodiments, the ratio is at least about 15:1. In some embodiments, the ratio is at least about 16:1. In some embodiments, the ratio is at least about 17:1. In some embodiments, the ratio is at least about 18:1. In some embodiments, the ratio is at least about 19:1. In some embodiments, the ratio is at least about 20:1. In some embodiments, the ratio is at least about 25:1. In some embodiments, the ratio is at least about 30:1. In some embodiments, the ratio is at least about 35:1. In some embodiments, the ratio is at least about 40:1. In some embodiments, the ratio is at least about 45:1. In some embodiments, the ratio is at least about 50:1. In some embodiments, the ratio is at least about 55:1. In some embodiments, the ratio is at least about 60:1.

[0191] In some embodiments, the ratio of VP protein subunits in the mosaic virus particle stoichiometrically reflects, but is not necessarily, the ratio of modified cap genes to reference cap genes. As a non-limiting exemplary embodiment, a mosaic capsid formed according to the present methods can be considered to have a ratio of modified capsid protein to reference capsid protein similar to, but not necessarily, the ratio (wt:wt) of the nucleic acids encoding the same used to produce the mosaic capsid. In some embodiments, the mosaic capsid comprises a protein subunit ratio of about 1:59 to about 59:1.

[0192] A further embodiment of the present invention is a method for modifying the tropism of a virus, the method comprising: (a) inserting a nucleic acid encoding an amino acid sequence into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the amino acid sequence; and / or (b) culturing packaging cells under conditions sufficient for the production of viral particles, wherein the packaging cells comprise the nucleic acid. A further embodiment of the present invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising: (a) expressing under suitable conditions a nucleic acid encoding a modified viral capsid protein described herein (optionally together with nucleotides encoding a reference capsid protein), wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair; (b) isolating the expressed capsid protein comprising the first member of the specific binding pair of step (a), or a capsid comprising the same; and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under suitable conditions to allow formation of an isopeptide bond between the first and second members, wherein the second cognate member of the specific binding pair is fused to a targeting ligand.

[0193] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid containing the nucleotide of interest. In some embodiments, the method further comprises isolating the self-complementing adeno-associated viral particles from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral particles from the cell lysate. In some embodiments, the method further comprises (a) removing cellular debris, (b) treating the supernatant containing the viral particles with a nuclease, e.g., DNase I and MgCl2, (c) concentrating the viral particles, (d) purifying the viral particles, or (e) any combination of (a)-(d).

[0194] Packaging cells useful for producing the viral particles described herein include, for example, animal cells that are permissive for the virus, or cells modified to be permissive for the virus, or packaging cell constructs using a transforming agent such as, for example, calcium phosphate. Non-limiting examples of packaging cell lines useful for producing the viral particles described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), SV40 Large T-antigen-containing HEK-293 cells (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raji (CCL-86), epithelial glioblastoma-astrocytoma cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH Examples of such cells include 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, and CAP-T cells.

[0195] L929 cells, the FLY virus packaging cell line reviewed in Cosset et al. (1995) J Virol 69, 7430-7436, NS0 (mouse myeloma) cells, human amniotic cells (e.g., CAP, CAP-T), yeast cells (including but not limited to S. cerevisiae, Pichia pastoris), plant cells (including but not limited to tobacco NT1, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g., High5)), or bacterial cells (including but not limited to E. coli).

[0196] For additional packaging cells and systems, packaging techniques and particles for packaging nucleic acid genomes into pseudotyped viral particles, see, e.g., Polo, et al., Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express the viral components or transiently transfecting cells with plasmids.

[0197] Further embodiments include methods comprising contacting a modified Cap protein described herein with a targeting vector under conditions sufficient to operably link the modified Cap protein with the targeting vector, e.g., conditions sufficient to promote association of the targeting vector to the modified Cap protein, e.g., via chemical bonding and / or association of a first and second member of a specific binding pair, wherein the first member is inserted into the modified Cap protein and the first member and targeting vector are fused to the second member of the specific binding pair.

[0198] Further embodiments include methods of redirecting viruses and / or delivering reporter or therapeutic genes to target cells, including methods for transducing cells in vitro (e.g., ex vivo) or in vivo, comprising contacting a target cell with a viral particle comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the target cell is in vitro (e.g., ex vivo). In other embodiments, the target cell is in vivo in a subject, e.g., a human.

[0199] target cell A wide variety of cells can be targeted for delivery of a nucleotide of interest using the modified viral particles disclosed herein. Target cells will generally be selected based on the nucleotide of interest and the desired effect.

[0200] In some embodiments, the nucleotide of interest can be delivered to enable the targeted cells to produce a protein that compensates for a defect in the organism, such as an enzyme deficiency or an immune deficiency, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that would normally produce the protein in the animal are targeted. In other embodiments, cells in the region where the protein would be most beneficial are targeted.

[0201] In other embodiments, the nucleotide of interest, such as the gene encoding siRNA, can inhibit the expression of a specific gene in target cells.The nucleotide of interest can, for example, inhibit the expression of a gene involved in the pathogen life cycle.Therefore, cells that are susceptible to infection by pathogens or infected by pathogens can be targeted.In other embodiments, the nucleotide of interest can inhibit the expression of a gene responsible for the production of toxins in target cells.

[0202] In other embodiments, the nucleotide of interest may encode a toxic protein that kills cells that express the toxic protein, in which case tumor cells or other unwanted cells may be targeted.

[0203] In yet another embodiment, the nucleotide of interest encodes a therapeutic protein.

[0204] Once a specific population of target cells in which expression of a nucleotide of interest is desired is identified, a target receptor that is specifically expressed on that population of target cells is selected. The target receptor may be expressed only in that population of cells, or to a greater extent in that population of cells than in other populations of cells. The more specific the expression, the more specifically delivery can be directed to the target cells. Depending on the context, the desired amount of specificity of the marker (and therefore of gene delivery) may vary. For example, when introducing a toxic gene, high specificity is most preferred to avoid killing non-targeted cells. For expression of a protein for harvesting or expression of a secreted product where a global effect is desired, less marker specificity may be required.

[0205] As discussed above, the target receptor can be any receptor for which a targeting ligand can be identified or generated. Preferably, the target receptor is a peptide or polypeptide, such as a receptor. However, in other embodiments, the target receptor can be a carbohydrate or other molecule that can be recognized by a binding partner. If the binding partner, e.g., ligand, of the target receptor is known, the ligand can be used as an affinity molecule. However, if the binding molecule is unknown, an antibody against the target receptor can be generated using standard procedures. The antibody can then be used as a targeting ligand.

[0206] Thus, target cells can be selected based on a variety of factors, including, for example, (1) the intended use (e.g., therapy, expression of harvested proteins, and conferring disease resistance), and (2) the expression of a marker with a desired amount of specificity.

[0207] The target cells are not limited in any way and include both germline cells and cell lines, and somatic cells and cell lines. When the target cells are germline cells, the target cells are preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES).

[0208] Therapeutic Formulations and Administration Also described herein are pharmaceutical compositions comprising the antigen-binding molecules described herein. In some embodiments, the pharmaceutical compositions of the present invention can be formulated with suitable carriers, excipients, and other agents that improve transportation, delivery, tolerability, etc. Numerous suitable formulations can be found in a formulary 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, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0209] The dose of an antigen-binding molecule administered to a patient may vary depending on the patient's age and physique, the target disease, condition, route of administration, etc. 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 in a single dose of typically about 0.01 to about 20 mg per kg of body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering bispecific antigen-binding molecules can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation, and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0210] Various delivery systems are known and can be used to administer the pharmaceutical compositions described herein, including, for example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0211] The pharmaceutical compositions described herein can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are easily adapted for delivery of the pharmaceutical compositions described herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices come pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0212] A number of reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions described herein. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25™ pen, HUMALOG™ pen, HUMALIN70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN, to name just a few. STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having application in the subcutaneous delivery of the pharmaceutical compositions described herein include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name a few.

[0213] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit Ref Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled release system can be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0214] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), and the like. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, and the like. The injectable solutions prepared in this manner are preferably filled into appropriate ampoules.

[0215] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for adapting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antigen-binding molecule contained therein is generally about 5 to about 500 mg per dosage form in unit dose. In particular, for injections, the antibody is preferably contained in an amount of about 5 to about 100 mg, and for other dosage forms, it is preferably contained in an amount of about 10 to about 250 mg.

[0216] Its therapeutic and diagnostic uses Also disclosed herein are methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-hCACNG1 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCACNG1 antibody (e.g., an anti-hCACNG1 antibody or ADC comprising any of the HCVR / LCVR or CDR sequences described in Table 1 herein). The therapeutic composition may comprise any of the anti-hCACNG1 antibodies, antigen-binding fragments thereof, or ADCs disclosed herein, and a pharmaceutically acceptable carrier or diluent.

[0217] Antibodies, antigen-binding fragments thereof, or antibody-drug conjugates, including the anti-hCACNG1 antibodies described herein, may be useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with skeletal muscle tissue. For example, the antibodies and ADCs described herein may be useful for the treatment of muscle wasting disorders (e.g., cachexia, glucocorticoid-induced muscle loss, heart failure-induced muscle loss, HIV wasting, disuse, aging, etc.) and / or muscular dystrophy / myopathy.

[0218] The anti-hCACNG1 antibodies described herein have various utilities. For example, in some embodiments, the anti-hCACNG1 antibodies described herein can be used as a diagnostic assay for CACNG1, for example, to detect its expression in specific cells, tissues, etc., for example, to identify / label skeletal muscle fibers. Various diagnostic and prognostic assay techniques known in the art can be used, such as competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays performed in either heterogeneous or homogeneous phases (Zola (1987) Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. pp. 147-1581). The antibody used in the assay can be labeled with a detectable moiety. The detectable moiety must be capable of generating a detectable signal, either directly or indirectly. Any method known in the art for conjugating an antibody to a detectable moiety may be employed.

[0219] In another embodiment, a method for treating a disease, such as a muscle wasting disorder, is provided. The method can include providing an antibody or a CACNG1 antigen-binding fragment thereof, as described above, to a subject in need of such treatment. [Example]

[0220] Example 1: Exemplary CACNG1 Antibodies Generation of anti-human CACNG1 antibodies Anti-human CACNG1 antibodies were obtained by immunizing mice (eg, engineered mice containing DNA encoding a human immunoglobulin heavy chain variable region and a human kappa light chain variable region) with human CACNG1.

[0221] After immunization, splenocytes were harvested from each mouse and either (1) fused with mouse myeloma cells to preserve their viability, forming hybridoma cells, and screened for human CACNG1 specificity, or (2) sorted for B cells (as described in US2007 / 0280945(A1)) using either human CACNG1 fragments as sorting reagents to bind and identify reactive antibodies (antigen-positive B cells).

[0222] Chimeric antibodies against human CACNG1 were first isolated having human variable regions and murine constant regions using VELOCIMMUNE technology as described, for example, in U.S. Pat. No. 7,105,348, U.S. Pat. No. 8,642,835, and U.S. Pat. No. 9,622,459, each of which is incorporated herein by reference.

[0223] For some antibodies, for testing purposes, the murine constant region was replaced with a desired human constant region, such as a wild-type human CH or a modified human CH (e.g., an IgG1, IgG2, or IgG4 isotype), and a light chain constant region (CL) to generate a fully human anti-hCACNG1 antibody or antigen-binding portion thereof. While the constant region selected can vary according to the specific use, the high-affinity antigen-binding and target specificity properties reside in the variable region.

[0224] Certain biological properties of exemplary anti-human CACNG1 antibodies generated according to the methods of this example are detailed in the Examples set forth below.

[0225] Amino acid and nucleic acid sequences of the heavy and light chain variable regions of anti-hCACNG1 antibody Table 1 lists the sequence identifiers of the nucleic acid (NA) sequences encoding the heavy or light chain variable regions (HCVRs or LCVRs, respectively) or heavy or light chain CDRs (HCDRs and LCDRs, respectively) of selected anti-hCACNG1 proteins used to generate the therapeutic anti-hCACNG1 proteins disclosed herein, along with the sequence identifiers of the amino acid (AA) sequences in parentheses. [Table 1] 31929 / 10728(wild type hIgG1) / 14647(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO: 1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTACAGCGTCTGGAATCACCTTCAGAAATTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTGGTATGATGGAAGTAATAAGTACT ATGCAGACTCCGTGAAGGCCGTTTCACCATCTCCGGAGACAATTCCAAGGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAAGGGGCACTATAAGAACAGCTGCCCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 2) QVQLVESGGGVVQPGRSLRLSCTASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISGDNSKVYLQMNSLRAEDTAVYYCARRGTIRTAAPFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 3) GGA ATC ACC TTC AGA AAT TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 4) GITFRNYG HCDR2 nucleic acid sequence (SEQ ID NO: 5) ATG TGG TAT GAT GGA AGT AAT AAG HCDR2 amino acid sequence (SEQ ID NO: 6) MWYDGSNK HCDR3 nucleic acid sequence (SEQ ID NO: 7) GCG AGA AGG GGC ACT ATA AGA ACA GCT GCC CCT TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO: 8) ARRGTIRTAAPFDY LCVR nucleic acid sequence (SEQ ID NO: 9) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO: 11) CAG AGC ATT AGC AGC TAT LCDR1 amino acid sequence (SEQ ID NO: 12) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO: 13) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 14) AAS LCDR3 nucleic acid sequence (SEQ ID NO: 15) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO: 16) QQSYSTPPIT HC nucleic acid sequence (SEQ ID NO: 193) CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA HC amino acid sequence (SEQ ID NO: 194) [ka] *Underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase. See, e.g., SEQ ID NO: 269 LC nucleic acid sequence (SEQ ID NO: 195) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 196) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10715(wild type hIgG1) / 14570(IgG1 N180Q): HCVR nucleic acid sequence (SEQ ID NO: 17) CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGCGACCCTGTCCCGCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAACTGGATCCGCCAGTCCCAGGGAAGGGGCTGGAATGGATTGGGGAAATCCTTCATAGTGGAAGAACCAACTACA ACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGGGAAGGATAGCAGCTCGTCACGGCTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 18) QVQLQQWGAGLLKPSATLSRTCAVYGGSFSGYYWNWIRQSPGKGLEWIGEILHSGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAGRIAARHGWFDPWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 19) GGT GGG TCC TTC AGT GGT TAC TAC HCDR1 amino acid sequence (SEQ ID NO: 20) GGSFSGYY HCDR2 nucleic acid sequence (SEQ ID NO: 21) ATC CTT CAT AGT GGA AGA ACC HCDR2 amino acid sequence (SEQ ID NO: 22) ILHSGRT HCDR3 nucleic acid sequence (SEQ ID NO: 23) GCG GGA AGG ATA GCA GCT CGT CAC GGC TGG TTC GAC CCC HCDR3 amino acid sequence (SEQ ID NO: 24) AGRIAARHGWFDP LCVR nucleic acid sequence (SEQ ID NO: 25) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTACATCTGTAGGAGACAGAGTCACCATCTCTTGTCGGGCGAGTCAGGATATTCGCAAGTGGTTAGCCTGGTATCAACAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTATGCTACATCCAGTT TGCAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATTTTGCAACTTACTTTTGTCAACAGGCTAACAGTTTCCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA LCVR amino acid sequence (SEQ ID NO: 26) DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIK LCDR1 nucleic acid sequence (SEQ ID NO: 27) CAG GAT ATT CGC AAG TGG LCDR1 amino acid sequence (SEQ ID NO: 28) QDIRKW LCDR2 nucleic acid sequence (SEQ ID NO: 29) GCT ACA TCC LCDR2 amino acid sequence (SEQ ID NO: 30) ATS LCDR3 nucleic acid sequence (SEQ ID NO: 31) CAA CAG GCT AAC AGT TTC CCG TTC ACT LCDR3 amino acid sequence (SEQ ID NO: 32) QQANSFPFT HC nucleic acid sequence (SEQ ID NO: 197) GTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT GGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA HC amino acid sequence (SEQ ID NO: 198) [ka] *Underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase. See, e.g., SEQ ID NO: 269 LC nucleic acid sequence (SEQ ID NO: 199) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTACATCTGTAGGAGACAGAGTCACCATCTCTTGTCGGGCGAGTCAGGATATTCGCAAGTGGTTAGCCTGGTATCAACAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTATGCTACATCCAGTTT GCAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATTTTGCAACTTACTTTTGTCAACAGGCTAACAGTTTCCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 200) DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10717(wild type hIgG1) / 14572(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO: 33) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGCATGATGGAAGTGATAAATATT ATGTAGACTCCGTGAAGGGCCGATTCTCCATCGCCAGAGACAATTCCAAGAACACGCTTTATCTGCAAATGAATAGTCTGAGAGTCGAGGACACGGGTATATATTACTGTGCGAGAAGGGGTATACGTGGAACCGTTTTGACCACTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 34) QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDKYYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 35) GGA TTC ACC TTC AGT ACA TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 36) GFTFSTYG HCDR2 nucleic acid sequence (SEQ ID NO: 37) ATT TGG CAT GAT GGA AGT GAT AAA HCDR2 amino acid sequence (SEQ ID NO: 38) IWHDGSDK HCDR3 nucleic acid sequence (SEQ ID NO: 39) GCG AGA AGG GGT ATA CGT GGA ACC GTT TTT GAC CAC HCDR3 amino acid sequence (SEQ ID NO: 40) ARRGIRGTVFDH LCVR nucleic acid sequence (SEQ ID NO: 41) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATT TAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA LCVR amino acid sequence (SEQ ID NO: 42) DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 43) CAG AGT ATT AGT AAC AAG LCDR1 amino acid sequence (SEQ ID NO: 44) QSISNK LCDR2 nucleic acid sequence (SEQ ID NO: 45) AAG GCG TCT LCDR2 amino acid sequence (SEQ ID NO: 46) KAS LCDR3 nucleic acid sequence (SEQ ID NO: 47) CAA CAG TAT AAT AGT TAT TCG TGG ACG LCDR3 amino acid sequence (SEQ ID NO: 48) QQYNSYSWT HC nucleic acid sequence (SEQ ID NO: 201) HC amino acid sequence (SEQ ID NO: 202) [ka] *Underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase. See, e.g., SEQ ID NO: 269 LC nucleic acid sequence (SEQ ID NO: 203) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATTT AGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 204) DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10716(wild type hIgG1) / 14571(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO: 49) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCAAATAATTACTACTGGACCTGGCTCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGTTATATCTATTACAGTGGGAGCGCCAAC TACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTAAATTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGGGGCGGTCAAGTACTTCCGGCATTGGGCCAGGGCACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 50) QVQLQESGPGLVKPSETLSLTTCTVSGDSINNYYWTWLRQPPGKGLEWIGYIYYSGSANYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARGAVKYFRHWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 51) GGT GAC TCC ATC AAT AAT TAC TAC HCDR1 amino acid sequence (SEQ ID NO: 52) GDSINNYY HCDR2 nucleic acid sequence (SEQ ID NO: 53) ATC TAT TAC AGT GGG AGC GCC HCDR2 amino acid sequence (SEQ ID NO: 54) IYYSGSA HCDR3 nucleic acid sequence (SEQ ID NO: 55) GCG AGA GGG GCG GTC AAG TAC TTC CGG CAT HCDR3 amino acid sequence (SEQ ID NO: 56) ARGAVKYFRH LCVR nucleic acid sequence (SEQ ID NO: 57) GAAATTGTGTTGACGCAGTCTCCGGGCACCCTCTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTATTAACCACAACAACTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAAC AGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGAAGTGTATTCTTGTCAGCAGTATGGTAGCTTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA LCVR amino acid sequence (SEQ ID NO: 58) EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 59) CAG ACT ATT AAC CAC AAC AAC LCDR1 amino acid sequence (SEQ ID NO: 60) QTINHNN LCDR2 nucleic acid sequence (SEQ ID NO: 61) GGT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 62) GAS LCDR3 nucleic acid alignment (alignment number 63) CAG CAG TAT GGT AGC TTG CCG CTC ACT LCDR3 アミノ acid arrangement (allocation number 64) QQYGSLPLT HC nucleic acid array (array number 205) HC amino acid sequence (SEQ ID NO: 206) [ka] *Underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase. See, e.g., SEQ ID NO: 269 LC nucleic acid sequence (SEQ ID NO: 207) GAAATTGTGTTGACGCAGTCTCCGGGCACCCTCTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTATTAACCACAACAACTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAAC AGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGAAGTGTATTCTTGTCAGCAGTATGGTAGCTTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 208) EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10783(wild type hIgG1) / 14574(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO: 65) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGATTGATGGAAGTAATAAATATTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGGTATAGTAGTAGCTGCCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 66) QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWIDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGGIVVAAPFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 67) GGA TTC ACC TTC AGT AGC TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 68) GFTFSSYG HCDR2 nucleic acid sequence (SEQ ID NO: 69) ATA TGG ATT GAT GGA AGT AAT AAA HCDR2 amino acid sequence (SEQ ID NO: 70) IWIDGSNK HCDR3 nucleic acid sequence (SEQ ID NO: 71) GCG AGA AGG GGG GGT ATA GTA GTA GCT GCC CCC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO: 72) ARRGGIVVAAPFDY LCVR nucleic acid sequence (SEQ ID NO: 73) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO: 74) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO: 75) CAG AGC ATT AGC AGC TAT LCDR1 amino acid sequence (SEQ ID NO: 76) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO: 77) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 78) A A S LCDR3 nucleic acid sequence (SEQ ID NO: 79) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO: 80) Q Q S Y S T P P I T HC nucleic acid sequence (SEQ ID NO: 209) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGATTGATGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGGGTATAGTAGTAGCTGCCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCA AGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCAAAACCCAAGGACACTCTCATGATCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC TCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA HC amino acid sequence (SEQ ID NO: 210) [ka] *Underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase. See, e.g., SEQ ID NO: 269 LC nucleic acid sequence (SEQ ID NO: 211) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 212) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31944 HCVR nucleic acid sequence (SEQ ID NO: 81) CAG GTG CAG TTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GAA GCG TCT GGA ATC ACC TTC AGA AAC TAT GGC ATG CAC TGG GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG GCA GTT ATG TGG TAT GAT GGA AGT AAT AAA TAC TAC GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG GTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAA GAC ACG GCT GTG TAT TAC TGT GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA HCVR amino acid sequence (SEQ ID NO: 82) QVQLVESGGGVVQPGRSLRLSCEASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARRGHIATAAPFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 83) GGA ATC ACC TTC AGA AAC TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 84) GITFRNYG HCDR2 nucleic acid sequence (SEQ ID NO: 85) atg tgg tat gat gga agt aat aaa HCDR2 amino acid sequence (SEQ ID NO: 86) MWYDGSN HCDR3 nucleic acid sequence (SEQ ID NO: 87) GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO: 88) ARRGHIATAAPFD LCVR nucleic acid sequence (SEQ ID NO: 89) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCCGTAGGAGACAGAGTCACCATCAGTTGCCGGGCAAGTCAGAGCATTAGTAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO: 90) DIQMTQSPSSLSASVGDRVTISCRASQSISSYLNWYQQKPGKAPKVLMYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO: 91) CAG AGC ATT AGT AGT TAT LCDR1 amino acid sequence (SEQ ID NO: 92) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO: 93) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 94) AAS LCDR3 nucleic acid sequence (SEQ ID NO: 95) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO: 96) QQSYSTPPIT HC nucleic acid sequence (SEQ ID NO: 213) CAG GTG CAG TTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GAA GCG TCT GGA ATC ACC TTC AGA AAC TAT GGC ATG CAC TGG GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG GCA GTT ATG TGG TAT GAT GGA AGT AAT AAA TAC TAC GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG GTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAA GAC ACG GCT GTG TAT TAC TGT GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGA HC amino acid sequence (SEQ ID NO: 214) QVQLVESGGG VVQPGRSLRL SCEASGITFR NYGMHWVRQA PGKGLEWVAV MWYDGSNKYY ADSVKGRFTI SRDNSKNTVY LQMNSLRAED TAVYYCARRG HIATAAPFDY WGQGTLVTVS S AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK LC nucleic acid sequence (SEQ ID NO: 215) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCCGTAGGAGACAGAGTCACCATCAGTTGCCGGGCAAGTCAGAGCATTAGTAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA LC amino acid sequence (SEQ ID NO: 216) DIQMTQSPSS LSASVGDRVT ISCRASQSIS SYLNWYQQKP GKAPKVLMYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPPITFG QGTRLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC 31265(wild type hIgG1) / 5972(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO: 97) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTACAGCGTCTGGATTCACCTTCCGTTCCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGTCAGTTATTTGGATTGATGGAAATAATATATACTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAAGACTGGCTATAACATCAGCTGCCCCCTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 98) QVQLVESGGGVVQPGRSLRLSCTASGFTFRSYGMHWVRQAPGKGLEWVSVIWIDGNNIYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARRLAITSAAPFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 99) GGA TTC ACC TTC CGT TCC TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 100) GFTFRSYG HCDR2 nucleic acid sequence (SEQ ID NO: 101) ATT TGG ATT GAT GGA AAT AAT ATA HCDR2 amino acid sequence (SEQ ID NO: 102) IWIDGNNI HCDR3 nucleic acid sequence (SEQ ID NO: 103) GCG AGA AGA CTG GCT ATA ACA TCA GCT GCC CCC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO: 104) ARRLAITSAAPFDY LCVR nucleic acid sequence (SEQ ID NO: 105) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO: 106) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO: 107) CAG AGC ATT AGC AGC TAT LCDR1 amino acid sequence (SEQ ID NO: 108) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO: 109) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 110) AAS LCDR3 nucleic acid sequence (SEQ ID NO: 111) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO: 112) QQSYSTPPIT HC nucleic acid sequence (SEQ ID NO: 217) HC amino acid sequence (SEQ ID NO: 218) [ka] *Underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase. See, e.g., SEQ ID NO: 269 LC nucleic acid sequence (SEQ ID NO: 219) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 220) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31941 HCVR nucleic acid sequence (SEQ ID NO: 113) CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACGCCTTCACCACCTATGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGATGGATCAGCGCTTACAATGGAAATACAAACTATGCAGAGAAG GTCCAGGGCAGATTCACCATGACCACAGACACATCCACGAATACAGCCTACATGGAGCTGAGGAGCCTGAGATCCGACGACACGGCCGTGTATTTCTGTGCGAGAAAGGGTCACTATGGTTCGGGGACTTATTATAACCCCTTTGGTTTTGATTTTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA HCVR amino acid sequence (SEQ ID NO: 114) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 115) ggt tac gcc ttc acc acc tat ggt HCDR1 amino acid sequence (SEQ ID NO: 116) GYAFTTYG HCDR2 nucleic acid sequence (SEQ ID NO: 117) atc agc gct tac aat gga aat aca HCDR2 amino acid sequence (SEQ ID NO: 118) ISAYNGN HCDR3 nucleic acid sequence (SEQ ID NO: 119) GCG AGA AAG GGT CAC TAT GGT TCG GGG ACT TAT TAT AAC CCC TTT GGT TTT GAT TTT HCDR3 amino acid sequence (SEQ ID NO: 120) CARKGHYGSGTYYNPFGFD LCVR nucleic acid sequence (SEQ ID NO: 121) GAAATTATGTTGATGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGACATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAG LCVR amino acid sequence (SEQ ID NO: 122) EIMLMQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYYGSPWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 123) cag agt gtt agc agc agc tac LCDR1 amino acid sequence (SEQ ID NO: 124) QSVSSSY LCDR2 nucleic acid sequence (SEQ ID NO: 125) ggt gca tcc LCDR2 amino acid sequence (SEQ ID NO: 126) GA LCDR3 nucleic acid sequence (SEQ ID NO: 127) cag cag tat tat ggc tca cct tgg acg LCDR3 amino acid sequence (SEQ ID NO: 128) CQQYYGSPW HC nucleic acid sequence (SEQ ID NO: 221) HC amino acid sequence (SEQ ID NO: 222) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFG FDFWGQGTMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK LC nucleic acid sequence (SEQ ID NO: 223) GAAATTATGTTGATGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGACATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAGCGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA LC amino acid sequence (SEQ ID NO: 224) EIMLMQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATDIP DRFSGSGSGT DFTLTISRLE PEDFAVYFCQ QYYGSPWTFG QGTKVEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC 7660 HCVR nucleic acid sequence (SEQ ID NO: 129) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAAACTCTCCTGTACAGCCTCTGGGTTGACCCTCAGTGACTCTGCTATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTTGGCCGTATAAGAAATAAGGCTAATAGGTACGCGACA GAATATGCTGCGTCGGTGAAAGGCAGGTTCACCATTTCAAGAGATGATTCAAAGAACACGGCGTATCTACAAATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTATTGTACTAGAAAACTGGAAGATTTTCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 130) EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 131) GGG TTG ACC CTC AGT GAC TCT GCT HCDR1 amino acid sequence (SEQ ID NO: 132) GLTLSDSA HCDR2 nucleic acid sequence (SEQ ID NO: 133) ATA AGA AAT AAG GCT AAT AGG TAC GCG ACA HCDR2 amino acid sequence (SEQ ID NO: 134) IRNKANRYAT HCDR3 nucleic acid sequence (SEQ ID NO: 135) ACT AGA AAC TGG AAG ATT TTC CTC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO: 136) TRNWKIFLFDY LCVR nucleic acid sequence (SEQ ID NO: 137) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGACTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCAAATACTTAGCCTGGTTCCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGACCAGTGGCATCCCCGACAGGATCAGTGGCAGTGGGTCAGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGAAGTTCACCCTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA LCVR amino acid sequence (SEQ ID NO: 138) EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 139) CAG AGT GTT GGC AGC AAA TAC LCDR1 amino acid sequence (SEQ ID NO: 140) QSVGSKY LCDR2 nucleic acid sequence (SEQ ID NO: 141) GGT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 142) GAS LCDR3 nucleic acid sequence (SEQ ID NO: 143) CAG CAG TAT GGA AGT TCA CCC TGG ACG LCDR3 amino acid sequence (SEQ ID NO: 144) QQYGSSPWT HC nucleic acid sequence (SEQ ID NO: 225) HC amino acid sequence (SEQ ID NO: 226) EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK LC nucleic acid sequence (SEQ ID NO: 227) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGACTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCAAATACTTAGCCTGGTTCCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGACCAGTGGCATCCCCGACAGGATCAGTGGCAGTGGGTCAGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGAAGTTCACCCTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 228) EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 9909 HCVR nucleic acid sequence (SEQ ID NO: 145) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAACAACTATGGCATGAGCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTGGTAGTGGTGGTACCACATTCTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGGCAAAGGAGGATATTGTAGTAGTAGCGGCTGCCGTCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 146) EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMDVWGQGTTVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 147) GGA TTC ACC TTT AAC AAC TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 148) GFTFNNYG HCDR2 nucleic acid sequence (SEQ ID NO: 149) ATT AGT GGT AGT GGT GGT ACC ACA HCDR2 amino acid sequence (SEQ ID NO: 150) SGSGGT HCDR3 nucleic acid sequence (SEQ ID NO: 151) GGC AAA GGA GGA TAT TGT AGT AGT AGC GGC TGC CGT CAC TAC GGT ATG GAC GTC HCDR3 amino acid sequence (SEQ ID NO: 152) CGKGGYCSSSGCRH LCVR nucleic acid sequence (SEQ ID NO: 153) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGACCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAATAATTATATATACTGGTACCAGCGGCTCCCAGGAACGACCCCCAAACTCCTCATCTATAGGAATAATCAGCGG CCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACACCCTGAGTGGGTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA LCVR amino acid sequence (SEQ ID NO: 154) QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNYIYWYQRLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDTLSGYVFGTGTKVTVL LCDR1 nucleic acid sequence (SEQ ID NO: 155) AGC TCC AAC ATC GGA AAT AAT TAT LCDR1 amino acid sequence (SEQ ID NO: 156) SSNIGNNY LCDR2 nucleic acid sequence (SEQ ID NO: 157) agg aat aat LCDR2 amino acid sequence (SEQ ID NO: 158) RN LCDR3 nucleic acid sequence (SEQ ID NO: 159) GCA GCA TGG GAT GAC ACC CTG AGT GGG TAT GTC LCDR3 amino acid sequence (SEQ ID NO: 160) CAAWDDTLSGY HC nucleic acid sequence (SEQ ID NO: 229) HC amino acid sequence (SEQ ID NO: 230) EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMD VWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGP TIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK LC nucleic acid sequence (SEQ ID NO: 231) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAATAATTATATATACTGGTACCAGCGGCTCCCAGGAACGACCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACACCCTGAGTGGGTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA LC amino acid sequence (SEQ ID NO: 232) QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYIYWYQRLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDTLSGYVFGTGTKVTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC 10713 (wild-type hIgG1) / 14573 (hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO: 161) GAGGTGCAGCTGGTGGAGTCTGGGGGAAACTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCAGCCATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTACTGGTAGAGGTTTTGACACACACTACG CTGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACATTTCCAAAAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAAAGGTCTCTATGATTCGGGGAATTATTATATCGATTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 162) EVQLVESGGNLVQPGGSLRLSCAASGFTFTSHAMNWVRQAPGKGLEWVSVITGRGFDTHYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAKGLYDSGNYYIDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 163) GGA TTC ACC TTT ACC AGC CAT GCC HCDR1 amino acid sequence (SEQ ID NO: 164) GFTFTSHA HCDR2 nucleic acid sequence (SEQ ID NO: 165) ATT ACT GGT AGA GGT TTT GAC ACA HCDR2 amino acid sequence (SEQ ID NO: 166) ITGRGFDT HCDR3 nucleic acid sequence (SEQ ID NO: 167) GCG AAA GGT CTC TAT GAT TCG GGG AAT TAT TAT ATC GAT TAC HCDR3 amino acid sequence (SEQ ID NO: 168) AKGLYDSGNYYIDY LCVR nucleic acid sequence (SEQ ID NO: 169) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTTTCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGAC CCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATCTCAGCCTGAGTTTCAATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA LCVR amino acid sequence (SEQ ID NO: 170) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDLSLSFNWVFGGGTKLTVL LCDR1 nucleic acid sequence (SEQ ID NO: 171) AGC TCC AAC ATT GGG AAT AAT TAT LCDR1 amino acid sequence (SEQ ID NO: 172) SSNIGNNY LCDR2 nucleic acid sequence (SEQ ID NO: 173) GAC AAT AAT LCDR2 amino acid sequence (SEQ ID NO: 174) DNN LCDR3 nucleic acid sequence (SEQ ID NO: 175) GGA ACA TGG GAT CTC AGC CTG AGT TTC AAT TGG GTG LCDR3 amino acid sequence (SEQ ID NO: 176) GTWDLSLSFNWV HC nucleic acid sequence (SEQ ID NO: 233) HC amino acid sequence (SEQ ID NO: 234) [ka] *Underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase. See, e.g., SEQ ID NO: 269 LC nucleic acid sequence (SEQ ID NO: 235) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTTTCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATA AGCGACCCTCAGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATCTCAGCCTGAGTTTCAATTGGGTGTTCGGCGGAGGGACCAA GCTGACCGTCCTAGGCCAGCCCAAGGCCGCCCCCTCCGTGACCCTGTTCCCCCTCCTCCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCTCCGACTTCTACCCCGGCGCCGTGACCGTGGCCTGGAAGGCCGACTCCTCCCCGTG AAGGCCGGCGTGGAGACCACCACCCCCTCCAAGCAGTCCAACAACAAGTACGCCGCCTCCTCCTACCTGTCCCTGACCCCCGAGCAGTGGAAGTCCCACCGGTCCTACTCCTGCCAGGTGACCCACGAGGGCTCCACCGTGGAGAAGACCGTGGCCCCCA CCGAGTGCTCCTGA LC amino acid sequence (SEQ ID NO: 236) QSVLTQPPSVSAAPGQKVTISCSSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDLSLSFNWVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 7854 HCVR nucleic acid sequence (SEQ ID NO: 177) CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACGCCTTCACCACCTATGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGATGGATCAGCGCTTACAATGGAAATACAAACTATGCAGAGAAG GTCCAGGGCAGATTCACCATGACCACAGACACATCCACGAATACAGCCTACATGGAGCTGAGGAGCCTGAGATCCGACGACACGGCCGTGTATTTCTGTGCGAGAAAGGGTCACTATGGTTCGGGGACTTATTATAACCCCTTTGGTTTTGATTTTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA HCVR amino acid sequence (SEQ ID NO: 178) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 179) GGT TAC GCC TTC ACC ACC TAT GGT HCDR1 amino acid sequence (SEQ ID NO: 180) GYAFTTYG HCDR2 nucleic acid sequence (SEQ ID NO: 181) ATC AGC GCT TAC AAT GGA AAT ACA HCDR2 amino acid sequence (SEQ ID NO: 182) ISAYNGNT HCDR3 nucleic acid sequence (SEQ ID NO: 183) GCG AGA AAG GGT CAC TAT GGT TCG GGG ACT TAT TAT AAC CCC TTT GGT TTT GAT TTT HCDR3 amino acid sequence (SEQ ID NO: 184) ARKGHYGSGTYYNPFGFDF LCVR nucleic acid sequence (SEQ ID NO: 185) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCTTTGTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA LCVR amino acid sequence (SEQ ID NO: 186) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFALYFCQQYYGSPWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 187) CAG AGT GTT AGC AGC AGC TAC LCDR1 amino acid sequence (SEQ ID NO: 188) QSVSSSY LCDR2 nucleic acid sequence (SEQ ID NO: 189) GGT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 190) GAS LCDR3 nucleic acid sequence (SEQ ID NO: 191) CAG CAG TAT TAT GGC TCA CCT TGG ACG LCDR3 amino acid sequence (SEQ ID NO: 192) QQYYGSPWT HC nucleic acid sequence (SEQ ID NO: 237) HC amino acid sequence (SEQ ID NO: 238) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGT MVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK LC nucleic acid sequence (SEQ ID NO: 239) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCTTTGTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 240) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGAS...

Claims

1. 1. A recombinant adeno-associated virus (AAV) particle comprising: (i) An AAV capsid protein modified with an antigen-binding protein that specifically binds to human calcium voltage-dependent channel accessory subunit gamma 1 (hCACNG1), wherein the antigen-binding protein is selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 84-86-88-92-94-96, and 100-102-104-108-11. an AAV capsid protein comprising an anti-hCACNG1 antibody or an antigen-binding fragment thereof, the AAV capsid protein comprising a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from the group consisting of SEQ ID NOs: 0-112, 116-118-120-124-126-128, SEQ ID NOs: 132-134-136-140-142-144; SEQ ID NOs: 148-150-152-156-158-160, SEQ ID NOs: 164-166-168-172-174-176, and SEQ ID NOs: 180-182-186-188-190-192; (ii) a nucleotide of interest comprising a sequence including a coding sequence encoding microdystrophin, fukutin-related protein (FKRP), or myotubularin (MTM1); the nucleotide of interest is encapsulated by an AAV capsid comprising the AAV capsid protein modified with an antigen-binding protein that specifically binds to the hCACNG1; Optionally, the modified AAV capsid protein comprises a first member of a protein:protein binding pair and a second member of a protein:protein binding pair, the second member of the protein:protein binding pair comprising the anti-hCACNG1 antibody or antigen-binding fragment, and the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are associated to direct the tropism of the viral particle toward hCACNG1.

2. The recombinant AAV particle of claim 1, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR or VH) and / or a light chain variable region (LCVR or VL).

3. 3. The recombinant AAV particle of claim 2, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof is selected from the group consisting of a human or humanized antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bispecific T-cell engager (BiTE), a triabody, and chemically modified derivatives thereof.

4. The recombinant AAV particle according to any one of claims 1 to 3, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof comprises a fragment antigen-binding region (Fab).

5. The recombinant AAV particle of any one of claims 1 to 3, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof comprises a single-chain variable fragment (scFv).

6. The recombinant AAV particle of claim 5, wherein the scFv comprises domains arranged in the following direction from N-terminus to C-terminus: HCVR-LCVR.

7. The recombinant AAV particle of claim 5, wherein the scFv comprises domains arranged in the following direction from N-terminus to C-terminus: LCVR-HCVR.

8. The recombinant AAV particle according to any one of claims 5 to 7, wherein the scFv variable regions are connected by a linker.

9. The recombinant AAV particle of claim 8, wherein the linker is a peptide linker.

10. The recombinant AAV particle of claim 9, wherein the peptide linker is -(GGGGS)n- (SEQ ID NO: 268), where n is 1 to 10.

11. The anti-hCACNG1 antibody or antigen-binding fragment thereof is about 1 x 10 －７ K of M Ｄ The recombinant AAV particle according to any one of claims 1 to 10, which binds to hCACNG1 with a higher affinity than that of the recombinant AAV particle.

12. The anti-hCACNG1 antibody or antigen-binding fragment thereof is about 10 x 10 －８ ~Approx. 1×10 －１０ K Ｄ The recombinant AAV particle according to any one of claims 1 to 11, which binds to hCACNG1 at

13. The anti-hCACNG1 antibody or antigen-binding fragment thereof is about 5 x 10 －９ ~Approx. 1×10 －１０ K Ｄ The recombinant AAV particle according to any one of claims 1 to 11, which binds to hCACNG1 at

14. The recombinant AAV particle of any one of claims 1 to 13, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair having at least 90% sequence identity to an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, and 178 / 186.

15. (a) the first member of the protein:protein binding pair comprises SpyTag, Isopeptag, SnoopTag, SpyTag002, SpyTag003, or a biologically equivalent portion or variant thereof; (b) the second member of the protein:protein binding pair is (i) SpyCatcher, KTag, Pirin-C, SnoopCatcher, SpyCatcher002, SpyCatcher003, or any biologically equivalent portion or variant thereof, and (ii) comprising the anti-hCACNG1 antibody or an antigen-binding fragment thereof; (c) the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are linked by an isopeptide bond.

16. (a) the first member of the protein:protein binding pair comprises a SpyTag, or any biologically equivalent portion or variant thereof; (b) the second member of the protein:protein binding pair comprises SpyCatcher fused to the anti-hCACNG1 antibody or antigen-binding fragment thereof, or any biologically equivalent portion or variant thereof.

17. 17. The recombinant AAV particle of any one of claims 1 to 16, comprising a first and / or second linker operably linking the first member of the protein:protein binding pair to a viral capsid protein.

18. The recombinant AAV particle of claim 17, wherein the first and second linkers are not identical.

19. The recombinant AAV particle of claim 17, wherein the first and second linkers are identical.

20. The recombinant AAV particle according to any one of claims 17 to 19, wherein the first linker is 10 amino acids in length and / or the second linker is 10 amino acids in length.

21. the modified AAV capsid proteins comprise a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein; the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises an insertion of a first member of a protein:protein binding pair and / or the anti-hCACNG1 antibody or antigen-binding fragment thereof; 21. The recombinant AAV particle of any one of claims 1 to 20, wherein the portion of the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprising the insertion of the first member of a protein:protein binding pair and / or the anti-hCACNG1 antibody or antigen-binding fragment thereof further comprises an amino acid sequence that is at least 90% identical to the corresponding capsid protein of a wild-type AAV.

22. the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein, in addition to the insertion of a first member of a protein:protein binding pair and / or the anti-hCACNG1 antibody or antigen-binding fragment thereof; (i) amino acid substitutions, insertions, or deletions; (ii) a chimeric amino acid sequence, or 22. The recombinant AAV particle of claim 21, further comprising: (iii) any combination of (i) and (ii).

23. 23. The recombinant AAV particle of claim 22, wherein the amino acid substitution, insertion, or deletion reduces the natural tropism of the viral particle and / or creates a detectable label.

24. The recombinant AAV particle of any one of claims 1 to 23, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, a non-primate AAV listed in Table 2, and any chimera thereof.

25. The recombinant AAV particle according to any one of claims 1 to 24, wherein the AAV is AAV2.

26. 26. The recombinant AAV particle of any one of claims 1 to 25, wherein the recombinant AAV particle comprises a modified AAV2 VP1 capsid protein comprising a first member of a protein:protein binding pair inserted at amino acid position 1-453 and / or 1-587, and optionally linked to an AAV sequence via a linker on one or both sides.

27. the recombinant AAV particle comprises a modified AAV2 VP1 capsid protein comprising the first member of the protein:protein binding pair inserted at position G453, optionally via a linker; 27. The recombinant AAV particle of claim 26, wherein optionally, the modified AAV2 VP1 capsid protein further comprises a mutation selected from R585A, R588A, R484A, R487A, K532A, and any combination thereof.

28. the recombinant AAV particle comprises a mosaic AAV capsid comprising a second set of AAV2 VP1 capsid proteins that lacks the first member of the protein:protein binding pair; Optionally, the second set of AAV2 VP1 capsid proteins comprises a mutation selected from R585A, R588A, R484A, R487A, K532A, and any combination thereof.

29. The recombinant AAV particle according to any one of claims 1 to 24, wherein the AAV is AAV9.

30. 30. The recombinant AAV particle of claim 29, wherein the viral capsid comprises a modified AAV9 VP1 capsid protein comprising a first member of a specific binding pair inserted at position 1-453 or 1-589, optionally via a linker.

31. the recombinant AAV particles comprise a modified AAV9 VP1 capsid protein comprising a first member of a protein:protein binding pair inserted at position G453, optionally via a linker; Optionally, the modified AAV9 VP1 capsid protein further comprises a mutation selected from N272A, W503A, and a combination thereof.

32. the recombinant AAV particle is a mosaic virus capsid comprising a second set of AAV9 VP1 capsid proteins lacking the first member of the protein:protein binding pair; Optionally, the second set of AAV9 VP1 capsid proteins comprises a mutation selected from N272A, W503A, and a combination thereof.

33. The recombinant AAV particle according to any one of claims 1 to 24, wherein the AAV is a non-primate AAV.

34. The recombinant AAV particle of claim 33, wherein the non-primate AAV is an avian AAV (AAAV).

35. 35. The recombinant AAV particle of claim 34, wherein the AAV capsid comprises a modified AAAV VP1 capsid protein comprising the first member of the protein:protein binding pair inserted at position 1-444 or 1-580, optionally via a linker.

36. The recombinant AAV particle of claim 33, wherein the non-primate AAV is a squamate AAV.

37. The recombinant AAV particle of claim 36, wherein the Squamata AAV is a bearded dragon AAV.

38. 38. The recombinant AAV particle of claim 37, wherein the AAV capsid comprises a modified bearded dragon AAV VP1 capsid protein comprising the first member of the protein:protein binding pair inserted at position 1-573 or 1-436, optionally via a linker.

39. The recombinant AAV particle of claim 33, wherein the non-primate AAV is a non-primate mammalian AAV.

40. The recombinant AAV particle of claim 39, wherein the non-primate mammalian AAV is a sea lion AAV.

41. 41. The recombinant AAV particle of claim 40, wherein the AAV capsid comprises a modified sea lion AAV VP1 capsid protein comprising the first member of the protein:protein binding pair inserted, optionally via a linker, at a position selected from the group consisting of 1-429, 1-430, 1-431, 1-432, 1-433, 1-434, 1-436, 1-437, and 1-565.

42. 42. The recombinant AAV particle of any one of claims 1 to 41, wherein the recombinant AAV particle comprises a mosaic AAV capsid, optionally comprising: (i) a first plurality of reference capsid proteins, each of which is not associated with the anti-hCACNG1 antibody or antigen-binding fragment thereof; and (ii) a second plurality of capsid proteins, each of which is associated with the anti-hCACNG1 antibody or antigen-binding fragment thereof; and optionally, the mosaic AAV particle comprises the first plurality of reference capsid proteins and the second plurality of capsid proteins in a ratio of 1:

7.

43. The recombinant AAV particle of any one of claims 1 to 42, wherein the target nucleotide encodes microdystrophin.

44. The recombinant AAV particle of any one of claims 1 to 43, wherein the target nucleotide comprises the sequence set forth as SEQ ID NO:

270.

45. The recombinant AAV particle of any one of claims 1 to 42, wherein the target nucleotide encodes human FKRP.

46. 46. ​​The recombinant AAV particle of any one of claims 1 to 42 and 45, wherein the target nucleotide comprises the sequence set forth as SEQ ID NO:

271.

47. The recombinant AAV particle of any one of claims 1 to 42, wherein the target nucleotide encodes human MTM1.

48. 48. The recombinant AAV particle of any one of claims 1 to 42 and 47, wherein the target nucleotide comprises the sequence set forth as SEQ ID NO:

272.

49. 10. A method of treating Duchenne muscular dystrophy in a patient in need thereof, comprising administering to the patient recombinant AAV particles of any one of claims 1 to 44, optionally at a concentration of 3 x 10 １３ vg / kg (e.g., 2 x 10 １４ ) to said patient.

50. 1. A method of treating limb-girdle muscular dystrophy in a patient in need thereof, comprising administering recombinant AAV particles according to any one of claims 1 to 42 and 45 to 46, optionally at a concentration of 3 x 10 １３ vg / kg (e.g., 2 x 10 １４ ) to said patient.

51. 1. A method of treating myotubular myopathy in a patient in need thereof, comprising administering recombinant AAV particles according to any one of claims 1 to 42 and 47 to 48, optionally in a dose of 3 x 10 １３ vg / kg (e.g., 2 x 10 １４ ) to said patient.

52. 1. A method of treating muscle wasting or a genetic muscle disorder in a subject in need thereof, comprising: The recombinant AAV particles according to any one of claims 1 to 42 are optionally cultured at a concentration of 3 x 10 １３ administering to the subject a dose of more than 100 mg / kg of ribozyme; The method, wherein the nucleotide of interest 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.

53. 1. Optionally 3×10 for the manufacture of a medicament for administration to a subject in the treatment of muscle wasting or a genetic muscle disorder. １３ vg / kg (e.g., 2 x 10 １４ 43. Use of recombinant AAV particles according to any one of claims 1 to 42, at a dose of

54. 54. The method of claim 52 or the use of claim 53, wherein the muscle wasting or genetic 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.

55. 3 x 10 １３ vg / kg (e.g., 2 x 10 １４ ) to the subject, (i) a significant increase in the level of a liver enzyme (e.g., ALT) 1, 3, 5, 7, 15, and / or 21 days after administration compared to the corresponding level of the liver enzyme (e.g., ALT) in said subject prior to said administration; (ii) a significant increase in the level of one or more complement components (e.g., Bb, C3a, sC5b-9) 1, 3, 5, 7, 15, and / or 21 days after administration compared to the corresponding level of one or more complement components (e.g., Bb, C3a, sC5b-9) in said subject prior to said administration; (iii) a significant decrease in the level of platelet count at 1, 3, 5, 7, 15, and / or 21 days after administration compared to the corresponding platelet count in said subject before said administration; (iv) a significant increase in red blood cell distribution width (RDW) at 1, 3, 5, 7, 15, and / or 21 days after administration compared to the corresponding RDW in said subject prior to said administration; (v) a significant increase in the level of serum creatinine at 1, 3, 5, 7, 15, and / or 21 days after administration compared to the corresponding level of serum creatinine in said subject before said administration; or (vi) any combination of (i) to (v).

56. 56. The method or use of claim 55, wherein the subject is a non-human primate.

57. 56. The method or use of claim 55, wherein the subject is a human.

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