Treatment of muscle related disorders with anti-human CACNG1 antibodies

Abstract

Adeno-associated virus (AAV) particles that are redirected with an antibody against CACNG1 and carry a nucleotide of therapeutic interest are provided. Also provided are methods of making and using the AAV particles, e.g., for treating a patient in need thereof or for manufacturing a medicament for treating a patient in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 484,675, filed on February 13, 2023, U.S. Provisional Application No. 63 / 494,119, filed on April 4, 2023, and U.S. Provisional Application No. 63 / 583,724, filed on September 19, 2023, the disclosures of which are hereby incorporated by reference in their entireties.

[0003] Reference to a sequence listing submitted as an XML file

[0004] The xml formatted Sequence Listing entitled "11459WO01 Sequence Listing XML" is incorporated herein by reference in its entirety, was created on February 8, 2024, and is 280 Kb. Technical Field

[0005] The present application generally relates to human antibodies and antigen-binding fragments of human antibodies that bind to human CACNG1 (hCACNG1), and methods of use thereof, for example, in methods of treating a condition in a patient in need thereof. The present application also relates to antigen-binding molecules comprising at least an antigen-binding fragment of an anti-hCACNG1 antibody, wherein the complexing of the antigen-binding molecule with CACNG1 mediates internalization of the antigen-binding molecule / CACNG1 complex. The present application further relates to viral vectors conjugated to anti-hCACNG1 antibodies (or antigen-binding molecules comprising an antigen-binding fragment of an anti-hCACNG1 antibody), wherein these viral vectors comprise a therapeutic nucleotide of interest, and the conjugates can be used to treat muscle-related conditions. Background Art

[0006] Skeletal muscle is the largest organ in the human body, accounting for approximately 40% of total body weight. It is one of the three major muscle tissues in the human body. Each skeletal muscle is composed of thousands of muscle fibers bound together by a connective tissue sheath. Individual bundles of muscle fibers within skeletal muscle are called fiber fascicles. The outermost connective tissue sheath surrounding the entire muscle is called the epimysium. The connective tissue sheath covering each fiber bundle is called the perimysium, and the innermost sheath surrounding a single muscle fiber is called the endomysium. Each muscle fiber is composed of multiple myofibrils, each containing multiple myofilaments.

[0007] When bundled together, all myofibrils are arranged in a unique striped pattern to form the sarcomere, the basic contractile unit of skeletal muscle. The two most important myofilaments are actin filaments and myosin filaments, which are uniquely arranged to form the various stripes on skeletal muscle.

[0008] The main functions of skeletal muscle are achieved through its intrinsic excitation-contraction coupling process. When the muscle is attached to the skeletal tendon, the contraction of the muscle causes the movement of the skeleton, allowing the execution of specific movements. Skeletal muscle also provides structural support and helps maintain the body's posture. Skeletal muscle also serves as a storage source of amino acids, which can be used by different organs of the body to synthesize organ-specific proteins. Skeletal muscle also serves as a disposal site for glucose in the form of muscle glycogen. Skeletal muscle also plays an important role in maintaining a constant body temperature and serves as an energy source during periods of starvation. Therefore, skeletal muscle plays a key role in movement, body temperature regulation, and controlling systemic metabolism.

[0009] In many muscle diseases, as well as during normal aging, skeletal muscle tissue decreases in size and function, leading to impaired functional mobility and, in the case of severe muscle disease, long-term disability and early death.

[0010] Treatments for muscle wasting and genetic muscle diseases typically consist of broad therapies, such as testosterone therapy for muscle wasting and glucocorticoids for muscular dystrophy. The non-targeted delivery of these therapies reduces the efficiency of uptake by specific muscles and also causes significant deleterious off-target effects in other organs.

[0011] There is a need in the art for novel anti-human antibodies that can bind muscle-specific markers and enable internalization of therapeutic payloads by muscle cells. Summary of the Invention

[0012] 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 are particularly useful for specifically directing the internalization of nucleic acids encoding therapeutic proteins, for example, into skeletal muscle cells.

[0013] Virus particles as described herein are particularly suitable for nucleotide specific targeting introduction into muscle cells, because virus capsids as described herein or virus capsid proteins include targeting ligands in conjunction with muscle cell specific surface proteins. In some embodiments, virus capsids or virus capsid proteins include binding to a right first member, and this first member is combined with its binding to a right homologous second member, wherein the second member is connected to a targeting ligand (for example, fused with this targeting ligand) in conjunction with muscle cell specific surface proteins. In some embodiments, the targeting ligand is optionally operably connected to the second member via a joint, for example, fused with this second member. In some embodiments, the targeting ligand can be a binding moiety, for example, a natural ligand, an antibody, a multispecific binding molecule, etc. In some embodiments, the targeting ligand is an antibody or part thereof. In some embodiments, the targeting ligand is an antibody comprising variable domains and heavy chain constant domains of muscle-specific surface proteins in conjunction with muscle cells. In some embodiments, the targeting ligand is an antibody comprising variable domains and IgG heavy chain constant domains of muscle-specific surface proteins in conjunction with target cells. In some embodiments, the targeting ligand is an antibody comprising a variable domain and an IgG heavy chain constant domain that binds to a muscle-specific surface protein on a target cell, wherein the IgG heavy chain constant domain is, for example, operably linked to a protein (e.g., a second member of a protein: protein binding pair) that forms an isopeptide covalent bond with the first member via a linker. In some embodiments, the capsid protein described herein comprises a first member and a second member, the first member comprising a SpyTag that is operably linked to a viral capsid protein and covalently linked to a SpyTag, the second member comprising a SpyCatcher linked to a targeting ligand, the targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein the SpyCatcher and IgG heavy chain domain are linked via an amino acid linker, e.g., 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 HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set, which comprises the amino acid sequence 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 HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set as shown in any one of SEQ ID NOs: 1-240.

[0014] In some embodiments, viral particles as described herein, such as AAV particles retargeted with anti-CACNG1 antibodies or fragments thereof as described herein, comprise nucleotides, such as target nucleotides. In some embodiments, the target nucleotide encodes a reporter gene. In some embodiments, the target nucleotide encodes microdystrophin, such as human microdystrophin, for example, for use in methods for treating Duchenne muscular dystrophy or a model thereof and / or for the manufacture of a drug for treating Duchenne muscular dystrophy or a model thereof. In some embodiments, the target nucleotide encodes Fukutin-related protein (FKRP), such as human FKRP, for example, for use in methods for treating limb-girdle muscular dystrophy or a model thereof and / or for the manufacture of a drug for treating limb-girdle muscular dystrophy or a model thereof. In some embodiments, the target nucleotide encodes myotubularin (MTM1), such as human MTM1, for example, for use in methods for treating myotubular myopathy or a model thereof and / or for the manufacture of a drug for treating myotubular myopathy or a model thereof.

[0015] Exemplary target nucleotide molecules as described herein can comprise the sequence shown in SEQ ID NO: 270, for example, 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. Exemplary target nucleotide molecules as described herein can comprise the sequence shown in SEQ ID NO: 271, for example, 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. Exemplary target nucleotide molecules as described herein can comprise the sequence shown in SEQ ID NO: 272, for example, for use in methods 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017] Figures 1A-1D In vitro and ex vivo evaluation of CACNG1 antibody properties are shown. Mouse and human myotubes were used as in vitro models of muscle to evaluate CACNG1 antibody cell binding ( Figures 1A-1B ) and internalization ( Figure 1C Living myotubes were incubated with anti-CACNG1 antibodies followed by fluorophore-conjugated secondary detection to assess antibody binding ( Figures 1A-1BMyotubes were incubated with anti-CACNG1 antibodies (e.g., REGN7854 and other anti-CACNG1 antibodies described herein) and then incubated with a duocarmycin-conjugated secondary (2° Ab - cytotoxic drug) to assess antibody internalization via a cell killing assay ( Figure 1C ). In live CACNG1 Hu / Hu Mouse single muscle fiber ( Figure 1D , left) and unfixed muscle tissue cross-section ( Figure 1D , right panel) immunostaining with anti-CACNG1 antibody showed the localization of CACNG1 on the surface of myofibroblasts.

[0018] Figure 2 Data are provided regarding acetylcholine-induced calcium flux (relative light units; y-axis) in human myotubes following incubation with anti-hCACNG1 antibodies (REGN5972, REGN10728, or H2aM31944N) at varying concentrations (0.01 μM, 0.1 μM, 1 μM, and 10 μM; x-axis), isotype control antibodies (REGN3892, REGN1945, or REGN1097), or with 20 μM nicardipine as a positive control for calcium blockade. The anti-hCACNG1 antibodies tested herein did not inhibit acetylcholine-induced calcium flux in human myotubes at these concentrations.

[0019] Figure 3 Provided are methods for expressing CACNG1 in wild-type ("WT") mice, mice homozygous for a CACNG1 deletion ("KO"), or mice expressing only human CACNG1 ("CACNG1 Hu / Hu Fluorescent immunohistochemical images of isolated single muscle fibers taken at 20x magnification after isolation from CACNG1; incubation with anti-human CACNG1 antibodies (H1M31941N or REGN5972) or isotype control antibodies (REGN653 or REGN1945); and labeling with fluorescent-conjugated secondary antibodies. CACNG1 antibodies bind to CACNG1 Hu / Hu myofibers, whereas the isotype control antibody did not bind.

[0020] Figure 4 Provided are methods for expressing CACNG1 in wild-type ("WT") mice, mice homozygous for a CACNG1 deletion ("KO"), or mice expressing only human CACNG1 ("CACNG1 Hu / HuSingle plane confocal fluorescence immunohistochemistry images of isolated single muscle fibers taken at 20x magnification after isolation from 5% CACNG1 cells and incubation with anti-human CACNG1 antibody (REGN10728) conjugated with Alexa 647 (A647) fluorophore or isotype control antibody (REGN4439) for 30 minutes, 4 hours, or 8 hours. Confocal imaging showed that after 30 minutes of incubation, the fluorophore-conjugated CACNG1 antibody bound to CACNG1. Hu / Hu The surface of myofibers, and after 4 and 8 hours of incubation, a portion of the CACNG1 antibody was internalized and detected within the myofibers. No fluorophore-conjugated isotype control antibody was detected in CACNG1. Hu / Hu Incorporated or internalized in muscle fibers.

[0021] Figure 5 Shown are the levels of androgen receptor activation expressed in relative light units (RLU; y-axis) after incubation for 24 hours of an LNCaP cell line modified to express luciferase upon androgen receptor (AR) activation (AR.Luc) with varying concentrations (Log[concentration (M)]; x-axis) of: 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. In this assay, only unconjugated DHT was shown to activate the androgen receptor, while CACNG1 antibodies conjugated to DHT did not show any appreciable androgen receptor activation in this cell line that does not express hCACNG1.

[0022] Figures 6A-6I Shown is the effect of incubating an hCACNG1-expressing LNCaP cell line (hCACNG1.AR.Luc), modified to also express luciferase upon androgen receptor (AR) activation, for 24 hours with various concentrations (Log[concentration (M)]; x-axis) of Figures 6A-6C )、48 hours( Figures 6D-6F ) or 72 hours ( Figures 6G-6I), the level of androgen receptor activation expressed in relative light units (RLU; y-axis): dihydrotestosterone (DHT) alone (M608; unconjugated DHT); anti-hCACNG1 antibodies (REGN14570, REGN14571, REGN14572, REGN14573, REGN14574, or REGN14647) conjugated to DHT (M3004) via a VC-PAB linker; or anti-FelD isotype control antibody (REGN3892) conjugated to DHT (M3004) via a VC-PAB linker. Several CACNG1 antibody-DHT conjugates activated the androgen receptor in this hCACNG1-expressing cell line, and although the efficacy and potency of androgen receptor activation were lower than those of unconjugated DHT at 24 hours after treatment, the activation of the androgen receptor was sustained at 48 and 72 hours compared to unconjugated DHT.

[0023] Figure 7 Provided are cryo-fluorescence tomography images of mice 6 days after systemic injection of 10 mg / kg of anti-hCACNG1 antibodies conjugated to Alexa 647 (REGN10728 or REGN5972) or an isotype control antibody conjugated to Alexa 647 (REGN4439).

[0024] Figures 8A-8G Provided are the results of tail vein injection of 10 mg / kg of anti-human CACNG1 antibody (REGN5972 or REGN10728) conjugated with Alexa 647 (A647) or isotype control antibody (REGN4439) to express only human CACNG1 ("CACNG1 Hu / Hu ”) and the gastrocnemius / plantaris / soleus muscles of mice sacrificed 6 days after injection ( Figure 8A ), tibialis anterior ( Figure 8B ), diaphragm( Figure 8C )、Tongue muscle( Figure 8D ), triceps( Figure 8E ), Trapezius( Figure 8F ) or pelvic floor muscles ( Figure 8G Fluorescent immunohistochemistry images of sections (Figure 5A) taken at 20x magnification. 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 muscle from isotype control and saline-injected mice.

[0025] Figures 9A-9DProvided are the results of tail vein injection of 10 mg / kg of anti-human CACNG1 antibody (REGN5972 or REGN10728) conjugated with Alexa 647 (647) or isotype control antibody (REGN4439) to express only human CACNG1 ("CACNG1 Hu / Hu ”) and the liver of mice sacrificed 6 days after injection ( Figure 9A ),kidney( Figure 9B ),spleen( Figure 9C ) or brown adipose tissue ( Figure 9D ) Fluorescent immunohistochemistry images of sections taken at 20× magnification. Neither fluorophore-conjugated CACNG1 antibody showed detectable signal in these organs, and Alexa 647 levels were similar to isotype and saline-injected controls.

[0026] Figure 10 Provided are schematics depicting an exemplary experimental timeline (top) and micrographs showing the distribution of CACNG1 antibodies to the soleus muscle under sedentary and exercise conditions at doses of 10 mg / kg or 50 mg / kg (high) (bottom). CACNG1 distribution changes with exercise and dose.

[0027] Figure 11 Provides Duchenne muscular dystrophy (D2-mdx), limb-girdle muscular dystrophy (Fkrp P448L Cryo-fluorescence tomography images of a mouse model of myotubular myopathy (MTM1 KO) or myotubular myopathy (MTM1 KO) that was injected systemically with 5 × 10 12 Two weeks after administration, the animals were sacrificed after administration of either wild-type AAV9 particles containing 10 viral genomes / kg or AAV9 particles containing the W503A mutation, retargeted with anti-hCACNG1 antibody (REGN10717), and expressing eGFP under the control of the CAG promoter.

[0028] Figure 12A A schematic diagram is provided for the treatment of D2-mdx mice with AAV expressing a nucleotide of interest encoding micro-dystrophin (μDys) under the control of the CK8 promoter. Figure 12B Provided are the 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 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 (y-axis; compared to levels from mice injected with WT AAV9). Figure 12CThe left panel provides (i) Western blots detecting μDys or β-actin in quadriceps muscles from D2-mdx mice injected with phosphate-buffered saline (PBS) containing wild-type (WT) AAV9 particles encoding the μDys nucleotide of interest or AAV9 particles with the N272A mutation retargeted with anti-hCACNG1 antibody (REGN10717) containing the μDys nucleotide of interest, and (ii) also provides graphs providing abundance levels of the proteins. Figure 12C The right panel provides immunohistochemical images of gastrocnemius muscle taken from untreated wild-type (WT) or D2-mdx mice, or after injection of wild-type (WT) AAV9 particles containing the nucleotide of interest encoding μDys or AAV9 particles with the N272A mutation, retargeted with an anti-hCACNG1 antibody (REGN10717), containing the nucleotide of interest encoding μDys, and after staining for dystrophin. Figure 12D The left panel provides the percent change in serum creatine kinase (CK) (y-axis; compared to pre-injection baseline levels) 4 weeks after injection of D2-mdx mice with either wild-type (WT) AAV9 particles containing a μDys-encoding nucleotide in phosphate-buffered saline (PBS) or AAV9 particles with the N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) containing a μDys-encoding nucleotide, and Figure 12D The right graph provides maximum grip strength (grams; y-axis) 12 weeks after D2-mdx mice were 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 an anti-hCACNG1 antibody (REGN10717), containing the nucleotide of interest encoding μDys.

[0029] Figure 13A Provided for the treatment of Fkrp with AAV P448L Schematic representation of a mouse AAV expressing a target nucleotide encoding human FKRP (hFKRP) under the control of the CK7 promoter. Figure 13B Provided are Fkrp antibodies injected with phosphate-buffered saline (PBS) containing wild-type (WT) AAV9 particles comprising a nucleotide of interest encoding hFKRP or AAV9 particles with an N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) comprising a nucleotide of interest encoding hFKRP. P448L Levels of hFKRP mRNA expressed in quadriceps, gastrocnemius, diaphragm, and liver of mice (y-axis; compared to levels from mice injected with WT AAV9). Figure 13CThe left panel provides images of untreated wild type (WT) or Fkrp P448L Immunohistochemical images of the diaphragm of mice, either 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 an anti-hCACNG1 antibody (REGN10717), containing a nucleotide of interest encoding hFKRP, and after incubation with IIH6 (which stains glycosylated α-dystroglycan), laminin, and DAPI; and Figure 13C The right panel provides the intensity of IIH6 (y-axis) in arbitrary units (upper panel) or as a percentage of the area within the laminin region (lower panel) of these animals. Figure 13D Provides Fkrp P448L Maximum treadmill distance (meters; y-axis) run by mice seven weeks after injection with phosphate-buffered saline (PBS), wild-type (WT) AAV9 particles containing the nucleotide of interest encoding hFKRP, or AAV9 particles with the N272A mutation, retargeted with anti-hCACNG1 antibody (REGN10717), containing the nucleotide of interest encoding hFKRP.

[0030] Figure 14A A schematic diagram is provided for treating MTM1 knockout (KO) mice with AAV expressing a nucleotide sequence encoding human MTM1 (hMTM1) under the control of the desmin promoter. Figure 14B Presented are the levels of hMTM1 mRNA expressed in the quadriceps, gastrocnemius, diaphragm, and liver of MTM1 KO mice injected with phosphate-buffered saline (PBS) containing wild-type (WT) AAV9 particles encoding the hMTM1 nucleotide of interest or AAV9 particles with the N272A mutation retargeted with an anti-hCACNG1 antibody (REGN10717) containing the hMTM1 nucleotide of interest (y-axis; compared to levels from mice injected with WT AAV9). Figure 14C The left panel provides immunohistochemical images of soleus muscles taken 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 an anti-hCACNG1 antibody (REGN10717), containing a nucleotide of interest encoding hMTM1, and after incubation with laminin and DAPI; and Figure 14CThe right panel provides the percentage of MTM1 KO mice that survived (up to 60 days) when injected on day 32 with PBS, wild-type (WT) AAV9 particles containing the nucleotide of interest encoding hMTM1, or AAV9 particles with the N272A mutation, retargeted with an anti-hCACNG1 antibody (REGN10717), containing the nucleotide of interest encoding hMTM1.

[0031] Figure 15 Provided are the results of a prospective co-administration of Fkrp after injection of wild-type AAV9 particles or AAV9 particles with the N272A mutation, retargeted with anti-hCACNG1 antibody (REGN10717), expressing the eGFP target nucleotide driven by the CAG promoter. P488L Images of cardiac GFP expression in mice (left); and Fkrp expression after injection of PBS, wild-type AAV9 particles containing the nucleotide of interest encoding hFKRP under the control of the CK7 promoter, or AAV9 particles with the N272A mutation, retargeted with an anti-hCACNG1 antibody (REGN10717), and containing the nucleotide of interest encoding hFKRP. P488L Levels of hFKRP mRNA in mouse hearts (right panel).

[0032] Figure 16 Provided are the rationale and study protocol for determining whether cardiac transduction by AAV9 particles can be preserved upon robust skeletal muscle retargeting by conjugating CACNG1 antibodies to non-detargeted AAV9 capsids.

[0033] Figures 17A-17C C57BL / 6 healthy mice ( Figure 17A ) or D2-mdx mice ( Figures 17B-17C ) Images of the liver, quadriceps, or heart after injection of: wild-type AAV9 particles encapsulating the nucleotide of interest encoding eGFP under the control of the CAG promoter; AAV9 particles retargeted with anti-hCACNG1 antibody (REGN10717) containing a detargeting mutation (e.g., W503A) and encapsulating the nucleotide of interest encoding eGFP under the control of the CAG promoter; or WT AAV9 particles (without the detargeting mutation) retargeted with anti-hCACNG1 antibody (REGN10717) and encapsulating the nucleotide of interest encoding eGFP under the control of the CAG promoter, with 2×10 12 vg / mouse (high), 4×10 11 vg / mouse (medium) or 8×10 10 vg / mouse (low) at different doses. Figure 17C Depicts the Figure 17B Same tissue, but at higher magnification.

[0034] Figures 18A-18B Shown is the injection of 2×10 12 vg / mouse (high), 4×10 11 vg / mouse (medium) or 8×10 10 After the following items of vg / mice (low), C57BL / 6 healthy mice ( Figure 18A ) or D2-mdx mice ( Figure 18B ), relative to the housekeeping gene Rplp0 in the liver, heart, or quadriceps of: wild-type AAV9 particles encapsulating the eGFP nucleotide of interest under the control of the CAG promoter (AAV WT); WT AAV9 particles (without a detargeting mutation) retargeted with an anti-hCACNG1 antibody and encapsulating the eGFP nucleotide of interest under the control of the CAG promoter (AAV WT+anti-CACNG1); AAV9 particles retargeted with an anti-hCACNG1 antibody (REGN10717), containing a detargeting mutation (e.g., W503A), and encapsulating the eGFP nucleotide of interest under the control of the CAG promoter (AAV W503A+anti-CACNG1); or phosphate-buffered saline (PBS) (x-axis).

[0035] Figures 19A-19B Shown is the injection of 2×10 12 vg / mouse (high), 4×10 11 vg / mouse (medium) or 8×10 10 After the following items of vg / mice (low), C57BL / 6 healthy mice ( Figure 19A ) or D2-mdx mice ( Figure 19B ), relative to the housekeeping gene Rplp0 in the gastrocnemius, quadriceps, diaphragm, soleus, tibialis anterior, or tongue muscles of: wild-type AAV9 particles encapsulating the nucleotide of interest encoding eGFP under the control of the CAG promoter (AAV WT); WT AAV9 particles (without a detargeting mutation) retargeted with an anti-hCACNG1 antibody and encapsulating the nucleotide of interest encoding eGFP under the control of the CAG promoter (AAV WT+anti-CACNG1); AAV9 particles retargeted with an anti-hCACNG1 antibody (REGN10717), containing a detargeting mutation (e.g., W503A), and encapsulating the nucleotide of interest encoding eGFP under the control of the CAG promoter (AAV W503A+anti-CACNG1); or phosphate-buffered saline (PBS) (x-axis).

[0036] Figure 20An illustrative schematic diagram is provided for refining the detargeting and retargeting of AAV9 viral particles by manipulating the AAV capsid, retargeting antibodies, or both. This modular design provides the flexibility to adjust the degree of detargeting, and the addition of antibodies to direct viral particles to new tissues and cell types, which can be finely tuned for the treatment of specific diseases.

[0037] Figure 21A An illustrative schematic diagram (not to scale) of the single-stranded (ss) viral genome is provided, which comprises, from 5' to 3': 141 base pair inverted terminal repeats (ITRs), a CAGG promoter, a sequence encoding enhanced green fluorescent protein (GFP), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a 32 base pair barcode, a human (h) growth hormone (GH) poly A tail, and a 141 base pair ITR. Figure 21B A bar graph is provided that demonstrates the Figure 21A Compared to wild-type AAV9 virus particles (AAV9) containing the viral genome depicted in Figure 21A After the AAV9 virus particles containing the viral genome depicted in (containing capsid mutations such as N272A, W503A, etc.) (these virus particles each have a unique barcode and are re-targeted with anti-CACNG1 antibodies), transduction to various muscles in vivo in non-human primates (cynomolgus monkeys) is enhanced. Figure 21A As described in , each candidate AAV is packaged with a unique barcoded genome. After IV administration of the barcoded pool of 12 candidates, the specified tissues are collected, and the relative abundance of each barcode in the total RNA purified from each tissue is assessed using next-generation sequencing (NGS). The percentage of NGS readings (Y-axis) of each barcode and associated capsid mapped to the following tissues (X-axis) is shown: a subset of liver (left lobe) and skeletal muscle (diaphragm, biceps, biceps, extensor digitorum longus (EDL), gastrocnemius, intercostal muscle, soleus, tibialis anterior, transverse abdominal muscle, triceps, vastus lateralis, psoas, tongue muscle); the percentage is normalized relative to the virus pool injected. The data presented here are the mean values ​​of two animals in the study. Figure 21C showed that systemically delivered detargeted AAV9 conjugated to anti-CACNG1 demonstrated antibody-dependent transduction of skeletal muscle in nonhuman primates. Figure 21B Data plotted as relative expression of mRNA compared to wild-type AAV9 expression (y-axis) depict enhanced transduction of the diaphragm, psoas, triceps, and intercostal muscles in response to anti-CACNG1 antibodies #3 and #5.

[0038] Figures 22A-22C Shown is the injection of 2×10 14vg / kg of wild-type AAV9 or AAV9 W503A expressing eGFP under the control of the CAG promoter, the liver enzymes (ALT; Figure 22A ) and complement pathway biomarkers (sC5b-9; Figure 22B ) and markers of thrombotic microangiopathy (platelet count; Figure 22C ) serum levels that were seropositive (serum(+)) or seronegative (serum(-)) for AAV9 at the indicated time points (x-axis). As expected, administration of wild-type AAV9 particles resulted in an increase in ALT ( Figure 22A ), sC5b-9 (a marker of the terminal membrane attack complex of complement) was elevated ( Figure 22B ) and decreased platelet count ( Figure 22C ), whereas administration of AAV9 W503A particles showed ALT levels similar to those of cynomolgus monkeys receiving saline only (negative control) ( Figure 22A ), sC5b-9 level ( Figure 22B ) and platelet count ( Figure 22C These data suggest that liver-detargeted AAV9 W503A particles offer a safety advantage over liver-tropic wild-type AAV serotypes.

[0039] Figures 23A-23C Figure 2 shows thrombocytopenia as a marker of the thrombotic microangiopathy (TMA) triad in nonhuman primates (cynomolgus macaques) following injection of wild-type AAV9 or AAV9W503A expressing eGFP under the control of the CAG promoter. Figure 23A ; platelet count), hemolytic anemia ( Figure 23B ; red blood cell distribution width) and impaired renal filtration ( Figure 23C ; serum creatinine), these serum levels were seropositive (serum (+)) or seronegative (serum (-)) for AAV9 at the specified time points (x-axis). A decrease in platelet count indicates transient thrombocytopenia, an increase in red blood cell distribution width is a marker of schistocytes, which indicates mild, transient hemolytic anemia, and an increase in serum creatinine level is a marker of impaired renal filtration, which indicates mild, transient acute kidney injury. Monkeys administered with wild-type AAV9 displayed some symptoms of the TMA triad, but monkeys administered with AAV9 W503A did not show these symptoms.

[0040] Figures 24A-24B shows a line graph ( Figure 24A ) and bar charts ( Figure 24B), which depict wild-type mice treated with PBS (50500 vehicle) and AAV9 particles containing the P448L point mutation in fukutin-related protein (FKRP) as a model for limb-girdle muscular dystrophy type 2I treated with PBS and pluronic acid (vehicle) or different doses (4E12 vg / kg, 1E13 vg / kg, and 5E13 vg / kg) of AAV9 particles. P448L / P448L ), humanized laminin subunit α2 (LAMA2; Lama2 HU / HU ) and humanized dystroglycan 1 (DAG1; DAG1 HU / HU Serum creatine kinase levels in mice ) were elevated after transfection with AAV9 particles containing the N272A mutation, retargeted with anti-hCACNG1 antibody (REGN10717), and encapsidating the nucleotide encoding human FKRP (hFKRP) under the control of the CK7 promoter. DETAILED DESCRIPTION

[0041] Provided herein are novel anti-human CACNG1 antibodies and monovalent antigen-binding fragments thereof that can be used to mediate the internalization of CACNG1. Anti-human CACNG1 antibodies and monovalent antigen-binding fragments thereof can be used as part of multispecific antigen-binding proteins and / or multidomain therapeutic proteins and / or as antibody-drug conjugates for, for example, the treatment of diseases.

[0042] The description herein is not limited to the particular embodiments, compositions, methods, and experimental conditions described, as such embodiments, compositions, methods, and conditions may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0043] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing as described herein, some preferred methods and materials are now described. All publications cited herein are incorporated herein by reference in their entirety for description. 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.

[0044] The term "about", when used to refer to a specific recited value, means that the value may vary from the recited value by no more than 1%. For example, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0045] Voltage-dependent calcium channels are typically composed of five subunits. The protein encoded by the CACNG1 gene represents one of these subunits. "CACNG1" includes the protein encoded by the CACNG1 gene and is 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 a functionally diverse eight-member protein subfamily within the PMP-22 / EMP / MP20 family and is located in the same cluster as two family members that function as transmembrane AMPA receptor regulatory proteins (TARPs). CACNG1 is highly 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 in length. The exemplary sequence of the human CACNG1 gene is designated as NCBI accession number NM_0007582.2 (SEQ ID NO: 241). An exemplary human CACNG1 protein is designated UniProt Accession No. 070578 (SEQ ID NO: 242).

[0046] The phrases "antibodies that bind to CACNG1" or "anti-hCACNG1 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize a single CACNG1 molecule. Antibodies and antigen-binding fragments thereof as 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 a transmembrane domain or are otherwise not associated with cell membranes.

[0047] The expression "cell surface expressed CACNG1" refers to one or more CACNG1 proteins that are expressed on the surface of cells in vitro or in vivo, such that at least a portion of the CACNG1 protein is exposed on the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. "Cell surface expressed CACNG1" may include or consist of a CACNG1 protein expressed on the surface of a cell that normally expresses the CACNG1 protein. Alternatively, "cell surface expressed CACNG1" may include or consist of a CACNG1 protein 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.

[0048] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies.

[0049] The term "antibody" refers to any antigen-binding molecule or molecular complex comprising at least one complementary determining region (CDR) that specifically binds to or interacts with a specific antigen (e.g., CACNG1). As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds) and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain: CL. The VH and VL regions can be subdivided into hypervariable regions (referred to as complementary determining regions (CDRs)), interspersed with more conserved regions (referred to as framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody refers to an antibody with at least 10 -9 M, at least 10 -10 M, at least 10 - 11 M, or at least 10 -12 The binding affinity of M to those antibodies, as determined by surface plasmon resonance, e.g., BIACORE TM The term "antibody" may encompass any type of antibody, such as, for example, monoclonal or polyclonal. Furthermore, the antibody may be of any origin, such as, for example, mammalian or non-mammal. In one embodiment, the antibody may be mammalian or avian. In another embodiment, the antibody may be of human origin and may further be a human monoclonal antibody.

[0050] The term "antibody" also includes the antigen binding fragment (Fb) of a full antibody molecule. The term "antigen binding portion thereof" or "Fb" of an antibody includes any naturally occurring, enzymatically available, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The Fb of an antibody can be derived from a full antibody molecule using any suitable standard technique (such as proteolytic digestion or the recombinant genetic engineering technology of the operation and expression of the DNA encoding the variable domains and optionally the constant domains of an antibody). Such DNA is known, and / or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA sequencing can be performed, and chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration, or to introduce codons, produce cysteine ​​residues, modify, add or delete amino acid, etc.

[0051] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs) such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment."

[0052] The antigen-binding fragment of an antibody generally includes at least one variable domain. The variable domain can have any size or amino acid composition and will generally include at least one CDR adjacent to or in frame with one or more framework sequences. H Domain and V L In the antigen-binding fragments associated with the structural domains, V H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomer V H or VL domain.

[0053] In certain embodiments, an antigen-binding fragment of an antibody may contain 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 present within an antigen-binding fragment of an antibody as described herein include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ;(viii)V L -C H 1; (ix) V L -C H 2;(x)V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C L . In any configuration of variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or may be connected by a full-length or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of an antibody as described herein may comprise any of the variable and constant domain configurations listed above, coupled to each other and / or to one or more monomeric V domains. H or V LThe domains are non-covalently associated (eg, through disulfide bonds) as homodimers or heterodimers (or other multimers).

[0054] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will generally comprise at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be made suitable for use in the context of antigen-binding fragments of an antibody as described herein.

[0055] In certain embodiments, the anti-hCACNG1 antibodies as described herein are human antibodies. The term "human antibody" refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies as described herein may include, for example, in CDRs and particularly in CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations 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 transplanted onto human framework sequences.

[0056] In some embodiments, antibodies as described herein can be recombinant human antibodies.The term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or separated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies separated from recombinant human antibody combinatorial libraries (described further below), antibodies separated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, produced or separated by any other means involving splicing human immunoglobulin gene sequences to 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 undergo in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, undergo in vivo somatic mutagenesis) and therefore the V H and V L The amino acid sequence of the region is derived from human germline V H and V L Sequences and sequences related thereto that may not naturally occur within the human antibody germline repertoire in vivo.

[0057] Human antibodies may 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, in which the dimer is held together by interchain heavy chain disulfide bonds. In the second general form, the dimer is not connected via interchain disulfide bonds, and the molecule of approximately 75-80 kDa is composed of covalently coupled light and heavy chains (half antibodies). These forms are extremely difficult to separate, even after affinity purification.

[0058] The frequency of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed with the human IgG1 hinge. Antibodies as described herein can be modified in the hinge, C H 2 or C H Region 3 may have one or more mutations, which may be desirable, for example, in production to increase the yield of a desired antibody form.

[0059] Antibodies as described herein can be isolated antibodies. 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 the antibody naturally occurs or is naturally produced, can be considered an "isolated antibody." Isolated antibodies also include in situ antibodies within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or separation step. According to certain embodiments, the isolated antibody can be substantially free of other cellular materials and / or chemicals.

[0060] Also described herein are single-arm antibodies that bind to CACNG1. The term "single-arm antibody" refers to an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The single-arm antibodies described herein may comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Table 1.

[0061] Compared to the corresponding germline sequence from which the antibody is derived, the anti-hCACNG1 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, public antibody sequence databases. Also described herein are antibodies and antigen-binding fragments thereof derived from any one of the amino acid sequences disclosed herein, wherein one or more amino acids within the one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or mutated to the corresponding residues of another human germline sequence, or mutated to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can easily produce a variety of antibodies and antigen-binding fragments starting from the heavy and light chain variable region sequences disclosed herein, comprising one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention.In another embodiment, the present invention relates to the antibody of the present invention. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. In some embodiments, antibodies or antigen-binding fragments as described herein are obtained in this general manner.

[0062] Also described herein are anti-hCACNG1 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, having one or more conservative substitutions. For example, some embodiments include anti-hCACNG1 antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences set forth in Table 1 herein.

[0063] "Biologically equivalent portions," "biologically equivalent variants," and the like of the reference nucleic acid sequences or polypeptide sequences disclosed herein include those sequences that exhibit similar biological activities as the reference nucleic acid sequence or reference polypeptide sequence. A biologically equivalent portion or variant of a reference nucleic acid sequence includes a nucleic acid that is shorter than the reference nucleic acid, which encodes a polypeptide that is the same as the polypeptide 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 that are at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the sequence from which the portion is derived, but less than the full-length polypeptide or nucleic acid molecule. "Portion" encompasses any contiguous segment of amino acids or nucleotides sufficient to determine the reference polypeptide or nucleic acid molecule from which the portion is derived. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 20 amino acids or 60 nucleotides that are 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 25 amino acids or 75 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 30 amino acids or 90 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 35 amino acids or 105 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 40 amino acids or 120 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 45 amino acids or 135 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence.In some embodiments, a portion comprises at least 50 amino acids or 150 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 60 amino acids or 180 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 70 amino acids or 210 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 80 amino acids or 240 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence. In some embodiments, a portion comprises at least 100 amino acids or 300 nucleotides that are at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to a reference polypeptide or nucleic acid sequence.

[0064] In a non-limiting example, biologically equivalent variants of nucleic acid sequences as disclosed herein can be developed via codon optimization of nucleic acid sequences." Codon optimization " utilizes the degeneracy of codons, as shown by the diversity of the three base pair codon combinations of a specified amino acid, and generally includes replacing at least one codon of a native sequence with a codon that is more frequently used or has the highest frequency of use in a host cell gene, while maintaining the native amino acid sequence to modify the nucleic acid sequence to enhance the process of expression in a specific host cell. For example, the nucleic acid encoding Cas9 protein can be modified to replace a codon with a higher frequency of use compared to a naturally occurring nucleic acid sequence in a given prokaryotic or eukaryotic cell, including 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, at "Codon Usage Database". These tables can be modified in a variety of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, which is incorporated herein by reference in its entirety for all purposes. Computer algorithms for codon optimization of specific sequences for expression in specific hosts are also available (see, e.g., Gene Forge). It will be understood by those skilled in the art that the nucleic acid sequences disclosed herein encompass variants thereof, including those variants that differ due to the degeneracy and / or codon optimization of the genetic code, and those variants of the same or substantially similar amino acid sequences encoding biologically equivalent polypeptides.

[0065] The phrase "bispecific antibody" includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, wherein each heavy chain specifically binds to different epitopes on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally lower than the affinity of the first heavy chain for the second epitope by at least one to two or three or four orders of magnitude, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be prepared, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, the nucleic acid sequence encoding the heavy chain variable sequences that recognize different epitopes of the same antigen can be fused with the nucleic acid sequence encoding different heavy chain constant regions, and such sequences can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs, followed by (N-terminus to C-terminus) a CHI 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 can associate with each heavy chain and can bind to one or more of the epitopes bound by the heavy chain antigen-binding region, or can associate with each heavy chain and enable one or both heavy chains to bind to one or both epitopes.

[0066] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain constant region sequences from any organism and, unless otherwise indicated, includes a heavy chain variable domain. Unless otherwise indicated, a heavy chain variable domain includes three heavy chain CDRs and four FR regions. The fragments of the heavy chain include CDRs, CDRs and FRs and combinations thereof. A typical heavy chain has (from N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain and a CH3 domain after the variable domain. The functional fragments of the heavy chain include fragments that can specifically recognize an antigen (e.g., with a KD recognition antigen in the micromolar, nanomolar or picomolar range), can be expressed and secreted from a cell and include at least one CDR.

[0067] The phrase "light chain" includes immunoglobulin light chain constant region sequences from any organism, and unless otherwise indicated, includes human kappa and lambda light chains. Unless otherwise indicated, a light chain variable (VL) domain typically includes three light chain CDRs and four framework (FR) regions. Typically, a full-length light chain includes a VL domain and a light chain constant domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from amino-terminal to carboxyl-terminal. Potentially useful light chains include, for example, light chains that do not selectively bind to a first antigen or a second antigen that is selectively bound by an antigen-binding protein. Suitable light chains include light chains that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or on a computer chip), wherein these light chains do not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of an antigen-binding protein. Suitable light chains include light chains that can bind to one or two epitopes bound by the antigen-binding region of an antigen-binding protein.

[0068] The phrase "variable domain" includes the amino acid sequence of an immunoglobulin light chain or heavy chain (modified as needed), which comprises the following amino acid regions from N-terminus to C-terminus (unless otherwise specified): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The "variable domain" includes the amino acid sequence of a classical domain (VH or VL) that folds into a double beta-sheet structure, wherein the beta sheets are connected by disulfide bonds between residues in the first and second beta sheets.

[0069] The phrase "complementarity determining region" or the term "CDR" includes an amino acid sequence encoded by the nucleic acid sequence of the immunoglobulin genes of an organism, which typically (i.e., in wild-type animals) appears between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). A CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and is, for example, encoded by a primary or mature B cell or T cell. In some cases (e.g., for 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 a B cell nucleic acid sequence, for example, due to splicing or joining sequences (e.g., VDJ recombination to form a heavy chain CDR3).

[0070] 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, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected 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 arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241:544-546) consisting of the VH domain; (vi) isolated CDRs; and (vii) scFv, which consists of the two domains, VL and VH, of the Fv fragment, connected by a synthetic linker to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule. Other forms of single chain antibodies, such as diabodies, are also encompassed under the term "antibody" (see, eg, Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0071] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least a functional portion of an immunoglobulin CH2 and CH3 region. "Functional portion" refers to a CH2 and CH3 region that can bind to an Fc receptor (e.g., FcγR; or FcRn, i.e., the neonatal Fc receptor) and / or can participate in complement activation. A CH2 and CH3 region is non-functional if it contains deletions, substitutions, and / or insertions or other modifications that render it incapable of binding to any Fc receptor and incapable of activating complement.

[0072] Fc-containing proteins may comprise modifications in the immunoglobulin domain, including modifications that affect one or more effector functions of the binding protein (e.g., modifications that affect FcγR binding, FcRn binding and thereby affect half-life and / or CDC activity). With respect to EU numbering of immunoglobulin constant regions, such modifications include, but are not limited to, the following modifications and combinations thereof: 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.

[0073] For example, but not by way of limitation, the binding protein is an Fc-containing protein and exhibits enhanced serum half-life (compared to the same Fc-containing protein without the modification) and has modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., L / R / S1 / 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).

[0074] As used herein, the term "antigen binding protein" refers to a polypeptide or protein (one or more polypeptides compounded in a functional unit) that specifically recognizes an epitope on an antigen (such as a cell-specific antigen and / or target antigen as described herein). Antigen binding proteins can be multispecific. The term "multispecific" with respect to antigen binding proteins means that the protein recognizes different epitopes whether on the same antigen or on different antigens. A multispecific antigen binding protein as described herein can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides covalently or non-covalently associated with each other. The term "antigen binding protein" includes antibodies as described herein or fragments thereof, which can be connected or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally connected (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities (such as a protein or fragment thereof) to produce a bispecific or multispecific antigen binding molecule with a second binding specificity.

[0075] The term "protein" means any amino acid polymer having more than about 20 amino acids covalently linked by amide bonds. A protein contains one or more amino acid polymer chains, commonly referred to in the art as a "polypeptide." 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 to form cystine) may be present in some proteins. These covalent bonds may 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, protamine, etc. 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).

[0076] As used herein, "protein" includes biotherapeutic proteins, recombinant proteins for 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, etc. Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), 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).

[0077] As used herein, the term "epitope" refers to a portion of an antigen that is recognized by a multispecific antigen-binding polypeptide. A single antigen (such as an antigenic polypeptide) may have more than one epitope. An epitope may be defined as structural or functional. A functional epitope is typically a subset of a structural epitope and is defined as those residues that directly contribute to the affinity of the interaction between the antigen-binding polypeptide and the antigen. An epitope may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant of a chemically active surface group (such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group) as a molecule, and in certain embodiments, may have specific three-dimensional structural characteristics and / or charge-to-mass ratio characteristics. An epitope formed by adjacent amino acids is typically retained when exposed to a denaturing solvent, while an epitope formed by tertiary folding is typically lost when treated with a denaturing solvent.

[0078] The term "domain" refers to any portion of a protein or polypeptide that has a specific function or structure. Preferably, the domain as described herein binds to a cell-specific or target antigen. As used herein, cell-specific antigen-binding domains or target antigen-binding domains, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen.

[0079] The terms "hemibody" or "half antibody" are used interchangeably to refer to half of an antibody, which essentially contains one heavy chain and one light chain. Antibody heavy chains can form dimers, so the heavy chain of one half can associate with a heavy chain associated with a different molecule (e.g., another half) or with a heavy chain of another Fc-containing polypeptide. Two slightly different Fc-domains can "heterodimerize", such as in the formation of bispecific antibodies or other heterodimers, trimers, tetramers, etc. See Vincent and Murini, "Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies and antibody drug conjugates," 7 Biotechnol. J. 1444-1450 (20912); and Shimamoto et al., "Peptibodies: A flexible alternative format to antibodies," 4 (5) MAbs 586-91 (2012).

[0080] 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 to 25 amino acids. The ScFv polypeptide may also include other amino acid sequences, such as CL or CH1 regions. ScFv molecules can be made by phage display, or can be prepared by subcloning heavy and light chains directly from hybridomas or B cells. Regarding methods for preparing scFv fragments by phage display and antibody domain cloning, Ahmad et al., Clinical and Developmental Immunology, Vol. 2012, Article ID 98025, is incorporated herein by reference.

[0081] Adeno-associated virus (AAV)

[0082] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. AAV is a small, non-enveloped single-stranded DNA virus. Typically, the wild-type AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames (ORFs), rep and cap. The wild-type rep reading frame encodes four proteins with molecular weights of 78 kD ("Rep78"), 68 kD ("Rep68"), 52 kD ("Rep52"), and 40 kD ("Rep40"). Rep78 and Rep68 are transcribed by the p5 promoter, and Rep52 and Rep40 are transcribed by the p19 promoter. These proteins are primarily used to regulate the transcription and replication of the AAV genome. The wild-type cap reading frame encodes three structural (capsid) viral proteins (VP) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2), and 61-62 kD (VP3). More than 80% of the total protein in the AAV virion (capsid) comprises VP3; the relative abundance of VP1, VP2, and VP3 is found in mature virions at approximately 1:1:10, although a ratio of 1:1:8 has been reported. Padron et al. (2005) J. Virology 79:5047-58.

[0083] The genomic sequences of various serotypes of AAV, as well as natural inverted terminal repeats (ITRs), Rep proteins, and capsid subunit sequences 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 numbers NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of these documents are incorporated herein by reference to teach AAV nucleic acid and amino acid sequences. See also, e.g., 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. (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; Moris et al. (2004) Virology 33:375-383; U.S. Patent Application No. 20170130245; International Patent Application No. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303, each of which is incorporated herein by reference in its entirety. Table 2 herein provides the sequences of various non-primate AAVs.

[0084] "AAV" includes all subtypes and naturally occurring and modified forms well known in the art. AAV includes primate AAV (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV rh10 (AAV rh10), AAV h10 (AAV h10), AAV hu11 (AAVhu11), AAV rh32.33 (AAV 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 AAVs, such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, horse AAV, goat AAV, and sheep AAV, etc.), squamates AAV (e.g., snake AAV, iguana AAV), etc. "Primate AAV" refers to AAV generally isolated from primates. Similarly, "non-primate AAV" refers to AAV isolated from non-primates.

[0085] As used herein, "[specific] AAV" related 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 specific AAV (e.g., PLA2 region, VP1-u region, VP1 / VP2 common region, VP3 region), a nucleotide sequence (e.g., ITR sequence), such as the cap gene or capsid protein of AAV, encompasses variants of the gene or polypeptide, including variants comprising the minimum number of nucleotides or amino acids required to retain one or more biological functions, in addition to genes or polypeptides comprising the nucleic acid sequence or amino acid sequence of a specific AAV as set forth herein, respectively. As used herein, a variant gene or variant polypeptide includes a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence of a gene or polypeptide of a specific AAV as set forth herein, wherein the difference generally does not alter at least one biological function of the gene or polypeptide, and / or the phylogenetic characteristics of the gene or polypeptide, for example, the difference may be due to degeneracy of the genetic code, segregation variation, length of the sequence, etc. For example, as used herein, rep genes and cap genes can encompass rep and cap genes that are different from the wild-type genes, because genes can encode one or more Rep proteins and Cap proteins, respectively. In some embodiments, the Rep gene encodes at least Rep78 and / or Rep68. In some embodiments, the cap gene includes those genes that may be different from the wild-type, wherein one or more alternative start codons or the sequence between one or more alternative start codons are removed so that the cap gene encodes only a single cap protein, for example, wherein the VP2 and / or VP3 start codons are removed or replaced so that the cap gene encodes a functional VP1 capsid protein instead of a VP2 capsid protein or a VP3 capsid protein. Therefore, as used herein, the rep gene encompasses any sequence that encodes a functional rep protein. The cap gene encompasses any sequence that encodes at least one functional cap gene.

[0086] It is well known that the wild-type cap gene expresses all three VP1, VP2, and VP3 capsid proteins from a single open reading frame of the cap gene under the control of the p40 promoter present in the repORF. The terms "capsid protein," "Cap protein," and the like include proteins that are part of the viral capsid. For adeno-associated viruses, the capsid proteins are typically referred to as VP1, VP2, and / or VP3 and can be encoded by a single cap gene. For AAV, the three AAV capsid proteins are essentially generated in an overlapping manner from alternative translation start codon usage in the capORF, although all three proteins use a common stop codon. The ORF of the wild-type cap gene encodes the following three alternative start codons and one "common stop codon" from 5' to 3': "VP1 start codon," "VP2 start codon," and "VP3 start codon." The largest viral protein, VP1, is typically encoded from the VP1 start codon to the "common stop codon." VP2 is typically encoded from the VP2 start codon to the common stop codon. VP3 is typically encoded from the VP3 start codon to the common stop codon. VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3. The capsid of VP1 is the capsid of VP2 and VP3.

[0087] In some embodiments, one or more Cap proteins 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 more than one ORF comprising nucleotide sequences encoding any combination of VP1, VP2, and / or VP3 using separate nucleotide sequences operably linked to at least one expression control sequence for expression in packaging cells, each of which produces one or more of the VP1, VP2, and / or VP3 capsid proteins of the present invention. In some embodiments, the VP capsid proteins of the present invention may be expressed individually from an ORF comprising a nucleotide sequence encoding any one of VP1, VP2, or VP3 using separate nucleotide sequences operably linked to one expression control sequence for expression in viral replicating cells, each of which produces only one of the VP1, VP2, or VP3 capsid proteins. In another embodiment, the VP protein can be expressed from an ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins, which are operably linked to at least one expression control sequence for expression in a viral replicating cell, which produces each of the VP1, VP2, and VP3 capsid proteins. Thus, although the amino acid positions provided herein may be provided relative to the VP1 capsid protein of a reference AAV, one skilled in the art will be able to separately and readily determine the position of the same amino acid within the VP2 and / or VP3 capsid proteins of an AAV, as well as the corresponding positions of amino acids in different AAVs.

[0088] The phrase "inverted terminal repeats" or "ITRs" includes 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. The ITRs serve as origins of replication for viral DNA synthesis and are essential cis components for the production of AAV particles (e.g., packaging into AAV particles).

[0089] AAV ITRs include recognition sites for the replication proteins Rep78 or Rep68. The "D" region of the ITR contains a DNA nicking site where DNA replication initiates and provides directionality for the nucleic acid replication step. AAVs that replicate in mammalian cells typically contain two ITR sequences.

[0090] A single ITR can be engineered with Rep binding sites on both strands of the "A" region and on two symmetrical D regions on each side of the ITR palindrome. Such engineered constructs on double-stranded circular DNA templates allow nucleic acid replication initiated by Rep78 or Rep68 to proceed in both directions. A single ITR is sufficient for AAV replication of circular particles. In the method for producing the AAV viral particles of the present invention, the rep coding sequence encodes a Rep protein or a Rep protein equivalent that is capable of binding to the ITR included on the transfer plasmid.

[0091] When expressed by a packaging cell with an appropriate Rep protein, the Cap protein of the present invention can encapsidate a transfer plasmid comprising a target nucleotide and an even number of two or more ITR sequences. In some embodiments, the transfer plasmid comprises one ITR sequence. In some embodiments, the transfer plasmid comprises two ITR sequences.

[0092] Either Rep78 and / or Rep68 binds to a unique and known site on the ITR hairpin sequence and acts to disrupt and unwind the hairpin structure on the end of the AAV genome, thereby providing access to the replication machinery of the virally replicating cell. It is well known that Rep proteins can be expressed from more than one ORF comprising nucleotide sequences encoding any combination of Rep78, Rep68, Rep52 and / or Rep40 by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in virally replicating cells, each of which produces one or more of the Rep78, Rep68, Rep52 and / or Rep40 Rep proteins. Alternatively, Rep proteins can be expressed individually from an ORF comprising nucleotide sequences encoding any one of Rep78, Rep68, Rep52, or Rep40, operably linked to an expression control sequence for expression in a packaging cell, each of which produces a Rep78, Rep68, Rep52, or Rep40 Rep protein. In another embodiment, Rep proteins can be expressed from an ORF comprising nucleotide sequences encoding Rep78 and Rep52 Rep proteins, operably linked to at least one expression control sequence for expression in a viral replicating cell, each of which produces a Rep78 and Rep52 Rep protein.

[0093] In the method of producing the AAV virions (e.g., viral particles) of the present invention, the rep coding sequence and the cap gene of the present invention can be provided in a single packaging plasmid. However, the skilled artisan will recognize that such conditions are not necessary. Such viral particles may or may not contain a genome.

[0094] "Chimeric AAV capsid proteins" include AAV capsid proteins that comprise amino acid sequences, e.g., from two or more different AAVs and that are capable of forming and / or forming portions of AAV viral capsids / virions. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide comprising a plurality (e.g., at least two) nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a different AAV, and the plurality of nucleic acid sequences together encode a functional chimeric AAV capsid protein. The association of a chimeric capsid protein with a particular AAV indicates that the capsid protein comprises one or more portions of a capsid protein from that AAV and one or more portions of a capsid protein from a different AAV. For example, a chimeric AAV2 capsid protein comprises a capsid protein comprising one or more portions of the VP1, VP2, and / or VP3 capsid proteins of AAV2 and one or more portions of the VP1, VP2, and / or VP3 capsid proteins of a different AAV.

[0095] The term "portion" refers to at least 5 amino acids or at least 15 nucleotides that are 100% identical to the sequence from which the portion is derived, but less than the full-length polypeptide or nucleic acid molecule, see Penzes (2015) J. General Virol. 2769. "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 belongs to "[a particular] AAV" or is "substantially identical" to a particular AAV (e.g., non-primate AAV or remote AAV). In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides that are 100% identical to a sequence associated with a particular AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides that are 100% identical to a sequence associated with a particular AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides that are 100% identical to a sequence associated with a particular AAV. In some embodiments, a portion comprises at least 20 amino acids or 60 nucleotides that are 100% identical to a sequence associated with a particular AAV. In some embodiments, a portion comprises at least 25 amino acids or 75 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 30 amino acids or 90 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 35 amino acids or 105 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 40 amino acids or 120 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 45 amino acids or 135 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 50 amino acids or 150 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 60 amino acids or 180 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 70 amino acids or 210 nucleotides that are 100% identical to a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 80 amino acids or 240 nucleotides that are 100% identical to a sequence associated with a particular AAV. In some embodiments, a portion comprises at least 90 amino acids or 270 nucleotides that are 100% identical to a sequence associated with a particular AAV. In some embodiments, a portion comprises at least 100 amino acids or 300 nucleotides that are 100% identical to a sequence associated with a particular AAV.

[0096] Modified viral capsid proteins, viral particles, viral nucleic acids

[0097] In some embodiments, the Cap protein (e.g., a VP1 capsid protein as described herein, a VP2 capsid protein as described herein, and / or a VP3 capsid protein as described herein) is modified to include, for example, any one or a combination of insertion of a targeting ligand, chemical modification, a first member of a binding pair, a detectable label, a point mutation, and the like.

[0098] Typically, modifications to a gene or polypeptide of a particular AAV or variant thereof result in a nucleic acid sequence or amino acid sequence that is different from the nucleic acid sequence or amino acid sequence set forth herein for the particular AAV, wherein the modification alters, confers, or removes one or more biological functions, but does not alter the phylogenetic characteristics of the gene or polypeptide as an AAV gene or AAV polypeptide. Modifications may include any one or a combination of the following: substitution of a sequence of a first AAV serotype with a sequence of a second AAV serotype to create a chimera; chemical modification; insertion of the first member of a binding pair and / or point mutations, etc., such that the natural tropism of the capsid protein is reduced or eliminated, the tropism of the capsid protein can be more easily redirected, and / or the capsid protein comprises a detectable label. Modifications as described herein generally do not alter, and preferably reduce, the low to no recognition of the modified capsid by pre-existing antibodies found in the general population, which antibodies are generated during infection with another AAV, e.g., with a serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or a combination thereof.

[0099] Targeting ligands

[0100] The modifications described herein may involve association (e.g., display, operable connection, binding) of a targeting ligand with a modified capsid protein and / or a capsid comprising a modified capsid protein. Typically, a targeting ligand as described herein binds to a surface protein expressed by a mammalian muscle cell, 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).

[0101] Antigen-binding molecules

[0102] Anti-hCACNG1 antibodies and antigen-binding fragments thereof as described herein can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of a target polypeptide, or can contain antigen-binding domains that are specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. Anti-hCACNG1 antibodies and antigen-binding fragments thereof as described herein can be linked or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities (such as another antibody or antibody fragment) to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[0103] As used herein, the expression "anti-hCACNG1 antibody" is intended to include monospecific anti-hCACNG1 antibodies as well as 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 has specificity for another target molecule. The CACNG1-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Table 1 herein.

[0104] 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 weakly binds to human CACNG1 and induces internalization of CACNG1 and antibodies bound thereto.

[0105] In certain exemplary embodiments, bispecific antigen binding molecules are bispecific antibodies.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 CDR of the first antigen-binding domain can be specified with the prefix "A1", and the CDR of the second antigen-binding domain can be specified with the prefix "A2". Therefore, the CDR of the first antigen-binding domain may be referred to as A1-HCDR1, A1-HCDR2, and A1-HCDR3 in this article; and the CDR of the second antigen-binding domain may be referred to as A2-HCDR1, A2-HCDR2, and A2-HCDR3 in this article.

[0106] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form a bispecific antigen binding molecule as described herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerization domain. The association of a 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 with the ability to associate with a second multimerization domain of the same or similar structure or structure. For example, a multimerization domain can be a domain comprising immunoglobulin C. H A non-limiting example of a multimeric component is the Fc portion of an immunoglobulin (comprising a C H 2-C H 3 domains), e.g., an Fc domain of IgG selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype of IgG within each isotype group.

[0107] Bispecific antigen binding molecules as described herein will typically comprise two multimerization domains, e.g., two Fc domains, each of which is individually part of a separate antibody heavy chain. The first multimerization domain and the second multimerization domain can have the same IgG isotype, such as, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first multimerization domain and the second multimerization domain can have different IgG isotypes, such as, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0108] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length that contains at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short peptide containing cysteine. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0109] Any bispecific antibody format or technology can be used to prepare bispecific antigen-binding molecules as described herein. For example, an antibody or fragment thereof with a first antigen-binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other molecular entities, such as another antibody or antibody fragment with a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats include, but are not limited to, for example, scFv-based or double antibody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs-in-holes, common light chains (e.g., common light chains with knobs-in-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab 2 Bispecific formats (for review of the aforementioned formats, see, eg, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0110] In the context of bispecific antigen binding molecules as described herein, a multimerization domain (e.g., an Fc domain) may comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring form of an Fc domain. For example, a bispecific antigen binding molecule may comprise one or more modifications in the Fc domain that result in a modified binding interaction (e.g., enhanced or weakened) between the modified Fc domain and FcRn. In one embodiment, the bispecific antigen binding molecule comprises one or more modifications in the Fc domain. H 2 or C HRegion 3 comprises a modification that increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at 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 or 308P).

[0111] Also described herein are those comprising a first C H 3 domains and the second Ig C H 3-domain bispecific antigen-binding molecule, wherein the first and second Ig C H The three domains differ from each other by at least one amino acid, and wherein 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. In one embodiment, the first Ig C H 3 domains bind protein A and the second Ig C H The 3 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, for example, U.S. Patent No. 8,586,713. HAdditional modifications within 3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of an IgG1 antibody; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU) in the case of an IgG2 antibody; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I) in the case of an IgG4 antibody.

[0112] In certain embodiments, the Fc domain can be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise Fc sequences derived from human IgG1, human IgG2, or human IgG4. H C in Zone 2 H 2 sequence, and C derived from human IgG1, human IgG2 or human IgG4 H 3 sequence. A chimeric Fc domain may also contain a chimeric hinge region. For example, a 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. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule described herein include, from N-terminus to C-terminus: [IgG4C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules described herein comprises, from N-terminus to C-terminus: [IgG1 C H

[0014] -[IgG1 upper hinge] -[IgG2 lower hinge] -[IgG4 CH2] -[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any antigen-binding molecule as described herein are described in U.S. Publication No. 2014 / 0243504, published on August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector functions.

[0113] In certain embodiments, the heavy chain of an antibody as described herein comprises a heavy chain constant region (CH) region comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID 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 (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.

[0114] In some embodiments, the heavy chain of an antibody as described herein comprises an Fc domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID 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.

[0115] Germline mutations

[0116] The anti-hCACNG1 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domain compared to the corresponding germline sequence from which the antibody was derived.

[0117] Anti-hCACNG1 antibodies and antigen-binding fragments thereof as disclosed herein can be derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody was derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"), and have weak or undetectable binding to the CACNG1 antigen. Several such exemplary antibodies that recognize CACNG1 are described in Table 1 herein.

[0118] In addition, the anti-hCACNG1 antibodies and antigen-binding fragments thereof disclosed herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once an antibody or antigen-binding fragment containing one or more germline mutations is obtained, it can be tested for one or more desired properties, such as improved binding specificity, weak 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.

[0119] 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, the variants having one or more conservative substitutions. For example, an anti-hCACNG1 antibody or antigen-binding fragment thereof as described herein may comprise a HCVR, LCVR, and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, or the like, conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences shown in Table 1 herein. Antibodies and antigen-binding fragments thereof as described herein may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains were derived, while maintaining or improving the desired weak to undetectable binding to, for example, CACNG1. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions will not significantly alter the functional properties of the protein, i.e., in the case of anti-hCACNG1 binding molecules, the amino acid substitutions maintain or improve the desired weak to undetectable binding affinity. Examples of amino acid groups containing side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy 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, i.e., 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 as 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.

[0120] Also described herein are anti-hCACNG1 antibodies and antigen-binding fragments thereof comprising an antigen-binding domain having an HCVR and / or CDR amino acid sequence substantially identical to any of the HCVR and / or CDR amino acid sequences disclosed herein, while maintaining or improving the desired weak affinity for the CACNG1 antigen. When referring to amino acid sequences, the terms "substantially identical" or "substantially identical" mean that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, the difference between non-identical residue positions is a conservative amino acid substitution. Where conservative substitutions in two or more amino acid sequences differ from each other, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331.

[0121] Sequence analysis software is usually used to measure the sequence similarity of polypeptides, which is also referred to as sequence identity. Protein analysis software uses similarity metrics about being assigned to various replacements, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine the sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from organisms of different species), or between wild-type proteins and mutant proteins thereof. See, for example, GCG version 6.1. FASTA (the program in GCG version 6.1) can also be used to compare polypeptide sequences using default or recommended parameters. FASTA (for example, FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlapping region between query and search sequence (Pearson (2000) the same). When comparing sequences as described herein with databases comprising a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, eg, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0122] Once an antigen binding domain containing one or more germline mutations is obtained, it is tested for reduced binding affinity using one or more in vitro assays. Typically, antibodies that recognize a specific antigen are screened for their purpose by testing for high (i.e., strong) binding affinity to the antigen.

[0123] Further modification of the antibodies as described herein by the methods described herein may achieve unexpected benefits, such as improved pharmacokinetic properties and low toxicity to patients.

[0124] Antibody binding properties

[0125] The term "binding" in the context of binding of an antibody, immunoglobulin, antibody binding fragment or Fc-containing protein to, for example, a predetermined antigen (such as a cell surface protein or fragment thereof) generally refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0126] For example, when measured by, for example, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using an antigen as a ligand and an antibody, Ig, antibody binding fragment, or Fc-containing protein as the analyte (or anti-ligand), binding affinity generally corresponds to K D The value is about 10 -7 M or less, such as about 10 -8 M or less, such as about 10 -9 M or less. Cell-based binding strategies, such as fluorescence activated cell sorting (FACS) binding assays, are also routinely used and provide binding characterization data for proteins expressed on the cell surface. FACS data correlate well with other methods such as radioligand competition 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).

[0127] Thus, the anti-hCACNG1 antibodies and antigen-binding fragments thereof described herein bind to a predetermined antigen or cell surface molecule (receptor) with an affinity corresponding to a K D The affinity of the binding site is at least ten times lower than its affinity for binding to nonspecific antigens (e.g., BSA, casein). D Antibodies with affinities equal to or less than ten times that of the nonspecific antigen can be considered to have no detectable binding, yet such antibodies can be paired with a second antigen-binding arm for use in generating bispecific antibodies as described herein.

[0128] The term "K D " or "KD" (in molar (M) units) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, or the dissociation equilibrium constant for the binding of an antibody or antibody binding fragment to an antigen. K D There is an inverse relationship between K and binding affinity, so K DThe smaller the value, the higher the affinity, i.e. the stronger. Therefore, the term "higher affinity" or "stronger affinity" relates to a higher ability to form an interaction, so K D In contrast, the term "lower affinity" or "weaker affinity" relates to a lower ability to form an interaction, so K D In some cases, a higher binding affinity (or K) of a particular molecule (e.g., an antibody) to its interacting partner molecule (e.g., antigen X) than the binding affinity of the molecule (e.g., an antibody) to another interacting partner molecule (e.g., antigen Y) is greater. D ) can be expressed as D value (lower or weaker affinity) divided by the smaller K D The binding ratio is determined based on the binding affinity of the target protein (higher or stronger affinity), for example, expressed as 5-fold or 10-fold the binding affinity, as the case may be.

[0129] The term "k d "(s-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody binding fragment. This value is also called k off value.

[0130] The term "k a "(M-1×s-1 or 1 / M) refers to the association rate constant of a specific antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0131] The term "K A "(M-1 or 1 / M) refers to the association equilibrium constant of a specific antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody binding fragment. The association equilibrium constant is calculated by dividing k a Divide by k d To obtain.

[0132] The term "EC50" or "EC 50 ” refers to the half maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC 50 Essentially represents the antibody concentration at which 50% of the maximal effect is observed. 50 The value is equal to the concentration of an antibody as described herein that gives half-maximal binding to cells expressing CACNG1 as determined by, for example, a FACS binding assay or an androgen receptor activated luciferase assay. 50 or an increase in the half-maximal effect concentration value, a decrease or weakening of binding is observed.

[0133] In one embodiment, decreased binding can be defined as an increased EC 50The concentration of antibody that enables binding to half the maximal number of target cells.

[0134] sequence variants

[0135] Compared to the corresponding germline sequences from which individual antigen binding domains are derived, anti-hCACNG1 antibodies and antigen-binding fragments as described herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. By comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, public antibody sequence databases, such mutations can be easily determined. Antigen-binding molecules as described herein may comprise antigen-binding domains derived from any one of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more frameworks and / or CDR regions are mutated to the corresponding residues from the germline sequence of the antibody, or mutated to the corresponding residues of another human germline sequence, or mutated to the conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can easily produce a variety of antibodies and antigen-binding fragments starting from the heavy and light chain variable region sequences disclosed herein, comprising one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L In other embodiments, all framework and / or CDR residues in the domain are mutated back to the residues present in the original germline sequence from which the antigen-binding domains were initially derived. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only the mutated residues present in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues present in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences from which the antigen-binding domains were initially derived). In addition, the antigen-binding domains can contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, wherein some individual residues are mutated to the corresponding residues of a specific germline sequence, while some other residues different from the original germline sequence are maintained or mutated to the corresponding residues of different germline sequences. Once an antigen binding domain containing one or more germline mutations is obtained, it can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antigen binding molecules are described herein that comprise one or more antigen binding domains obtained in this general manner.

[0136] Also described herein are antigen binding molecules in which one or two antigen binding domains comprise variants of any one of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein, and these variants have one or more conservative substitutions. For example, an antigen binding molecule as described herein may comprise an antigen binding domain having a HCVR, LCVR and / or CDR amino acid sequence, and these HCVR, LCVR and / or CDR amino acid sequences have, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative to any one of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted by another amino acid residue containing a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Typically, conservative amino acid substitutions will not significantly change the functional properties of the protein. Examples of amino acid groups containing side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy 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, i.e., 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.

[0137] Antigen binding molecules as described herein may include antigen binding domains having HCVR, LCVR and / or CDR amino acid sequences substantially identical to any one of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. When referring to amino acid sequences, the term "substantially identical" or "substantially identical" means that two amino acid sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the program GAP or BESTFIT using the default gap weights for optimal alignment. Preferably, the difference in the non-identical residue positions is a conservative amino acid substitution. When the conservative substitutions of two or more amino acid sequences are different from each other, the sequence identity percentage or degree of similarity can be adjusted upwards to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol.Biol.24:307-331, which is incorporated herein by reference.

[0138] Sequence analysis software is usually used to measure the sequence similarity of polypeptides, which is also referred to as sequence identity. Protein analysis software uses similarity metrics about being assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine the sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from organisms of different species), or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. FASTA (a program in GCG version 6.1) can also be used to compare polypeptide sequences using default or recommended parameters. FASTA (for example, FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlapping region between query and search sequence (Pearson (2000) the same). When a sequence is compared with a database comprising a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, eg, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0139] pH-dependent binding

[0140] Also described herein are anti-hCACNG1 antibodies and antigen-binding fragments thereof having pH-dependent binding properties. For example, an anti-hCACNG1 antibody as described herein may exhibit reduced binding to CACNG1 at acidic pH compared to neutral pH. Alternatively, an anti-hCACNG1 antibody as described herein may exhibit enhanced binding to CACNG1 at acidic pH compared to neutral pH. The expression "acidic pH" includes a pH value of 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 less. The expression "neutral pH" means 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.

[0141] In some cases, "binding is reduced at acidic pH compared to neutral pH" refers to the K of the antibody binding to its antigen at acidic pH. D The K of the antibody binding to its antigen at neutral pH D For example, if an antibody or antigen-binding fragment thereof exhibits an acidic / neutral K of about 3.0 or greater, D For the purposes described herein, an antibody or antigen-binding fragment thereof may be considered to "exhibit reduced binding to CACNG1 at acidic pH compared to neutral pH" if the ratio of the acidic / neutral K to the neutral pH of the antibody or antigen-binding fragment is greater than or equal to 1. D The ratio may 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.

[0142] Antibodies with pH-dependent binding properties can be obtained, for example, by screening an antibody population for reduced (or enhanced) binding to a specific antigen at acidic pH compared to neutral pH. Additionally, antibodies with pH-dependent properties can be generated by modifying the antigen-binding domain at the amino acid level. For example, by replacing one or more amino acids of the antigen-binding domain (e.g., within the CDRs) with histidine residues, antibodies can be obtained that have reduced antigen binding at acidic pH relative to neutral pH.

[0143] Antibodies containing Fc variants

[0144] In some embodiments, anti-hCACNG1 antibodies and antigen-binding fragments thereof (including multispecific antigen-binding molecules and multi-domain therapeutic proteins comprising anti-hCACNG1 antibodies or antigen-binding fragments thereof) are provided, which comprise an Fc domain comprising one or more mutations that enhance or reduce binding of the antibody to the FcRn receptor at, for example, acidic pH compared to neutral pH. For example, an antibody as described herein may comprise a C-terminal domain of the Fc domain. H 2 or C H In the 3rd region, a mutation is included, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome with a pH range of about 5.5 to about 6.0). When administered to an animal, such mutations can result in an increase in the serum half-life of the antibody. Non-limiting examples of such Fc modifications include, for example, position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at 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 or 308P).

[0145] For example, the anti-hCACNG1 antibodies and antigen-binding fragments described herein can comprise an Fc domain comprising one or more mutation pairs or groups selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations, as well as other mutations within the antibody variable domains disclosed herein, are encompassed within the description herein.

[0146] Biological properties of antibodies and bispecific antigen-binding molecules

[0147] Also described herein is an antibody and antigen-binding fragment thereof that binds to human CACNG1 with high, medium or low affinity, depending on the therapeutic context and the specific targeting properties desired. For example, in the context of a bispecific antigen-binding molecule, wherein one arm binds to CACNG1 and the other arm binds to a target antigen (e.g., a tumor-associated antigen), it may be desirable that the target antigen-binding arm binds to the target antigen with high affinity, while the anti-hCACNG1 arm binds to CACNG1 only with medium or low affinity. In this way, preferential targeting of antigen-binding molecules to cells expressing the target antigen can be achieved while avoiding general / non-targeted CACNG1 binding and the adverse side effects associated therewith.

[0148] Also described herein are antibodies, antigen-binding fragments thereof, and bispecific antibodies that bind to human CACNG1 with weak (i.e., low) affinity or even undetectable affinity. In some embodiments, the antibodies and antigen-binding fragments thereof as described herein bind to human CACNG1 with a K of greater than about 100 nM. D Binds to human CACNG1 (e.g., at 37° C.) as measured by surface plasmon resonance. In some embodiments, an antibody or antigen-binding fragment as described herein binds to human CACNG1 with 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. D , or binds CACNG1 with undetectable affinity as measured by surface plasmon resonance (e.g., mAb capture or antigen capture format) or a substantially similar assay.

[0149] Epitope mapping and related technologies

[0150] The epitope on CACNG1 to which the anti-hCACNG1 antibodies and antigen-binding fragments thereof described herein bind can be composed of a single contiguous sequence of 3 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 can be composed 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 (called a paratope) in the variable region of an antibody molecule. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope can include a sugar, phosphoryl, or sulfonyl moiety on the antigen.

[0151] Various techniques known to those of ordinary skill in the art can be used to determine whether an antigen binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays (such as Antibodies , Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)), alanine scanning mutagenesis analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463) and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids in polypeptides that interact with the antigen-binding domain of an antibody is hydrogen / deuterium exchange detected by mass spectrometry. In general, hydrogen / deuterium exchange methods involve deuterium labeling of the target protein, followed by binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except the antibody-protected residues (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues corresponding to specific amino acids that interact with the antibody. See, for example, 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.

[0152] 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 shown in Table 1 herein). Likewise, 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 shown in Table 1 herein).

[0153] By using conventional methods known in the art, it is easy to determine whether a specific antigen binding molecule (e.g., an antibody) or its antigen binding domain is bound to the same epitope as a reference antigen binding molecule as described herein, or competes for binding with a reference antigen binding molecule as described herein. For example, in order to determine whether a test antibody is bound to the same epitope on CACNG1 as a reference bispecific antigen binding molecule as described herein, first allow the reference bispecific molecule to bind to the CACNG1 protein. Next, the ability of the test antibody to bind to the CACNG1 molecule is assessed. If the test antibody is able to bind to CACNG1 after saturation binding with the reference bispecific antigen binding molecule, the following conclusion can be drawn: the test antibody is bound to different epitopes of CACNG1 with the reference bispecific antigen binding molecule. On the other hand, if the test antibody cannot be bound to the CACNG1 molecule after saturation binding with the reference bispecific antigen binding molecule, the test antibody can bind to the same epitope on CACNG1 as the epitope bound by the reference bispecific antigen binding molecule as described herein. Then other routine experiments (e.g., peptide mutations and binding analysis) can be performed to confirm whether the observed lack of test antibody binding is due to being bound to the same epitope with reference to the bispecific antigen binding molecule, or whether steric blocking (or another phenomenon) is the cause of the observed lack of binding. Such experiments can be carried out using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody binding assays available in the art. According to some embodiments described herein, if, for example, an excess of 1, 5, 10, 20 or 100 times of a kind of antigen-binding proteins suppresses the binding of another antigen-binding protein by at least 50%, but preferably 75%, 90% or even 99% (as measured by competitive binding assays), then two antigen-binding proteins are bound to identical (or overlapping) epitopes (see, for example, Junghans et al., Junghans et al., Cancer Res. 1990: 50: 1495-1502). Alternatively, two antigen-binding proteins are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other antigen-binding protein. Two antigen-binding proteins are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other antigen-binding protein.

[0154] In order to determine whether an antibody or its antigen-binding domain competes for binding with a reference antigen-binding molecule, the above-mentioned binding method is performed in two directions: in the first direction, the reference antigen-binding molecule is allowed to bind to the CACNG1 protein under saturation conditions, and then the binding of the test antibody to the CACNG1 molecule is evaluated. In the second direction, the test antibody is allowed to bind to the CACNG1 molecule under saturation conditions, and then the binding of the reference antigen-binding molecule to the CACNG1 molecule is evaluated. If only the first (saturated) antigen-binding molecule is able to bind to the CACNG1 molecule in both directions, the following conclusion is drawn: the test antibody and the reference antigen-binding molecule compete for binding to CACNG1. As will be understood by those of ordinary skill 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 antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0155] Preparation of antigen-binding domains and construction of binding molecules

[0156] Antigen binding domains with specificity for a particular antigen can be prepared by any antibody production technology known in the art. Once two different antigen binding domains with specificity for two different antigens (e.g., CACNG1 and target antigen) are obtained, they can be appropriately arranged relative to each other to produce bispecific antigen binding molecules as described herein using conventional methods. (Discussion of exemplary bispecific antibody forms that can be used to construct bispecific antigen binding molecules as described herein is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy chain and light chain) of antigen binding molecules as described herein are derived from chimeric, humanized or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMMUNE can be used. TM The VELOCIMMUNE technology is used to prepare one or more of the heavy and / or light chains of the antigen binding molecules described herein. TM The invention relates to a method for producing antibodies against a specific antigen (e.g., CACNG1) by using a human variable region and a mouse constant region. The antibodies are characterized and selected for their desired properties, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate fully human heavy and / or light chains that can be incorporated into antigen-binding molecules as described herein.

[0157] Genetically engineered animals can be used to prepare human bispecific antigen binding molecules. For example, genetically modified mice that can not rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, wherein the mice only express one or two human light chain variable domains encoded by human immunoglobulin sequences, and these human immunoglobulin sequences are operably linked to the mouse κ constant genes at endogenous mouse κ locus. Such genetically modified mice can be used to separate heavy chain and light chain variable regions to produce fully human bispecific antigen binding molecules. Therefore, fully human bispecific antigen binding molecules include two different heavy chains associated with the same light chain. (See, for example, US2011 / 0195454). Fully human refers to an antibody or its antigen-binding fragment or immunoglobulin domain, which includes an amino acid sequence encoded by a DNA derived from a human sequence, and the human sequence is on the total length of each polypeptide of an antibody or its antigen-binding fragment or immunoglobulin domain. In some cases, fully human sequences are derived from human endogenous proteins. In other cases, fully human proteins or protein sequences include chimeric sequences, and wherein each component sequence is derived from a human sequence. While not being bound by any one theory, chimeric proteins or chimeric 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.

[0158] Bispecific antigen-binding molecules can be constructed using a single heavy chain having a modified Fc domain that eliminates its binding to Protein A, thereby enabling purification methods that produce heterodimeric proteins. See, e.g., U.S. Patent No. 8,586,713. Thus, a bispecific antigen-binding molecule comprises a first C H 3 domains and the second IgC H 3 domains, in which the first and second Ig C H The three domains differ from each other by at least one amino acid, and wherein 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. In one embodiment, the first Ig C H 3 domains bind protein A and the second Ig C H The 3 domain contains a mutation / modification 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 (by IMGT; Y436F by EU).

[0159] Bioequivalents

[0160] Also described herein are antigen binding molecules having amino acid sequences that differ from the amino acid sequences of the exemplary molecules disclosed herein but that retain the ability to bind to CACNG1. Such variant molecules may comprise one or more amino acid additions, deletions, or substitutions when compared to the parent sequence, but exhibit biological activity that is substantially equivalent to that of the described bispecific antigen binding molecules.

[0161] Also described are any bioequivalent antigen binding molecules with exemplary antigen binding molecules shown herein.If for example two kinds of antigen binding proteins or antibody are when using identical molar dosage with single dose or multiple doses under similar experimental conditions, absorption rate and degree do not demonstrate the drug equivalents or drug substitutes of significant differences, then they are considered to be bioequivalent.If some antigen binding proteins are equivalent in absorption degree, but are not equivalent in absorption rate, then they will be considered to be equivalents or drug substitutes, but because this difference of absorption rate is intentional and is reflected in labeling, so can be considered to be bioequivalent, these antibodies are not necessary for for example reaching effective drug concentration in vivo in long-term use, and are considered to not have clinical significance for the particular drug studied.

[0162] In one embodiment, two antigen binding proteins are bioequivalent if they do not have clinically meaningful differences in safety, purity, and potency.

[0163] In one embodiment, two antigen binding proteins are bioequivalent if a patient can undergo one or more switches between the reference product and the biological product without an expected increase in the risk of adverse reactions, including clinically significant changes in immunogenicity, or reduced effectiveness, compared to continuous therapy without switching between such products.

[0164] In one embodiment, two antigen binding proteins are bioequivalent if both act by one or more common mechanisms of action for one or more conditions of use, to the extent such mechanisms are known.

[0165] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence metrics include, for example, (a) in vivo studies in humans or other mammals, in which the concentration of the antibody or its metabolites in blood, plasma, serum or other biological fluids is measured over time; (b) in vitro studies that correlate with and are reasonably predictive of in vivo human bioavailability data; (c) in vivo studies in humans or other mammals, in which the appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding protein.

[0166] Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not required for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antigen-binding proteins can include variants of the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation properties of the molecule (e.g., mutations that eliminate or remove glycosylation).

[0167] Species selectivity and species cross-reactivity

[0168] In some embodiments, an antigen binding molecule as described herein binds to human CACNG1 but not to CACNG1 from other species. Also described herein are antigen binding molecules that bind to human CACNG1 and to CACNG1 from one or more non-human species.

[0169] In some embodiments, an antigen binding molecule as described herein that binds to human CACNG1 may or may not bind, as the case may be, 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.

[0170] 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 region of an antibody (e.g., isolated complementary determining regions (CDRs) such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. As used herein, other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "targeting ligand." In non-limiting embodiments, an anti-CACNG1 targeting ligand that binds to CACNG1 and can be used to retarget the viral capsid as described herein comprises an scFv. As a non-limiting example, a V that can be used to retarget a viral capsid as described herein L -(Gly4Ser)3-V HThe scFv sequence of the 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 HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set having 90%, 95%, 97%, 98%, 99% or 100% identity to any of the amino acid sequences of a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 and / or a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set as shown in any one of SEQ ID NOs: 1-240, respectively.

[0171] Targeting ligands that bind to mammalian muscle cell-specific surface proteins can be associated with (e.g., displayed by, operably linked to, or bound to) modified AAV capsid proteins and resulting AAV capsids according to well-known methods, e.g., by direct insertion of the targeting ligand (e.g., using recombinant methods) according to well-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), supra. Well-known chemical linkers can be used to couple a targeting ligand that binds to a mammalian muscle cell-specific surface protein to a modified AAV capsid protein and the resulting AAV capsid, for example, wherein the AAV capsid protein can be chemically modified to contain a dibenzocycloalkynyl group or an azide group, and optionally wherein a targeting ligand as described herein is attached to the dibenzocycloalkynyl group or the azide group, see, for example, US2022 / 028234, which is incorporated herein by reference in its entirety; wherein the targeting ligand is covalently attached to a primary amino acid group of the AAV capsid protein, for example, via a -CSNH- bond. In some embodiments, the modified capsid as described herein comprises a targeting ligand, such as an anti-CACNG1 antibody or a binding portion thereof, directly inserted therein or coupled thereto according to well-known direct recombination methods.

[0172] Combination pair

[0173] In some embodiments, a targeting ligand that binds to a mammalian muscle cell-specific surface protein can be associated with (e.g., displayed by, operably linked to, or associated with) a modified AAV capsid protein and the resulting AAV capsid according to an indirect recombinant approach, wherein the AAV capsid protein is modified to comprise a first member of a binding pair (e.g., a heterologous scaffold), and optionally wherein the first member of the binding pair is linked (e.g., covalently or non-covalently bound) to a second homologous member of the binding pair (e.g., an adaptor), further optionally wherein the second homologous member of the binding pair is fused to the targeting ligand. Non-limiting and exemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12: 248-265.

[0174] Thus, in some embodiments, modifications of the capsid protein as described herein include those modifications that typically result from modifications at the genetic level, for example, via modifications of the cap gene, such as insertion of the first member of a binding pair (e.g., a protein:protein binding pair, a protein:nucleic acid binding pair), a detectable label, etc., for display of the Cap protein.

[0175] In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member forms a binding pair with a metal ion (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, Strep II, HA, L14, 4C-RGD, LH, and protein A.

[0176] In some embodiments, the binding pair comprises an enzyme:nucleic acid binding pair. In some embodiments, the first member comprises an HUH endonuclease or an HUH tag, and the second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises an HUH tag. See, e.g., US2021 / 0180082, which is incorporated herein by reference in its entirety.

[0177] In some embodiments, the capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

[0178] 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.

[0179] In some embodiments, the first member is a B cell epitope, for example, between about 1 and about 35 amino acids in length, and forms a binding pair with an antibody paratope (e.g., an immunoglobulin variable domain). In some embodiments, the capsid protein of the present invention can be modified to include a detectable label as the first member of the binding pair. Many detectable labels are known in the art. (See, for example: Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of modified proteins" Protein Expression and Purification 11: 1-16, Terpe et al. (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60: 523-533, and references therein). Detectable labels include, but are not limited to, the following: binding to immobilized divalent cations (e.g., Ni 2+ ), a polyhistidine detectable tag (e.g., His-6, His-8, or His-10) that binds to immobilized avidin (e.g., on a polypeptide sequence that is biotinylated 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 corresponding antibodies), a FLASH tag (a highly detectable tag coupled to a specific arsenic 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, a maltose binding protein (MBP) that binds to immobilized amylose, an 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 detectable labels that are recognized by an antibody paratope, wherein the detectable label and the antibody paratope form a protein:protein binding pair.

[0180] In some embodiments, the capsid protein of the present invention comprises a first member of a protein:protein binding pair comprising a detectable label, which can also be used to detect and / or isolate the Cap protein and / or as a 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 to a targeting ligand comprising a multispecific binding protein that can bind to both a detectable label and a target expressed by a cell of interest. In some embodiments, the Cap protein of the present invention comprises a first member of a protein:protein binding pair comprising c-myc (the use of detectable labels as first members of protein binding pairs is described, for example, in WO2019006043, which is incorporated herein by reference in its entirety).

[0181] In some embodiments, the capsid protein comprises a protein: a first member of a protein binding pair, wherein the protein: protein binding pair forms a covalent isopeptide bond. In some embodiments, the peptide: peptide binding pair first member is covalently bound to the peptide: peptide binding pair homologous second member via an isopeptide bond, and optionally wherein the peptide: peptide binding pair homologous second member is fused to a targeting ligand that binds to a target expressed by a cell of interest. In some embodiments, the protein: protein binding pair can be selected from the group consisting of: SpyTag: SpyCatcher, SpyTag002: SpyCatcher002, SpyTag003: SpyCatcher003, SpyTag: KTag, Isopeptag: pilin-C, and SnoopTag: SnoopCatcher. In some embodiments, the first member is SpyTag (or its biologically equivalent portion or variant), and the protein (second homologous member) is SpyCatcher (or its biologically equivalent portion or variant). In some embodiments, the first member is a SpyTag (or a biologically equivalent portion or variant thereof) and the protein (second homologous member) is a KTag (or a biologically equivalent portion or variant thereof). In some embodiments, the first member is a KTag (or a biologically equivalent portion or variant thereof) and the protein (second homologous member) is a SpyTag (or a biologically equivalent portion or variant thereof). In some embodiments, the first member is a SnoopTag (or a biologically equivalent portion or variant thereof) and the protein (second homologous member) is a SnoopCatcher (or a biologically equivalent portion or variant thereof). In some embodiments, the first member is an Isopeptag (or a biologically equivalent portion or variant thereof) and the protein (second homologous member) is Pilin-C (or a biologically equivalent portion or variant thereof). In some embodiments, the first member is SpyTag002 (or a biologically equivalent portion or variant thereof) and the protein (second homologous member) is SpyCatcher002 (or a biologically equivalent portion or variant thereof). In some embodiments, wherein the first member is SpyTag003 (or a biologically equivalent portion or variant thereof), and the protein (second homologous member) is SpyCatcher003 (or a biologically equivalent portion or variant thereof). In some embodiments, the Cap protein of the present invention comprises SpyTag or a biologically equivalent portion or variant thereof. The use of the first member of a protein:protein binding pair is described in WO2019006046, which is incorporated herein by reference in its entirety.

[0182] As used herein, the phrase "operably connected" comprises the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other, or otherwise coordinate with each other to participate in biological events, which juxtaposition enables or allows such interactions and / or coordination. As just one example, when a regulatory element (e.g., an expression control sequence) in a nucleic acid is positioned relative to a coding sequence so that its presence or absence can affect the expression and / or activity of the coding sequence, it is said to be "operably connected" to the coding sequence. In many embodiments, "operably connected" involves covalent attachment of related components or elements to each other. Those skilled in the art will readily appreciate that, in some embodiments, covalent attachment is not required to achieve effective operable attachment. For example, proteins that are operably connected together can, for example, associate with each other via covalent or non-covalent bonds. As non-limiting examples, capsid proteins as described herein can be operably connected to a targeting ligand, wherein the capsid protein is non-covalently bound to the targeting ligand, or is covalently bound to the targeting ligand, optionally with or without a scaffold and / or adapter between the capsid protein and the targeting ligand. As another example, in some embodiments, the nucleic acid regulatory element operably linked to the controlled coding sequence is adjacent to the target nucleotide. Alternatively or additionally, in some embodiments, one or more such regulatory elements act in trans or remotely to control the target coding sequence. In some embodiments, as used herein, the term "regulatory element" refers to a polynucleotide sequence that is necessary and / or sufficient to affect the expression and processing of the connected coding sequence. In some embodiments, the regulatory element can be or comprise appropriate transcription initiation, termination, promoter and / or enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and / or in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more regulatory elements are preferentially or exclusively active in a specific host cell or organism or type thereof. As just one example, in prokaryotes, regulatory elements typically can include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, in many embodiments, regulatory elements typically can include a promoter, an enhancer, and / or a transcription termination sequence. One of ordinary skill in the art will appreciate from the context that, in many embodiments, the term "regulatory element" refers to a component whose presence is essential for expression and processing, and in some embodiments, includes components whose presence is advantageous for expression (including, for example, a leader sequence, a targeting sequence, and / or a fusion partner sequence).

[0183] "Retargeting" or "redirecting" can include situations where a wild-type particle targets a few cells within a tissue and / or a few organs within an organism, and general targeting to a tissue or organ is reduced or eliminated by insertion of a heterologous amino acid, and retargeting to more specific cells in a tissue or a specific organ in an organism is achieved using a targeting ligand that binds to a marker expressed by a specific cell (e.g., via a targeting ligand). Such retargeting or redirecting can also include situations where a wild-type particle targets a tissue, and targeting to a tissue is reduced or eliminated by insertion of a heterologous amino acid, and retargeting to an entirely different tissue is achieved using a targeting ligand.

[0184] A "specific binding pair," "binding pair," "protein:protein binding pair," and the like comprise two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non-covalent bond between a first member epitope and a second member antigen binding portion of an antibody that recognizes the epitope; a covalent bond between proteins that are capable of forming isopeptide bonds; a split intein that recognizes each other and mediates the joining of flanking proteins and their own removal through the process of protein trans-splicing). In some embodiments, the term "cognate" refers to components that act together. Epitopes and their cognate antibodies, particularly epitopes that can also serve as detectable labels (e.g., c-myc), are well known in the art. Specific protein: protein binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32: 506 and include peptide: peptide binding pairs such as SpyTag: SpyCatcher, SpyTag002: SpyCatcher002; SpyTag: KTag; isopeptag: pilin C, SnoopTag: SnoopCatcher, and the like, and variants thereof, such as SpyTag003: SpyCatcher003. Typically, the first member of a protein: protein binding pair is a member of a protein: protein binding pair that is typically less than 30 amino acids in length and that forms a spontaneous covalent isopeptide bond with a second homologous protein, where the second homologous protein is typically larger, but may also be less than 30 amino acids in length, such as in the SpyTag: KTag system.

[0185] 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, viewed from another perspective, is not part of the protein backbone. An isopeptide bond can form within a single protein or between two peptides or between a peptide and a protein. Thus, an isopeptide bond can form intramolecularly within a single protein or intermolecularly, i.e., between two peptide / protein molecules, such as between two peptide linkers. Typically, an isopeptide bond can form between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue or the terminal carboxyl group of a protein or peptide chain, or between the α-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid. Each residue in the pair involved in the isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, an isopeptide bond can form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. In particular, an isopeptide bond can form between the side chain amine of lysine and the carboxyl group of asparagine or the carboxyl group of aspartic acid.

[0186] 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 from Streptococcus pyogenes. By splitting the domains, Zaveri et al. obtained a peptide "SpyTag" with the sequence AHIVMVDAYKPTK (SEQ ID NO: 243), which binds to its cognate protein "SpyCatcher" by forming an amide bond. The cognate protein is a 112-amino acid polypeptide having the amino acid sequence shown 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 was engineered by cleaving the beta strand containing the reactive lysine from SpyCatcher, resulting in a protein with the amino acid sequence ATHIKFSKRD (SEQ ID NO: 245): the first member of the 10-residue protein binding pair, KTag. The SpyTag002: SpyCatcher002 system is described in Keeble et al. (2017) Angew Chem Int Ed Engl 56: 16521-25, which is incorporated herein by reference. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK as shown in SEQ ID NO: 255 and binds to SpyCatcher002. SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK as shown in SEQ ID NO: 259 and binds to SpyCatcher003.

[0187] 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, is cleaved to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher generates a specific spontaneous isopeptide bond between the complementary proteins. Veggiani (2016), supra.

[0188] The isopeptag:pilin-C specific binding pair is derived from the major pilin protein Spy0128 from Streptococcus pyogenes (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE as set forth in SEQ ID NO:254 and binds to pilin-C (residues 18-299 of Spy0128). Incubation of SnoopTag and SnoopCatcher generates a specific spontaneous isopeptide bond between the complementary proteins. Zakeir and Howarth (2010), supra.

[0189] The term "detectable label" includes a polypeptide sequence that is a member of a specific binding pair, for example, a polypeptide sequence that specifically binds to another polypeptide sequence (e.g., an antibody paratope) with high affinity via a non-covalent bond. Exemplary and non-limiting detectable labels include hexahistidine tags, FLAG tags, Strep II tags, streptavidin binding peptide (SBP) tags, calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), S- tags, HA tags, and myc tags from c-myc (SEQ ID NO: 246). (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8; the document is 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 that do not naturally contain the B1 epitope can be modified herein to include the B1 epitope. Typically, the AAV capsid proteins described herein can comprise a sequence having substantial homology to a B1 epitope within the last 10 amino acids of the capsid protein. Thus, in some embodiments, the non-primate AAV capsid proteins of the invention can be modified with one but fewer than five point mutations within the last 10 amino acids of the capsid protein such that the AAV capsid protein comprises the B1 epitope.

[0190] The term "target cell" includes any cell in which expression of a nucleic acid of interest is desired. Preferably, the target cell exhibits a receptor on its surface that allows targeting of the cell via a targeting ligand, as described below.

[0191] The terms "transduction" or "infection" refer to the introduction of a nucleic acid into the nucleus of a target cell by a viral particle. Terms related to transduction, such as "transduction efficiency," refer to the fraction (e.g., percentage) of cells that express the target nucleotide after incubation with a given number of viral particles containing the target nucleotide. Well-known methods for determining transduction efficiency include flow cytometry of cells transduced with a fluorescent reporter gene, RT-PCR for expression of the target nucleotide, and the like.

[0192] Typically, the "reference" viral capsid protein / capsid / particle is identical to the test viral capsid protein / capsid / particle, except for the change in the effect to be tested. For example, to determine the effect of inserting the first member of a specific binding pair into a test viral particle, for example, on transduction efficiency, the transduction efficiency of the test viral particle (in the absence or presence of an appropriate targeting ligand) can be compared with the transduction efficiency of a reference viral particle (in the absence or presence of an appropriate targeting ligand, if desired), which is identical to the test viral particle in every respect (e.g., additional point mutations, target nucleotides, number of viral particles and target cells, etc.) except for the presence of the first member of the specific binding pair. In some embodiments, the reference viral capsid protein is a reference viral capsid protein capable of forming a capsid with a second viral capsid protein, the second viral capsid protein being modified to include at least the first member of the protein:protein binding pair, wherein the reference viral capsid protein does not include the first member of the protein:protein binding pair, preferably wherein the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.

[0193] In some embodiments, the first member of the protein: protein binding pair and / or the detectable label is operably linked to a Cap protein of the invention (translated in frame with, chemically linked to, and / or displayed by) via a first linker or a second linker, e.g., an amino acid spacer of at least one amino acid in length. In some embodiments, the first member of the protein: protein binding pair is flanked by a first linker and / or a second linker, e.g., a first amino acid spacer and / or a second amino acid spacer, each of which is at least one amino acid in length.

[0194] In some embodiments, the first joint and / or the second joint are different. In some embodiments, the length of the first joint and / or the second joint is each independently one or two amino acids. In some embodiments, the length of the first joint and / or the second joint is each independently one, two, or three amino acids. In some embodiments, the length of the first joint and / or the second joint is each independently one, two, three, or four amino acids. In some embodiments, the length of the first joint and / or the second joint is each independently one, two, three, four, or five amino acids. In some embodiments, the length of the first joint and / or the second joint is each independently one, two, three, four, or five amino acids. In some embodiments, the length of the first joint and / or the second joint is each independently one, two, three, four, five, or six amino acids. In some embodiments, the length of the first joint and / or the second joint is each independently one, two, three, four, five, six, or seven amino acids. In some embodiments, the length of the first joint and / or the second joint is each independently one, two, three, four, five, six, seven, or eight amino acids. In some embodiments, the length of the first linker and / or the second linker is each independently one, two, three, four, five, six, seven, eight, or nine amino acids. In some embodiments, the length of the first linker and / or the second linker is each independently one, two, three, four, five, six, seven, eight, nine, or ten amino acids. In some embodiments, the length of the first linker and / or the second linker is each independently one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids.

[0195] In some embodiments, the first linker and the second linker are of the same sequence and / or length, and each is one amino acid in length. In some embodiments, the first linker and the second linker are of the same length, and each is one amino acid in length. In some embodiments, the first linker and the second linker are of the same length, and each is two amino acids in length. In some embodiments, the first linker and the second linker are of the same length, and each is three amino acids in length. In some embodiments, the first linker and the second linker are of the same length, and each is four amino acids in length, for example, the linker is GLSG (SEQ ID NO: 248). In some embodiments, the first linker and the second linker are of the same length, and each is five amino acids in length. In some embodiments, the first linker and the second linker are of the same length, and each is six amino acids in length, for example, the first linker and the second linker each comprise the sequence of GLSGSG (SEQ ID NO: 249). In some embodiments, the first linker and the second linker are of the same length, and each is seven amino acids in length. In some embodiments, the first linker and the second linker are of the same length, and each is eight amino acids in length, for example, the first linker and the second linker each comprise the sequence of GLSGLSGS (SEQ ID NO: 250). In some embodiments, the first linker and the second linker are the same length and are each nine amino acids in length. In some embodiments, the first linker and the second linker are the same length and are each ten amino acids in length, for example, the first linker and the second linker each comprise the sequence of GLSGLSGLSG (SEQ ID NO: 251) or GLSGGSGLSG (SEQ ID NO: 252). In some embodiments, the first linker and the second linker are the same length and are each more than ten amino acids in length.

[0196] Typically, the length of the first member of a protein:protein binding pair amino acid sequence as described herein (e.g., comprising the first member of a specific binding pair, by itself or in combination with one or more linkers) is between about 5 amino acids and about 50 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is at least 5 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 6 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 7 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 8 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 9 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 10 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 11 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 12 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 13 amino acids. In some embodiments, the length of the first member of a protein:protein binding pair amino acid sequence is 14 amino acids. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 15 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 16 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 17 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 18 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 19 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 20 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 21 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 22 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 23 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 24 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 25 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 26 amino acids in length. In some embodiments, the first member of the protein:protein binding pair amino acid sequence is 27 amino acids in length.In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 28 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 29 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 30 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 31 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 32 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 33 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 34 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 35 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 36 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 37 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 38 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 39 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 40 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 41 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 42 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 43 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 44 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 45 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 46 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 47 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 48 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 49 amino acids in length. In some embodiments, the first member of a protein:protein binding pair amino acid sequence is 50 amino acids in length.

[0197] Modified capsid comprising a modified capsid protein

[0198] In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein is a mosaic capsid, for example, comprising at least two groups of VP1, VP2 and / or VP3 proteins, each group of proteins being encoded by a different cap gene. Mosaic capsids herein generally refer to a mosaic of a first viral capsid protein modified to comprise a first member of a binding pair and a second corresponding viral capsid protein lacking the first member of the binding pair. With regard to mosaic capsids, the second viral capsid protein lacking the first member of the binding pair can be referred to as a reference capsid protein encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP1, VP2 and / or VP3 capsid protein modified with the first member of the protein pair is not a mosaic capsid protein, the VP1, VP2 and / or VP3 reference capsid protein can comprise an amino acid sequence identical to the amino acid sequence of the viral VP1, VP2 and / or VP3 capsid protein modified with the first member of the binding pair, except that 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, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP1 reference capsid protein corresponds to a viral VP1 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP2 reference capsid protein corresponds to a viral VP2 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some mosaic capsid embodiments comprising a chimeric VP1, VP2 and / or VP3 capsid protein that has been further modified to comprise a first member of a binding pair, the reference protein can be a corresponding capsid protein, a portion of which forms a portion of the chimeric capsid protein. As a non-limiting example, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP1 capsid protein modified to include a first member of a binding pair can further comprise, as a reference capsid protein, an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member. Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP2 capsid protein modified to include a first member of a binding pair can further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member.In some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP3 capsid protein modified to include a first member of a binding pair can further comprise, 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 can be any capsid protein that lacks the first member of the binding pair and is capable of forming a capsid with the first capsid protein modified with the first member of the binding pair.

[0199] Typically, mosaic particles can be produced by transfecting a mixture of modified Cap genes and reference Cap genes at a specified ratio into production cells. The protein subunit ratio in the particle (e.g., the ratio of modified VP protein:unmodified VP protein) can, but does not necessarily, stoichiometrically reflect the ratio of at least two species: a 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., a modified cap gene:reference cap gene transfected into packaging cells. In some embodiments, the protein subunit ratio in the particle does not stoichiometrically reflect the ratio of modified cap gene:reference cap gene transfected into packaging cells.

[0200] In some mosaic virus particle embodiments, the protein subunit ratio ranges from about 1:59 to about 59:1. In some mosaic virus particle embodiments, the protein subunits are at least about 1:1 (e.g., the mosaic virus particle comprises about 30 modified capsid proteins and about 30 reference capsid proteins). In some mosaic virus particle embodiments, the protein subunit ratio is at least about 1:2 (e.g., the mosaic virus particle comprises about 20 modified capsid proteins and about 40 reference capsid proteins). In some mosaic virus particle embodiments, the protein subunit ratio is at least about 3:5. In some mosaic virus particle embodiments, the protein subunit ratio is at least about 1:3 (e.g., the mosaic virus particle comprises about 15 modified capsid proteins and about 45 reference capsid proteins). In some mosaic virus particle embodiments, the protein subunit ratio 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 about 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 about 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.

[0201] In some non-mosaic viral particle embodiments, the protein subunit ratio can be 1:0, wherein each capsid protein of the non-mosaic viral particle is modified with the first member of the binding pair. In some non-mosaic viral particle embodiments, the protein subunit ratio can be 0:1, wherein each capsid protein of the non-mosaic viral particle is not modified with the first member of the binding pair.

[0202] Insertion site

[0203] Due to the high conservation of at least the large segments and the large members of closely related family members, the corresponding insertion sites of AAVs other than the listed AAVs can be identified by performing amino acid alignments or by comparing the capsid structures. See, for example, 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 for exemplary alignments of different AAV capsid proteins, each of which is incorporated herein by reference in its entirety. For example, Mietzcsh et al. (2019) in Figure 7 An overlay of bands from different dependent parvoviruses is provided, depicting variable regions VR I to VR IX. Using such structural analysis as described herein, along with sequence analysis, one can determine which amino acids within the variable region correspond to the amino acid sequence of an AAV that can accommodate insertion of, for example, a targeting ligand, a first member of a binding pair, and / or a detectable label as described herein.

[0204] Typically, the targeting ligand, the first member of the binding pair, and / or the detectable label can be inserted into the variable region or variable loop of the AAV capsid protein, the GH loop of the AAV capsid protein, or the like.

[0205] In some embodiments, the first member of the binding pair and / or the detectable label is inserted into the VP1 capsid protein of a non-primate AAV after an amino acid position corresponding to an amino acid position selected from the group consisting of: G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the first member of the binding pair and / or the detectable label is inserted into the VP1 capsid protein of a 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 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, and 1-716 of the VP1 capsid protein of AAV2 (Wu et al. (2000) J. Virol. 74:8635-8647). The modified viral capsid protein as described herein can be a non-primate capsid protein comprising a first member of a binding pair and / or a detectable label inserted into a position corresponding to a position of an AAV2 capsid protein selected from the group consisting of: 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 for non-primate AAVs include sites corresponding to I-587 or I-590 of AAV1, I-589 of AAV1, I-585 of AAV3, I-584 or I-585 of AAV4, and I-575 or I-585 of AAV5. In some embodiments, the modified viral capsid protein as described herein can be a non-primate capsid protein comprising a targeting ligand, a first member of a binding pair, and / or a detectable label inserted into a position corresponding to a position selected from the group consisting of: I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5), and combinations thereof.

[0206] In some embodiments, the first member of the binding pair and / or the detectable label is inserted into the VP1 capsid protein of a non-primate AAV after the amino acid position corresponding to the amino acid position selected from the group consisting of: I444 of avian AAV capsid protein VP1, I580 of avian AAV capsid protein VP1, I573 of bearded dragon AAV capsid protein VP1, I436 of bearded dragon AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I433 of sea lion AAV capsid protein VP1, I434 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I437 of sea lion AAV capsid protein VP1, and I565 of sea lion AAV capsid protein VP1.

[0207] The nomenclature herein is I-###, I#, etc., which refers to the insertion site (I) designated by ### relative to the amino acid number of the VP1 protein of the AAV capsid protein, however, such insertion may be located directly at the N-terminus or the C-terminus, preferably at the N-terminus or C-terminus of the 5 amino acids of a given amino acid, preferably at the C-terminus of one amino acid in a sequence of 3, more preferably 2, especially 1 amino acids of the N-terminus or C-terminus of a given amino acid. In addition, the positions mentioned herein are relative to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and point mutations thereof) of the VP2 and VP3 capsid proteins encoded by the capsid gene can be easily identified by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid gene.

[0208] Thus, insertion into the corresponding position of the encoding nucleic acid at one of these sites of the cap gene results in insertion into VP1, VP2, and / or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Thus, for AAV2, for example, according to this nomenclature, insertion between amino acids 1 and 138 is insertion into VP1 only, insertion between 138 and 203 is insertion into VP1 and VP2, and insertion between 203 and the C-terminus is insertion into VP1, VP2, and VP3, as is the case for insertion site 1-587. Thus, the present invention encompasses structural genes of AAV having corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0209] Also provided herein are nucleic acids encoding the VP3 capsid protein of the present invention. AAV capsid proteins can, but are not necessarily, encoded by overlapping reading frames of the same gene with staggered start codons. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the present invention does not also encode the VP2 capsid protein or the VP1 capsid protein of the present invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the present invention may also encode the VP2 capsid protein of the present invention, but does not also encode the VP1 capsid of the present invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the present invention may also encode the VP2 capsid protein of the present invention and the VP1 capsid of the present invention.

[0210] In some embodiments, a viral capsid comprising a modified viral capsid protein having a first member and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is capable of infecting specific cells, for example, having an enhanced ability to target and bind to specific cells compared to a control viral capsid, the control viral capsid being identical to the modified viral capsid protein, for example, comprising a control capsid protein, except for the lack of one or both of the first and second members of the binding pair. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the 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, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair linked to a targeting ligand) exhibits a transduction efficiency that is 10% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 20% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 30% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 40% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 50% higher than the transduction efficiency of the control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) exhibits a transduction efficiency that is 60% higher than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) exhibits a transduction efficiency that is 70% higher than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) exhibits a transduction efficiency that is 75% higher than the transduction efficiency of a control viral capsid.In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 80% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 85% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 90% higher than the transduction efficiency of the control capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 95% higher than the transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein as described herein bound to a first member and a second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.

[0211] In some embodiments, a viral capsid comprising a modified viral capsid protein having a first member and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is capable of infecting specific cells, for example, having an enhanced ability to target and bind to specific cells compared to a control viral capsid, the control viral capsid being identical to the modified viral capsid protein, for example, comprising a control capsid protein, except for the lack of one or both of the first and second members of the binding pair. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the 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, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair linked to a targeting ligand) exhibits a transduction efficiency that is 10% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 20% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 30% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 40% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 50% higher than the transduction efficiency of the control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) exhibits a transduction efficiency that is 60% higher than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) exhibits a transduction efficiency that is 70% higher than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) exhibits a transduction efficiency that is 75% higher than the transduction efficiency of a control viral capsid.In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 80% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 85% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 90% higher than the transduction efficiency of the control capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 95% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is 99% higher than the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is at least 1.5 times that of the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is at least 2 times that of the transduction efficiency of the control viral capsid. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) exhibits a transduction efficiency that is at least 3 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) is at least 4 times that of the transduction efficiency of a control viral capsid. In some embodiments, the transduction efficiency exhibited by a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) is at least 5 times that of the transduction efficiency of a control viral capsid. In some embodiments, the transduction efficiency exhibited by a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) is at least 6 times that of the transduction efficiency of a control viral capsid.In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 7 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 8 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 9 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 10 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 20 times that of the transduction efficiency of the control capsid. In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 30 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 40 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by the viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair connected to the targeting ligand) is at least 50 times that of the transduction efficiency of the control viral capsid. In some embodiments, the transduction efficiency exhibited by a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) is at least 60 times that of the transduction efficiency of a control viral capsid. In some embodiments, the transduction efficiency exhibited by a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) is at least 70 times that of the transduction efficiency of a control viral capsid. In some embodiments, the transduction efficiency exhibited by a viral capsid comprising a modified viral capsid protein as described herein (which is bound to the first and second members of the binding pair attached to the targeting ligand) is at least 80 times that of the transduction efficiency of a control viral capsid.In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein that is bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 90 times greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein that is bound to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100 times greater than the transduction efficiency of a control viral capsid. In some embodiments, viral particles of the invention comprising a viral capsid protein having the amino acid sequence of a capsid protein of a non-primate AAV, a distant AAV, or a combination thereof, and optionally comprising a first member and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.), are better able to evade neutralization by pre-existing antibodies in serum isolated from a human patient than an appropriate control viral particle (e.g., comprising a viral capsid of an AAV serotype, a portion of which is included in the viral capsid of the invention, e.g., as part of a viral capsid protein, which viral capsid protein comprises the amino acid sequence of a capsid protein of a non-primate AAV, a distant AAV, or a combination thereof), which control viral particle also optionally comprises a first member and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.). In some embodiments, a viral particle of the invention comprising a viral capsid protein having the amino acid sequence of a capsid protein of a non-primate AAV, a distant AAV, or a combination thereof requires at least twice as much total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) as compared to an appropriate control viral particle, e.g., (e.g., the IC50 value of the viral particle of the invention is at least twice the IC50 value of the control viral particle).

[0212] In some embodiments of the present 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, which can be conjugated to the surface of a bead (e.g., for purification) or expressed by a target cell. Thus, the multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor targets the viral particle. Such "targeting" or "direction" can include a situation where wild-type viral particles target several cells within a tissue and / or several organs within an organism, reduce or eliminate the broad targeting of the tissue or organ by inserting a detectable label, and utilize multispecific binding molecules to achieve re-targeting of more specific cells in the tissue or more specific organs in the organism. Such re-targeting or redirection can also include a situation where wild-type viral particles target a tissue, reduce or eliminate the targeting of the tissue by inserting a detectable label, and utilize multispecific binding molecules to achieve re-targeting of completely different tissues. As described herein, the antibody paratope generally comprises at least a complementary determining region (CDR) that specifically recognizes a detectable marker, for example, a CDR3 region of a heavy chain and / or light chain variable domain. In some embodiments, the multispecific binding molecule comprises an antibody (or portion thereof) comprising an antibody paratope that specifically binds to a detectable marker. For example, the multispecific binding molecule can comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or the single domain light chain variable region comprises an antibody paratope that specifically binds to a detectable marker. In some embodiments, the multispecific binding molecule can comprise an Fv region, for example, the multispecific binding molecule can comprise an scFv, which comprises an antibody paratope that specifically binds to a detectable marker. In some embodiments, the multispecific binding molecule as described herein comprises an antibody paratope that specifically binds to c-myc (SEQ ID NO: 246).

[0213] One embodiment of the present invention is a multimeric structure comprising a modified viral capsid protein of the present invention. The multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 modified viral capsid proteins as described herein, and these modified viral capsid proteins comprise the first member of the specific binding pair. It can form a conventional viral capsid (empty viral particle) or a viral particle (capsid that encapsidates the target nucleotide). The formation of a viral particle comprising a viral genome is a highly preferred feature of using a modified viral capsid as described herein.

[0214] A further embodiment of the present invention is the use of at least one modified viral capsid protein and / or nucleic acid encoding the same, preferably at least one multimeric structure (e.g., a viral particle) for producing a nucleotide of interest and for transferring the nucleotide of interest into a target cell.

[0215] Methods of use and preparation

[0216] The other embodiment of modified viral capsid as described herein is that it is used for delivering target nucleotide (for example, reporter gene or therapeutic gene) to target cell.Generally, packaging target nucleotide includes replacing the AAV genome between AAV ITR sequences with target gene to produce transfer plasmid, which is then encapsulated in AAV capsid according to well-known method.Therefore, modified viral capsid as described herein can encapsulate transfer plasmid and / or target nucleotide, which can generally include 5' and 3' reverse terminal repeat (ITR) sequences (which can be under the control of viral or non-viral promoters) of the flank of a part for target gene (for example, reporter gene or therapeutic gene) or target gene.According to the method for packaging AAV virus particles as well-known, modified viral capsid, 5' ITR and 3' ITR do not need to have identical AAV serotype.In one embodiment, transfer plasmid and / or target nucleotide comprise from 5' to 3': 5' ITR, promoter, gene (for example, reporter gene and / or therapeutic gene) and 3' ITR.

[0217] The target genes disclosed herein include, but are not limited to, genes encoding micro-dystrophin, FKRP, and MTM1, for example, genes encoding human micro-dystrophin, human FKRP, and human MTM1. A non-limiting sequence encoding micro-dystrophin is shown in SEQ ID NO: 270. A non-limiting sequence encoding FKRP is shown in SEQ ID NO: 271. A non-limiting sequence encoding MTM1 is shown in SEQ ID NO: 272. The target genes described herein also include, but are not limited to, biologically equivalent portions or variants of the genes disclosed herein. For example, the target gene may comprise a biologically equivalent portion or variant of the sequence shown in SEQ ID NO: 270. The target gene may comprise a biologically equivalent portion or variant of the sequence shown in SEQ ID NO: 271. The target gene may comprise a biologically equivalent portion or variant of the sequence shown in SEQ ID 272.

[0218] A consideration in the design of AAV transfer plasmids is that the wild-type AAV genome is approximately 4.7 kb. Therefore, this paper includes well-known strategies for providing packaging of target nucleotides that exceed the packaging capacity of individual AAVs. Such strategies include, but are not limited to, dual-vector strategies that utilize ITR-mediated recombination to express target genes larger than the wild-type AAV genome by transcriptional splicing across intermolecular recombination ITRs from two complementary vector genomes, vector recombination by homology, RNA trans-splicing, and / or protein "trans-splicing" achieved via split intein design. See, e.g., Nakai, H. et al. (2000) Nat. Biotechnol. 18:527–532; Sun, L. (2000) Nat. Med. 6:599–602 (2000); Ghosh, A. et al. (2008) Mol. Ther. 16:124–130 (2008); Lai, Y (2005) Nat. Biotechnol. 23:1435–1439; Chew, WL et al. (2016) Nat. Methods 13:868–874; Li, J. (2008) Hum. Gene Ther. 19:958–964, each of which is incorporated herein by reference in its entirety.

[0219] Dual AAV vector strategies for transferring large genes into target cells have been described and rely on different mechanisms, including but not limited to trans-splicing, inclusion of overlapping regions in the dual vectors, and hybrids of the two. Tornabene and Trapani (2020) Human Gene Ther. 31:47-56; see also U.S. Patent No. 8,236,557, each of which is incorporated herein by reference in its entirety.

[0220] The trans-splicing method utilizes the ability of AAV ITR sequences to be tandemly connected to reconstruct the full-length genome, where each of the two or more viral capsids encapsulates one of the two or more transfer plasmids, each of which contains a portion of the gene of interest. For example, in the dual-vector method, the two transfer plasmids can be designed as follows: the 5'-transfer plasmid contains a promoter, the 5' portion of the coding sequence of the gene of interest, and a splice donor (SD) signal; the 3'-transfer plasmid contains a splice acceptor (SA) signal, the 3' portion of the gene of interest, and a polyA signal. After the ITR-mediated concatenation of the two AAV genomes, the SD and SA signals will allow splicing of the recombinant genome.

[0221] Large genes of interest can also be fragmented when using the overlapping region approach. In the overlapping region approach, the 5' and 3' portions (thus the 5' transfer plasmid and the 3' transfer plasmid) share recombination sequences, e.g., regions of homology, e.g., each portion contains overlapping sequences. The gene of interest is then intact in the target cell through homologous recombination mediated by recombinogenic sequences (e.g., regions of homology / overlapping).

[0222] In the hybrid method, the 5'-transfer plasmid and the 3'-transfer plasmid each contain a highly recombinant sequence, where the recombinant sequence is 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 through ITR-mediated concatenation and splicing and / or through homologous recombination.

[0223] Trans-splicing at the RNA or protein level can also be exploited. In the RNA trans-splicing approach, two transfer plasmids can encode the 5' and 3' fragments of the pre-mRNA of a large gene, respectively, and share an intronic hybridization domain that facilitates trans-splicing, resulting in the ligation of the two half-transcripts into a complete full-length mRNA.

[0224] Protein trans-splicing occurs after translation and is catalyzed by an intermediate protein called a split intein. Split intein is expressed as two independent polypeptides (N-intein and C-intein) at the ends of two host proteins. N-intein and C-intein polypeptides remain catalytically inactive before meeting each other. When meeting each other, each intein accurately excises itself from the host protein while mediating the connection of N- and C- host polypeptides via peptide bonds. Split intein uses have been used for AAV-based delivery of therapeutic target genes in muscle, liver, and retinal diseases. For example, when co-delivering two half mini-dystrophin cDNAs fused with N- and C-intein coding sequences, effective production of two polypeptides is shown. Li et al. (2008) Hum Gene Ther 19:958-64. Similarly, AAV split intein has been widely used for expression and connection of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases.

[0225] The above-mentioned dual vector method is well known in the art. See, for example, Tornabene and Trapani (2020), supra; U.S. Patent No. 8,236,557. Therefore, in some embodiments, the modified viral capsid described herein encapsulates a target nucleotide, wherein the target nucleotide comprises a portion of a target gene. In some embodiments, the target nucleotide comprising a portion of a target gene further comprises a splice donor signal or a splice acceptor signal and / or a recombination sequence. In some embodiments, the target nucleotide comprising a portion of a target gene comprises an intron hybridization domain coding sequence. In some embodiments, the target nucleotide comprising a portion of a target gene comprises an N-intein or C-intein coding sequence.

[0226] The design of transfer plasmid / destination nucleotide includes one or more regulatory elements, such as promoter and / or enhancer element, that will control the expression of target gene.The limiting examples of useful promoter include such as cytomegalovirus (CMV) promoter, spleen lesion forming virus (SFFV) promoter, elongation factor 1 alpha (EF1a) promoter (1.2kb EF1a promoter or 0.2kb EF1a promoter), chimeric EF1a / IF4-promoter, phosphoglycerate kinase (PGK) promoter and its biological equivalent part or variant.Internal enhancer also can be present in viral construct, to increase the expression of target gene.For example, CMV enhancer (people such as Karasuyama 1989.J.Exp.Med.169:13, this document is incorporated herein by reference) can be used.In some embodiments, tissue-specific regulatory element, such as muscle-specific promoter and / or regulatory element, can be used for driving the expression of target gene, such as muscle-specific promoter of other genes expressed in muscle based on skeletal muscle α-actin, muscle creatine kinase and desmin gene. A non-limiting example of an actin gene that is ubiquitous in adult muscle is the human skeletal muscle α-actin gene (HSA). The HSA promoter and its biologically equivalent parts or variants, as well as other regulatory regions of homologous chicken, rat and cattle genes have been used in transgenic animal models and AAV-mediated gene transfer in vitro and in vivo. Skopenkova et al. Acta Naturae 13:47-58. In some embodiments, an enhancer (e.g., a CMV enhancer) can be used in combination with the promoter of an actin gene (e.g., a chicken β-actin promoter) and its biologically equivalent parts or variants. The use of muscle-specific regulatory elements based on the muscle creatine kinase gene (MCK) has also been used for 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 the target nucleotide herein include an enhancer and / or promoter of MCK, or a biologically equivalent portion or variant thereof, wherein the enhancer and / or promoter of MCK drives the expression of the 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 the target nucleotide herein include an enhancer and / or promoter element that recruits RNA polymerase II, wherein the enhancer and / or promoter of MCK (or a biologically equivalent portion or variant thereof) drives the expression of the gene of interest.In some embodiments, the transfer plasmid and / or target nucleotide herein comprises an enhancer and / or promoter element that recruits RNA polymerase III, wherein the enhancer and / or promoter of MCK (or its biologically equivalent portion or variant) drives the expression of the gene of interest. In some embodiments, the transfer plasmid and / or target nucleotide herein comprises a desmin promoter or its biologically equivalent portion or variant. In some embodiments, the transfer plasmid and / or target nucleotide herein comprises a human myosin heavy chain gene (αMHC) promoter or its biologically equivalent portion or variant. In some embodiments, the transfer plasmid and / or target nucleotide herein comprises an MLC promoter or its biologically equivalent portion or variant, such as a CMV-IE enhancer linked to a rat MLC promoter. In some embodiments, the transfer plasmid and / or target nucleotide herein comprises a ΔUSEx3 promoter or its biologically equivalent portion or variant, which is based on the human troponin I (TNN1) gene. In some embodiments, the transfer plasmid and / or target nucleotide herein comprises an unc45b promoter or its biologically equivalent portion or variant.

[0227] In some embodiments, bidirectional promoters and / or vectors have also been used to deliver dual therapeutic gene cassettes. An example of this is the ubiquitous promoter of bidirectional chicken β-actin, which drives the simultaneous expression of the hexosaminidase α-subunit and β-subunit of the HexA enzyme, two corresponding genes involved in Tay-Sachs and Sandhoff disease. Lahey et al. (2020) Mol.Ther.28:2150–2160, which is incorporated herein by reference in its entirety. In some embodiments, the transfer plasmid and / or target nucleotide herein comprises a bidirectional promoter, wherein the bidirectional promoter drives the expression of two different target genes.

[0228] A variety of reporter genes (or detectable moieties) can be encapsidated in a polymeric structure comprising modified viral capsid proteins as described herein. Exemplary reporter genes include, for example, beta-galactosidase (encoded 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. Methods described herein demonstrate the use of reporter genes to encode green fluorescent protein to construct targeting particles, but, it will be understood by the skilled person after reading this disclosure that viral particles as described herein can be produced in the absence of reporter genes or with any reporter genes known in the art.

[0229] A variety of therapeutic genes can also be encapsidated in a multimeric structure comprising a modified viral capsid protein as described herein, for example, 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.

[0230] Another embodiment of the present invention is a method for preparing a modified capsid protein, the method comprising the steps of:

[0231] a. expressing the nucleic acid encoding the modified capsid protein under appropriate conditions, and

[0232] b. Isolating the expressed capsid protein of step a).

[0233] In some embodiments, a viral particle as described herein comprises a chimeric capsid, e.g., a capsid comprising a capsid protein that is genetically modified as described herein (in the absence or presence of a covalent bond to a targeting ligand) at a certain ratio to a reference capsid protein. Methods for preparing such a chimeric viral particle include:

[0234] a. Under suitable conditions at a ratio of at least about 60:1 to about 1:60, for example, 2:1, 1:1, 3:5,

[0235] The nucleic acid encoding the modified capsid protein and the nucleotide encoding the reference capsid protein are expressed in a ratio of 1:2, 1:3, etc. (wt / wt), and

[0236] b. Isolating the expressed capsid protein of step a).

[0237] In some embodiments, the compositions described herein comprise, or the methods described herein combine, a modified cap gene: a reference cap gene (or a combination 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.

[0238] In some embodiments, the ratio of VP protein subunits in the mosaic virus particles can, but need not, stoichiometrically reflect the ratio of modified cap gene:reference cap gene. As a non-limiting exemplary embodiment, the ratio of modified capsid protein:reference capsid protein of the mosaic capsids formed according to this method can be considered, but not necessarily, to be similar to the ratio (wt:wt) of the nucleic acids encoding the same used to generate the mosaic capsids. In some embodiments, the mosaic capsids comprise a protein subunit ratio of about 1:59 to about 59:1.

[0239] Another embodiment of the present invention is a method for altering the tropism of a virus, comprising the steps of: (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 to produce viral particles, wherein the packaging cells comprise the nucleic acid. Another embodiment of the invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising the steps of: (a) expressing a nucleic acid encoding a modified viral capsid protein as described herein (and optionally together with nucleotides encoding a reference capsid protein) under suitable conditions, 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 or a capsid comprising the capsid protein of step (a), and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under conditions suitable to allow formation of an isopeptide bond between the first member and the second member, wherein the second cognate member of the specific binding pair is fused to the targeting ligand.

[0240] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising a target nucleotide. In some embodiments, the method further comprises isolating self-complementary adeno-associated virus particles from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating single-stranded adeno-associated virus particles from the cell lysate. In some embodiments, the method further comprises (a) removing cell debris; (b) treating the supernatant containing the virus particles with a nuclease, such as DNase I and MgCl2; (c) concentrating the virus particles; (d) purifying the virus particles; and (e) any combination of (a)-(d).

[0241] Packaging cells that can be used to produce the viral particles described herein include, for example, virus-permissive animal cells or cells modified to be virus-permissive; or packaging cell constructs, for example, using a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines that can be used to produce the viral particles described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells containing the SV40 large T-antigen (HEK-293T or 293T), HEK293T / 17 cells, the human sarcoma cell line HT-1080 (CCL-121), the lymphoblastoid cell line Raji (CCL-86), the glioblastoma-astrocytoma epithelioid cell line U87-MG (HTB-14), the T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells, etc.

[0242] L929 cells, the FLY virus packaging cell system summarized in Cosset et al. (1995) J Virol 69, 7430-7436, NSO (murine myeloma) cells, human amniocytes (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., High 5)), or bacterial cells (including but not limited to E. coli).

[0243] For additional packaging cells and systems, packaging techniques and particles for packaging nucleic acid genomes into pseudotyped viral particles, see, for example, Polo et al., Proc Natl Acad Sci USA, (1999) 96: 4598-4603. Packaging methods include the use of packaging cells that permanently express viral components or by transiently transfecting cells with plasmids.

[0244] Additional embodiments include methods comprising contacting a modified Cap protein as described herein with a targeting vector under conditions sufficient to operably link the modified Cap protein to the targeting vector, e.g., under conditions sufficient to promote association of the targeting vector with the modified Cap protein, e.g., via chemical ligation and / or association of a first member and a second member of a specific binding pair, wherein the first member is inserted into the modified Cap protein first member, and the targeting vector is fused to the second member of the specific binding pair.

[0245] Additional embodiments include methods for redirecting viruses to target cells and / or delivering reporter genes or therapeutic genes to target cells, including methods for transducing cells in vitro (e.g., ex vivo) or in vivo, the method comprising the following steps: contacting target cells with viral particles comprising capsids as described herein, wherein the capsids comprise targeting ligands that specifically bind to receptors expressed by target cells. In some embodiments, the target cells are in vitro (e.g., ex vivo). In other embodiments, the target cells are in a subject, such as a human.

[0246] target cells

[0247] A variety of cells can be targeted for delivery of the nucleotide of interest using modified viral particles as disclosed herein. The target cell will generally be selected based on the nucleotide of interest and the desired effect.

[0248] In some embodiments, the nucleotide of interest can be delivered to enable target cells to produce a protein that compensates for a defect in the organism, such as an enzyme defect or an immune deficiency, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that normally produce the protein in an animal are targeted. In other embodiments, cells located in an area where the protein would be most beneficial are targeted.

[0249] In other embodiments, the target nucleotide (such as a gene encoding an siRNA) can inhibit the expression of a specific gene in the target cell. The target nucleotide can, for example, inhibit the expression of a gene involved in the life cycle of a pathogen. Thus, cells susceptible to or infected by a pathogen can be targeted. In other embodiments, the target nucleotide can inhibit the expression of a gene responsible for producing a toxin in the target cell.

[0250] In other embodiments, the nucleotide of interest may encode a toxic protein that kills the cells in which the nucleotide of interest is expressed. In this case, tumor cells or other unwanted cells may be targeted.

[0251] In other embodiments, the nucleotide of interest encodes a therapeutic protein.

[0252] Once a specific target cell population in which expression of the target nucleotide is desired is identified, a target receptor specifically expressed on the target cell population is selected. The target receptor may be expressed only on this cell population or to a greater extent than other cell populations. The more specific the expression, the more specific the delivery can be directed to the target cell. Depending on the context, the specific amount of the marker (and therefore gene delivery) can vary. For example, in order to introduce a toxic gene, high specificity is most preferred to avoid killing non-targeted cells. In order to express a protein for harvest, or to express a secretory product where a global impact is desired, less marker specificity may be required.

[0253] 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 a binding partner, such as a ligand, is known for the target receptor, it can be used as an affinity molecule. However, if a binding molecule is unknown, antibodies to the target receptor can be generated using standard procedures. The antibody can then be used as a targeting ligand.

[0254] Thus, target cells can be selected based on a variety of factors, including, for example, (1) application (eg, therapy, expression of a protein to be harvested, and conferring disease resistance) and (2) expression of a specific marker in a desired amount.

[0255] The target cell is not limited in any way and includes both germline cells and cell lines and somatic cells and cell lines. When the target cell is a germline cell, the target cell is preferably selected from the group consisting of a single cell embryo and embryonic stem (ES) cells.

[0256] Therapeutic formulations and administration

[0257] Also described herein are pharmaceutical compositions comprising antigen binding molecules as described herein. In some embodiments, pharmaceutical compositions can be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, etc. Numerous suitable formulations can be found in formularies known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0258] The dosage of the antigen binding molecules applied to the patient can vary according to the patient's age and body shape, target disease, illness, route of administration, etc. Preferred dosage is generally calculated according to body weight or body surface area. When the antigen binding molecules as described herein are used for therapeutic purposes in adult patients, it is generally advantageous to administer the antigen binding molecules as described herein intravenously with a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the disease, the frequency and duration of treatment can be adjusted. The effective dose and schedule of the bispecific antigen binding molecules used can be determined empirically; For example, patient progress can be monitored by regular assessment, and dosage can be adjusted accordingly. In addition, methods well known in the art can be used to scale the species of dosage (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

[0259] Various delivery systems are known and can be used to administer the pharmaceutical compositions described herein, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endosomes (see, for example, 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 the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents. Administration can be systemic or local.

[0260] The pharmaceutical compositions described herein can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions described herein. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge becomes empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. In fact, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0261] Many reusable pen delivery devices and autoinjector delivery devices are useful for subcutaneous delivery of the pharmaceutical compositions described herein. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TMPen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), etc. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions described herein include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM pen (Abbott Labs, Abbott Park IL), etc.

[0262] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed near the target of the composition, thus requiring only a small portion of the systemic dose (see, for example, Goodson, 1984, in 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.

[0263] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by well-known methods. For example, the above-mentioned antibody or its salt can be dissolved, suspended or emulsified in a sterile aqueous medium or an oily medium for injection routinely to prepare an injectable preparation. As aqueous media for injection, there are for example normal saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizing agents (such as alcohol (for example, ethanol), polyols (for example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (polyoxyethylene (50mol) adducts of hydrogenated castor oil)], etc.). As oily medium, for example sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizing agents, such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in a suitable ampoule.

[0264] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared into a dosage form suitable for a unit dose that conforms to the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, for other dosage forms, the amount of the aforementioned antibody contained is preferably about 5 to about 100 mg and about 10 to about 250 mg.

[0265] Its therapeutic and diagnostic uses

[0266] 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 an ADC comprising any of the HCVR / LCVR or CDR sequences shown 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.

[0267] Antibodies, antigen-binding fragments thereof, or antibody-drug conjugates comprising anti-hCACNG1 antibodies as described herein are particularly useful for treating, preventing, and / or ameliorating any disease or condition associated with skeletal muscle tissue. For example, antibodies and ADCs as described herein can be used to treat muscle wasting conditions (e.g., cachexia, glucocorticoid-induced muscle loss, heart failure-induced muscle loss, HIV consumption, disuse, aging, etc.) and / or muscular dystrophy / myopathy.

[0268] Anti-hCACNG1 antibodies as described herein have multiple uses. For example, in some embodiments, anti-hCACNG1 antibodies as described herein can be used for diagnostic assays of CACNG1, such as detecting its expression in specific cells, tissues, etc., for example, as reagents for identifying / marking 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 heterogeneous or homogeneous phases (Zola (1987) Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. pp. 147-1581). The antibodies used in the assay can be labeled with a detectable moiety. The detectable moiety should be capable of producing a detectable signal directly or indirectly. Any method known in the art for conjugating an antibody to a detectable moiety can be used.

[0269] In another embodiment, a method for treating a disease such as muscle wasting disorder is provided. The method may include providing the antibody or CACNG1 antigen-binding fragment thereof as described above to a subject in need of such treatment.

[0270] Example 1: Exemplary CACNG1 Antibodies

[0271] Generation of anti-human CACNG1 antibodies

[0272] Anti-human CACNG1 antibodies are obtained by immunizing mice (eg, engineered mice containing DNA encoding human immunoglobulin heavy chain and human kappa light chain variable regions) with human CACNG1.

[0273] After immunization, spleen cells were harvested from each mouse and either (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells that were screened for human CACNG1 specificity, or (2) B cell sorting was performed using human CACNG1 fragments as sorting reagents to bind and identify reactive antibodies (antigen-positive B cells) (as described in US 2007 / 0280945 A1).

[0274] Chimeric antibodies against human CACNG1 having human variable regions and mouse constant regions were initially isolated using, for example, VELOCIMMUNE technology as described in US Pat. No. 7,105,348; US Pat. No. 8,642,835; and US Pat. No. 9,622,459, each of which is incorporated herein by reference.

[0275] In some antibodies, for testing purposes, the mouse constant region is replaced with a desired human constant region, such as a wild-type human CH or a modified human CH (e.g., IgG1, IgG2, or IgG4 isotype) and a light chain constant region (CL) to generate a fully human anti-hCACNG1 antibody or antigen-binding portion thereof. Although the constant region selected can vary depending on the specific application, the high-affinity antigen binding and target specificity characteristics are present in the variable region.

[0276] Certain biological properties of exemplary anti-human CACNG1 antibodies produced according to the methods of this example are described in detail in the Examples shown below.

[0277] Amino acid and nucleic acid sequences of the heavy and light chain variable regions of anti-hCACNG1 antibodies

[0278] Table 1 lists the sequence identifiers of the nucleic acid (NA) sequences encoding the heavy chain or light chain variable regions (HCVR or LCVR, respectively) or heavy chain or light chain CDRs (HCDR and LCDR, respectively) of selected anti-hCACNG1 antibodies used to generate the therapeutic anti-hCACNG1 proteins disclosed herein, as well as the sequence identifiers of the amino acid (AA) sequences in parentheses.

[0279] Table 1: Anti-hCACNG1 sequence identifiers

[0280]

[0281] 31929 / 10728 (wild-type hIgG1) / 14647 (hIgG1 N180Q)

[0282] HCVR nucleic acid sequence (SEQ ID NO: 1)

[0283] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTACAGCGTCTGGAATCACCTTCAGAAATTATGGCATGCACTGGGTCCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTGGTATGATGGAAGTAATAAGTACT ATGCAGACTCCGTGAAGGGCCGTTTCACCATCTCCGGAGACAATTCCAAGGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAAGGGGCACTATAAGAACAGCTGCCCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCCTCA

[0284] HCVR amino acid sequence (SEQ ID NO: 2)

[0285] QVQLVESGGGVVQPGRSLRLSCTASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISGDNSKVYLQMNSLRAEDTAVYYCARRGTIRTAAPFDYWGQGTLVTVSS

[0286] HCDR1 nucleic acid sequence (SEQ ID NO: 3)

[0287] GGA ATC ACC TTC AGA AAT TAT GGC

[0288] HCDR1 amino acid sequence (SEQ ID NO: 4)

[0289] GITFRNYG

[0290] HCDR2 nucleic acid sequence (SEQ ID NO: 5)

[0291] ATG TGG TAT GAT GGA AGT AAT AAG

[0292] HCDR2 amino acid sequence (SEQ ID NO: 6)

[0293] MWYDGSNK

[0294] HCDR3 nucleic acid sequence (SEQ ID NO: 7)

[0295] GCG AGA AGG GGC ACT ATA AGA ACA GCT GCC CCT TTT GAC TAC

[0296] HCDR3 amino acid sequence (SEQ ID NO: 8)

[0297] ARRGTIRTAAPFDY

[0298] LCVR nucleic acid sequence (SEQ ID NO: 9)

[0299] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0300] LCVR amino acid sequence (SEQ ID NO: 10)

[0301] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0302] LCDR1 nucleic acid sequence (SEQ ID NO: 11)

[0303] CAG AGC ATT AGC AGC TAT

[0304] LCDR1 amino acid sequence (SEQ ID NO: 12)

[0305] QSISSY

[0306] LCDR2 nucleic acid sequence (SEQ ID NO: 13)

[0307] GCT GCA TCC

[0308] LCDR2 amino acid sequence (SEQ ID NO: 14)

[0309] AAS

[0310] LCDR3 nucleic acid sequence (SEQ ID NO: 15)

[0311] CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC

[0312] LCDR3 amino acid sequence (SEQ ID NO: 16)

[0313] QQSYSTPPIT

[0314] HC nucleic acid sequence (SEQ ID NO: 193)

[0315]

[0316] HC amino acid sequence (SEQ ID NO: 194)

[0317] QVQLVESGGGVVQPGRSLRLSCTASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISGDNSKVYLQMNSLRAEDTAVYYCARRGTIRTAAPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0318]

[0319] *Underlined and bolded asparagine (N) may be mutated to glutamine (Q) for conjugation of transglutaminase, see e.g. SEQ ID NO: 269

[0320] LC nucleic acid sequence (SEQ ID NO: 195)

[0321] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0322] LC amino acid sequence (SEQ ID NO: 196)

[0323] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0324] 10715 (wild-type hIgG1) / 14570 (IgG1 N180Q):

[0325] HCVR nucleic acid sequence (SEQ ID NO: 17)

[0326] CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGCGACCCTGTCCCGCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAACTGGATCCGCCAGTCCCCAGGGAAGGGGCTGGAATGGATTGGGGAAATCCTTCATAGTGGAAGAACCAACTACA ACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGGGAAGGATAGCAGCTCGTCACGGCTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0327] HCVR amino acid sequence (SEQ ID NO: 18)

[0328] QVQLQQWGAGLLKPSATLSRTCAVYGGSFSGYYWNWIRQSPGKGLEWIGEILHSGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAGRIAARHGWFDPWGQGTLVTVSS

[0329] HCDR1 nucleic acid sequence (SEQ ID NO: 19)

[0330] GGT GGG TCC TTC AGT GGT TAC TAC

[0331] HCDR1 amino acid sequence (SEQ ID NO: 20)

[0332] GGSFSGYY

[0333] HCDR2 nucleic acid sequence (SEQ ID NO: 21)

[0334] ATC CTT CAT AGT GGA AGA ACC

[0335] HCDR2 amino acid sequence (SEQ ID NO: 22)

[0336] ILHSGRT

[0337] HCDR3 nucleic acid sequence (SEQ ID NO: 23)

[0338] GCG GGA AGG ATA GCA GCT CGT CAC GGC TGG TTC GAC CCC

[0339] HCDR3 amino acid sequence (SEQ ID NO: 24)

[0340] A G R I A A R H G W F D P

[0341] LCVR nucleic acid sequence (SEQ ID NO: 25)

[0342] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTACATCTGTAGGAGACAGAGTCACCATCTCTTGTCGGGCGAGTCAGGATATTCGCAAGTGGTTAGCCTGGTATCAACAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTATGCTACATCCAGTTTGCAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATTTTGCAACTTACTTTTGTCAACAGGCTAACAGTTTCCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0343] LCVR amino acid sequence (SEQ ID NO: 26)

[0344] DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIK

[0345] LCDR1 nucleic acid sequence (SEQ ID NO: 27)

[0346] CAG GAT ATT CGC AAG TGG

[0347] LCDR1 amino acid sequence (SEQ IDNO: 28)

[0348] Q D I R K W

[0349] LCDR2 nucleic acid sequence (SEQ ID NO: 29)

[0350] GCT ACA TCC

[0351] LCDR2 amino acid sequence (SEQ ID NO: 30)

[0352] ATS

[0353] LCDR3 nucleic acid sequence (SEQ ID NO: 31)

[0354] CAA CAG GCT AAC AGT TTC CCG TTC ACT

[0355] LCDR3 amino acid sequence (SEQ ID NO: 32)

[0356] QQANSFPFT

[0357] HC nucleic acid sequence (SEQ ID NO: 197)

[0358]

[0359] HC amino acid sequence (SEQ ID NO: 198)

[0360] QVQLQQWGAGLLKPSATLSRTCAVYGGSFSGYYWNWIRQSPGKGLEWIGEILHSGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAGRIAARHGWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0361]

[0362] *Underlined and bolded asparagine (N) may be mutated to glutamine (Q) for conjugation of transglutaminase, see e.g. SEQ ID NO: 269

[0363] LC nucleic acid sequence (SEQ ID NO: 199)

[0364] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTACATCTGTAGGAGACAGAGTCACCATCTCTTGTCGGGCGAGTCAGGATATTCGCAAGTGGTTAGCCTGGTATCAACAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTATGCTACATCCAGTTTGCAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATTTTGCAACTTACTTTTGTCAACAGGCTAACAGTTTCCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0365] LC amino acid sequence (SEQ ID NO:200)

[0366] DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0367] 10717 (wild-type hIgG1) / 14572 (hIgG1 N180Q)

[0368] HCVR Nucleic Acid Sequence (SEQ ID NO: 33)

[0369] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGCATGATGGAAGTGATAAATATTATGTAGACTCCGTGAAGGGCCGATTCTCCATCGCCAGAGACAATTCCAAGAACACGCTTTATCTGCAAATGAATAGTCTGAGAGTCGAGGACACGGGTATATATTACTGTGCGAGAAGGGGTATACGTGGAACCGTTTTTGACCACTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA

[0370] HCVR Amino Acid Sequence (SEQ ID NO: 34)

[0371] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDKYYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS

[0372] HCDR1 Nucleic Acid Sequence (SEQ ID NO: 35)

[0373] GGA TTC ACC TTC AGT ACA TAT GGC

[0374] HCDR1 Amino Acid Sequence (SEQ ID NO: 36)

[0375] G F T F S T Y G

[0376] HCDR2 Nucleic Acid Sequence (SEQ ID NO: 37)

[0377] ATT TGG CAT GAT GGA AGT GAT AAA

[0378] HCDR2 Amino Acid Sequence (SEQ ID NO: 38)

[0379] I W H D G SD K

[0380] HCDR3 nucleic acid sequence (SEQ ID NO: 39)

[0381] GCG AGA AGG GGT ATA CGT GGA ACC GTT TTT GAC CAC

[0382] HCDR3 amino acid sequence (SEQ ID NO: 40)

[0383] ARRGIRGTVFDH

[0384] LCVR nucleic acid sequence (SEQ ID NO:41)

[0385] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATT TAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0386] LCVR amino acid sequence (SEQ ID NO: 42)

[0387] DIQMTQSPSTLSASSVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK

[0388] LCDR1 nucleic acid sequence (SEQ ID NO:43)

[0389] CAG AGT ATT AGT AAC AAG

[0390] LCDR1 amino acid sequence (SEQ ID NO:44)

[0391] QSISNK

[0392] LCDR2 nucleic acid sequence (SEQ ID NO:45)

[0393] AAG GCG TCT

[0394] LCDR2 amino acid sequence (SEQ ID NO:46)

[0395] KAS

[0396] LCDR3 nucleic acid sequence (SEQ ID NO:47)

[0397] CAA CAG TAT AAT AGT TAT TCG TGG ACG

[0398] LCDR3 amino acid sequence (SEQ ID NO: 48)

[0399] QQYNSYSWT

[0400] HC nucleic acid sequence (SEQ ID NO: 201)

[0401]

[0402] HC amino acid sequence (SEQ ID NO: 202)

[0403] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDKYYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0404]

[0405] *Underlined and bolded asparagine (N) may be mutated to glutamine (Q) for conjugation of transglutaminase, see e.g. SEQ ID NO: 269

[0406] LC nucleic acid sequence (SEQ ID NO: 203)

[0407] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0408] LC amino acid sequence (SEQ ID NO: 204)

[0409] DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0410] 10716 (wild-type hIgG1) / 14571 (hIgG1 N180Q)

[0411] HCVR nucleic acid sequence (SEQ ID NO: 49)

[0412] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCAATAATTACTGGACCTGGCTCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGTTATATCTATTACAGTGGGAGCGCCAAC TACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTAAATTCTGTGACCGCTGCGGACACGGGCCGTGTATTACTGTGCGAGAGGGGCGGTCAAGTACTTCCGGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA

[0413] HCVR amino acid sequence (SEQ ID NO: 50)

[0414] QVQLQESGPGLVKPSETLSLTCTVSGDSINNYYWTWLRQPPGKGLEWIGYIYYSGSANYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARGAVKYFRHWGQGTLVTVSS

[0415] HCDR1 nucleic acid sequence (SEQ ID NO: 51)

[0416] GGT GAC TCC ATC AAT AAT TAC TAC

[0417] HCDR1 amino acid sequence (SEQ ID NO: 52)

[0418] GDSINNYY

[0419] HCDR2 nucleic acid sequence (SEQ ID NO: 53)

[0420] ATC TAT TAC AGT GGG AGC GCC

[0421] HCDR2 amino acid sequence (SEQ ID NO: 54)

[0422] IYYSGSA

[0423] HCDR3 nucleic acid sequence (SEQ ID NO: 55)

[0424] GCG AGA GGG GCG GTC AAG TAC TTC CGG CAT

[0425] HCDR3 amino acid sequence (SEQ ID NO: 56)

[0426] ARGAVKYFRH

[0427] LCVR nucleic acid sequence (SEQ ID NO:57)GAAATTGTGTTGACGCAGTCTCCGGGCACCCTCTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTATTAACCACAACAACTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC AACAGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGAAGTGTATTCTTGTCAGCAGTATGGTAGCTTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0428] LCVR amino acid sequence (SEQ ID NO: 58)

[0429] EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEIK

[0430] LCDR1 nucleic acid sequence (SEQ ID NO: 59)

[0431] CAG ACT ATT AAC CAC AAC AAC

[0432] LCDR1 amino acid sequence (SEQ ID NO: 60)

[0433] QTINHNN

[0434] LCDR2 nucleic acid sequence (SEQ ID NO:61)

[0435] GGT GCA TCC

[0436] LCDR2 amino acid sequence (SEQ ID NO: 62)

[0437] GAS

[0438] LCDR3 nucleic acid sequence (SEQ ID NO: 63)

[0439] CAG CAG TAT GGT AGC TTG CCG CTC ACT

[0440] LCDR3 amino acid sequence (SEQ ID NO: 64)

[0441] QQYGSLPLT

[0442] HC nucleic acid sequence (SEQ ID NO: 205)

[0443]

[0444] HC amino acid sequence (SEQ ID NO: 206)

[0445] QVQLQESGPGLVKPSETLSLTCTVSGDSINNYYWTWLRQPPGKGLEWIGYIYYSGSANYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARGAVKYFRHWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0446]

[0447] *Underlined and bolded asparagine (N) may be mutated to glutamine (Q) for conjugation of transglutaminase, see e.g. SEQ ID NO: 269

[0448] LC nucleic acid sequence (SEQ ID NO: 207)

[0449] GAAATTGTGTTGACGCAGTCTCCGGGCACCCTCTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTATTAACCACAACAACTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAACAGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGAAGTGTATTCTTGTCAGCAGTATGGTAGCTTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0450] LC amino acid sequence (SEQ ID NO: 208)

[0451] EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0452] 10783 (wild-type hIgG1) / 14574 (hIgG1 N180Q)

[0453] HCVR nucleic acid sequence (SEQ ID NO: 65)

[0454] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGATTGATGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGGGTATAGTAGTAGCTGCCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0455] [[ID=⑥]]HCVR amino acid sequence (SEQ ID NO: 66)[[ID=⑦]] [[ID=⑧]]

[0456] [[ID=⑨]]QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWIDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGGIVVAAPFDYWGQGTLVTVSS[[ID=⑩]] [[ID=⑪]]

[0457] [[ID=⑫]]HCDR1 nucleic acid sequence (SEQ ID NO: 67)[[ID=⑬]] [[ID=⑭]]

[0458] [[ID=⑮]]GGA TTC ACC TTC AGT AGC TAT GGC[[ID=⑯]] [[ID=⑰]]

[0459] [[ID=⑱]]HCDR1 amino acid sequence (SEQ ID NO: 68)[[ID=⑲]] [[ID=⑳]]

[0460] [[ID=㉑]]G F T F S S Y G[[ID=㉒]] [[ID=㉓]]

[0461] [[ID=㉔]]HCDR2 nucleic acid sequence (SEQ ID NO: 69) [[ID=㉕]] [[ID=㉖]]

[0462] [[ID=㉗]]ATA TGG ATT GAT GGA AGT AAT AAA[[ID=㉘]] [[ID=㉙]]

[0463] [[ID=㉚]]HCDR2 amino acid sequence (SEQ ID NO: (70))[[ID=㉛]] [[ID=㉜]]

[0464] [[ID=㉝]]I W ID G S N K Note: There seems to be a small error in the original text where "SEQ ID NO: (70)" in the translation of line 30 has an extra parenthesis. It should be "SEQ ID NO: 70" as per the pattern in the other sequences. I've translated it as "SEQ ID NO: (70)" to follow your instruction of preserving all content exactly as-is.

[0465] HCDR3 nucleic acid sequence (SEQ ID NO:71)

[0466] GCG AGA AGG GGG GGT ATA GTA GTA GCT GCC CCC TTT GAC TAC

[0467] HCDR3 amino acid sequence (SEQIDNO:72)

[0468] A R R G G I V V A A P F D Y

[0469] LCVR nucleic acid sequence (SEQ ID NO:73)

[0470] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0471] LCVR amino acid sequence (SEQ ID NO:74)

[0472] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0473] LCDR1 nucleic acid sequence (SEQ IDNO:75)

[0474] CAG AGC ATT AGC AGC TAT

[0475] LCDR1 amino acid sequence (SEQIDNO:76)

[0476] Q S I S S Y

[0477] LCDR2 nucleic acid sequence (SEQ ID NO: 77)

[0478] GCT GCA TCC

[0479] LCDR2 amino acid sequence (SEQ ID NO: 78)

[0480] AAS

[0481] LCDR3 nucleic acid sequence (SEQ ID NO: 79)

[0482] CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC

[0483] LCDR3 amino acid sequence (SEQ ID NO:80)

[0484] QQSYSTPPIT

[0485] HC nucleic acid sequence (SEQ ID NO: 209)

[0486] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGATTGATGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGGGTATAGTAGTAGCTGCCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCA

[0487] AGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

[0488] HC amino acid sequence (SEQ ID NO: 210)

[0489] QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWIDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGGIVVAAPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0490]

[0491] *Underlined and bolded asparagine (N) may be mutated to glutamine (Q) for conjugation of transglutaminase, see e.g. SEQ ID NO: 269

[0492] LC nucleic acid sequence (SEQ ID NO: 211)

[0493] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0494] LC amino acid sequence (SEQ ID NO: 212)

[0495] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31944

[0497] HCVR nucleic acid sequence (SEQ ID NO:81)

[0498] CAG GTG CAG TTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCCCTG AGA CTC TCC TGT GAA GCG TCT GGA ATC ACC TTC AGA AAC TAT GGC ATG CAC TGGGTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG GCA GTT ATG TGG TAT GAT GGAAGT AAT AAA TAC TAC GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AATTCC AAG AAC ACG GTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAA GAC ACG GCT GTGTAT TAC TGT GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC TGG GGCCAG GGA ACC CTG GTC ACC GTC TCC TCA

[0499] HCVR amino acid sequence (SEQ ID NO:82)

[0500] QVQLVESGGGVVQPGRSLRLSCEASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARRGHIATAAPFDYWGQGTLVTVSS

[0501] HCDR1 nucleic acid sequence (SEQ ID NO: 83)

[0502] GGA ATC ACC TTC AGA AAC TAT GGC

[0503] HCDR1 amino acid sequence (SEQ ID NO: 84)

[0504] GITFRNYG

[0505] HCDR2 nucleic acid sequence (SEQ ID NO: 85)

[0506] atg tgg tat gat gga agt aat aaa

[0507] HCDR2 amino acid sequence (SEQ ID NO: 86)

[0508] MWYDGSN

[0509] HCDR3 nucleic acid sequence (SEQ ID NO: 87)

[0510] GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC

[0511] HCDR3 amino acid sequence (SEQ ID NO: 88)

[0512] ARRGHIATAAPFD

[0513] LCVR nucleic acid sequence (SEQ ID NO: 89)

[0514] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCCGTAGGAGACAGAGTCACCATCAGTTGCCGGCAAGTCAGAGCATTAGTAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0515] LCVR amino acid sequence (SEQ ID NO: 90)

[0516] DIQMTQSPSSSLSASVGDRVTISCRASQSISSYLNWYQQKPGKAPKVLMYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0517] LCDR1 nucleic acid sequence (SEQ ID NO:91)

[0518] CAG AGC ATT AGT AGT TAT

[0519] LCDR1 amino acid sequence (SEQ ID NO:92)

[0520] QSISSY

[0521] LCDR2 nucleic acid sequence (SEQ ID NO:93)

[0522] GCT GCA TCC

[0523] LCDR2 amino acid sequence (SEQ ID NO:94)

[0524] AAS

[0525] LCDR3 nucleic acid sequence (SEQ ID NO:95)

[0526] CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC

[0527] LCDR3 amino acid sequence (SEQ ID NO:96)

[0528] QQSYSTPPIT

[0529] HC nucleic acid sequence (SEQ ID NO: 213)

[0530] CAG GTG CAG TTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCCCTG AGA CTC TCC TGT GAA GCG TCT GGA ATC ACC TTC AGA AAC TAT GGC ATG CAC TGGGTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG GCA GTT ATG TGG TAT GAT GGAAGT AAT AAA TAC TAC GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AATTCC AAG AAC ACG GTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAA GAC ACG GCT GTGTAT TAC TGT GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC TGG GGCCAG GGA ACC CTG GTC ACC GTC TCC TCA

[0531] GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGA

[0532] HC amino acid sequence (SEQ ID NO: 214)

[0533] QVQLVESGGG VVQPGRSLRL SCEASGITFR NYGMHWVRQA PGKGLEWVAVMWYDGSNKYYADSVKGRFTI SRDNSKNTVY LQMNSLRAED TAVYYCARRGHIATAAPFDY WGQGTLVTVS S

[0534] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0535] LC nucleic acid sequence (SEQ ID NO: 215)

[0536] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCCGTAGGAGACAGAGTCACCATCAGTTGCCGGGCAAGTCAGAGCATTAGTAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA

[0537] LC amino acid sequence (SEQ ID NO: 216)

[0538] DIQMTQSPSS LSASVGDRVT ISCRASQSIS SYLNWYQQKP GKAPKVLMYAASSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPPITFG QGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC

[0539] 31265 (wild-type hIgG1) / 5972 (hIgG1 N180Q)

[0540] HCVR nucleic acid sequence (SEQ ID NO:97)

[0541] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTACAGCGTCTGGATTCACCTTCCGTTCCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGTCAGTTATTTGGATTGATGGAAATAATATATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAAGACTGGCTATAACATCAGCTGCCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0542] HCVR amino acid sequence (SEQ ID NO:98)

[0543] QVQLVESGGGVVQPGRSLRLSCTASGFTFRSYGMHWVRQAPGKGLEWVSVIWIDGNNIYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARRLAITSAAPFDYWGQGTLVTVSS

[0544] HCDR1 nucleic acid sequence (SEQ ID NO:99)

[0545] GGA TTC ACC TTC CGT TCC TAT GGC

[0546] HCDR1 amino acid sequence (SEQ ID NO:100)

[0547] G F T F R S Y G

[0548] HCDR2 nucleic acid sequence (SEQ ID NO:101)

[0549] ATT TGG ATT GAT GGA AAT AAT ATA

[0550] HCDR2 amino acid sequence (SEQ ID NO:102)

[0551] IW ID GNNI

[0552] HCDR3 nucleic acid sequence (SEQ ID NO: 103)

[0553] GCG AGA AGA CTG GCT ATA ACA TCA GCT GCC CCC TTT GAC TAC

[0554] HCDR3 amino acid sequence (SEQ ID NO: 104)

[0555] ARRLAITSAAPFDY

[0556] LCVR nucleic acid sequence (SEQ ID NO: 105)

[0557] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0558] LCVR amino acid sequence (SEQ ID NO: 106)

[0559] DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0560] LCDR1 nucleic acid sequence (SEQ ID NO: 107)

[0561] CAG AGC ATT AGC AGC TAT

[0562] LCDR1 amino acid sequence (SEQ ID NO: 108)

[0563] QSISSY

[0564] LCDR2 nucleic acid sequence (SEQ ID NO: 109)

[0565] GCT GCA TCC

[0566] LCDR2 amino acid sequence (SEQ ID NO: 110)

[0567] AAS

[0568] LCDR3 nucleic acid sequence (SEQ ID NO: 111)

[0569] CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC

[0570] LCDR3 amino acid sequence (SEQ ID NO: 112)

[0571] QQSYSTPPIT

[0572]

[0573] HC amino acid sequence (SEQ ID NO: 218)

[0574] QVQLVESGGGVVQPGRSLRLSCTASGFTFRSYGMHWVRQAPGKGLEWVSVIWIDGNNIYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARRLAITSAAPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

[0575]

[0576]

[0577] *Underlined and bolded asparagine (N) may be mutated to glutamine (Q) for conjugation of transglutaminase, see e.g. SEQ ID NO: 269

[0578] LC nucleic acid sequence (SEQ ID NO: 219)

[0579] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0580] LC amino acid sequence (SEQ ID NO: 220)

[0581] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31941

[0583] HCVR nucleic acid sequence (SEQ ID NO: 113)

[0584] CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACGCCTTCACCACCTATGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGATGGATCAGCGCTTACAATGGAAATACAAACTATGCAGAGAAG GTCCAGGGGCAGATTCACCATGACCACAGACACATCCACGAATACAGCCTACATGGAGCTGAGGAGCCTGAGATCCGACGACACGGCCGTGTATTTCTGTGCGAGAAAGGGTCACTATGGTTCGGGGACTTATTATAACCCCTTTGGTTTTGATTTTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

[0585] HCVR amino acid sequence (SEQ ID NO: 114)

[0586] QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSS

[0587] HCDR1 nucleic acid sequence (SEQ ID NO: 115)

[0588] ggt tac gcc ttc acc acc tat ggt

[0589] HCDR1 amino acid sequence (SEQ ID NO: 116)

[0590] GYAFTTYG

[0591] HCDR2 nucleic acid sequence (SEQ ID NO: 117)

[0592] atc agc gct tac aat gga aat aca

[0593] HCDR2 amino acid sequence (SEQ ID NO: 118)

[0594] ISAYNGN

[0595] HCDR3 nucleic acid sequence (SEQ ID NO: 119)

[0596] GCG AGA AAG GGT CAC TAT GGT TCG GGG ACT TAT TAT AAC CCC TTT GGT TTTGAT TTT

[0597] HCDR3 amino acid sequence (SEQ ID NO: 120)

[0598] CARKGHYGSGTYYNPFGFD

[0599] LCVR nucleic acid sequence (SEQ ID NO: 121)

[0600] GAAATTATGTTGATGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGACATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAG

[0601] LCVR amino acid sequence (SEQ ID NO: 122)

[0602] EIMLMQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYYGSPWTFGQGTKVEIK

[0603] LCDR1 nucleic acid sequence (SEQ ID NO: 123)

[0604] cag agt gtt agc agc agc tac

[0605] LCDR1 amino acid sequence (SEQ ID NO: 124)

[0606] QSVSSSY

[0607] LCDR2 nucleic acid sequence (SEQ ID NO: 125)

[0608] ggt gca tcc

[0609] LCDR2 amino acid sequence (SEQ ID NO: 126)

[0610] GA

[0611] LCDR3 nucleic acid sequence (SEQ ID NO: 127)

[0612] cag cag tat tat ggc tca cct tgg acg

[0613] LCDR3 amino acid sequence (SEQ ID NO: 128)

[0614] CQQYYGSPW

[0615] HC nucleic acid sequence (SEQ ID NO: 221)

[0616]

[0617] HC amino acid sequence (SEQ ID NO:222)

[0618] QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0619] LC nucleic acid sequence (SEQ ID NO:223)

[0620] GAAATTATGTTGATGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGACATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAGCGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA

[0621] LC amino acid sequence (SEQ ID NO: 224)

[0622] EIMLMQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIYGASSRATDIPDRFSGSGSGT DFTLTISRLE PEDFAVYFCQ QYYGSPWTFG QGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC 7660 <第1633号>[0001633]<第1634号>[0001634]HCVR nucleic acid sequence (SEQ ID NO: 129)

[0625] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAAACTCTCCTGTACAGCCTCTGGGTTGACCCTCAGTGACTCTGCTATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTTGGCCGTATAAGAAATAAGGCTAATAGGTACGCGACAGAATATGCTGCGTCGGTGAAAGGCAGGTTCACCATTTCAAGAGATGATTCAAAGAACACGGCGTATCTACAAATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTATTGTACTAGAAACTGGAAGATTTTCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0626] HCVR amino acid sequence (SEQ ID NO: 130)

[0627] EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWGQGTLVTVSS

[0628] HCDR1 nucleic acid sequence (SEQ ID NO: 131)

[0629] GGG TTG ACC CTC AGT GAC TCT GCT

[0630] HCDR1 amino acid sequence (SEQ ID NO: 132)

[0631] G L T L S D S A

[0632] HCDR2 nucleic acid sequence (SEQ ID NO: 133)

[0633] ATA AGA AAT AAG GCT AAT AGG TAC GCG ACA

[0634] HCDR2 amino acid sequence (SEQ ID NO: 134)

[0635] IRNKANRYAT

[0636] HCDR3 nucleic acid sequence (SEQ ID NO: 135)

[0637] ACT AGA AAC TGG AAG ATT TTC CTC TTT GAC TAC

[0638] HCDR3 amino acid sequence (SEQ ID NO: 136)

[0639] TRNWKIFLFDY

[0640] LCVR nucleic acid sequence (SEQ ID NO: 137)

[0641] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGACTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCAAATACTTAGCCTGGTTCCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGACCAGTGGCATCCCCGACAGGATCAGTGGCAGTGGGTCAGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGAAGTTCACCCTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0642] LCVR amino acid sequence (SEQ ID NO: 138)

[0643] EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0644] LCDR1 nucleic acid sequence (SEQ ID NO: 139)

[0645] CAG AGT GTT GGC AGC AAA TAC

[0646] LCDR1 amino acid sequence (SEQ ID NO: 140)

[0647] QSVGS KY

[0648] LCDR2 nucleic acid sequence (SEQ ID NO: 141)

[0649] GGT GCA TCC

[0650] LCDR2 amino acid sequence (SEQ ID NO: 142)

[0651] GAS

[0652] LCDR3 nucleic acid sequence (SEQ ID NO: 143)

[0653] CAG CAG TAT GGA AGT TCA CCC TGG ACG

[0654] LCDR3 amino acid sequence (SEQ ID NO: 144)

[0655] QQYGSSPWT

[0656] HC nucleic acid sequence (SEQ ID NO: 225)

[0657]

[0658] HC amino acid sequence (SEQ ID NO: 226)

[0659] EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0660] LC nucleic acid sequence (SEQ ID NO: 227)

[0661] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGACTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCAAATACTTAGCCTGGTTCCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGACCAGTGGCATCCCCGACAGGATCAGTGGCAGTGGGTCAGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGAAGTTCACCCTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0662] LC amino acid sequence (SEQ ID NO: 228)

[0663] EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 9909

[0665] HCVR nucleic acid sequence (SEQ ID NO:145)

[0666] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAACAACTATGGCATGAGCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTGGTAGTGGTGGTACCACATTCTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGGCAAAGGAGGATATTGTAGTAGTAGCGGCTGCCGTCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0667] HCVR amino acid sequence (SEQ ID NO:146)

[0668] EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMDVWGQGTTVTVSS

[0669] HCDR1 nucleic acid sequence (SEQ ID NO:147)

[0670] GGA TTC ACC TTT AAC AAC TAT GGC

[0671] HCDR1 amino acid sequence (SEQ ID NO:148)

[0672] GFTFNNYG

[0673] HCDR2 nucleic acid sequence (SEQ ID NO:149)

[0674] ATT AGT GGT AGT GGT GGT ACC ACA

[0675] HCDR2 amino acid sequence (SEQ ID NO:150)

[0676] SGSGGT

[0677] HCDR3 nucleic acid sequence (SEQ ID NO: 151)

[0678] GGC AAA GGA GGA TAT TGT AGT AGT AGC GGC TGC CGT CAC TAC GGT ATG GACGTC

[0679] HCDR3 amino acid sequence (SEQ ID NO: 152)

[0680] CGKGGYCSSSGCRH

[0681] LCVR nucleic acid sequence (SEQ ID NO: 153)

[0682] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCCTTGTTCTGGAAGCAGCTCCAACATCGGAAATAATTATATACTGGTACCAGCGGCTCCCAGGAACGACCCCCAAACTCCTCATCTATAGGAATAATCAGCGG CCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACACCCTGAGTGGGTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA

[0683] LCVR amino acid sequence (SEQ ID NO: 154)

[0684] QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYIYWYQRLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDTLSGYVFGTGTKVTVL

[0685] LCDR1 nucleic acid sequence (SEQ ID NO: 155)

[0686] AGC TCC AAC ATC GGA AAT AAT TAT

[0687] LCDR1 amino acid sequence (SEQ ID NO: 156)

[0688] SSNIGNNY

[0689] LCDR2 nucleic acid sequence (SEQ ID NO: 157)

[0690] agg aat aat

[0691] LCDR2 amino acid sequence (SEQ ID NO: 158)

[0692] RN

[0693] LCDR3 nucleic acid sequence (SEQ ID NO: 159)

[0694] GCA GCA TGG GAT GAC ACC CTG AGT GGG TAT GTC

[0695] LCDR3 amino acid sequence (SEQ ID NO: 160)

[0696] CAAWDDTLSGY

[0697] HC nucleic acid sequence (SEQ ID NO: 229)

[0698]

[0699] HC amino acid sequence (SEQ ID NO: 230)

[0700] EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMDVWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0701] LC nucleic acid sequence (SEQ ID NO: 231)

[0702] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAATAATTATATATACTGGTACCAGCGGCTCCCAGGAACGACCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACACCCTGAGTGGGTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA

[0703] LC amino acid seque...

Claims

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-gated channel auxiliary subunit gamma 1 (hCACNG1), wherein the antigen binding protein comprises an anti-hCACNG1 antibody or an antigen binding fragment thereof, wherein the anti-hCACNG1 antibody or antigen binding fragment thereof comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from the group consisting of SEQ ID NO: 4-6-8-12-14-16, SEQ ID NO: 20-22-24-28-30-32, SEQ ID NO: 36-38-40-44-46-48, SEQ ID NO: 52-54-56-60-62-64, SEQ ID NO: 68-70-72-76-78-80, SEQ ID NO: 84-86-88-92-94-96, SEQ ID NO: NO:100-102-104-108-110-112, SEQ ID NO:116-118-120-124-126-128, SEQ ID NO:132-134-136-140-142-144, SEQ ID NO:148-150-152-156-158-160, SEQ ID NO:164-166-168-172-174-176 and SEQ ID NO:180-182-186-188-190-192, and (ii) a target nucleotide sequence comprising a sequence encoding a microdystrophin protein, a fukutin-related protein (FKRP), or a myotubularin protein (MTM1), wherein the target nucleotide is encapsidated by an AAV capsid, and the AAV capsid comprises the AAV capsid protein modified with the antigen binding protein that specifically binds to 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, and the second member of the protein:protein binding pair comprises the anti-hCACNG1 antibody or antigen-binding fragment, and wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair associate together to direct the tropism of the viral particle to 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. 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 divalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a dual scFv, (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 a binding fragment thereof, a bispecific T cell engager (BiTE), a trispecific antibody, and chemically modified derivatives thereof. 4 . The recombinant AAV particle of claim 1 , 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 from N-terminus to C-terminus in the following orientation: HCVR-LCVR.

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

8. The recombinant AAV particle of 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); and wherein n is 1-10.

11. The recombinant AAV particle of any one of claims 1 to 10, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof is present at a concentration of about 1×10 -7 M's K D or stronger affinity to hCACNG1.

12. The recombinant AAV particle of any one of claims 1 to 11, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof is present at a concentration of about 10×10 -8 to about 1×10 -10 K D Binds to hCACNG1.

13. The recombinant AAV particle of any one of claims 1 to 11, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof is present at a concentration of about 5×10 -9 to about 1×10 -10 K D Binds to hCACNG1.

14. The recombinant AAV particle of any one of claims 1 to 13, wherein the anti-hCACNG1 antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair having at least 90% sequence identity to a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 2 / 10, SEQ ID NO: 18 / 26, SEQ ID NO: 34 / 42, SEQ ID NO: 50 / 58, SEQ ID NO: 66 / 74, SEQ ID NO: 82 / 90, SEQ ID NO: 98 / 106, SEQ ID NO: 114 / 122, SEQ ID NO: 130 / 138, SEQ ID NO: 146 / 154, SEQ ID NO: 162 / 170, and SEQ ID NO: 178 / 186.

15. The recombinant AAV particle of any one of claims 1 to 14, wherein: (a) the protein: the first member of the protein binding pair comprises SpyTag, Isopeptag, SnoopTag, SpyTag002, SpyTag003, or any biologically equivalent portion or variant thereof, (b) the second member of the protein:protein binding pair comprises: (i) SpyCatcher, KTag, pilin-C, SnoopCatcher, SpyCatcher002, SpyCatcher003, or any biologically equivalent portion or variant thereof, and (ii) the anti-hCACNG1 antibody or antigen-binding fragment thereof, and (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. The recombinant AAV particle of claim 15, wherein: (a) the first member of the protein:protein binding pair comprises a SpyTag or any biologically equivalent portion or variant thereof, and (b) The second member of the protein:protein binding pair comprises SpyCatcher, or any biologically equivalent portion or variant thereof, fused to the anti-hCACNG1 antibody or antigen-binding fragment thereof.

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

18. The recombinant AAV particle of claim 17, wherein the first linker and the second linker are different.

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

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

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

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 produces 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, the non-primate AAVs listed in Table 2, and any chimeras thereof.

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

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 an insertion at amino acid position 1-453 and / or 1-587 and optionally linked via a linker on one or both sides to a first member of a protein:protein binding pair of an AAV sequence.

27. The recombinant AAV particle of claim 26, wherein 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, Optionally, the modified AAV2 VP1 capsid protein further comprises a mutation selected from the group consisting of R585A, R588A, R484A, R487A, K532A, and any combination thereof.

28. The recombinant AAV particle or composition of claim 26 or claim 27, wherein the recombinant AAV particle comprises a mosaic AAV capsid comprising a second set of AAV2 VP1 capsid proteins lacking the first member of the protein:protein binding pair, Optionally, wherein the second set of AAV2 VP1 capsid proteins comprises a mutation selected from the group consisting of R585A, R588A, R484A, R487A, K532A, and any combination thereof.

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

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 particle of claim 30, wherein the recombinant AAV particle comprises 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 the group consisting of N272A, W503A, and combinations thereof.

32. The recombinant AAV particle of claim 30 or claim 31 , wherein the recombinant AAV particle is a mosaic viral capsid comprising a second set of AAV9 VP1 capsid proteins lacking the first member of the protein:protein binding pair, Optionally, wherein the second set of AAV9 VP1 capsid proteins comprises a mutation selected from the group consisting of N272A, W503A, and combinations thereof.

33. The recombinant AAV particle of 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. 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 squamates AAV is a bearded dragon AAV.

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

42. The recombinant AAV particle of any one of claims 1 to 41, wherein the recombinant AAV particle comprises a mosaic AAV capsid, optionally wherein the mosaic AAV capsid comprises (i) a first plurality of reference capsid proteins, each reference capsid protein in the first plurality of reference capsid proteins not associated with the anti-hCACNG1 antibody or antigen-binding fragment thereof, and (ii) a second plurality of capsid proteins, each capsid protein in the second plurality of capsid proteins associated with the anti-hCACNG1 antibody or antigen-binding fragment thereof, optionally wherein 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 nucleotide of interest encodes micro-dystrophin.

44. The recombinant AAV particle of any one of claims 1 to 43, wherein the nucleotide of interest comprises the sequence shown in SEQ ID NO:

270.

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

46. ​​The recombinant AAV particle of any one of claims 1 to 42 and 45, wherein the nucleotide of interest comprises the sequence shown in SEQ ID NO:

271.

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

48. The recombinant AAV particle of any one of claims 1 to 42 and 47, wherein the nucleotide of interest comprises the sequence shown in SEQ ID NO:

272.

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

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

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

52. A method of treating muscle wasting or a genetic muscle disease in a subject in need thereof, the method comprising: The subject is administered the recombinant AAV particles of any one of claims 1 to 42, optionally at a dose greater than 3×10 13 The dose of vg / kg, The target nucleotide encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule or a shRNA molecule.

53. The recombinant AAV particle of any one of claims 1 to 42 is optionally present in a volume greater than 3×10 13 vg / kg (e.g., 2×10 14 ) in the preparation of a medicament for administration to a subject to treat muscle atrophy or genetic muscle disease.

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

55. The method or use of any one of claims 50 to 54, wherein the amount of 13 vg / kg (e.g., 2×10 14 ) to the subject does not result in: (i) the level of a liver enzyme (e.g., ALT) is significantly increased 1, 3, 5, 7, 15, and / or 21 days after administration, compared to the corresponding level of the liver enzyme (e.g., ALT) in the subject before the administration, (ii) the level of one or more complement components (e.g., Bb, C3a, sC5b-9) in the subject is significantly increased 1, 3, 5, 7, 15, and / or 21 days after administration, as compared to the corresponding level of one or more complement components (e.g., Bb, C3a, sC5b-9) in the subject before administration, (iii) the platelet count is significantly reduced at a level 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 before said administration, (v) the level of serum creatinine is significantly increased 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)-(v).

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

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

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