Anti-human CACNG1 antibody-drug conjugates and uses thereof

Abstract

Provided herein are antigen binding protein-drug conjugates and compositions thereof that are useful, for example, for target-specific delivery of therapeutic moieties, such as testosterone analogs and / or derivatives. Dihydrotestosterone analogs, antibody-drug conjugates, and compositions comprising glutaminyl modified antibodies and dihydrotestosterone analogs are provided.

Description

Technical Field

[0001] This application relates to conjugates comprising an anti-hCACNG1 antibody (or an antigen-binding molecule comprising an antigen-binding fragment of an anti-hCACNG1 antibody) and a therapeutic agent, said conjugates being used to treat a disease. This application also relates to methods for preparing and using conjugates comprising an anti-hCACNG1 antibody (or an antigen-binding molecule comprising an antigen-binding fragment of an anti-hCACNG1 antibody) and a therapeutic agent. sequence list

[0002] The XML-formatted sequence list titled “250298_000513_SL.xml”, created on October 31, 2023, and being 337,817 bytes, is incorporated herein by reference in its entirety. background

[0003] Skeletal muscle is the largest organ in the body, accounting for approximately 40% of total body weight. It is one of the three major types of muscle tissue in the human body. Each skeletal muscle consists of thousands of muscle fibers wrapped together by connective tissue sheaths. Individual bundles of muscle fibers in skeletal muscle are called fiber bundles. 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 each individual muscle fiber is called the endomysium. Each muscle fiber contains numerous myofibrils containing multiple myofilaments.

[0004] When bundled together, all the myofibrils arrange themselves in a unique striped pattern to form sarcomeres, which are the basic contractile units of skeletal muscle. The two most important myofibrils are actin and myosin filaments, which are uniquely arranged to form various bands on skeletal muscle.

[0005] The primary functions of skeletal muscle occur through its inherent excitation-contraction coupling process. When a muscle attaches to a skeletal tendon, the contraction of the muscle causes movement of that bone, thus allowing specific movements to occur. Skeletal muscle also provides structural support and helps maintain body posture. It also serves as a storage source of amino acids that can be used by different organs in the body to synthesize organ-specific proteins. Skeletal muscle also acts as the site of glucose metabolism in the form of muscle glycogen. Furthermore, skeletal muscle plays a central role in maintaining homeostasis and serves as an energy source during periods of starvation. Therefore, skeletal muscle plays a crucial role in movement, thermoregulation, and the control of systemic metabolism.

[0006] In many muscle diseases and during normal aging, the size and function of skeletal muscle tissue decrease, leading to impaired functional activity; and in cases of severe muscle diseases, it can result in long-term disability and premature death.

[0007] Treatment for muscle atrophy and hereditary muscle diseases typically consists of broad-spectrum therapies, such as testosterone therapy for muscle atrophy and glucocorticoids for muscle dystrophy. The non-targeted delivery of these therapies reduces the efficiency of uptake of specific muscles while also causing significant and harmful off-target effects on other organs.

[0008] There is a need in the field for novel anti-human antibodies and antibody-drug conjugate therapeutics that can bind to muscle-specific markers and enable muscle cells to internalize the therapeutic payload. Overview

[0009] This document describes antibody-drug conjugates comprising an antibody that binds to human CACNG1 or an antigen-binding fragment thereof. This document describes antibody-drug conjugates comprising an anti-hCACNG1 antibody as described herein, its antigen-binding fragment, and a payload (e.g., a therapeutic payload, such as a therapeutic small molecule). In some embodiments, (a) the anti-hCACNG1 antibody, the antigen-binding fragment, and / or the multispecific binding protein and (b) the payload are covalently attached via a linker, as discussed herein. In various embodiments, the anti-hCACNG1 antibody or antigen-binding fragment can be any of the anti-CACNG1 antibodies or fragments described herein.

[0010] In one aspect, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprising an antigen-binding protein that specifically binds to the human calcium voltage-gated channel helper subunit γ1 (hCACNG1), wherein the antigen-binding protein is conjugated directly or via a linker to at least one therapeutic agent.

[0011] In one embodiment, the antibody-drug conjugate has a structure according to formula (I): A – [L – P] y (I), Where A is an antigen-binding protein; L is absent or is a connector; P is a therapeutic agent, and y is an integer from 1 to 8.

[0012] In one embodiment, the anti-hCACNG1 antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR or VH) and / or a light chain variable region (LCVR or VL).

[0013] In one embodiment, the anti-hCACNG1 antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR or VH) and / or a light chain variable region (LCVR or VL).

[0014] In one embodiment, the anti-hCACNG1 antibody or its antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair having at least 90% sequence identity with an 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, SEQ ID NO: 178 / 186, SEQ ID NO: 294 / 301, and SEQ ID NO: 314 / 322.

[0015] In one embodiment, the therapeutic agent is dihydrotestosterone (DHT) or a prodrug or derivative thereof. In one embodiment, the therapeutic agent is selected from the group consisting of: having a structure DHT and with selectivity and The structure of the group composed of proDHT, R1 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R2 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R3 is selected from the following groups: H and C 1-3 Alkyl, and R4 is selected from the following groups: H and C 1-3 alkyl.

[0016] In one embodiment, the therapeutic agent is conjugated to an antigen-binding protein via a linker L.

[0017] In one implementation, n is 2 or 4.

[0018] In one embodiment, the connector has the following structure: -L1-B-L2-, where: L1 is the first linker unit that is covalently attached to the antigen-binding protein; B is a unit that either does not exist or is an adduct containing at least one group B', wherein the group B' is selected from -N3, , , and Where Q is C or N; L2 is absent or is a second linker unit covalently attached to unit B via at least one group B”, wherein group B’ and group B” form at least one adduct. The condition is that when B is absent, L2 is also absent and L1 is covalently attached to the therapeutic agent, and when L2 is present, L2 is covalently attached to the therapeutic agent.

[0019] In one implementation, L1 includes C 1-6 Alkyl, phenyl, -NH-, -C(O)-, -(CH2) u -NH-C(O)-、-(CH2) u -C(O)-NH-、-(CH2-CH2-O) v -、-(CH2) u -(O-CH2-CH2) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof; each of which may optionally be substituted by one or more of -S-, -S(O2)-, -C(O)-, -C(O2)- and CO2H; wherein the subscripts u and v are independent integers from 1 to 8.

[0020] In one implementation, L1 is .

[0021] In one implementation, B has a structure selected from the group consisting of: , , , and , where Q is C or N.

[0022] In one implementation, L2 has the following structure: -SP1-AA-SP2-(L2), where: SP1 is absent or is the first spacer unit; AA either does not exist or contains peptide units of 2 to 4 amino acids; SP2 either lacks a second spacer unit or is covalently attached to the therapeutic agent.

[0023] In one implementation, SP1 is absent or selected from the group consisting of: , , , , C 1-6 Alkyl group, -(CH2-CH2-O) v -、-(CH2-CH2-O) v -(CH2) u -、-(CH2-CH2-O) v -(CH2) u -C(O), -O-CH2-C(O)-NH, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -C(O)-NH, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -、-NH-(CH2) u -C(O)-、-NH-(CH2-CH2-O) v -、-NH-(CH2-CH2-O) v -C(O)-、-NH-(CH2-CH2-O) v -(CH2) u -、-NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof; where the subscripts u and v are independent integers from 1 to 8.

[0024] In one embodiment, the amino acid is a peptide unit comprising two to four amino acids selected from the group consisting of glycine, valine, phenylalanine, proline, glutamic acid, lysine, N,N-dipropyllysine, phenylalanine, and citrulline, and combinations thereof.

[0025] In one embodiment, AA is valine-citrulline, valine-alanine, valine-lysine, valine-N,N-dipropyllysine, phenylalanine-lysine, glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO:289)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO:290)), L-glutamic acid-valine-citrulline (… L EVC) and D-glutamic acid-valine-citrulline ( D EVC).

[0026] In one implementation, SP2 is absent or selected from the group consisting of: , , , and their combinations.

[0027] In one embodiment, the connector-therapeutic agent (LP) has a structure selected from the group consisting of:

[0028] In one embodiment, the linker attaches to the side chain of a glutamine residue of an antigen-binding protein.

[0029] In one embodiment, glutamine residues are naturally present in the CH2 or CH3 domain of the antigen-binding protein.

[0030] In one embodiment, glutamine residues are introduced into the antigen-binding protein by modifying one or more amino acids.

[0031] In one embodiment, the antibody-drug conjugate has a structure selected from the group consisting of:

[0032] Where Ab is the antigen-binding protein, and n is an integer from 1 to 4.

[0033] In another aspect, this disclosure provides compounds according to formula (L2-P) or (L2'-P): B”-SP1-AA-SP2-P (L2-P), H2N-SP1-AA-SP2-P) p (L2'-P), Or its pharmaceutically acceptable salt, wherein: B" is selected from the following groups: -N3, , , , and ; SP1 either does not exist or is selected as the first spacer unit of the following group: and C 1-6 Alkyl group, -(CH2-CH2-O) v -、-(CH2-CH2-O) v -(CH2) u -、-(CH2-CH2-O) v -(CH2) u -C(O), -O-CH2-C(O)-NH, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -C(O)-NH, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -、-NH-(CH2) u -C(O)-、-NH-(CH2-CH2-O) v -、-NH-(CH2-CH2-O) v -C(O)-、-NH-(CH2-CH2-O) v -(CH2) u -、-NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof; where the subscripts u and v are independent integers from 1 to 8; AA either does not exist or contains peptide units of 2 to 4 amino acids; SP2 either does not exist or is selected from the following groups of second spacer units: , , , , and their combinations, and P is a therapeutic agent selected from the group consisting of: having a structure DHT and with selectivity and The structure of the proDHT group is composed of groups, where R1 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R2 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R3 is selected from the following groups: H and C 1-3 Alkyl, and R4 is selected from the following groups: H and C 1-3 alkyl.

[0034] In one embodiment, the compound has a structure selected from the group consisting of:

[0035] On another aspect, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprising an antigen-binding protein that specifically binds to the human calcium voltage-gated channel helper subunit γ1 (hCACNG1), the antigen-binding protein conjugating to a linker-payload selected from the group consisting of: , and .

[0036] In another aspect, this disclosure provides a composition comprising a group of antibody-drug conjugates having a drug-antibody ratio (DAR) of about 0.5 to about 8.0 in any of the above embodiments.

[0037] In one embodiment, the composition has a DAR of about 1.0 to about 2.5.

[0038] In one embodiment, the composition has a DAR of about 2.0.

[0039] In one embodiment, the composition has a DAR of about 3.0 to about 4.5.

[0040] In one embodiment, the composition has a DAR of about 4.0.

[0041] In one embodiment, this disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate and a diluent, carrier, and / or excipient of any of the foregoing embodiments.

[0042] In another respect, this disclosure provides a pharmaceutical dosage form comprising an antibody-drug conjugate of any of the above embodiments or a pharmaceutical composition of any of the above embodiments.

[0043] In another aspect, this disclosure provides a method for treating a condition in a subject with a corresponding need, wherein the method includes administering to the subject an antibody-drug conjugate of any of the above embodiments, a pharmaceutical composition of any of the above embodiments, or a pharmaceutical dosage form of any of the above embodiments.

[0044] In one implementation, the condition is selected from the group consisting of: muscle atrophy and hereditary muscle diseases.

[0045] In one implementation, the condition is selected from the group consisting of: adult spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), anoctaminopathy, autoimmune neuropathy, Becker muscular dystrophy, Bethlem myopathy, calcium dystrophy, caveolinopathy, central core disease, Charcot-Marie-Tooth disease (CMT), congenital fiber type disproportion (CFTD), congenital muscular dystrophy, congenital myasthenia gravis (CMS), congenital myopathy, congenital myotonic dystrophy, dermatomyositis (DM), neuromuscular junction disorders, distalmyopathy with rimmed vacuoles (DMRV), and Duchenne muscular dystrophy. Muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy, facioscapuloperoneal muscular dystrophy (FSHD), and facioscapuloperoneal muscular dystrophy...FSPD, fibrodysplasia ossificans progressiva (FOP), glycogen storage disease type V (GSD V), GNE myopathy (GNEM), hereditary inclusion body myopathy (HIBM), hereditary inclusion body myopathy type 2 (HIBM2), hereditary motor and sensory neuropathy (HMSN), hereditary neuropathies, inclusion body myositis (IBM), infantile progressive spinal muscular atrophy, inflammatory neuropathies, infectious myelitis, intermediate spinal muscular atrophy, juvenile dermatomyositis, juvenile spinal muscular atrophy, Landouzy-Dejerine syndrome, limb-girdle muscular dystrophy (LGMD), McCardell disease, merosin-deficient congenital muscular dystrophy, metabolic myopathy, minicore myopathy, multiaxial void myopathy. Myopathy, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myofibril myopathy, myositis, progressive ossifying myositis (MOP), myotonic dystrophy, myotubular and other centronuclear myopathy, nemaline myopathy, nonaka myopathy, oculopharyngeal muscular dystrophy (OPMD), periodic paralysis, polymyositis (PM) and dermatomyositis (DM), quadriceps-sparing myopathy (QSM), sarcoglycanopathy, sarcopenia, SEPN1-related myopathy, spinal muscular atrophy (SMA), Steinert's disease, Ullrich congenital muscular dystrophy, VCP disease, and age-related progressive loss of muscle mass and strength.

[0046] In one implementation, the subject is an adult. In another implementation, the subject is over 65 years of age.

[0047] In one implementation, the subjects were under 18 years of age.

[0048] In another aspect, this disclosure provides a method for selectively delivering a compound into cells, wherein the compound is an antibody-drug conjugate of any of the above embodiments.

[0049] In another aspect, this disclosure provides a method for selectively targeting antigens on the cell surface with a compound, wherein the compound is any of the embodiments described above.

[0050] In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a muscle cell.

[0051] In one implementation, the antibody-drug conjugate is administered intravenously or subcutaneously.

[0052] In another aspect, this disclosure provides a method for internalizing a compound into muscle fibers, the method comprising contacting the muscle fibers with an antibody-drug conjugate of any of the above embodiments.

[0053] On the other hand, this disclosure provides a method for producing a compound having a structure according to formula (A) or a pharmaceutically acceptable salt thereof: Ab-(L1-B-L2-P) n (A), in: Ab is an antigen-binding protein according to any one of claims 1-14; L1 is the first linker that covalently binds to the side chain of the glutamine residue of Ab; B is the portion containing triazole; L2 is the second linker that covalently binds to the therapeutic agent P; P is a therapeutic agent selected from the group consisting of DHT and proDHT, and n is an integer from 1 to 8, where the method includes the following steps: a) In the presence of transglutaminase, contact A, which contains at least one glutamine residue, with at least one compound L1-B'; b) Contact the product of step a) with one or more equivalent amounts of compound B”-L2-P, wherein group B” is covalently attached to group B’. One of the groups B' and B" is selected from -N3 and ; and another of the groups B' and B” is selected from , and Where Z is C or N; and c) The resulting compound of formula (A) was isolated.

[0054] In one embodiment, A has glutamine residues at positions 295 (Q295) and 297 (N297Q).

[0055] In one implementation, L1-B' has a structure .

[0056] In one embodiment, compound B”-L2-P has a structure selected from the group consisting of:

[0057] In one embodiment, the compound of formula (A) has a structure selected from the group consisting of:

[0058] On the other hand, this disclosure provides a method for producing compounds having the structure according to formula (I): A – [L – P] y (I), Where A is an antigen-binding protein; L stands for connector; P is a therapeutic agent selected from the group consisting of DHT and proDHT, and y is an integer from 1 to 8, where the method includes the following steps: a) In the presence of transglutaminase, contacting A, which contains at least one glutamine residue, with at least one compound LP, wherein said compound LP has at least one terminal amine moiety, and b) The resulting compound of formula (I) after separation.

[0059] In one embodiment, compound LP has a structure selected from the group consisting of:

[0060] These and other aspects of the invention will become apparent to those skilled in the art after reading the following detailed description of the invention (including the appended claims). Attached Figure

[0061] This patent or application document contains at least one drawing shown in color. A published copy of this patent or application with color drawings will be provided by the competent authority upon request and payment of the necessary fees.

[0062] Figure 1Data on acetylcholine-induced calcium flux (relative optical units; y-axis) in human myotubular tubes after incubation with different concentrations (0.01 μM, 0.1 μM, 1 μM, and 10 μM; x-axis) of anti-hCACNG1 antibodies (anti-hCACNG1 Ab 1, anti-hCACNG1 Ab 2, or anti-hCACNG1 Ab 3) or isotype control antibodies (isotype control Ab 1, isotype control Ab 2, or isotype control Ab 3) or with 20 μM nicardipine as a positive control for calcium blocking are provided. The anti-hCACNG1 antibodies tested here did not inhibit acetylcholine-induced calcium flux in human myotubular tubes at these concentrations.

[0063] Figure 2 Provided from wild-type (“WT”) mice, CACNG1-deficient homozygous mice (“KO”) or mice expressing only human CACNG1 (“K”) CACNG1 Hu / Hu The muscle fibers were isolated; incubated with anti-human CACNG1 antibody (anti-hCACNG1 Ab 4 or anti-hCACNG1 Ab 1) or isotype control antibody (isotype control Ab 5 or isotype control Ab 2); and fluorescent immunohistochemical images of single muscle fibers taken ex vivo at 20x magnification after labeling with a fluorescently conjugated secondary antibody. (CACNG1 antibody and CACNG1) Hu / Hu The antibody binds to the muscle fibers, while the isotype control antibody does not bind.

[0064] Figure 3 Provided from wild-type (“WT”) mice, CACNG1-deficient homozygous mice (KO) or mice expressing only human CACNG1 (“ CACNG1 Hu / Hu The image shows a single-plane confocal immunohistochemical image of a muscle fiber isolated at 20x magnification after incubation for 30 minutes, 4 hours, or 8 hours with an anti-human CACNG1 antibody (anti-hCACNG1 Ab 2) or an isotype control antibody (isotype control Ab 4) conjugated to the Alexa 647 (A647) fluorophore. The confocal imaging revealed that after 30 minutes of incubation, the fluorophore-conjugated CACNG1 antibody bound to CACNG1. Hu / Hu On the surface of muscle fibers, and after 4 and 8 hours of incubation, a portion of the CACNG1 antibody was internalized and detected within the muscle fibers. No fluorophore-conjugated isotype control antibody was detected in CACNG1. Hu / Hu It binds to or is internalized in muscle fibers.

[0065] Figure 4Frozen fluorescence tomography images of mice 6 days after systemic injection of 10 mg / kg of an anti-hCACNG1 antibody conjugated to Alexa 647 (anti-hCACNG1Ab 2 or anti-hCACNG1 Ab 1) or an isotype control antibody conjugated to Alexa 647 are provided.

[0066] Figure 5 Mice expressing only human CACNG1 were provided after intravenous injection of 10 mg / kg of an anti-human CACNG1 antibody (anti-hCACNG1 Ab 1 or anti-hCACNG1 Ab 2) conjugated with the Alexa 647 (A647) fluorophore or an isotype control antibody (isotype control Ab 4) and sacrificed 6 days post-injection. CACNG1 Hu / Hu Tilt-and-slide fluorescent immunohistochemical images of gastrocnemius / plantar / soleus, triceps brachii, tibialis anterior, trapezius, diaphragm, pelvic floor muscles, or tongue sections at 20x magnification. Fluorocytically conjugated CACNG1 antibodies were detected in all these skeletal muscles, with anti-hCACNG1 Ab 2 exhibiting a stronger signal in muscle compared to anti-hCACNG1 Ab 1. Only low levels of fluorescence were detected in muscle from isotype control and saline-injected mice.

[0067] Figure 6 Mice expressing only human CACNG1 were provided after intravenous injection of 10 mg / kg of an anti-human CACNG1 antibody (anti-hCACNG1 Ab 1 or anti-hCACNG1 Ab 2) conjugated with the Alexa 647 (A647) fluorophore or an isotype control antibody (isotype control Ab 4) and sacrificed 6 days post-injection. CACNG1 Hu / Hu Tied-up fluorescent immunohistochemical images of liver, spleen, kidney, or brown adipose tissue sections at 20x magnification. Neither of the two fluorophore-conjugated CACNG1 antibodies showed a detectable signal in these organs, and Alexa 647 levels were similar to those of the isotype and saline-injected controls.

[0068] Figure 7 A schematic diagram depicting an exemplary experimental timeline (top image) and a photomicrograph showing the distribution of CACNG1 antibody to the soleus muscle under sedentary and exercise conditions at doses of 10 mg / kg or 50 mg / kg (high) are provided (bottom image). CACNG1 distribution varies with exercise and dose.

[0069] Figures 8A-8C Schematic diagrams of various ADC conjugation pathways are depicted. Figure 8A A schematic diagram of a two-step ADC coupling procedure based on this disclosure is provided. Figure 8BA schematic diagram of a one-step ADC coupling procedure based on this disclosure is provided. Figure 8C A schematic diagram of Cys-maleimide ADC conjugation is provided.

[0070] Figures 9A-9B The ES-MS of aCACNG1-(AL)4 was described. Figure 9A ) and schematic structure ( Figure 9B ).

[0071] Figures 10A-10C The ES-MS of aCACNG1-(AL-LP2)4 ADC was described. Figure 10A ), FelD1-(AL-LP2)4ADC's ES-MS ( Figure 10B ) and schematic structures of aCACNG1-(AL-LP2)4 Figure 10C ).

[0072] Figures 11A-11C Depicting aCACNG1-(AL)4 ( Figure 11A ), aCACNG1-(AL-LP3)4 ( Figure 11B ) and FelD1-(AL-LP3)4 ( Figure 11C ES-MS spectrum of ).

[0073] Figure 12 The results of the antagonist screening are shown.

[0074] Figure 13 The in vitro plasma stability of anti-hCACNG1 Ab 5-L2 in plasma was described.

[0075] Figure 14 The in vitro plasma stability of anti-hCACNG1 Ab 5-L3 in plasma was described.

[0076] Figure 15 The activation of androgen receptors in the LNCaP.hCANCG1.AR.Luc cell line was depicted 24 hours after treatment.

[0077] Figure 16The activation of the androgen receptor in the AR.Luc cell line after 24 hours of treatment was depicted. It shows the androgen receptor (AR) activation levels in relative light units (RLU; y-axis) after 24 hours of incubation with the LNCaP cell line (AR.Luc) modified to express luciferase upon androgen receptor activation and incubated at different concentrations (Log[concentration(M)]; x-axis) of the following: dihydrotestosterone (DHT) alone (unconjugated DHT), anti-hCACNG1 antibody conjugated to DHT (L2) via VC-PAB linker (anti-hCACNG1 Ab 6, anti-hCACNG1 Ab 7, anti-hCACNG1 Ab 8, anti-hCACNG1 Ab 9, anti-hCACNG1 Ab 10 or anti-hCACNG1 Ab 5); or anti-FelD isotype control antibody conjugated to DHT (L2) via VC-PAB linker (isotype control Ab 1). In this assay, only unconjugated DHT showed activation of the androgen receptor, while CACNG1 antibodies conjugated with DHT did not show any perceptible androgen receptor activation in the cell line that does not express hCACNG1.

[0078] Figure 17 The activation of androgen receptors in the hCACNG1.AR.Luc cell line was depicted at 24, 48, and 72 hours after treatment (48 and 72 hours). It shows the androgen receptor (AR) activation levels, expressed in relative light units (RLU; y-axis), after incubation for 24, 48, or 72 hours with different concentrations (Log[concentration(M)]; x-axis) of the LNCaP cell line (hCACNG1.AR.Luc) expressing hCACNG1 and modified to also express luciferase upon androgen receptor activation, at different concentrations (Log[concentration(M)]; x-axis): dihydrotestosterone (DHT) alone (unconjugated DHT), anti-hCACNG1 antibodies conjugated to DHT (L2) via VC-PAB linkers (anti-hCACNG1 Ab 6, anti-hCACNG1 Ab 7, anti-hCACNG1 Ab 8, anti-hCACNG1 Ab 9, anti-hCACNG1 Ab 10, or anti-hCACNG1 Ab 5); or anti-FelD isotype control antibody conjugated to DHT (L2) via VC-PAB linkers (isotype control Ab 1). Several CACNG1 antibody-DHT conjugates activated androgen receptors in the hCACNG1-expressing cell line, and although the efficacy and potency of androgen receptor activation were lower at 24 hours post-treatment than with unconjugated DHT, androgen receptor activation was maintained at 48 and 72 hours compared with unconjugated DHT.

[0079] describe This article provides novel anti-human CACNG1 antibodies, their monovalent antigen-binding fragments, and antibody-drug conjugates (ADCs) that can be used to mediate CACNG1 internalization. Anti-human CACNG1 antibodies, their monovalent antigen-binding fragments, and ADCs containing them 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.

[0080] The description herein is not limited to the specific embodiments, compositions, methods, and experimental conditions described, as such embodiments, compositions, methods, and conditions can vary. The terminology used herein is for the purpose of describing specific embodiments only and is not restrictive.

[0081] While any methods and materials similar to or equivalent to those described herein may be used in the practices or tests as described herein, some preferred methods and materials are now described. All publications cited herein are incorporated herein by reference for their complete 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.

[0082] The term “about” when used to refer to a specific set of numerical values ​​means that the value may differ from the set of values ​​by no more than 1%. For example, the expression “about 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0083] Voltage-dependent calcium channels typically contain five subunits. CACNG1 The gene-encoded protein represents one of these subunits. "CACNG1" includes subunits composed of... CACNG1 CACNG1 is a gene-encoded protein and one of two known γ-subunit proteins. It is part of the skeletal muscle 1,4-dihydropyridine-sensitive calcium channel and is an integrated membrane protein that functions in excitation-contraction coupling. CACNG1 is part of a functionally diverse eight-member subfamily of proteins belonging to the PMP-22 / EMP / MP20 family and is located in a cluster of two family members that function as transmembrane AMPA receptor regulatory proteins (TARP). CACNG1 is highly and specifically expressed in skeletal muscle. The gene encoding human CACNG1 (… CACNG1 It is located on the long arm of chromosome 17. CACNG1 It contains 4 exons and is approximately 12,244 bases long. Human. CACNG1 An exemplary sequence of the gene is designated as NCBI accession number NM_000727.4 (SEQ ID NO:241). An exemplary human CACNG1 protein is designated as NCBI accession number NP_000718 (SEQ ID NO:242).

[0084] The phrase "antibody binding CACNG1" or "anti-hCACNG1 antibody" includes antibodies that specifically recognize a single CACNG1 molecule and their antigen-binding fragments. Antibodies and their antigen-binding fragments as described herein can bind soluble CACNG1 and / or CACNG1 expressed on the cell surface. Soluble CACNG1 includes native CACNG1 protein as well as recombinant CACNG1 protein variants lacking transmembrane domains or not associated with the cell membrane.

[0085] The term "cell surface expressed CACNG1" refers to one or more CACNG1 proteins expressed on the cell surface in vitro or in vivo, such that at least a portion of the CACNG1 protein is exposed to the extracellular space of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface expressed CACNG1" may include or consist of the CACNG1 protein expressed on the surface of cells that normally express the CACNG1 protein. Alternatively, "cell surface expressed CACNG1" may include or consist of the CACNG1 protein expressed on the surface of cells that do not normally express human CACNG1 on their cell surface but have been engineered to express CACNG1 on their surface.

[0086] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies.

[0087] The term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CACNG1). As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, and their polymers (e.g., IgM). Each heavy chain includes 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 includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains a domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions scattered within them called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: 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 Those antibodies that bind to M with affinity, such as those that do so via surface plasmon resonance, for example, BIACORE. TM Alternatively, it can be measured by solution affinity ELISA. The term "antibody" can cover any type of antibody, such as, for example, monoclonal or polyclonal. Furthermore, antibodies can be of any origin, such as, for example, mammalian or non-mammal. In one embodiment, the antibody can be mammalian or avian. In another embodiment, the antibody can be of human origin and can also be a human monoclonal antibody.

[0088] The term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. The terms "antigen-binding portion" and "antigen-binding fragment" of an antibody, etc., encompass any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody can be derived from a complete antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including manipulating and expressing DNA encoding variable and optional constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable conformation, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0089] 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 of the hypervariable region of a mimic antibody (e.g., a separated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included in the term "antigen-binding fragment".

[0090] Antibody antigen-binding fragments typically include at least one variable domain. Variable domains can be of any size or amino acid composition and will generally include at least one CDR adjacent to or within a frame having one or more frame sequences. In the presence of V... L V of domain association H In the antigen-binding fragment of the domain, V H and V L Domains can be positioned relative to each other in any suitable arrangement. For example, variable regions can be dimers and contain V. H -V H V H -V L or V L -V L Dimer. Optionally, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0091] In some embodiments, the antigen-binding fragment of an antibody may include at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding 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-CH 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 connected via fully or partially hinged or linker regions. Hinge regions may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, forming flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies as described herein may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, which are connected to each other and / or with one or more monomers V H or V L Non-covalent association of structural domains (e.g., via disulfide bonds).

[0092] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two distinct variable domains, each capable of specifically binding to a single antigen or different epitopes on the same antigen. Any form of multispecific antibody, including the exemplary bispecific antibody forms disclosed herein, can be adapted using conventional techniques available in the art for use in the context of antigen-binding fragments of antibodies as described herein.

[0093] In some embodiments, the anti-hCACNG1 antibody, as described herein, is a human antibody. The term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies as described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro through random or site-specific mutagenesis or in vivo through somatic mutations), such as those in the CDR and particularly in CDR3. However, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human framework sequence.

[0094] In some embodiments, the antibody as described herein may be a recombinant human antibody. The term “recombinant human antibody” is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells (further described below), antibodies isolated from recombinant, combined human antibody libraries (further described below), antibodies isolated from animals (e.g., mice) that are transgenic for the human immunoglobulin gene (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means including splicing the human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using animals transgenic for the human Ig sequence, in vivo somatic cell mutagenesis), and therefore the V of the recombinant antibody… H District and V L The amino acid sequence of the region is such that, although it originates from and is related to human lineage V, it is... H and V L Sequence-related, but may not naturally exist in the human antibody germline library in vivo.

[0095] Human antibodies can exist in two general forms, both of which are associated with hinge heterogeneity. In one general form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150 kDa–160 kDa, where the dimers are held together by interchain heavy chain disulfide bonds. In the second general form, the dimers are not linked by interchain disulfide bonds and form a molecule of approximately 75 kDa–80 kDa (a half-antibody) consisting of covalently coupled light and heavy chains. These forms are extremely difficult to separate even after affinity purification.

[0096] 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. Single amino acid substitutions 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 levels typically observed with the human IgG1 hinge. As described herein, antibodies can exhibit this form at the hinge, C... H 2 or C H Region 3 contains one or more mutations, which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[0097] As described herein, antibodies can be isolated antibodies. "Isolated antibody" means an antibody that has been identified and isolated and / or recovered from at least one component of its natural environment. For example, an antibody that has been isolated or removed from at least one component of an organism or from tissues or cells in which the antibody is naturally present or produced can be considered "isolated antibody." Isolated antibodies also include antibodies in situ within recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0098] This article also describes 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. Single-arm antibodies as described herein may contain any HCVR / LCVR or CDR amino acid sequence listed in Table 1.

[0099] Compared to the corresponding germline sequences of the derived antibodies, the anti-hCACNG1 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the frame regions and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. Antibodies and their antigen-binding fragments derived from any of the amino acid sequences disclosed herein are also described, wherein one or more amino acids in one or more frame regions and / or CDR regions are mutated to corresponding residues in the germline sequence of the derived antibody, or mutated to corresponding residues in another human germline sequence, or mutated to conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In some embodiments, V H and / or V LAll frame and / or CDR residues within the domain are mutated back to residues found in the original germline sequence of the derived antibody. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, mutated residues present only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues present only in CDR1, CDR2, or CDR3. In other embodiments, one or more frame and / or one or more CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated). Furthermore, antibodies as described herein may contain any combination of two or more germline mutations within the frame and / or CDR regions, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. Once obtained, the antibody and antigen-binding fragment containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as applicable), reduced immunogenicity, etc. In some embodiments, the antibody or antigen-binding fragment as described herein is obtained in this general manner.

[0100] This document also describes anti-hCACNG1 antibodies comprising variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conserved substitutions. For example, some embodiments include anti-hCACNG1 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequences listed in Table 1 herein.

[0101] The phrase "bispecific antibody" refers to antibodies capable of selectively binding to two or more epitopes. Bispecific antibodies typically comprise two distinct heavy chains, each specifically binding to a different epitope—either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will typically be at least one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by a 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, a nucleic acid sequence encoding a variable sequence of a heavy chain that recognizes different epitopes of the same antigen can be fused with a nucleic acid sequence encoding a constant region of a different heavy chain, and such sequences can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a (N-terminal to C-terminal) CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and be able to bind one or more 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 one or two epitopes.

[0102] The phrase "heavy chain" or "immunoglobulin heavy chain" includes the constant region sequence of the immunoglobulin heavy chain from any organism and, unless otherwise specified, includes the heavy chain variable domain. Unless otherwise specified, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. Segments of the heavy chain include CDRs, CDRs, and FRs, and combinations thereof. A typical heavy chain has a CH1 domain, a hinge, a CH2 domain, and a CH3 domain (from N-terminus to C-terminus) following the variable domain. Functional segments of the heavy chain include segments capable of specifically recognizing antigens (e.g., recognizing antigens with KD in the micromolar, nanomolar, or picomolar range), capable of being expressed and secreted from cells, and containing at least one CDR.

[0103] The phrase "light chain" includes the constant region sequence of immunoglobulin light chains from any organism, and, unless otherwise specified, includes human κ and λ light chains. Unless otherwise specified, the light chain variable (VL) domain typically comprises three light chain CDRs and four frame (FR) regions. Typically, a full-length light chain comprises a VL domain from the amino terminus to the carboxyl terminus and a light chain constant domain, said VL domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Potentially useful light chains include, for example, light chains that do not selectively bind to a first or second antigen selectively bound by an antigen-binding protein. Suitable light chains include those that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or computer-simulated libraries), wherein the light chain substantially does not interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Suitable light chains include light chains that can bind one or both epitopes bound by the antigen-binding region of an antigen-binding protein.

[0104] The phrase "variable domain" comprises the amino acid sequence (modified as needed) of the immunoglobulin light or heavy chain, which contains the following amino acid regions in order from the N-terminus to the C-terminus (unless otherwise specified): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. "Variable domain" also comprises the amino acid sequence capable of folding into a typical domain (VH or VL) with a double β-sheet structure, wherein the β-sheets are linked by disulfide bonds between residues of the first and second β-sheets.

[0105] The phrase "complementarity-determining region" or the term "CDR" includes an amino acid sequence encoded by the nucleic acid sequence of an organism's immunoglobulin gene, which typically (i.e., in wild-type animals) appears between two frame regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). A CDR can be encoded by, for example, germline sequences or rearranged or unrearranged sequences, and by, for example, naive or mature B cells or T cells. 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, as a result of sequence splicing or joining (e.g., VDJ recombination to form the heavy chain CDR3).

[0106] 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 covered by the term "antibody fragment" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of an antibody single arm; (v) dAb fragments consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); (vi) isolated CDR and (vii) scFv, consisting of two domains of an Fv fragment, with VL and VH linked by a synthetic linker to form a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule. Other forms of single-chain antibodies, such as biantibodies, are also covered under the term “antibody” (see, for example, Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0107] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least one functional portion of the CH2 and CH3 regions of immunoglobulins. "Functional portion" refers to the CH2 and CH3 regions that can bind to Fc receptors (e.g., FcγR or FcRn, i.e., neonatal Fc receptors) and / or participate in complement activation. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions or other modifications that prevent them from binding to any Fc receptors and from activating complement, then the CH2 and CH3 regions are non-functional.

[0108] Fc-containing proteins may include modifications in the immunoglobulin domain, including modifications that affect one or more effector functions of the bound protein (e.g., modifications affecting FcγR binding, FcRn binding, and thus affecting half-life and / or CDC activity). Referring to the EU numbers for the immunoglobulin constant region, 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.

[0109] For example, but not limited to, the binding protein is an Fc-containing protein that exhibits an enhanced serum half-life (compared to the same Fc-containing protein without the said modification) and has modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In another instance, the modifications may 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); 307 and / or 308 modifications (e.g., 308F or 308P).

[0110] 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 epitopes on antigens (such as cell-specific antigens and / or target antigens described herein). Antigen-binding proteins can be multispecific. The term "multispecific" with respect to antigen-binding proteins means that the protein recognizes different epitopes 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 or fragments thereof as described herein, which can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, antibodies or fragments thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as proteins or fragments thereof, to produce bispecific or multispecific antigen-binding molecules with a second binding specificity.

[0111] The term "protein" refers to any amino acid polymer having more than 20 amino acids covalently linked via 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) can be present in some proteins. These covalent links can be within a single polypeptide chain or between two separate polypeptide chains. For example, disulfide bridges are essential for the proper structure and function of insulin, immunoglobulins, protamines, 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).

[0112] As used herein, “protein” includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, etc. Proteins can be produced using recombinant cell-based production systems, such as bacculovirus systems, yeast systems (e.g., Pichia pastoris species), etc. Pichia(sp) and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation,” 28 Biotechnol Genet Eng Rev. 147-75 (2012).

[0113] As used herein, the term "epitope" refers to the portion of an antigen recognized by a multispecific antigen-binding polypeptide. A single antigen (such as an antigenic polypeptide) may have more than one epitope. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and are defined as those residues that directly contribute to the affinity between the antigen-binding polypeptide and the antigen. Epitopes can also be conformational, i.e., containing nonlinear amino acids. In some embodiments, epitopes may include determinant clusters, which are chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, epitopes may have specific three-dimensional structural features and / or specific charge features. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally lost upon treatment with denaturing solvents.

[0114] The term "domain" refers to any part of a protein or polypeptide that has a specific function or structure. Preferably, as described herein, a domain binds to a cell-specific or target antigen. As used herein, cell-specific antigen or target antigen-binding domains, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to antigens.

[0115] The interchangeable terms “half-body” or “half-antibody” refer to one half of an antibody, which essentially contains a heavy chain and a light chain. The antibody heavy chain 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 another Fc-containing polypeptide. Two slightly different Fc domains can “heterodimerize”, as in the formation of bispecific antibodies or other heterodimers, β-trimers, β-tetramers, etc. See Vincent and Murini, “Current strategies in antibody engineering: Fc engineering and pH-dependent antibody 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).

[0116] The term "single-chain variable fragment" or "scFv" refers to a single-chain fusion polypeptide containing both the variable region (VH) of the immunoglobulin heavy chain and the variable region (VL) of the immunoglobulin light chain. In some embodiments, the VH and VL are linked by a linker sequence of 10 to 25 amino acids. ScFv polypeptides may also include other amino acid sequences, such as CL or CH1 regions. ScFv molecules can be prepared by phage display or by direct subcloning of the heavy and light chains from hybridomas or B cells. For the method of preparing scFv fragments by phage display and antibody domain cloning, Ahmad et al., Clinical and Developmental Immunology, 2012, article ID 98025, are incorporated herein by reference.

[0117] The term "treat" or "treatment" for a state, disorder, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of the development of at least one clinical or subclinical symptom of the state, disorder, or condition in a subject who may be susceptible to or prone to the state, disorder, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; or (2) suppressing the state, disorder, or condition, i.e., preventing, reducing, or delaying the development of the disease or a relapse of the disease or at least one clinical or subclinical symptom of the disease; or (3) alleviating the disease, i.e. causing the resolution of at least one of the state, disorder, or condition or clinical or subclinical symptoms of the disease. The benefit to the subject treated is statistically significant or at least perceptible to the patient or physician. In some embodiments, the treatment includes methods of ablating cells in a manner that indirectly affects the disease. In some embodiments, the treatment includes depleting immune cells as a hematopoietic modulatory protocol prior to the therapy.

[0118] As used herein, “subject,” “patient,” “individual,” or “animal” refers to humans, veterinary animals (e.g., cats, dogs, cattle, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0119] As used herein, the term "effective" when applied to dosage or amount means an amount of compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject with appropriate need. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject based on species, age and general condition, severity of the condition being treated, one or more specific drugs used, mode of administration, etc.

[0120] The phrase "pharmaceuticalally acceptable salt" as used in conjunction with the compositions described in this disclosure refers to any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those described in Berge et al., "Pharmaceutical Salts". J. Pharm. SciThose disclosed in ., 1977, 66:1 are incorporated herein by reference. Examples of salts include, but are not limited to, salts of acid origin, salts of base origin, organic salts, inorganic salts, amine salts, and alkali metal or alkaline earth metal salts, including, but not limited to, calcium salts, magnesium salts, potassium salts, sodium salts, and salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. In some instances, the payloads described herein (e.g., rifamycin analogs described herein) comprise tertiary amines, wherein the nitrogen atom in the tertiary amine is an atom of the payload bonded to a linker or linker-spacer. In such cases, the tertiary amine bonded to the payload creates a quaternary amine in the linker-payload molecule. The positive charge on a quaternary ammonium can be balanced by an anti-charged ion (such as chlorine, bromine, iodine, or any other suitable charged portion, such as those described herein).

[0121] "Comprising," "containing," or "including" means that at least the specified compound, element, particle, or method step is present in the composition, article, or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if such other compounds, materials, particles, or method steps have the same function as the specified compound, material, particle, or method step.

[0122] The compounds disclosed herein include those generally described herein, and are further described by the classes, subclasses and species disclosed herein. Unless otherwise indicated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements are identified according to the periodic table, CAS version, Handbook of Chemistry and Physics, 75th edition. Additionally, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th edition, eds. Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0123] As used herein, the term "alkyl" is given its common meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, the straight-chain alkyl or branched alkyl group has about 1 to 20 carbon atoms in its main chain (e.g., straight chain C1–C1). 20 The branch is C2–C 20 The alkyl group may have about 1 to 10 carbon atoms or about 1 to 6 carbon atoms. In some embodiments, the cycloalkyl ring has about 3 to 10 carbon atoms in its ring structure, wherein such a ring is monocyclic or bicyclic, and optionally has about 5, 6, or 7 carbon atoms in the ring structure. In some embodiments, the alkyl group may be a lower alkyl group, wherein the lower alkyl group contains 1 to 4 carbon atoms (e.g., a straight-chain lower alkyl group is C1–C4).

[0124] As used herein, the term "alkenyl" refers to an alkyl group as defined herein that has one or more double bonds.

[0125] As used herein, the term "alkynyl" refers to an alkyl group as defined herein that has one or more triple bonds.

[0126] The term “heteroatom” refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; any quaternized form of basic nitrogen; or substituted nitrogen of a heterocycle).

[0127] The term "halogen" refers to F, Cl, Br, or I; the term "halogen" refers to a halogen group or substituent, namely -F, -Cl, -Br, or -I.

[0128] The term “adduct” (e.g., “adduct of group B”) in this disclosure covers any portion of a product that includes an addition reaction (e.g., an addition reaction of group B’), regardless of the synthetic steps taken to produce that portion.

[0129] The term "covalent attachment" refers to the formation of a covalent bond, a chemical bond involving two atoms sharing one or more electron pairs. Covalent bonding can include various interactions, including but not limited to σ-bonding, π-bonding, metal-to-metal bonding, agostic interactions, bend bonds, and three-center two-electron bonds. When a first group is described as "capable of covalent attachment" to a second group, it means that the first group can form a covalent bond with the second group directly or indirectly, for example, by using a catalyst or under specific reaction conditions. Non-limiting examples of groups capable of covalent attachment to each other include, for example, amines and carboxylic acids (forming amide bonds), dienes and dienophiles (via the Diels-Alder reaction), and azides and alkynes (forming triazoles via 1,3-cycloaddition reactions).

[0130] As described herein, compounds of this disclosure may comprise "optionally substituted" portions. Generally, the term "substituted," whether or not preceded by the term "optionally," means that one or more hydrogens of the specified portion are substituted by suitable substituents. Unless otherwise indicated, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from a particular group, the substituents may be the same or different at each position. The combinations of substituents contemplated in this disclosure are preferably those that result in the formation of stable or chemically viable compounds. The term "stable," as used herein, means a compound that remains substantially unchanged when subjected to conditions permissible for its production, detection, and, in some embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein.

[0131] Unless otherwise stated, the structures described herein are also intended to include all isomers of the structure (e.g., enantiomers, diastereomers, and geometric (or conformations)); for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, individual stereochemical isomers of the compounds of the present invention, as well as enantiomers, diastereomers, and geometric (or conformational) mixtures, are within the scope of this disclosure.

[0132] Unless otherwise stated, all tautomer forms of the compounds disclosed herein are within the scope of this disclosure.

[0133] Additionally, unless otherwise stated, the structures described herein also refer to compounds that differ only in the presence of atoms enriched in one or more isotopes. For example, those that, in addition to replacing hydrogen with deuterium or tritium, or those that use... 11 C- or 13 C- or 14Compounds having the structure of this invention, other than C-enriched carbon substitutes for carbon, are within the scope of this disclosure.

[0134] It should also be understood that referring to one or more method steps does not preclude the existence of other method steps or intermediate method steps between those explicitly identified steps. Similarly, it should be understood that referring to one or more components of an apparatus or system does not preclude the existence of other components or intermediate components between those explicitly identified components.

[0135] Unless otherwise stated, all crystalline forms of the compounds and their salts disclosed herein are also within the scope of this disclosure. The compounds of this disclosure can be isolated in a variety of amorphous and crystalline forms, including but not limited to anhydrous, hydrated, non-solventized, or solvated forms. Exemplary hydrates include hemihydrates, monohydrates, dihydrates, etc. In some embodiments, the compounds of this disclosure are anhydrous and non-solventized. “Anhydrous” means that the crystalline form of the compound contains substantially no bound water in its crystal lattice structure, i.e., the compound does not form crystalline hydrates.

[0136] As used herein, "crystallization form" refers to a specific lattice configuration of a crystalline substance. Different crystalline forms of the same substance typically possess different lattices (e.g., unit cells), resulting in distinct physical properties characteristic of each form. In some cases, different lattice configurations exhibit different water or solvent contents. Different crystalline lattices can be identified using solid-state characterization methods such as X-ray powder diffraction (PXRD). Other characterization methods, such as differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor adsorption (DVS), and solid-state NMR, further aid in identifying crystalline forms and determining stability and solvent / water content.

[0137] The crystalline forms of a substance include both solvated (e.g., hydrated) and non-solvated (e.g., anhydrous) forms. The hydrated form is a crystalline form that includes water within a crystalline lattice. The hydrated form can be a stoichiometric hydrate, where water exists in the lattice at a given water / molecule ratio, such as a hemihydrate, monohydrate, dihydrate, etc. The hydrated form can also be non-stoichiometric, where the water content is variable and depends on external conditions such as humidity.

[0138] In some embodiments, the compounds of this disclosure are substantially separated. "Substantially separated" means that a particular compound is at least partially separated from impurities. For example, in some embodiments, the compounds of this disclosure include less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2.5%, less than about 1%, or less than about 0.5% of impurities. Impurities generally include anything that is not substantially separated from the compound, including, for example, other crystalline forms and other substances.

[0139] Certain groups, parts, substituents, and atoms are depicted with wavy lines. Wavy lines may intersect one or more bonds or cap one or more bonds. Wavy lines indicate the group, part, substituent, or atom through the atoms it is bonded to. For example, a phenyl group substituted with a propyl group as depicted below: It has the following structure: .

[0140] All amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as shown in 37 CFR § 1.822 (B)(J).

[0141] The amino acid sequences of antibodies can be numbered using any known numbering scheme, including those described by Kabat et al. (“Kabat” scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat scheme. However, the choice of numbering scheme is not intended to imply the absence of differences in the sequence, and those skilled in the art can readily confirm the sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise stated, when referring to residues in the constant region of the antibody heavy chain, the “EU numbering scheme” is generally used (e.g., as reported by Kabat et al., ibid.).

[0142] The term "glutamine acyl-modified antibody" refers to an antibody of this disclosure having at least one covalent link from a glutamine side chain to a primary amine compound. In certain embodiments, the primary amine compound is linked via an amide link on the glutamine side chain. In some embodiments, the glutamine is endogenous glutamine. In other embodiments, the glutamine is an endogenous glutamine that is reactive through peptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the peptide). In yet another embodiment, the glutamine is a peptide engineered with a tag containing an acyl donor glutamine (e.g., a glutamine-containing peptide tag, Q-tag, or TGase recognition tag).

[0143] The term "TGase recognition tag" refers to an amino acid sequence comprising a recipient glutamine residue, which, upon incorporation (e.g., attachment) into a polypeptide sequence, is recognized by TGase under suitable conditions, and results in TGase crosslinking through a reaction between the amino acid side chain within the amino acid sequence and the reaction partner. The recognition tag may be a peptide sequence not naturally present in the polypeptide comprising the TGase recognition tag. In some embodiments, the TGase recognition tag comprises at least one Gln. In some embodiments, the TG recognition tag comprises the amino acid sequence XXQX, where X is any amino acid (e.g., conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp, or unconventional amino acids). In some embodiments, the tag containing the acyl donor glutamine includes an amino acid sequence selected from the group consisting of: LLQGG (SEQ ID NO: 270), LLQG (SEQ ID NO: 271), LSLSQG (SEQ ID NO: 272), GGGLLQGG (SEQ ID NO: 273), GLLQG (SEQ ID NO: 274), GSPLAQSHGG (SEQ ID NO: 275), GLLQGGG (SEQ ID NO: 276), GLLQGG (SEQ ID NO: 277), GLLQ (SEQ ID NO: 278), LLQLLQGA (SEQ ID NO: 279), LLQGA (SEQ ID NO: 280), LLQYQGA (SEQ ID NO: 281), LLQGSG (SEQ ID NO: 282), LLQYQG (SEQ ID NO: 283), LLQLLQG (SEQ ID NO: 284), SLLQG (SEQ ID NO: 285), LLQLQ (SEQ ID NO: 286), LLQLLQ (SEQ ID NO: 287), and LLQGR (SEQ ID NO: 288). See, for example, WO2012059882, the entire contents of which are incorporated herein by reference.

[0144] The term "non-glycosylated antibody" refers to an antibody that does not contain glycosylated sequences that could interfere with the transglutamine reaction, such as antibodies that do not have a glycosylated group at N297 on one or more heavy chains. In a particular embodiment, the antibody heavy chain has an N297 mutation. In other words, according to the EU numbering system disclosed by Kabat et al., the antibody is mutated to no longer have an asparagine residue at position 297. In a particular embodiment, the antibody heavy chain has an N297Q or N297D mutation. Such antibodies can be prepared by site-directed mutagenesis to remove or disable the glycosylated sequence, or by site-directed mutagenesis to insert a glutamine residue at a site other than any interfering glycosylation site or any other interfering structure. Such antibodies can also be isolated from natural or artificial sources. Non-glycosylated antibodies also include antibodies containing T299 or S298P or other mutations or combinations of mutations that result in the absence of glycosylation.

[0145] The term "deglycosylated antibody" refers to an antibody in which a glycosyl group is removed to facilitate transglutaminase-mediated conjugation. The sugar includes, but is not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation occurs at residue N297. In some embodiments, the removal of the glycosyl group is enzymatic (including, but not limited to, via PNGase).

[0146] As used herein, the terms “conjugated protein” or “conjugated antibody” refer to a protein or antibody covalently linked to one or more chemical moieties. The chemical moieties may include amine compounds of the present disclosure. Connectors (L) and payloads (D) suitable for use with the present disclosure are described in detail herein. In a particular embodiment, a conjugated antibody including a therapeutic moieties is an antibody-drug conjugate (ADC), also known as an antibody-payload conjugate or antibody-connector-payload conjugate.

[0147] The term “drug-to-antibody ratio” or (DAR) is the average of the therapeutic portions (e.g., drugs) conjugated with the binder of this disclosure.

[0148] In some embodiments, the term "connector-antibody ratio" or (LAR), also represented by a lowercase 1, is the average number of reactive primary amine compounds conjugated with the binders of this disclosure. Such binders, such as antibodies, can be conjugated with primary amine compounds comprising, for example, suitable azides or alkynes. The resulting binder, functionalized with an azide or alkyne, can then be reacted with a therapeutic portion comprising the corresponding azide or alkyne via a 1,3-cycloaddition reaction.

[0149] The phrase “pharmaceutically acceptable amount” means an amount that is effective or sufficient in treating, reducing, alleviating, or modulating the effects or symptoms of at least one health problem in a subject with an appropriate need. For example, a pharmaceutically acceptable amount of an antibody or antibody-drug conjugate is an effective amount for modulating a biological target using the antibody or antibody-drug conjugate provided herein. Suitable pharmaceutically acceptable amounts include, but are not limited to, up to about 0.001% and any amount in between, such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the antibody or antibody-drug conjugate provided herein.

[0150] The phrase "reaction pH" refers to the pH of the reaction after all reactants or components have been added.

[0151] Bispecific antigen-binding molecules Anti-hCACNG1 antibodies and their antigen-binding fragments as described herein can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific to different epitopes of a single target polypeptide, or can contain antigen-binding domains specific to 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 their antigen-binding fragments as described herein can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce bispecific or multispecific antibodies with a second or additional binding specificity.

[0152] The term "anti-hCACNG1 antibody" as used herein is intended to encompass both monospecific anti-hCACNG1 antibodies and bispecific antibodies comprising a CACNG1-binding arm and a "target"-binding arm. Therefore, this document describes bispecific antibodies in which one arm of the immunoglobulin binds to human CACNG1, and the other arm of the immunoglobulin is specific for another target molecule. The CACNG1-binding arm may contain any HCVR / LCVR or CDR amino acid sequence as listed in Table 1 of this document.

[0153] In some embodiments, the CACNG1 binding arm binds to human CACNG1 and induces the internalization of CACNG1 and the antibody bound thereto. In some embodiments, the CACNG1 binding arm weakly binds to human CACNG1 and induces the internalization of CACNG1 and the antibody bound thereto.

[0154] In some exemplary embodiments, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising first and second antigen-binding domains, the CDR of the first antigen-binding domain may be designated with the prefix "A1", and the CDR of the second antigen-binding domain may be designated with the prefix "A2". Thus, the CDR of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDR of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0155] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly linked to each other to form a bispecific antigen-binding molecule as described herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be linked to a separate multimerizing domain. Association of one multimerizing domain with the other multimerizing domain promotes association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. A “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or composition. For example, a multimerizing domain could be containing immunoglobulin C. H 3-domain polypeptides. A non-limiting example of a polymerized component is the Fc region of an immunoglobulin (containing C... H 2-C H 3 domains), such as the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype of IgG within each isotype group.

[0156] Bispecific antigen-binding molecules as described herein typically comprise two multimerizing domains, such as two Fc domains, each a separate portion of a separate antibody heavy chain. The first and second multimerizing domains may have the same IgG isotype, such as, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerizing domains may have different IgG isotypes, such as, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0157] In some embodiments, the polymerizing domain is an Fc fragment or an amino acid sequence of about 200 amino acids containing at least one cysteine ​​residue. In other embodiments, the polymerizing domain is a cysteine ​​residue or a short cysteine-containing peptide. Other polymerizing domains include peptides or polypeptides comprising leucine zippers, helical ring motifs, or coiled helical motifs or thereof.

[0158] Any bispecific antibody form or technique can be used to prepare bispecific antigen-binding molecules as described herein. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific forms include, but are not limited to, for example, bispecific forms based on scFv or dual antibodies, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knots-into-holes, common light chains (e.g., common light chains with knots-into-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab. 2 Bispecific form (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein, a review of the aforementioned forms).

[0159] In the context of bispecific antigen-binding molecules as described herein, the polymerized domain (e.g., the Fc domain) may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type, naturally occurring Fc domain. For example, a bispecific antigen-binding molecule may contain one or more modifications to the Fc domain that result in altered (e.g., enhanced or weakened) binding interactions between the modified Fc domain and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises C H 2 or C HModifications in region 3, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes with 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); positions 250 and 428 (e.g., L or F); positions 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).

[0160] This article also describes the inclusion of the first C. H 3 structural domains and second Ig C H A bispecific antigen-binding molecule with three domains, wherein the first and second IgC H The three domains differ from each other by at least one amino acid, and the difference of at least one amino acid reduces the binding of the bispecific antibody to protein A compared to bispecific antibodies lacking amino acid differences. In one embodiment, the first IgC H 3-domain binding to protein A, and second IgC H Domain 3 contains mutations that reduce or eliminate protein A binding, such as the H95R modification (identified by IMGT exon numbering; identified by EU numbering as H435R). Second C H 3 may also include a Y96F modification (via IMGT; or Y436F via EU). See, for example, U.S. Patent No. 8,586,713. This may be possible in the second C. HOther modifications found within 3 include: in the case of IgG1 antibody, D16E, L18M, N44S, K52N, V57M, and V82I (via IMGT; via EU, D356E, L358M, N384S, K392N, V397M, and V422I); in the case of IgG2 antibody, N44S, K52N, and V82I (via IMGT; via EU, N384S, K392N, and V422I); and in the case of IgG4 antibody, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (via IMGT; via EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I).

[0161] In some implementations, the Fc domain can be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may contain sequences derived from human IgG1, human IgG2, or human IgG4 C. H Part or all of Zone 2, C H 2 sequence, and part or all of C derived from human IgG1, human IgG2 or human IgG4 H 3. Sequence. The chimeric Fc domain may also include a chimeric hinge region. For example, the chimeric hinge may comprise a combination of an "upper hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4 and a "lower hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule listed herein, from the N-terminus to the C-terminus, include: [IgG4 C 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 antigen-binding molecule listed herein, from the N-terminus to the C-terminus, includes: [IgG1 C H [1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any antigen-binding molecule as described herein are described in U.S. Publication 2014 / 0243504, published August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains having these general structural arrangements and their variants can have altered Fc receptor binding, which in turn affects Fc effector function.

[0162] In some embodiments, the antibody heavy chain, as described herein, includes a heavy chain constant region (CH) containing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same amino acid sequence as 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, SEQ ID NO:255, and SEQ ID NO:330. In some embodiments, the heavy chain constant region (CH) contains 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, SEQ ID NO:255, or SEQ ID NO:330.

[0163] In some embodiments, the antibody heavy chain, as described herein, comprises an Fc domain containing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same amino acid sequence as 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.

[0164] Germplasm mutation Compared to the corresponding germline sequences of the derived antibody, the anti-hCACNG1 antibody disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the frame region and / or CDR region of the heavy chain variable domain.

[0165] As disclosed herein, the anti-hCACNG1 antibodies and their antigen-binding fragments can be derived from any amino acid sequence disclosed herein, with one or more amino acids in one or more frames and / or CDR regions mutated to corresponding residues in the germline sequence of the derived antibody, or mutated to corresponding residues in another human germline sequence, or mutated to conserved amino acid substitutions of corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”), and exhibiting weak or undetectable binding to the CACNG1 antigen. Several such exemplary antibodies recognizing CACNG1 are described in Table 1 herein.

[0166] Furthermore, the anti-hCACNG1 antibodies and their antigen-binding fragments disclosed herein can contain any combination of two or more germline mutations within the frame region and / or CDR region, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. Once obtained, the antibody or antigen-binding fragment containing one or more germline mutations 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 antibody or antigen-binding fragment as described herein is obtained in this general manner.

[0167] This document also describes anti-hCACNG1 antibodies and their antigen-binding fragments, which comprise variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conserved substitutions. For example, an anti-hCACNG1 antibody or its antigen-binding fragment as described herein may comprise an HCVR, LCVR, and / or CDR amino acid sequence with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequence listed in Table 1 herein. Compared to corresponding germline sequences deriving a single antigen-binding domain, antibodies and their antigen-binding fragments as described herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy and light chain variable domains, while maintaining or improving a desired weak to undetectable binding to, for example, CACNG1. A “conserved amino acid substitution” is the substitution of one amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein; that is, in the case of resistance to hCACNG1 binding molecules, amino acid substitutions maintain or improve the desired weak to undetectable binding affinity. Examples of amino acid groups with side chains possessing 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: cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. “Moderately conservative” substitution is any change with a non-negative value in the PAM250 log-likelihood matrix.

[0168] This document also describes anti-hCACNG1 antibodies and their antigen-binding fragments, comprising antigen-binding domains having HCVR and / or CDR amino acid sequences substantially identical to any 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 the same” mean that two amino acid sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when aligned optimally, such as by using the default gap weights in the GAP or BESTFIT procedure. Preferably, dissimilar residue positions are distinguished by conserved amino acid substitutions. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or similarity can be adjusted upwards to correct for the conservatism of the substitutions. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331.

[0169] Sequence similarity of peptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related peptides, such as homologous peptides from different biological species or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (the program in GCG version 6.1) with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments of the best overlapping regions between the query and search sequences and a percentage of sequence identity (Pearson (2000) ibid.). Another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters when comparing sequences described herein with databases containing large numbers of sequences from different organisms. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) NucleicAcids Res. 25:3389-402.

[0170] Once obtained, the reduced binding affinity of the antigen-binding domain containing one or more germline mutations is tested using one or more in vitro assays. Typically, antibodies that recognize a specific antigen are screened for their intended purpose by testing for high (i.e., strong) binding affinity to the antigen.

[0171] Further modification of antibodies as described herein using the methods described herein can yield unexpected benefits, such as improved pharmacokinetic properties and lower toxicity to patients.

[0172] Antibody binding properties In the context of the binding of antibodies, immunoglobulins, antibody-binding fragments, or Fc-containing proteins to, for example, a predetermined antigen (such as a cell surface protein or a fragment thereof), the term "binding" generally refers to an interaction or association between at least two entities or molecular structures, such as antibody-antigen interactions.

[0173] For example, when using antigens as ligands and antibodies, Ig, antibody-binding fragments, or Fc-containing proteins as analytes (or anti-ligands) to determine binding affinity using surface plasmon resonance (SPR) technology in instruments such as the BIAcore 3000, the binding affinity typically corresponds to approximately 10. -7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller K D Values. Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used, providing binding characterization data on proteins expressed on the cell surface. FACS data show good correlation with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods 1997, 201(2):223-31; Geuijen, CA et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0174] Therefore, anti-hCACNG1 antibodies and their antigen-binding fragments, as described herein, bind to predetermined antigens or cell surface molecules (receptors) and have a K0 value that is at least ten times lower than their affinity for binding nonspecific antigens (e.g., BSA, casein). D Affinity for the value. Corresponding to K D Antibodies with an affinity equal to or less than 10 times that of a nonspecific antigen can be considered to have undetectable binding; however, such antibodies can pair with a second antigen binding arm to produce bispecific antibodies as described herein.

[0175] The term "K" in moles (M) units D "KD" or "antibody-antigen interaction" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment bound to an antigen. D There is an inverse relationship between K and binding affinity, therefore K DThe smaller the value, the higher the affinity, i.e., the stronger. Therefore, the terms "higher affinity" or "stronger affinity" refer to a higher capacity to form an interaction, and thus K... D Smaller values, conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, and therefore K D The value is relatively large. In some cases, a molecule (e.g., an antibody) has a higher binding affinity (or Kb) to its interacting partner molecule (e.g., antigen X) compared to the binding affinity of a specific molecule (e.g., an antibody) to another interacting partner molecule (e.g., antigen Y). D This can be expressed as by increasing the size of K. D The value (lower or weaker affinity) divided by the smaller K D The binding ratio is determined by (higher or stronger affinity), for example, expressed as 5 times or 10 times greater binding affinity, depending on the case.

[0176] The term "k" d "(sec⁻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 known as k. off value.

[0177] The term "k" a "(M-1x sec-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.

[0178] 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 determined by k. a Divide by k d get.

[0179] The term "EC50" or "EC" 50 "" refers to the half-maximal effective concentration, which includes the antibody concentration that induces half the response between baseline and maximum response after a specified exposure time. 50 Essentially, this represents the concentration of the antibody at which 50% of its maximum effect is observed. In some implementations, EC... 50 The value is equal to the antibody concentration as described herein, which allows the antibody to achieve half-maximal binding to cells expressing CACNG1, as determined by, for example, FACS binding assays or androgen receptor activated luciferase assays. Therefore, EC 50 When the half-maximum effective concentration value increases, a decrease or weaker binding is observed.

[0180] In one implementation, the reduced binding can be defined as the increased EC. 50 Antibody concentration, which enables it to bind to half the maximum number of target cells.

[0181] Sequence variants Compared to the corresponding germline sequence of a single antigen-binding domain, the anti-hCACNG1 antibody and antigen-binding fragments described herein may contain one or more amino acid substitutions, insertions, and / or deletions in the frame and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules described herein may contain an antigen-binding domain derived from any exemplary amino acid sequence disclosed herein, wherein one or more amino acids in one or more frame and / or CDR regions are mutated to one or more corresponding residues of the germline sequence of the derived antibody, or one or more corresponding residues of another human germline sequence, or conserved amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce numerous antibody and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In some embodiments, V H Domain and / or V L All frame and / or CDR residues within the domain are mutated back to residues present in the original germline sequence of the originally derived antigen-binding domain. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, mutated residues present only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues present only in CDR1, CDR2, or CDR3. In other embodiments, one or more frame and / or one or more CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence of the originally derived antigen-binding domain). Furthermore, the antigen-binding domain may comprise any combination of two or more germline mutations within the frame and / or CDR regions, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. Once obtained, one or more desired properties of the antigen-binding domain containing one or more germline mutations can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. This article describes antigen-binding molecules containing one or more antigen-binding domains obtained in this general manner.

[0182] This document also describes antigen-binding molecules in which one or both antigen-binding domains comprise variants of any HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conserved substitutions. For example, an antigen-binding molecule as described herein may comprise an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conserved amino acid substitutions relative to any HCVR, LCVR, and / or CDR amino acid sequence disclosed herein. A “conserved amino acid substitution” is the substitution of one amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Typically, conserved amino acid substitutions will not significantly alter the functional properties of the protein. Examples of amino acid groups with side chains possessing 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: cysteine ​​and methionine. Preferred conserved 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 with 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 with a non-negative value in the PAM250 log-likelihood matrix.

[0183] The antigen-binding molecules described herein may comprise an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence substantially identical to any HCVR, LCVR, and / or CDR amino acid sequence disclosed herein. When referring to amino acid sequences, the terms “substantially identical” or “substantially the same” mean that two amino acid sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when aligned using the default gap weights via procedures such as GAP or BESTFIT. Preferably, dissimilar residue positions are distinguished by conserved amino acid substitutions. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the sequence identity percentage or similarity may be adjusted upwards to correct for the conservatism of the substitutions. Methods for making such adjustments 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.

[0184] Sequence similarity of peptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software uses similarity metrics assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related peptides, such as homologous peptides from different biological species or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (the program in GCG version 6.1) with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments of the best overlapping regions between the query and search sequences and a percentage of sequence identity (Pearson (2000) ibid.). Another preferred algorithm when comparing sequences to databases containing large numbers of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402, each incorporated herein by reference.

[0185] pH-dependent binding This article also describes anti-hCACNG1 antibodies and their antigen-binding fragments that exhibit pH-dependent binding characteristics. For example, anti-hCACNG1 antibodies as described herein may exhibit reduced binding to CACNG1 at acidic pH compared to neutral pH. Alternatively, anti-hCACNG1 antibodies as described herein may exhibit enhanced binding to CACNG1 at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values ​​less than about 6.2, such as 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 smaller. The expression "neutral pH" means a pH from about 7.0 to about 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0186] In some cases, "compared to neutral pH, binding is reduced at acidic pH..." refers to the K+ binding of antibodies to their antigens at acidic pH. D The K value of antibody binding to its antigen at neutral pH D The ratio of values ​​is used to express this (or vice versa). For example, if an antibody or its antigen-binding fragment exhibits an acidic / neutral K value of approximately 3.0 or greater. D For the purposes described herein, an antibody or its antigen-binding fragment may be considered to exhibit “reduced binding to CACNG1 at acidic pH compared to neutral pH.” In some exemplary embodiments, the acidic / neutral pH of the antibody or antigen-binding fragment as described herein is... D The ratio can be approximately 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.

[0187] Antibodies exhibiting pH-dependent binding characteristics can be obtained, for example, by screening antibody populations to obtain antibodies that exhibit reduced (or enhanced) binding to a specific antigen at acidic pH compared to neutral pH. Additionally, modifications to the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For instance, by substituting one or more amino acids of the antigen-binding domain (e.g., within the CDR) with histidine residues, antibodies exhibiting reduced antigen binding at acidic pH relative to neutral pH can be obtained.

[0188] Antibodies containing Fc variants In some embodiments, anti-hCACNG1 antibodies and their antigen-binding fragments (including multispecific antigen-binding molecules and multidomain therapeutic proteins containing anti-hCACNG1 antibodies or their antigen-binding fragments) are provided, comprising an Fc domain containing one or more mutations that, for example, enhance or reduce antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the antibodies described herein may contain C0 of the Fc domain. H 2 or C H Mutations in region 3, where the mutation increases the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes with a pH range of about 5.5 to about 6.0). When administered to animals, such mutations can lead to an increase in the serum half-life of the antibody. Non-limiting examples of such Fc modifications include, for example, modifications at 250 bits (e.g., E or Q); 250 bits and 428 bits (e.g., L or F); 252 bits (e.g., L / Y / F / W or T), 254 bits (e.g., S or T), and 256 bits (e.g., S / R / Q / E / D or T); or modifications at 428 bits and / or 433 bits (e.g., H / L / R / S / P / Q or K) and / or 434 bits (e.g., H / F or Y); or modifications at 250 bits and / or 428 bits; or modifications at 307 bits or 308 bits (e.g., 308F, V308F) and 434 bits. 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).

[0189] For example, the anti-hCACNG1 antibody and antigen-binding fragment described herein may include an Fc domain containing one or more mutation pairs or groups selected from the following: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are covered within the scope of this description.

[0190] Biological characteristics of antibodies and bispecific antigen-binding molecules This article also describes an antibody and its antigen-binding fragment that binds to human CACNG1 with high, medium, or low affinity, depending on the desired therapeutic context and specific targeting characteristics. For example, in the context of a bispecific antigen-binding molecule, where one arm binds to CACNG1 and the other arm binds to a target antigen (e.g., a tumor-associated antigen), it might be desirable for the target antigen-binding arm to bind to the target antigen with high affinity, while the anti-hCACNG1 arm binds to CACNG1 with only medium or low affinity. In this way, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved, while avoiding general / untargeted CACNG1 binding and the associated adverse side effects.

[0191] This document also describes antibodies, their antigen-binding fragments, and bispecific antibodies that bind to human CACNG1 with weak (i.e., low) or even undetectable affinity. In some embodiments, antibodies and their antigen-binding fragments, as described herein, bind with a K+ greater than about 100 nM. D Binding to human CACNG1 (e.g., at 37°C), as measured by surface plasmon resonance. In some embodiments, the antibody or antigen-binding fragment as described herein is in 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 it may bind to CACNG1 with undetectable affinity, as measured by surface plasmon resonance (e.g., mAb capture or antigen capture) or substantially similar assays.

[0192] Epitope plotting and related techniques The epitopes on CACNG1 targeted by the anti-hCACNG1 antibodies and their antigen-binding fragments described herein can consist of a single, continuous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of the CACNG1 protein. Alternatively, an epitope can consist of more than one non-continuous amino acid (or amino acid sequence) of CACNG1. The term "epitaph" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, and is called a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions of an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include portions of a sugar, phosphoryl group, or sulfonyl group on an antigen.

[0193] Various techniques known to those skilled in the art can be used to determine whether an antibody's antigen-binding domain interacts with "one or more amino acids" in a peptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays, such as... Antibodies The alanine scanning mutation analysis, peptide blotting analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis described in Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) are examples. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer, 2000, Protein Science 9:487-496). Another method for identifying amino acids within a peptide that interact with the antigen-binding domain of an antibody is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterating the protein of interest, followed by binding the antibody to the deuterated protein. Next, the protein / antibody complex is transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which retains the deuterium label). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis, revealing the deuterated residues corresponding to the specific amino acids interacting 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.

[0194] This document also describes anti-hCACNG1 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies containing any amino acid sequences as listed in Table 1 of this document). Similarly, this document also describes anti-hCACNG1 antibodies that compete with any of the specific exemplary antibodies described herein (e.g., antibodies containing any amino acid sequences as listed in Table 1 of this document) for binding to CACNG1.

[0195] By using conventional methods known in the art, it is readily possible to determine whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as or competes with a reference antigen-binding molecule as described herein. For example, to determine whether a test antibody binds to the same epitope on CACNG1 as the reference bispecific antigen-binding molecule described herein, the reference bispecific molecule is first allowed to bind to the CACNG1 protein. Next, the ability of the test antibody to bind to the CACNG1 molecule is evaluated. If the test antibody is able to bind to CACNG1 after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the CACNG1 epitope bound by the test antibody is different from that bound by the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CACNG1 molecule after saturation binding with the reference bispecific antigen-binding molecule, the test antibody may bind to the same epitope on CACNG1 as the reference bispecific antigen-binding molecule described herein. Further routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to some embodiments described herein, such as those measured in competitive binding assays, if, for example, an excess of 1, 5, 10, 20, or 100 times of one antigen-binding protein inhibits the binding of another antigen-binding protein by at least 50%, but preferably 75%, 90%, or even 99%, then the two antigen-binding proteins bind to the same (or overlapping) epitope (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, if an amino acid mutation in the antigen that substantially reduces or eliminates the binding of one antigen-binding protein also reduces or eliminates the binding of the other antigen-binding protein, then the two antigen-binding proteins are considered to bind to the same epitope. If a subset of amino acid mutations that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of another antigen-binding protein, then the two antigen-binding proteins are considered to have "overlapping epitopes".

[0196] To determine whether an antibody or its antigen-binding domain competes with a reference antigen-binding molecule for binding, the above 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, in both directions, only the first (saturated) antigen-binding molecule is able to bind to the CACNG1 molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CACNG1. As will be understood by those skilled in the art, the antibody competing for binding with the 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.

[0197] Preparation of antigen-binding domains and construction of bispecific molecules Antigen-binding domains specific to a particular antigen can be prepared using any antibody generation technique known in the art. Once obtained, the two different antigen-binding domains specific to two different antigens (e.g., CACNG1 and the target antigen) can be appropriately arranged relative to each other to generate a bispecific antigen-binding molecule as described herein using conventional methods. (A discussion of exemplary bispecific antibody forms that can be used to construct bispecific antigen-binding molecules as described herein is provided elsewhere herein.) In some embodiments, one or more of the individual components (e.g., heavy and light chains) of the antigen-binding molecule 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, one or more of the heavy and / or light chains of the antigen-binding molecule as described herein can be prepared using the VELOCIMMUNE™ technology. Using the VELOCIMMUNE™ technology (or any other human antibody generation technique), a high-affinity chimeric antibody against a specific antigen (e.g., CACNG1) having a human variable region and a mouse constant region is initially isolated. The antibody is characterized and selected for desired characteristics, including affinity, selectivity, epitopes, etc. The mouse constant region was replaced with the desired human constant region to generate a fully human heavy chain and / or light chain that can be incorporated into antigen-binding molecules as described herein.

[0198] Genetically engineered animals can be used to prepare human bispecific antigen-binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operatively linked to the mouse κ constant gene at the endogenous mouse κ locus. Such genetically modified mice can be used to isolate heavy and light chain variable regions to produce fully human bispecific antigen-binding molecules. Thus, a fully human bispecific antigen-binding molecule comprises two distinct heavy chains associated with the same light chain (see, for example, US 2011 / 0195454). Fully human refers to an antibody or its antigen-binding fragment or immunoglobulin domain containing an amino acid sequence encoded by DNA derived from a human sequence along the full length of each polypeptide of the antibody or its antigen-binding fragment or immunoglobulin domain. In some cases, the fully human sequence is derived from a human endogenous protein. In other cases, the fully human protein or protein sequence comprises a chimeric sequence in which each component sequence is derived from a human sequence. While not bound by any particular theory, chimeric proteins or chimeric sequences are typically designed to minimize the generation of immunogenic epitopes at the junctions of component sequences, for example, compared to any wild-type human immunoglobulin region or domain.

[0199] A bispecific antigen-binding molecule can be constructed using a heavy chain having a modified Fc domain that eliminates its binding to protein A, thereby enabling a purification method for producing a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. Therefore, a bispecific antigen-binding molecule contains a first C H 3 structural domains and second Ig C H 3 structural domains, where the first and second IgC H The three domains differ from each other by at least one amino acid, and the difference of at least one amino acid reduces the binding of the bispecific antibody to protein A compared to bispecific antibodies lacking amino acid differences. In one embodiment, the first IgC H 3-domain binding to protein A, and second IgC H Domain 3 contains mutations / modifications that reduce or eliminate protein A binding, such as the H95R modification (identified by IMGT exon numbering; identified by EU numbering as H435R). Second C H 3 may also include Y96F modification (via IMGT; via EU for Y436F).

[0200] Bioequivalence This document also describes antigen-binding molecules having an amino acid sequence different from that of the exemplary molecules disclosed herein but retaining the ability to bind CACNG1. When compared with the parental sequence, such variant molecules may contain one or more additions, deletions, or substitutions of amino acids, but exhibit biological activity substantially equivalent to that of the bispecific antigen-binding molecules described herein.

[0201] Antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules described herein are also described. For example, if two antigen-binding proteins or antibodies are pharmaceutical equivalents or substitutes, and their absorption rates and extents do not show significant differences when administered at the same molar dose under similar experimental conditions, whether as a single dose or multiple doses, they are considered bioequivalent. If some antigen-binding proteins are equivalent in extent of absorption but not in rate of absorption, they will be considered equivalents or substitutes, but may still be considered bioequivalent because such differences in absorption rates are intentional and reflected on the label, are not necessary for achieving effective in vivo drug concentrations for example, with prolonged use, and are considered medically insignificant for the specific pharmaceutical product under investigation.

[0202] In one implementation, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

[0203] In one implementation, the two antigen-binding proteins are bioequivalent if a patient can switch between the reference product and the biological product once or more without an expected increase in the risk of side effects, including clinically significant changes in immunogenicity or reduced effectiveness, compared to continued treatment without such switching.

[0204] In one implementation, if two antigen-binding proteins act on one or more common use conditions through one or more common mechanisms of action, then the two antigen-binding proteins are bioequivalent, and such mechanisms are known to some extent.

[0205] Bioequivalence can be demonstrated through in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo testing in humans or other mammals, in which changes in the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluids over time are measured; (b) in vitro testing that correlates with and reasonably predicts bioavailability data in humans; (c) in vivo testing in humans or other mammals, in which changes in the appropriate acute pharmacological effect of the antibody (or its target) over time are measured; and (d) in clinical trials that establish well-controlled safety, efficacy, or bioavailability or bioequivalence of antigen-binding proteins.

[0206] Bioequivalent variants of the exemplary bispecific antigen-binding molecules listed herein can be constructed, for example, by various substitutions or deletions of biologically unwanted terminal or internal residues or sequences. For instance, cysteine ​​residues not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges during renaturation. In other cases, bioequivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules listed herein that incorporate amino acid changes that modify the glycosylation characteristics of the molecule, such as mutations that eliminate or remove glycosylation.

[0207] Species selectivity and species cross-reactivity In some implementations, antigen-binding molecules, as described herein, bind to human CACNG1 but not to CACNG1 from other species. Antigen-binding molecules that bind to human CACNG1 and to CACNG1 from one or more non-human species are also described herein.

[0208] In some implementations, the antigen-binding molecule as described herein that binds to human CACNG1 may or may not bind, depending on the specific circumstances, to one or more of the following CACNG1: mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee.

[0209] Antibody-drug conjugates (ADCs) This article also describes antibody-drug conjugates (ADCs) comprising an anti-hCACNG1 antibody or an antigen-binding fragment thereof conjugated to a drug (e.g., a payload or molecular cargo (e.g., a small molecule and / or a therapeutic portion)). Anti-hCACNG1 antibodies or antigen-binding fragments thereof conjugated to a therapeutic portion are also provided. Generally, ADCs comprise: A – [L – P] y , where A is an antigen-binding molecule, such as an anti-hCACNG1 antibody, or a fragment thereof (e.g., a fragment of HCDR3 containing at least one HCDR3 amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1), L is a linker, P is a payload or molecular cargo, and y is an integer from 1 to 30.

[0210] In various embodiments, the ADC comprises an anti-hCACNG1 antibody or its antigen-binding fragment thereof, the anti-hCACNG1 antibody or its antigen-binding fragment comprising a CDR of an HCVR or LCVR having the amino acid sequence of the SEQ ID NO listed in Table 1 (e.g., SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 294 and 314 or 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 301 and 322), or a specific HCVR / LCVR pair (e.g., SEQ ID NO: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 294 / 301, and 314 / 322). In some cases, the anti-hCACNG1 antibody or fragment comprises a CDR having an amino acid sequence having the SEQ ID NO or sequence listed in Table 1 (e.g., SEQ ID NO: 4-6-8-12-AAS-16, 20-22-24-26-28-ATS-32, 36-38-40-44-KAS-48, 52-54-56-60-GAS-64, 68-70-72-76-AAS-80, 84-86-88-92-AAS-96, 100-102-104-108-AAS-112, 116-118-120-12 4-GA-128, 132-134-136-140-GAS-144, 148-150-152-156-RN-160, 164-166-168-172-DNN-176, 180-182-186-188-GAS-192 and 36-297-299-303-YNS-305; and 316-318-152-324-RNN-326). In some cases, anti-hCACNG1 antibodies or fragments comprise HCVRs and LCVRs having amino acid sequences with SEQ ID NOs listed in Table 1 (e.g., SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 294 and 314 and 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 301 and 322), or specific amino acid sequence pairs (e.g., SEQ ID NO: ). 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 294 / 301 and 314 / 322).

[0211] In some implementations, the payload or molecular cargo contains small molecules as therapeutic agents, such as those used to treat muscle atrophy or hereditary muscle diseases. Small molecules (SMs) can readily enter cells because of their low molecular weight (typically up to about 1 kDa). Once inside the cell, small molecules can affect other molecules, such as proteins, and can, for example, cause cancer cell death. This differs from many large molecular weight molecules such as antibodies. Examples of small molecules can be conjugated to anti-CACNG1 antigen-binding proteins to form anti-CACNG1:SM conjugates.

[0212] Therapeutic agents that can be used to treat muscle atrophy or hereditary muscle diseases include testosterone and its bioactive variants (e.g., dihydrotestosterone (DHT), or its prodrugs or derivatives), β2-adrenergic receptor agonists (e.g., clenbuterol), rapamycin or its analogues, MAPK inhibitors, or histone deacetylase inhibitors, etc.

[0213] In one embodiment, the therapeutic agent conjugated with the antibody of this disclosure is a structure having DHT.

[0214] In one embodiment, the therapeutic agent conjugated with the antibody of this disclosure is proDHT having the following structure: .

[0215] In one embodiment, the therapeutic agent conjugated with the antibody of this disclosure is proDHT having the following structure: , R1 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R2 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R3 is selected from the following groups: H and C 1-3 Alkyl, and R4 is selected from the following groups: H and C1-3 alkyl.

[0216] This document also provides antibody-radionium conjugates (ARCs) comprising an anti-hCACNG1 antibody conjugated to one or more radionuclides. Exemplary radionuclides that may be used in the context of this aspect of the disclosure include, but are not limited to, for example... 225 Ac、 212 Bi、 213 Bi、 131 I, 186 Re、 227 Th、 222 Rn、 223 Ra、 224 Ra and 90 Y.

[0217] Connector (L) In some embodiments provided herein, an ADC comprising, for example, an anti-hCACNG1 antigen-binding protein conjugated to a therapeutic agent (e.g., any therapeutic agent disclosed above) via a linker molecule is provided. A linker is any group or portion that links, connects, or bonds an antibody or antigen-binding protein described herein to a therapeutic moiety (e.g., a cytotoxic agent). Suitable linkers may be, for example... Antibody-Drug Conjugates and Immunotoxins Phillips, GL (eds.); Springer Verlag: New York, 2013; Antibody- Drug Conjugates Edited by Ducry, L.; Humana Press, 2013; Antibody-Drug ConjugatesWang, J., Shen, W.-C., and Zaro, JL, eds.; Springer International Publishing, 2015, the contents of which are incorporated herein by reference in their entirety. Generally, suitable binder linkers for the antibody conjugates described herein are those that are sufficiently stable to utilize the circulating half-life of the antibody and simultaneously capable of releasing their payload after the internalization of the antigen-mediated conjugate. Linkers can be cleavable or non-cleavable. Cleavable linkers include those that are cleaved by intracellular metabolism after internalization (e.g., via hydrolysis, reduction, or enzymatic reactions). Non-cleavable linkers include those that release the attached payload after internalization via lysosomal degradation of the antibody. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolyzably unstable linkers, enzymatically cleavable linkers, reductively unstable linkers, self-destructive linkers, and non-cleavable linkers. Suitable connectors also include, but are not limited to, the following or include those of the following: peptide, glucuronic acid, succinimide-thioether, polyethylene glycol (PEG) unit, hydrazone, maleimide-hexanoyl unit, dipeptide unit, valine-citrulline unit, and p-aminobenzyl (PAB) unit.

[0218] Any linker molecule or linker technology known in the art can be used to create or construct the ADC of this disclosure. In some embodiments, the linker is a cleavable linker. According to other embodiments, the linker is a non-cleavable linker. Exemplary linkers that can be used in the context of this disclosure include linkers comprising or composed of: for example, MC (6-maleimide hexanoyl), MP (maleimide propionyl), val-cit (valine-citrulline), val-ala (valine-alanine), val-gly (valine-glycine), dipeptide sites in protease-cleavable linkers, ala-phe (alanine-phenylalanine), dipeptide sites in protease-cleavable linkers, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimide-4-(2-pyridinylthio)valerate), SMCC (N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate), SIAB (N-succinimide-(4-iodoacetyl)aminobenzoate), and variations and combinations thereof. Further examples of connectors that can be used in the context of this disclosure are, for example, in US 7,754,681 and Ducry, Bioconjugate Chem., 2010. 21 The contents of references 5-13 and those cited therein are provided and are incorporated herein by reference in their entirety.

[0219] In some embodiments, the linker is stable under physiological conditions. In some embodiments, the linker is cleavable, for example, capable of releasing at least a portion of the payload in the presence of an enzyme or within a specific pH range or value. In some embodiments, the linker comprises an enzyme-cleavable portion. Illustrative enzyme-cleavable portions include, but are not limited to, peptide bonds, ester bonds, hydrazone bonds, and disulfide bonds. In some embodiments, the linker comprises a cathepsin-cleavable linker.

[0220] In some implementations, the joint includes a non-breakable portion.

[0221] Suitable linkers also include, but are not limited to, those that are chemically bound to two cysteine ​​residues of a single binder (e.g., an antibody). Such linkers can be used to mimic the disulfide bonds of an antibody, which are broken due to the conjugation process.

[0222] In some embodiments, the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteogenous, non-proteogenous, and L- or D-α-amino acids. In some embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or combinations thereof. In some embodiments, one or more side chains of the amino acid are attached to side chain groups, as described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the linker comprises lysine, valine, and citrulline. In some embodiments, the linker comprises lysine, valine, and alanine. In some embodiments, the linker comprises valine and alanine.

[0223] In some embodiments, the connector includes a self-destructing group. The self-destructing group can be any such group known to those skilled in the art. In a particular embodiment, the self-destructing group is p-aminobenzyl (PAB) or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those skilled in the art will recognize that the self-destructing group is capable of undergoing a chemical reaction, thereby releasing the remaining atoms of the connector from the payload.

[0224] In some implementations, the connector is:

[0225] in It is a bond that binds to an antibody or antigen-binding protein (e.g., via a lysine residue), and It is a bond that binds to the therapeutic payload (e.g., testosterone or its biological equivalent). In some embodiments, the connector is:

[0226] in It is a bond that binds to an antibody or antigen-binding protein (e.g., via a lysine residue), and It is a bond that binds to the therapeutic payload (e.g., testosterone or its biological equivalent). In some embodiments, the connector is: .

[0227] In some implementations, the connector is: .

[0228] In some implementations, the connector is derived from maleimide methyl-4-trans-cyclohexane carboxysuccinate: .

[0229] In some implementations, the connector is:

[0230] in It is a bond that binds to an antibody or antigen-binding protein (e.g., via a lysine residue), and It is a bond that binds to the therapeutic payload (e.g., testosterone or its biological equivalent variant).

[0231] In some implementations, the connector is:

[0232] in It is a bond that binds to an antibody or antigen-binding protein (e.g., via a lysine residue), and It is a bond that binds to the therapeutic payload (e.g., testosterone or its biological equivalent variant).

[0233] This disclosure includes ADCs, wherein a linker connects an anti-hCACNG1 antigen-binding protein, as described herein, to a therapeutic agent via attachment at a specific amino acid within an antibody or antigen-binding molecule. Exemplary amino acid attachments that may be used in the context of this aspect include, for example, lysine (see, for example, US 5,208,020; US 2010 / 0129314; Hollander et al.). Bioconjugate Chem. , 2008, 19:358-361; WO 2005 / 089808; US 5,714,586; and US 2013 / 0101546), cysteine ​​(see, for example, US 2007 / 0258987; WO 2013 / 055993; WO2013 / 055990; WO 2013 / 053873; WO 2013 / 053872; WO 2011 / 130598; US 2013 / 0101546; and US 7,750,116), selenocysteine ​​(see, for example, WO 2008 / 122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105 :12451-12456), formylglycine (see, for example, Carrico et al., Nat. Chem . Biol. , 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110 :46-51; and Rabuka et al., Nat. Protocols , 2012 , 10 (1052-1067), non-natural amino acids (see, for example, WO 2013 / 068874 and WO 2012 / 166559) and acidic amino acids (see, for example, WO 2012 / 05982). The linker can also be located via a linker with sugars (see, for example, US 2008 / 0305497, WO 2014 / 065661 and Ryan et al.), Food & Agriculture Immunol (, 2001, 13:127-130) and disulfide joints (see, for example, WO 2013 / 085925, WO2010 / 010324, WO 2011 / 018611 and Shaunak et al., ...) Nat. Chem. Biol (See, 2006, 2:312-313) This conjugation technique can also be used to directly conjugate to specific residues of antibodies or antigen-binding proteins (see, e.g., Schumacher et al., 2006, 2:312-313). J Clin Immunol(2016) 36(Suppl 1): 100). Site-specific conjugation techniques include, but are not limited to, glutamine conjugation via transglutaminase (see, for example, Schibli, Angew Chemie Inter Ed. 2010, 49, 9995). In some embodiments, residues of the antibody as described herein (e.g., residues in the heavy chain constant region of the antibody) may be substituted with glutamine to further facilitate glutamine conjugation via transglutaminase. As a non-limiting example, the human heavy chain constant region may be modified with N180Q substitution found in the human IgG1 heavy chain constant region sequence listed as SEQ ID NO:269. Such substitution provides a total of 4 glutamine residues for conjugation by transglutaminase.

[0234] In one aspect of this disclosure, the connector (L) has the following structure: -L1-B-L2-, where: L1 is the first linker unit that is covalently attached to the antigen-binding protein; B is a unit that either does not exist or is an adduct containing at least one group B', wherein the group B' is selected from -N3, , , ,and Where Q is C or N; L2 is absent or is a second linker unit covalently attached to unit B via at least one group B”, wherein group B’ and group B” form at least one adduct. The condition is that when B is absent, L2 is also absent and L1 is covalently attached to the therapeutic agent, and when L2 is present, L2 is covalently attached to the therapeutic agent.

[0235] In some implementations, L1 includes C 1-6 Alkyl, phenyl, -NH-, -C(O)-, -(CH2) u -NH-C(O)-、-(CH2) u -C(O)-NH-、-(CH2-CH2-O) v -、-(CH2) u -(O-CH2-CH2) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof; each of which may optionally be substituted by one or more of -S-, -S(O2)-, -C(O)-, -C(O2)- and CO2H; wherein the subscripts u and v are independent integers from 1 to 8.

[0236] In one implementation, L1 is .

[0237] In some implementations, B has a structure selected from the group consisting of: , , , and , where Q is C or N.

[0238] In some implementations, L2 has the following structure: -SP1-AA-SP2- (L2), where: SP1 is absent or is the first spacer unit; AA either does not exist or contains peptide units of 2 to 4 amino acids; SP2 either lacks a second spacer unit or is covalently attached to the therapeutic agent.

[0239] In some implementations, SP1 is absent or selected from the following groups: , , , , C 1-6 Alkyl group, -(CH2-CH2-O) v -、-(CH2-CH2-O) v -(CH2) u -、-(CH2-CH2-O) v -(CH2) u -C(O), -O-CH2-C(O)-NH, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -C(O)-NH, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -、-NH-(CH2) u -C(O)-、-NH-(CH2-CH2-O) v -、-NH-(CH2-CH2-O) v -C(O)-、-NH-(CH2-CH2-O) v -(CH2) u -、-NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u-NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof; where the subscripts u and v are independent integers from 1 to 8.

[0240] In some embodiments, the amino acid is a peptide unit comprising two to four amino acids selected from the following: glycine, valine, phenylalanine, proline, glutamic acid, lysine, N,N-dipropyllysine, phenylalanine, and citrulline, and combinations thereof.

[0241] In some embodiments, AA is valine-citrulline, valine-alanine, valine-lysine, valine-N,N-dipropyllysine, phenylalanine-lysine, glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO:289)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO:290)), L-glutamic acid-valine-citrulline ( L EVC) and D-glutamic acid-valine-citrulline ( D EVC).

[0242] In some implementations, SP2 is absent or selected from the following groups: , , , and their combinations.

[0243] In some implementations, the connector-therapeutic agent (LP) has a structure selected from the group consisting of:

[0244] In another aspect, this disclosure provides compounds according to formula (L2-P) or (L2'-P): B”-SP1-AA-SP2-P (L2-P), H2N-SP1-AA-SP2-P) p (L2'-P), Or its pharmaceutically acceptable salt, wherein: B" is selected from the following groups: -N3, , , , and ; SP1 either does not exist or is selected as the first spacer unit of the following group: and C 1-6 Alkyl group, -(CH2-CH2-O) v -、-(CH2-CH2-O) v -(CH2) u -、-(CH2-CH2-O) v -(CH2) u -C(O), -O-CH2-C(O)-NH, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -C(O)-NH, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -、-NH-(CH2) u -C(O)-、-NH-(CH2-CH2-O) v -、-NH-(CH2-CH2-O) v -C(O)-、-NH-(CH2-CH2-O) v -(CH2) u -、-NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof; where the subscripts u and v are independent integers from 1 to 8; AA either does not exist or contains peptide units of 2 to 4 amino acids; SP2 either does not exist or is selected from the following groups of second spacer units: , , , , and their combinations; and P is a therapeutic agent selected from the group consisting of: having a structure DHT and with selectivity and The structure of the group composed of proDHT, R1 is selected from the following groups: H, C 1-6 Alkyl group, (CH2)0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R2 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R3 is selected from the following groups: H and C 1-3 Alkyl, and R4 is selected from the following groups: H and C 1-3 alkyl.

[0245] In one embodiment, the compound according to formula (L2-P) or (L2'-P) has a structure selected from the group consisting of:

[0246] In one particular embodiment, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprising an antigen-binding protein as described above conjugated to a linker-payload selected from the group consisting of: and .

[0247] In one respect, this disclosure provides antibody-drug conjugates having structures selected from the group consisting of:

[0248] Where Ab is the anti-hCAGNG1 antigen-binding protein as described above, and n is an integer from 1 to 4.

[0249] ADC fabrication The antibody-drug conjugates described herein can be prepared using conjugation conditions known to those skilled in the art (see, for example, Doronina et al.). Nature Biotechnology2003, 21, 7, 778 (This document is incorporated herein by reference in its entirety). In some embodiments, anti-hCACNG1 antigen-binding protein drug conjugates are prepared by contacting an anti-hCACNG1 antigen-binding protein, as described herein, with a compound comprising a desired linker and a therapeutic agent, wherein the linker has a portion that reacts with the antibody or antigen-binding protein, for example, at a desired residue of the antibody or antigen-binding protein.

[0250] In some embodiments, this document provides a method for preparing antibody-drug conjugates, comprising causing an anti-hCACNG1 antigen-binding protein (including an azide-functionalized anti-hCACNG1 antigen-binding protein) as described herein to react with a drug having the following formula A 1 The compound comes into contact with transglutaminase:

[0251] A1, See, for example, U.S. Patent No. 9,676,871, which is incorporated herein by reference in its entirety. 1 The image shows the cathepsin cleavage site.

[0252] In some embodiments, the protein-drug conjugates of this disclosure are generated according to a two-step method, wherein step 1 is a transglutaminase-mediated site-specific conjugation and step 2 is a therapeutic agent (payload) conjugation reaction (e.g., a 1,3-cycloaddition reaction).

[0253] Step 1: Transglutaminase-mediated site-specific conjugation In some embodiments, the anti-hCACNG1 antigen-binding protein of this disclosure can be modified according to known methods to provide a glutamine acyl-modified protein. Techniques for conjugating antibodies and primary amine compounds are known in the art. This document employs a site-specific conjugation technique, using glutamine conjugation via glutamine transaminase to guide conjugation with glutamine (see, e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0254] The primary amine-containing compounds of this disclosure (e.g., linker L1) can be conjugated to one or more glutamine residues of an antigen-binding protein (e.g., anti-hCACNG1 antigen-binding protein) via transglutaminase-based chemical-enzymatic conjugation (see, for example, Dennler et al.). Protein Conjugate Chem(2014, 25, 569-578, and WO2017 / 147542). For example, in the presence of transglutaminase, one or more glutamine residues of the antibody may be conjugated to a primary amine linker compound. In short, in some embodiments, in the presence of transglutaminase, an anti-hCACNG1 antigen-binding protein having glutamine residues (e.g., Gln295, i.e., Q295 residues) according to this disclosure is treated with a primary amine-containing linker L1 as described above. In some embodiments, the antigen-binding protein is non-glycosylated. In some embodiments, the antigen-binding protein is deglycosylated.

[0255] In some embodiments, the antigen-binding protein includes at least one glutamine residue in at least one polypeptide chain sequence. In some embodiments, the binder comprises two heavy chain polypeptides, each having a Gln295 residue. In other embodiments, the binder includes one or more glutamine residues at sites other than heavy chain 295.

[0256] In some embodiments, conjugates such as antibodies can be prepared by site-directed mutagenesis to insert glutamine residues at a specific site without causing antibody function or binding failure. For example, this includes antibodies carrying one or more Asn297Gln (N297Q) mutations as described herein. In some embodiments, antibodies having Gln 295 residues and / or N297Q mutations contain one or more additional naturally occurring glutamine residues in their variable region, which can be accessed by transglutaminase and thus conjugate to a linker or linker-payload. Exemplary naturally occurring glutamine residues may be present, for example, at Q55 of the light chain. In such cases, conjugates such as antibodies conjugated via transglutaminase may have a higher than expected LAR value (e.g., a LAR higher than 4). Any such antibody can be isolated from natural or artificial sources.

[0257] In some embodiments of this disclosure, the adapter-antibody ratio, or LAR, is 1, 2, 3, 4, 5, 6, 7, or 8 adapter L1 molecules per antibody. In some embodiments, the LAR is from 1 to 8. In some embodiments, the LAR is from 1 to 6. In some embodiments, the LAR is from 2 to 4. In some cases, the LAR is from 2 to 3. In some cases, the LAR is from 0.5 to 3.5. In some embodiments, the LAR is about 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5. In some embodiments, the LAR is 2. In some embodiments, the LAR is 4.

[0258] Step 2: Payload coupling reaction In some embodiments, the linker L1 according to this disclosure includes at least one reactive group B' capable of further reaction after transglutamination. In these embodiments, the glutamine acyl-modified antigen-binding protein can be further reacted with a reactive payload compound or a reactive linker-payload compound (e.g., L2-P as disclosed herein) to form an antigen-binding protein-payload conjugate. More specifically, the reactive linker-payload compound L2-P may include a reactive group B' capable of reacting with the reactive group B' of linker L1. In some embodiments, the reactive group B' according to this disclosure includes a moiety capable of undergoing a 1,3-cycloaddition reaction. In some embodiments, the reactive group B' is an azide. In some embodiments, the reactive group B' includes an alkyne (e.g., a terminal alkyne or an internally strained alkyne). In some embodiments of this disclosure, the reactive group B' is compatible with the antigen-binding protein and transglutamination reaction conditions.

[0259] In some embodiments of this disclosure, the connector L1 molecule contains one reactive group B'. In some embodiments of this disclosure, the connector L1 molecule contains more than one reactive group B'.

[0260] In some embodiments, the reactive connector-payload L2-P comprises one payload molecule (n=1). In some other embodiments, the reactive connector-payload L2-P comprises two or more payload molecules (n≥2). In some embodiments, the reactive connector-payload L2-P comprises from 1 to 12 payload molecules, or from 1 to 10 payload molecules, or from 1 to 8 payload molecules, or from 1 to 6 payload molecules, or from 1 to 4 payload molecules, or from 1 to 2 payload molecules.

[0261] In some embodiments, the reactive linker-loador L2-P contains a single load molecule. When such an L2-P reacts with Ab-L1-B', the DAR will be approximately equal to the LAR of Ab-L1-B'. For example, if an L2-P containing a single load molecule reacts with Ab-L1-B having a LAR of 4 (e.g., transglutamination via Q295 and N297Q), the resulting protein-drug conjugate will have a DAR of 4.

[0262] In some embodiments, the reactive linker-loador L2-P contains two load molecules. When such an L2-P reacts with Ab-L1-B', the DAR will be approximately twice that of the LAR of Ab-L1-B'. For example, if an L2-P containing two load molecules reacts with Ab-L1-B' having a LAR of 4 (e.g., transglutamination via Q295 and N297Q), the resulting protein-drug conjugate will have a DAR of 8.

[0263] In some embodiments of this disclosure, the drug-to-antibody ratio, or DAR (e.g., abbreviated as lowercase n), is from about 1 to about 24, or from about 1 to about 20, or from about 1 to about 16, or from about 1 to about 12, or from about 1 to about 10, or from about 1 to about 8, or about 1, 2, 3, 4, 5, 6, 7, or 8 payload molecules per antibody. In some embodiments, the DAR is from 1 to 24. In some embodiments, the DAR is from 1 to 16. In some embodiments, the DAR is from 1 to 8. In some embodiments, the DAR is from 1 to 6. In some embodiments, the DAR is from 2 to 4. In some cases, the DAR is from 2 to 3. In some cases, the DAR is from 0.5 to 3.5. In some cases, the DAR is from 10 to 14. In some cases, the DAR is from 14 to 18. In some cases, the DAR is from 20 to 24.5. In some embodiments, the DAR is about 1, or about 1.5, or about 2, or about 2.5, or about 3, or about 3.5. In some embodiments, the DAR is 2. In some embodiments, the DAR is 4. In some embodiments, the DAR is 8. In some embodiments, the DAR is 12. In some embodiments, the DAR is 16. In some embodiments, the DAR is 24.

[0264] In one aspect, this disclosure provides a method for producing a compound having a structure according to formula (A) or a pharmaceutically acceptable salt thereof: Ab-(L1-B-L2-P) n (A), in: Ab is the anti-hCACNG1 antigen-binding protein described in this article; L1 is the first linker that covalently binds to the side chain of the glutamine residue of Ab; B is the portion containing triazole; L2 is the second linker that covalently binds to the therapeutic agent P; P is a therapeutic agent selected from the group consisting of DHT and proDHT, and n is an integer from 1 to 8, where the method includes the following steps: a) In the presence of transglutaminase, contact A, which contains at least one glutamine residue, with at least one compound L1-B'; b) Contact the product of step a) with one or more equivalent amounts of compound B”-L2-P, wherein group B” is covalently attached to group B’. One of the groups B' and B" is selected from -N3 and ; and another of the groups B' and B” is selected from , ,and Where Z is C or N; and c) The resulting compound of formula (A) was isolated.

[0265] In one embodiment, A has glutamine residues at positions 295 (Q295) and 297 (N297Q).

[0266] In one implementation, L1-B' has a structure selected from the group consisting of:

[0267] In one implementation, L1-B' has a structure .

[0268] In one embodiment, compound B”-L2-P has a structure selected from the group consisting of:

[0269] In one embodiment, the compound of formula (A) has a structure selected from the group consisting of:

[0270] On the other hand, this disclosure provides a method for generating an antigen-binding protein-drug conjugate in a single step. In such a method, in the presence of transglutaminase, the antigen-binding protein according to this disclosure (i.e., the anti-hCACNG1 antigen-binding protein described herein) is treated with a primary amine-containing linker-loador LP as described above. In some embodiments, the antigen-binding protein is non-glycosylated. In some embodiments, the antigen-binding protein is deglycosylated.

[0271] In one aspect, this disclosure provides a method for producing compounds having a structure according to formula (I): A – [L – P] y (I), Where A is an antigen-binding protein; L stands for connector; P is a therapeutic agent selected from the group consisting of DHT and proDHT, and y is an integer from 1 to 8, where the method includes the following steps: c) In the presence of transglutaminase, contacting A, which contains at least one glutamine residue, with at least one compound LP, wherein said compound LP has at least one terminal amine moiety, and d) The compound of formula (I) produced by separation.

[0272] In one embodiment, compound LP has a structure selected from the group consisting of:

[0273] Therapeutic preparations and administration This article also describes pharmaceutical compositions comprising antigen-binding molecules as described herein. In some embodiments, the pharmaceutical composition may be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in all formularies known to medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol in various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), J Pharm Sci Technol 52:238-311.

[0274] The dosage of the antigen-binding molecule administered to a patient can vary depending on the patient's age and size, target disease, condition, route of administration, etc. Preferred dosages are typically calculated based on body weight or body surface area. When the antigen-binding molecule described herein is used for therapeutic purposes in adult patients, it may be advantageous to administer the antigen-binding molecule described herein in a single intravenous 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. The frequency and duration of treatment can be adjusted according to the severity of the condition. Effective dosages and regimens for administering bispecific antigen-binding molecules can be determined empirically; for example, by periodically evaluating and monitoring patient progress and adjusting the dosage accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8 :1351).

[0275] Various delivery systems are known and can be used to administer pharmaceutical compositions as described herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered via any convenient route, such as by infusion or rapid concentration, by absorption through mucosal epithelium or mucosal lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0276] The pharmaceutical compositions described herein can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, regarding subcutaneous delivery, pen delivery devices are readily applicable in delivering pharmaceutical compositions as described herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. After all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. After the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0277] Many reusable pens and autoinjector delivery devices have applications in the subcutaneous delivery of pharmaceutical compositions as described herein. Examples include, but are not limited to, a few examples only, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lillyand Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of single-use pen delivery devices used in the subcutaneous delivery of the pharmaceutical compositions described herein include, but are not limited to, to name just a few, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA™ pen (Abbott Labs, Abbott ParkIL).

[0278] In some cases, the pharmaceutical composition can be delivered using a controlled-release system. In one embodiment, a pump can be used (see Langer, above; Sefton, 1987, CRC Crit. Ref. Biomed.Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115–138). Other controlled-release systems are discussed in a review in Langer, 1990, Science 249:1527–1533.

[0279] Injectable products can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable products can be prepared by methods known to the public. For example, injectable products can be prepared by, for instance, dissolving, suspending, or emulsifying the antibodies or their salts described above in a sterile aqueous medium or an oily medium conventionally used for injection. As an aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As an oily medium, sesame oil, soybean oil, etc., are used, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. Injectable preparations thus prepared are preferably filled in suitable ampoules.

[0280] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared in dosage forms suitable for the dosage of the active ingredient. Such dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is typically from about 5 mg to about 500 mg per dosage form; particularly in the form of injections, it is preferable to contain from about 5 mg to about 100 mg of the antibody, and for other dosage forms, from about 10 mg to about 250 mg.

[0281] Its therapeutic and diagnostic uses This document also discloses methods comprising administering a therapeutic composition to a subject in need of the treatment, the therapeutic composition comprising an anti-hCACNG1 antibody, its antigen-binding fragment, or an antibody-drug conjugate comprising an anti-hCACNG1 antibody (e.g., an anti-hCACNG1 antibody or an ADC comprising any HCVR / LCVR or CDR sequence listed in Table 1 of this document). The therapeutic composition may comprise any anti-hCACNG1 antibody, its antigen-binding fragment, or an ADC disclosed herein, and a pharmaceutically acceptable carrier or diluent.

[0282] Antibodies, their antigen-binding fragments, or antibody-drug conjugates containing anti-hCACNG1 antibodies, as described herein, are particularly useful for the treatment, prevention, and / or improvement of any disease or disorder associated with skeletal muscle tissue. For example, antibodies and ADCs as described herein can be used to treat muscle atrophy disorders (e.g., cachexia, glucocorticoid-induced muscle loss, heart failure-induced muscle loss, HIV atrophy, disuse, aging, etc.) and / or muscle dystrophy / myopathy.

[0283] Anti-hCACNG1 antibodies as described herein have a variety of 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 a reagent for identifying / labeling skeletal muscle fibers. Various diagnostic and prognostic assay techniques known in the art can be used, such as competitive binding assays in heterogeneous or homogeneous phases, direct or indirect sandwich assays, and immunoprecipitation assays (Zola (1987) Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., pp. 147-1581). The antibody used in the assay can be labeled with a detectable portion. The detectable portion should be able to generate a detectable signal directly or indirectly. Any method known in the art for conjugating an antibody to a detectable portion can be employed.

[0284] In another embodiment, a method for treating diseases such as muscular dystrophy is provided. The method may include the step of providing a subject requiring the treatment with an antibody as described above or a CACNG1 antigen-binding fragment thereof.

[0285] Example 1: Exemplary CACNG1 antibody Generation of anti-human CACNG1 antibodies Anti-human CACNG1 antibodies were obtained by immunizing mice with human CACNG1 (e.g., engineered mice containing DNA encoding the variable regions of the human immunoglobulin heavy chain and the human κ light chain).

[0286] Following immunization, spleen cells were harvested from each mouse and (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells for screening for specificity against human CACNG1, or (2) B cells were sorted using a fragment of human CACNG1 as a sorting reagent to bind and identify reactive antibodies (antigen-positive B cells) (as described in US 2007 / 0280945A1).

[0287] The human CACNG1 chimeric antibody, which initially has a human variable region and a mouse constant region, was initially isolated, for example, using the VELOCIMMUNE technique described in U.S. Patent Nos. 7,105,348, 8,642,835, and 9,622,459, each of which is incorporated herein by reference.

[0288] In some antibodies, for testing purposes, the mouse constant region is replaced with a desired human constant region, such as wild-type human CH or modified human CH (e.g., IgG1, IgG2, or IgG4 isotypes), and a light chain constant region (CL) to generate a fully human anti-hCACNG1 antibody or its antigen-binding portion. While the chosen constant region can vary depending on the specific application, high-affinity antigen binding and target-specific characteristics are present in the variable region.

[0289] Certain biological characteristics of the exemplary anti-human CACNG1 antibody generated according to the method of this embodiment are described in detail in the embodiments listed below.

[0290] The amino acid and nucleic acid sequences of the heavy and light chain variable regions of the anti-hCACNG1 antibody Table 1 lists the sequence identifiers or nucleic acid sequences (NAs) of the heavy or light chain variable regions (HCVR or LCVR, respectively) or heavy or light chain CDRs (HCDR and LCDR, respectively) used to generate the selected anti-hCACNG1 proteins disclosed herein, along with the sequences of the amino acid (AA) sequences in parentheses.

[0291] Table 1: Anti-hCACNG1 sequence identifiers or sequences

[0292]

[0293] Example 2: Binding kinetics of anti-CACNG1 monoclonal antibody to human CACNG1 nanodiscs in Biacore form at 25°C via antigen capture The equilibrium dissociation constant of human CACNG1 nanodisks (human CACNG1 nanodisks) embedded with C-terminal PADRE-Flag-His tags, bound to purified anti-hCACNG1 antibody, was determined using a Biacore T200 instrument with real-time surface plasmon resonance biosensor technology. K D The CM5 Biacore sensor surface was derivatized with a monoclonal mouse anti-His antibody (Cytiva; Marlborough, MA) via amine conjugation. All Biacore binding studies were performed in a buffer (HBS-N++ running buffer) containing 0.01 M HEPES, 0.15 M NaCl, 1 mM CaCl2, and 0.5 mM MgCl2 at pH 7.4. Different concentrations of anti-hCACNG1 antibody (ranging from 300 nM to 12 nM in 5-fold serial dilutions) prepared in HBS-N++ running buffer were injected into the captured human CACNG1 nanodisks at a flow rate of 30 µL / min. Antibody association was monitored for 2 min, while dissociation was monitored for 5 min in HBS-N++ running buffer. At the end of each cycle, the human CACNG1 nanodisk capture surface was regenerated using three 10-second injections of 10 mM Gly at pH 1.5. All binding kinetics experiments were performed at 25 °C.

[0294] Specific SPR-Biacore sensing maps were obtained using a dual-reference procedure. Dual reference was performed by first subtracting the signal from the reference surface (anti-His) from the signal on the experimental surface (anti-His) after each injection, thus removing the contribution of refractive index changes. Furthermore, run buffer injection was performed to allow subtraction of signal changes caused by the dissociation of the captured antibody from the conjugated anti-His surface. Kinetic association was determined by fitting the real-time sensing maps to a 1:1 binding model using Scrubber v2.0c curve fitting software. k a ) and dissociation k d Rate constant. Combined with dissociation equilibrium constant ( K D The dissociation half-life (t½) is calculated from the kinetic rate constant as follows: .

[0295] The kinetic results of the anti-hCACNG1 antibodies are presented in Table 2. As shown in Table 2, all antibodies bound to the human CACNG1 nanodiscs captured on the surface, with some antibodies binding with single-digit nM or triple-digit pM affinity.

[0296] Table 2: Kinetic and equilibrium binding parameters of anti-hCACNG1 antibody with surface-trapped human CACNG1 nanodiscs at 25 °C

[0297] Example 3: Screening and purification of CACNG1 antibodies in vitro and in vitro using human and mouse myotubes Forty-three purified CACNG1 antibodies from two immunization events were screened in vitro using human and mouse myotubes. Live myotubes were incubated with CACNG1 antibodies followed by a secondary assay for fluorophore conjugation to assess antibody binding. Robust binding was demonstrated in human myotubes viable with 25 nM anti-CACNG1 antibody followed by fluorophore conjugation secondary antibody assay, compared to isotype controls. Myotubes were incubated with CACNG1 antibodies followed by incubation with a pyruvate-conjugated secondary antibody to assess antibody internalization via cell killing.

[0298] CACNG1 Hu / Hu Immunostaining of CACNG1 in single muscle fibers and cross-sections of muscle tissue in mice confirmed that CACNG1 is expressed on the cell surface of muscle fibers.

[0299] Example 4: Binding of anti-hCACNG1 monoclonal antibody to mouse or human myotubes, and the effect of binding on calcium flux in human myoblasts. CACNG1 is the γ1 subunit of a skeletal muscle-specific L-type calcium channel (dihydropyridine receptor), although genetic deletion of CACNG1 is not shown to have a significant impact on skeletal muscle function. To determine whether antibodies binding to CACNG1 affect muscle function, calcium flux was measured in human myotubes incubated with CACNG1 antibodies to determine whether these antibodies affect acetylcholine-induced calcium release.

[0300] Human skeletal muscle myoblasts (Cook Myosite, Inc.) were seeded at 10,000 cells / well in 96-well plates and differentiated into myotubes for 7 days. On the last day of differentiation, the medium was replaced in each well with 50 μL of FLIPR Calcium 5 dye containing probenecid (Invitrogen) and 50 μL of assay buffer (0.1% BSA-DMEM). Prior to calcium flux assay, CACNG1 and isotype control antibodies were serially diluted in assay buffer and added to the cells, and incubated at 37°C in a 5% CO2 incubator for 1 hour. Nicardipine hydrochloride (Sigma) was added to untreated wells as a control for calcium channel blockade. After 1 hour, acetylcholine (Sigma) at a final concentration of 20 μM was added to induce myotube calcium release, and the assay was performed on a FLIPR Tetra (Molecular Devices, LLC).

[0301] As expected, nicardipine significantly reduced calcium release from human myotubular tubes, while the CACNG1 antibody or the isotype control antibody had no significant effect on calcium flux. Figure 1 Overall, this demonstrates that the anti-hCACNG1 antibody binds to and internalizes the antibody, but does not block calcium release from cultured human myotubes.

[0302] Example 5: In vitro binding and internalization of CACNG1 antibody in muscle fibers After confirming the binding of CACNG1 antibodies to human myotubes, the binding of antibody subsets to fully mature myofibrils was tested in vitro. This was achieved from wild-type mice, homozygous mice lacking CACNG1 (referred to as CACNG1 knockout mice), or homozygous mice expressing human CACNG1 instead of mouse CACNG1 (referred to as CACNG1 knockout mice). Hu / Hu Individual muscle fibers were isolated from the gastrocnemius muscle. The gastrocnemius muscle was removed, digested with collagenase, and individual muscle fibers were isolated, washed, and incubated overnight at 37°C and 5% CO2 in DMEM + 10% horse serum. After overnight incubation, the individual muscle fibers were incubated for 30 minutes with 100 nM of each CACNG1 antibody or allotype control antibody. The muscle fibers were then washed twice in DMEM + 10% horse serum and subsequently incubated for 30 minutes with 10 μg / mL fluorescently conjugated secondary antibody, washed twice in DMEM + 10% horse serum, and then fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes. The individual fibers were then washed twice with PBS, stained with Hoechst for 5 minutes, washed once more with PBS, transferred to a microscope slide, covered with a coverslip, and imaged using a Zeiss LSM 710 confocal microscope.

[0303] In addition, to determine whether the anti-human CACNG1 antibody, as described herein, could be in vitro fused to myofibrils, the Alexa 647 (A647) fluorophore was directly conjugated to both the CACNG1 antibody and an isotype control antibody. Individual myofibrils were isolated, washed, and incubated overnight. The next day, the myofibrils were incubated with 100 nM A647-conjugated antibody for 30 min, 4 h, or 8 h, and then washed twice with PBS. The myofibrils were then fixed with 4% PFA for 15 min, stained with Hoechst for 5 min, washed once with PBS, transferred to a microscope slide, covered with a coverslip, and imaged using a Zeiss LSM 710 confocal microscope.

[0304] Two CACNG1 antibodies, anti-hCACNG1 Ab 4 and anti-hCACNG1 Ab 1, showed in vitro interaction with CACNG1 Hu / Hu It binds to myofibrils but not to wild-type or CACNG1 knockout myofibrils. The isotype control antibody does not bind to CACNG1.Hu / Hu Muscle fibers or wild-type muscle fibers. Figure 3 ) Single-plane confocal imaging revealed that the fluorophore-conjugated CACNG1 antibody, anti-hCACNG1 Ab2, bound to the surface of myofibrils after 30 minutes of incubation, and was internalized into the myofibrils as early as 4 hours later. Figure 1 ) In summary, CACNG1 antibodies can bind to the surface of individual muscle fibers in vitro. Furthermore, tracking of fluorophore-conjugated CACNG1 antibodies showed initial binding to the muscle fiber surface, followed by internalization into the muscle fiber several hours later.

[0305] Example 6: CACNG1 antibody-DHT conjugate androgen reporter assay CACNG1 is the γ1 subunit of the dihydropyridine receptor, specifically expressed in skeletal muscle. Therefore, antibodies generated against CACNG1 can be used to specifically deliver conjugated therapeutic payloads to skeletal muscle to enhance therapeutic efficacy in muscle and reduce off-target toxicity. For example, conjugation of a CACNG1 antibody to dihydrotestosterone (DHT), a potent metabolite of testosterone, can allow androgen receptor signaling in muscle, leading to increased muscle mass and function. Here, CACNG1 antibodies conjugated to linkers with DHT payloads were tested in androgen receptor (AR) reporter cell lines to determine whether these antibody conjugates could specifically activate AR in CACNG1-expressing cells in vitro.

[0306] To evaluate signaling via AR, LNCaP cells were transfected with lentivirus (Qiagen; ARE.Luc Cignal Lenti) to generate a stable cell line expressing the AR-luciferase reporter (AR.Luc). A subset of these selected cells were transduced to express human CACNG1 and further selected; this cell line was named hCACNG1.AR.Luc.

[0307] For bioassays, AR.Luc or hCACNG1.AR.Luc cells were seeded at 5,000 cells / well in PDL-coated 96-well plates in OptiMEM and 0.5% carbon-peeled FBS. Cells were then incubated for 24, 48, or 72 hours with a CACNG1 antibody conjugated to DHT (via L2 linker-payload), an allotype control antibody, or unconjugated DHT alone. All antibodies were conjugated to DHT at a drug-antibody ratio (DAR) of ~4. After the corresponding time points, cells were lysed and incubated with One-GLO buffer, and luminescence was read on an Envision plate reader. Relative luminescence units (RLU) were plotted against logarithmic concentration in mol / L and adjusted for DAR.

[0308] Unconjugated DHT activated AR.Luc ( Figure 2 ) and hCACNG1.AR.Luc cell line ( Figure 3 The AR in both cell lines is activated, while DHT conjugated with the isotype control antibody (isotype control Ab 1-L2) does not activate AR in either of these cell lines. Several CACNG1 antibody-DHT conjugates activate AR only in the hCACNG1.AR.Luc cell line. Figure 3 AR is not activated in the AR.Luc cell line. Figure 2 Although the efficacy and potency of CACNG1 antibody-DHT conjugates for AR activation were lower than those for unconjugated DHT at 24 hours post-treatment, these conjugates maintained AR activation at 48 and 72 hours, while unconjugated AR signaling was significantly reduced at these time points. Overall, these data indicate that DHT conjugation to CACNG1 antibody allows for specific AR activation in hCACNG1-expressing cells, and that DHT conjugated to CACNG1 antibody maintains sustained AR signaling in vitro for several days in hCACNG1-expressing cells.

[0309] Example 7: Biodistribution within the body To determine whether CACNG1 antibodies could specifically target skeletal muscle in vivo, CACNG1 antibodies (anti-hCACNG1 Ab 1 and anti-hCACNG1 Ab 2) and an isotype control antibody (isotype control Ab 4) were conjugated with Alexa Fluor 647 fluorescent dye, or with a saline control, and injected intravenously into mice (n=1 / group) at a dose of 10 mg / kg. These mice were homozygous for human CACNG1 substitutes for mouse CACNG1 (referred to as CACNG1). Hu / HuSix days post-injection, mice were cryopreserved for systemic antibody distribution analysis using cryo-fluorescence tomography (Invicro). A separate group of mice (n=1 / group) injected with the same antibody (or saline control) were sacrificed six days post-injection and perfused with PBS. The following tissues were harvested for immunofluorescence analysis: tibialis anterior muscle, gastrocnemius / plantar / solecithus complex, diaphragm, tongue, pelvic floor muscles, triceps brachii, trapezius, liver, kidney, spleen, and brown adipose tissue. Tissues were embedded in optical coherence tomography (OCT) compounds, frozen in isopentane cooled in liquid nitrogen, and then frozen sections were mounted onto microscope slides at 10 μm. The tissue sections were then permeabilized with Triton X-100, blocked with 4% BSA, and incubated overnight with a rabbit-derived laminin antibody (Sigma). The following day, the sections were washed, stained with anti-rabbit Alexa 488 secondary antibody (Thermo Fisher), counterstained with Hoescht, washed, fixed with 4% PFA, washed, and mounted with Fluoromount-G (Thermo Fisher). The tissues were then imaged on a Zeiss Axioscan Z1 slide scanner to visualize the tissue distribution of the Alexa 647-conjugated antibody.

[0310] Alexa 647-conjugated CACNG1 antibodies, anti-hCACNG1 Ab 2, and anti-hCACNG1 Ab 1 showed clear signals in various skeletal muscles via cryo-fluorescence computed tomography, while the isotype control antibody showed no muscle uptake and accumulated mostly in the bladder. Figure 4 The saline-treated control group showed no perceptible fluorescent signal throughout the mice. Mice administered the CACNG1 antibody anti-hCACNG1 Ab 2 showed stronger fluorescent signals in muscle compared to anti-hCACNG1 Ab 1, although their overall muscle distribution patterns were similar.

[0311] Fluorescence imaging of tissue sections revealed uptake of Alexa 647-conjugated CACNG1 antibodies anti-hCACNG1 Ab 2 and anti-hCACNG1 Ab 1 in various skeletal muscles, including: gastrocnemius / plantar / solecus complex, diaphragm, tongue, pelvic floor muscles, triceps brachii, and trapezius. Figure 5 Similar to cryo-fluorescence computed tomography (CFCT) results, anti-hCACNG1 Ab2 showed a stronger overall signal in muscle sections compared to anti-hCACNG1 Ab1. Neither anti-hCACNG1 Ab2 nor anti-hCACNG1 Ab1 showed any clear staining in various non-muscle tissues, including: liver, kidney, spleen, and brown adipose tissue. Figure 6 ).

[0312] These in vivo biodistribution studies show that the fluorophore-conjugated CACNG1 antibody specifically targets skeletal muscle and is not absorbed by other non-muscle tissues.

[0313] Example 8: The distribution of CACNG1 antibody into muscle changes due to exercise and dosage To test methods to enhance the distribution of CACNG1 antibodies into muscle, CACNG1 was injected with... Hu / Hu Mice were administered 10 mg / kg or 50 mg / kg of CACNG1 antibody, and a subset of mice was brought close to the treadmill wheel. Figure 7 (See above figure). Wheel movement enhanced the distribution of CACNG1 antibody into the working soleus muscle, and a dose of 50 mg / kg also showed enhanced distribution throughout the soleus muscle. Figure 7 (See the image below).

[0314] Example 9 Payload and Joint - Payload Preparation Exemplary compounds (payload and connector-payload) according to this disclosure are depicted in Table 3 below.

[0315] Table 3. Structures of the compounds

[0316]

[0317]

[0318]

[0319] The chemical properties of exemplary compounds (payload and connector-payload) according to this disclosure are described in Table 4 below.

[0320] Table 4. Payload and Joint - Chemical Properties of Payload:

[0321]

[0322]

[0323] ProDHT: 2-Amino- N -({[(1 S ,2 S 7 S 10 R ,11 S ,14 S 15 S )-2,15-Dimethyl-5-oxotetracyclo[8.7.0.0] 2 ,7 .0 11 , 15 Heptadecan-14-yloxymethylacetamide (ProDHT)

[0324] The synthesis of ProDHT and other AA-ProDHT is described in Schemes 1 and 1a below.

[0325] Option 1. Synthesis of ProDHT

[0326] Option 1a. General synthesis of other AAProDHT

[0327] DHT (0.40 g, 1.4 mmol), compound 1 (0.51 g, 1.4 mmol), PPTS (36 mg, 0.14 mmol), and DCM (8 mL) were added to a sealed tube. The tube was sealed, and the reaction mixture was stirred at 50 °C for 16 hours, as monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (ammonium bicarbonate (10 mM) aqueous solution in 5%–95% acetonitrile) to give compound 2 (0.52 g, ESI m / z 621 (M + Na)) as a white solid. + The solution was dissolved in DMF (5 mL). Diethylamine (0.32 g, 4.3 mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 2 hours until Fmoc was completely removed according to LCMS. The resulting mixture was directly separated by reversed-phase rapid chromatography (5%-95% acetonitrile in aqueous ammonium bicarbonate (10 mM)) to give ProDHT (0.30 g, 58% yield) as a white solid. ESI m / z 399 (M + Na) + . 1 H NMR (400 MHz, DMSO d6 ) δ8.60-8.41 (m, 1H), 4.69-4.48 (m, 2H), 3.57-3.39 (m, 4H), 3.14 (s, 1H), 2.46-2.36 (m, 1H), 2.34-2.25 (m, 1H), 2.12-1.82(m, 4H), 1.79-1.71 (m, 1H), 1.65-1.58 (m, 1H), 1.56-1.03 (m, 12H), 0.97 (s,3H), 0.94-0.63 (m, 2H), 0.63 (s, 3H) ppm.

[0328] LP1 The synthesis of LP1 is described in Scheme 2 below.

[0329] Scheme 2. Synthesis of LP1

[0330] {4-[(2S)-2-[(2S)-2-amino-3-methylbutyramido]-5-(carbamoylamino)pentamido]phenyl}methylN-{2-[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}carbonyl)(methyl)amino]ethyl}-N-methylcarbamate (5)

[0331] A mixture of DHT (1.2 g, 4.1 mmol) and 4-nitrophenyl chloroformate (0.89 g, 4.4 mmol) in DCM (15 mL) was cooled to 0 °C, and then pyridine (0.38 g, 4.8 mmol) was added to the mixture at this temperature. The reaction mixture was stirred at room temperature for 16 h, as monitored by LCMS. The resulting mixture was diluted with DCM (150 mL), washed with saturated aqueous sodium bicarbonate solution (30 mL) and brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel rapid chromatography (30%–100% DCM in petroleum ether) to give compound 3 (1.6 g) as a grayish-white solid.

[0332] Add to a solution of compound 3 (0.91 g, 2.0 mmol) in DCM (10 mL) N -Boc- N , N'-Dimethylethylamine (0.75 g, 4.0 mmol) and DIPEA (0.52 g, 4.0 mmol) were added, and the reaction mixture was stirred at room temperature for 16 hours, as monitored by LCMS. The resulting mixture was diluted with DCM (150 mL), washed successively with aqueous citric acid solution (1 M, 40 mL × 2), saturated sodium bicarbonate solution (40 mL × 2), and saturated brine (40 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to give compound 4 (0.50 g) as a grayish-white solid.

[0333] TFA (0.5 mL) was added to a solution of compound 4 (0.20 g, 0.40 mmol) in DCM (5 mL), and the mixture was stirred at room temperature for 3 hours. The resulting mixture was concentrated under vacuum. The residue was dissolved in DMF (3 mL), and Fmoc-vcPAB-PNP (CAS: 863971-53-3, 0.30 g, 0.39 mmol) and DIPEA (0.5 mL) were added to the solution. The reaction mixture was stirred at room temperature for 16 hours, as monitored by LCMS. Piperidine (0.2 mL) was then added to the resulting solution in a single step, and the mixture was stirred at room temperature for another 2 hours until Fmoc was completely removed according to LCMS. The mixture was directly separated by preparative HPLC (TFA aqueous solution (0.1%) in 5%–95% acetonitrile) to give compound 5 as a white solid (50 mg, 6.0% of DHT in total yield). ESI m / z 810.3 (M + H) + .

[0334] {4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclic[10.4.0.0]} 4 , 9 [Hexadecane-1(12),4(9),5,7,13,15-hexen-10-yn-2-yl}-4-oxobutyramido)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutyramido]-5-(carbamoylamino)pentamido]phenyl}methyl N-{2-[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}carbonyl)(methyl)amino]ethyl}-N-methylcarbamate (LP1)

[0335] A solution of compound 5 (0.25 g, 31 μmol) in DMF (0.55 mL) was added with DIBAC-PEG4-acid (CAS: 1537170-85-6, 20 mg, 37 μmol), DIPEA (19 mg, 0.15 mmol), and HATU (17 mg, 45 μmol), and the reaction mixture was stirred at room temperature for 16 hours, as monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (5%–95% acetonitrile in aqueous bicarbonate solution (10 mM)) to give LP1 (13 mg, 31% yield) as a white solid. ESI m / z 672.9 (M / 2 + H) + . 1 H NMR (400 MHz, DMSO d6 ) δ 9.98 (s, 1H), 8.12 (d, J = 7.3 Hz, 1H), 7.91-7.24 (m, 13H), 5.98 (t, J = 5.5 Hz, 1H), 5.41 (s,2H), 5.08-4.88 (m, 3H), 4.45-4.15 (m, 3H), 3.64-3.55 (m, 3H), 3.50-3.42 (m,9H), 3.31-3.28 (m, 2H), 3.14-2.89 (m, 4H), 2.88-2.65 (m, 5H), 2.64-2.16 (m,20H), 2.13-1.83 (m, 6H), 1.81-1.15 (m, 14H), 1.01-0.63 (m, 14H) ppm.

[0336] LP2 and LP2A The synthesis of LP2 is described in Scheme 3, and the synthesis of LP2A is described in Scheme 3a below.

[0337] Scheme 3. Synthesis of LP2

[0338] Scheme 3A. Synthesis of LP2A

[0339] {4-[(2S)-2-[(2S)-2-amino-3-methylbutyramido]-5-(carbamoylamino)pentamido]phenyl}methylN-{[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxymethyl)carbamoyl]methyl]carbamate (6)

[0340] HOBt (18 mg, 0.13 mmol) and DIPEA (0.11 g, 0.81 mmol) were added to a solution of ProDHT (0.10 g, 0.27 mmol) in DMF (5 mL), and the mixture was stirred at room temperature for 10 min, followed by the addition of Fmoc-vcPAB-PNP (0.21 g, 0.27 mmol). The reaction mixture was stirred at room temperature for 4 h, as monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (TFA aqueous solution (0.1%) in 5%–95% acetonitrile) to give a white solid (0.17 g, ESI m / z 1027.5 (M + Na)). + The compound was dissolved in DMF (5 mL). Diethylamine (60 mg, 0.82 mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 2 hours, as monitored by LCMS. The resulting mixture was purified directly by reversed-phase rapid chromatography (0-100% acetonitrile in aqueous ammonium bicarbonate solution (10 mM)) to give compound 6 as a white solid (0.11 g, 52% yield). ESI m / z 783 (M + H) + .

[0341] {4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclic[10.4.0.0]} 4 , 9 [Hexadecane-1(12),4(9),5,7,13,15-hexen-10-yn-2-yl}-4-oxobutyramido)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutyramido]-5-(carbamoylamino)pentamido]phenyl}methyl N-{[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 ,15 [Heptadecanyl-14-yl]oxymethyl]carbamoyl]methyl]carbamate (LP2)

[0342] DIBAC-PEG4-NHS (0.14 g, 0.14 mmol) and DIPEA (53 mg, 0.41 mmol) were added to a stirred solution of compound 6 (0.11 g, 0.14 mmol) in DMF (4 mL), and the reaction mixture was stirred at room temperature for 1 h, as monitored by LCMS. The resulting mixture was directly separated by reversed-phase rapid chromatography (5%–95% acetonitrile in aqueous ammonium bicarbonate solution (10 mM)) to give LP2 (90 mg, 51% yield) as a white solid. ESI m / z 659 (M / 2 + H) + . 1 H NMR (400 MHz, DMSO d6 ) δ 10.00 (s, 1H), 8.56 (t, J = 6.7 Hz, 1H), 8.14(d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.78 (t, J = 5.5 Hz, 1H), 7.68 (dd, J = 7.3, 1.4 Hz, 1H), 7.65-7.58 (m, 3H), 7.52-7.39 (m, 4H), 7.38-7.33(m, 2H), 7.31-7.27 (m, 2H), 5.98 (t, J = 6.0 Hz, 1H), 5.42 (s, 2H), 5.03 (d, J= 14.0 Hz, 1H), 4.96 (s, 2H), 4.60-4.51 (m, 1H), 4.50-4.45 (m, 1H), 4.42-4.26 (m, 1H), 4.23-4.20 (m, 1H), 3.67-3.55 (m, 5H), 3.52-3.42 (m, 11H), 3.30-3.15 (m, 2H), 3.13-2.89 (m, 5H), 2.70-2.20 (m, 6H), 2.15-1.65 (m, 8H), 1.58-1.05 (m, 21H), 0.96 (s, 3H), 0.84 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.67 (s, 3H) ppm.

[0343] LP3A, LP3, LP31, LP32 and LP33 The synthesis of LP3A and LP3 is described in Scheme 4 below. The synthesis of LP31, LP32 and LP33 is described in Scheme 4a below.

[0344] Scheme 4. Synthesis of LP3

[0345] Scheme 4a. Synthesis of LP31, LP32 and LP33

[0346] Methyl(4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}methyl)carbamoyl]methyl}carbamoyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]-4-[1-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecanyl-15-amido]butyrate (7)

[0347] HOBt (6.0 mg, 44 μmol) and DIPEA (35 mg, 0.27 mmol) were added to a solution of ProDHT (35 mg, 93 μmol) in DMF (5 mL), and the mixture was stirred at room temperature for 10 min. Then, Fmoc-PEG4-Glu(OMe)-vcPAB-PNP (CAS: 2758874-82-5, synthesized according to WO2022015656, 0.11 g, 95 μmol) was added. The reaction mixture was stirred at room temperature for 4 h, as monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (TFA aqueous solution (0.1%) in 5%–95% acetonitrile) to give compound 7 (0.11 g, 85% yield) as a white solid. ESI m / z 1396 (M + H) + .

[0348] (4S)-4-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]oxy]methyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]butyric acid (LP3A)

[0349] A solution of compound 7 (0.11 g, 77 μmol) in THF (5 mL) was added to an aqueous solution of lithium hydroxide (1 M, 0.38 mL), and the mixture was stirred at room temperature for 2 hours, as monitored by LCMS. Volatile substances were removed under vacuum, and the residue was separated by reversed-phase rapid chromatography (5%–95% acetonitrile in TFA aqueous solution (0.01%)) to give compound LP3A (78 mg, 88% yield) as a white solid. ESI m / z 1160 (M + H) + .

[0350] (4S)-4-[1-(4-{2-azatricyclic[10.4.0.0]] 4 , 9[Hexadecane-1(12),4(9),5,7,13,15-hexen-10-yn-2-yl}-4-oxobutamido)-3,6,9,12-tetraoxapentadecan-15-amido]-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]oxy]methyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]butyric acid (LP3)

[0351] DIBAC-OSu (CAS: 1353016-71-3, commercially available, 41 mg, 67 μmol) and DIPEA (26 mg, 0.20 mmol) were added sequentially to a stirred mixture of LP3A (78 mg, 67 μmol) in DMF (4 mL), and the reaction mixture was stirred at room temperature for 1 hour, as monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (ammonium bicarbonate aqueous solution (10 mM) in 5%–95% acetonitrile) to give LP3 (30 mg, 31% yield) as a white solid. ESI m / z 723 (M / 2 + H) + . 1 H NMR (400 MHz, DMSO d6 ) δ 10.00 (s, 1H), 8.56 (t, J = 6.7 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.83-7.70 (m, 1H), 7.68(dd, J = 7.3, 1.4 Hz, 1H), 7.65-7.55 (m, 3H), 7.52-7.25 (m, 8H), 6.01 (br s,1H), 5.42 (s, 2H), 5.03 (d, J= 14.0 Hz, 1H), 4.96 (s, 2H), 4.60-4.51 (m,1H), 4.50-4.45 (m, 1H), 4.42-4.26 (m, 1H), 4.23-4.20 (m, 1H), 3.67-3.55 (m,5H), 3.52-3.20 (m, 15H), 3.13-2.89 (m, 5H), 2.70-2.40 (m, 9H), 2.30-1.84 (m,8H), 1.80-1.05 (m, 22H), 0.96 (s, 3H), 0.85 (d, J = 6.7 Hz, 3H), 0.83 (d, J =6.7 Hz (3H), 0.67 (s, 3H) ppm. (COOH not disclosed.) LP31, LP32 and LP33 (4S)-4-{1-[({[(1R,8S,9R)-bicyclo[6.1.0]non-4-yn-9-yl]methoxy}carbonyl)amino]-3,6,9,12-tetraoxapentadecan-15-amido}-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]oxy]methyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]butyric acid (LP31)

[0352] Except for the use of BCN-OSu instead of DIBAC-OSu, the joint-load LP31 (1.2 mg, 18% yield) was obtained as a white solid following a similar procedure to LP3. ESI m / z: 667.9 (M / 2 + H)+.

[0353] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 ,15 [Heptadecanyl-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]oxy]methyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamitoyl]-3,6,9,12-tetraoxapentadecanyl-15-amido]butyric acid (LP32)

[0354] Except for the use of COT-OSu instead of DIBAC-OSu, following a similar procedure to LP3, the junction-load LP32 (2.2 mg, 33% yield) was obtained as a white solid. ESI m / z: 661.9 (M / 2 + H)+.

[0355] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]oxy]methyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]-4-{1-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetamyl]-3,6,9,12-tetraoxapentadecanyl-15-amido]butyric acid (LP33)

[0356] Except for the use of AMAS instead of DIBAC-OSu, a similar procedure to that for LP3 was followed to obtain the junction-load LP33 (0.9 mg, 14% yield) as a white solid. ESI m / z: 648.3 (M / 2 + H)+.

[0357] LP4 The synthesis of LP4 is described in Scheme 5 below.

[0358] Scheme 5. Synthesis of LP4

[0359] tert-Butyl(4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butyrate (9)

[0360] HATU (0.38 g, 1.0 mmol) and DIPEA (0.26 g, 2.0 mmol) were added to a solution of compound Fmoc-PEG4-acid (CAS: 557756-85-1, 0.49 g, 1.0 mmol) in DMF (5 mL), and the reaction mixture was stirred at room temperature for 5 min. Compound 8 (CAS: 2757059-05-3, synthesized according to WO2021262910, 0.56 g, 1.0 mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 2 h, as monitored by LCMS. The resulting reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (100 mL x 2). The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to give crude compound 9, which was purified by silica gel rapid chromatography (8%–10% methanol in DCM) to give compound 9 as a colorless oil (0.50 g, 48% yield). ESI m / z: 1034.3 (M + H) + .

[0361] tert-Butyl(4S)-4-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butyrate (10)

[0362] To a solution of compound 9 (0.50 g, 0.48 mmol) in DMF (5 mL), piperidine (82 mg, 0.96 mmol) was added, and the reaction mixture was stirred at room temperature for 4 h, as monitored by LCMS. The resulting mixture was purified directly by reversed-phase rapid chromatography (TFA aqueous solution (0.03%) in 5%–95% acetonitrile) to give compound 10 (0.30 g, 67% yield, TFA salt) as a colorless oil. ESI m / z: 812.9 (M + H) + .

[0363] tert-Butyl(4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(1-{2-[4-(6-methyl-1,2,4,5-tetraazin-3-yl)phenyl]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido)butyrate (11)

[0364] Compound 10 (93 mg, 0.10 mmol, TFA salt) and DIPEA (26 mg, 0.20 mmol) were added to a solution of MeTz-PhAc-NHS (CAS: 1644644-96-1, 33 mg, 0.10 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature for 2 h, as monitored by LCMS. The resulting mixture was purified directly by reversed-phase rapid chromatography (TFA aqueous solution (0.03%) in 5%–95% acetonitrile) to give compound 11 (80 mg, 78% yield) as a red solid. ESI m / z: 1023.5 (M + H) + .

[0365] tert-Butyl(4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(1-{2-[4-(6-methyl-1,2,4,5-tetraazin-3-yl)phenyl]acetamido}-3,6,9,12-tetraoxapentadecan-15-amido)butyrate (12)

[0366] A solution of compound 11 (0.10 g, 98 μmol) in DMF (5 mL) was added with DIPEA (26 mg, 0.20 mmol), DMAP (12 mg, 98 μmol), and bis(4-nitrophenyl) carbonate (0.12 g, 0.40 mmol), and the reaction mixture was stirred at room temperature for 1 h, as monitored by LCMS. The resulting mixture was directly separated by reversed-phase rapid chromatography (TFA aqueous solution (0.03%) in 5%–95% acetonitrile) to give compound 12 (60 mg, 51% yield) as a red solid. ESI m / z: 1189.5 (M + H) + .

[0367] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(1-{2-[4-(6-methyl-1,2,4,5-tetraazin-3-yl)phenyl]acetamyl}-3,6,9,12-tetraoxapentadecan-15-amido)butyric acid(13)

[0368] A solution of HCl in ethyl acetate (4 N, 2.5 mL) was added to a solution of compound 12 (60 mg, 51 μmol) in acetonitrile (2.5 mL). The reaction mixture was stirred at room temperature for 1 h, as monitored by LCMS. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution until pH 6–7. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography (TFA aqueous solution (0.03%) in 0–100% acetonitrile) to give compound 13 (30 mg, 51% yield) as a red solid. ESI m / z: 1133.5 (M + H) + .

[0369] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]oxy]methyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]-4-(1-{2-[4-(6-methyl-1,2,4,5-tetraazin-3-yl)phenyl]acetamido]-3,6,9,12-tetraoxapentadecanyl-15-amido)butyric acid (LP4)

[0370] ProDHT (17 mg, 44 μmol), DIPEA (11 mg, 88 μmol), and HOBt (3.0 mg, 22 μmol) were added to a solution of compound 13 (50 mg, 44 μmol) in DMF (5 mL), and the reaction mixture was stirred at room temperature for 2 h, as monitored by LCMS. The resulting mixture was directly separated by reversed-phase rapid chromatography (TFA aqueous solution (0.03%) in 0–100% acetonitrile) to give LP4 (10 mg, 17% yield) as a red solid. ESI m / z: 1371 (M + H) + 1393(M + Na) + . 1 H NMR (400 MHz, DMSO d6 ) δ 10.05 (s, 1H), 8.55 (t, J = 6.0 Hz, 1H), 8.40 (d, J = 8.8 Hz, 2H), 8.30 (t, J = 6.0 Hz, 1H), 8.20 (d, J = 6.0 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 6.0 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H),7.27 (br s, 1H), 6.03-6.00 (m, 1H), 5.52 (s, 2H), 4.97 (s, 2H), 4.60-4.58 (m,1H), 4.55-4.45 (m, 3H), 4.20 (t, J= 6.0 Hz, 1H), 3.65-3.60 (m, 5H), 3.45-3.40 (m, 14H), 3.25-3.20 (m, 4H), 3.00 (s, 3H), 2.45-2.40 (m, 6H), 2.05-1.90(m, 8H), 1.85-1.30 (m, 10H), 1.25-1.20 (m, 8H), 1.00 (s, 3H), 0.90-0.80 (m,8H), 0.80 (s, 3H) ppm.

[0371] LP5A, LP5, LP51 and LP52 The synthesis of LP5 and LP5A is described in Scheme 6 below. The synthesis of LP51 and LP52 is described in Scheme 6a below.

[0372] Scheme 6. Synthesis of LP5A and LP5

[0373] Scheme 6a. Synthesis of LP51 and LP52

[0374] ({[(4-azidophenyl)methoxy]carbonyl}amino)methyl acetate (15)

[0375] To compound 14 (CAS: 179806-96-3, according to J. Chem. SOC., Perkin Trans. I, Synthesized on December 5-11, 1996: Lead tetraacetate (2.7 g, 6.0 mmol) and copper acetate (35 mg, 0.20 mmol) were added to a solution of 0.50 g (2.0 mmol) in THF (15 mL), and the reaction mixture was stirred at room temperature for 4 hours, as monitored by LCMS. The resulting mixture was diluted with water (100 mL) and extracted with ethyl acetate (150 mL x 2). The combined organic solutions were washed with brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography (0-15% ethyl acetate in petroleum ether) to give compound 15 (0.42 g, 80% yield) as a yellow solid. ESI m / z: 287.1 (M+Na) + .

[0376] (4-Azidephenyl)methyl N-({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]) 2 ,7 .0 11 , 15 [Heptadecanyl-14-yl]oxymethyl)carbamate (16)

[0377] A solution of compound 15 (0.18 g, 0.69 mmol) in anhydrous THF (25 mL) was added to a 4A molecular sieve and DHT (0.20 g, 0.69 mmol), and the mixture was stirred at room temperature for half an hour. Then, a solution of bis(trifluoromethane)sulfonamide (CAS: 82113-65-3, 0.27 g, 2.1 mmol) in anhydrous THF (5 mL) was added. The reaction mixture was stirred at room temperature for half an hour, as monitored by LC and TLC. Both LC and TLC showed the formation of a new product, and that DHT was no longer reduced (by LC, the ratio of DHT to the new peak was ~1:3). The reaction mixture was filtered, and the solution was diluted with water (100 mL) and extracted with DCM (100 mL × 3). The combined organic solutions were washed with brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography (0-70% acetonitrile in ammonium bicarbonate aqueous solution (10 mM)) to give compound 16 (0.22 g, 65% yield) as a white solid and recovered DHT (20 mg, 10% recovery yield). ESI m / z: no mass signal.

[0378] 1 H NMR (400 MHz, DMSO d6 ) δ 7.36 (d, J = 4.4 Hz, 2H), 7.01 (d, J = 8.4Hz, 2H), 5.11-5.08 (m, 2H), 4.75-4.64 (m, 2H), 2.44-2.25 (m, 3H), 2.12-1.98(m, 3H), 1.85-1.79 (m, 2H), 1.74-1.66 (m, 1H), 1.59-1.50 (m, 3H), 1.45-1.21(m, 8H), 1.15-1.05 (m, 1H), 1.03-1.00 (m, 3H), 0.96-0.84 (m, 2H), 0.77-0.74(m, 3H) ppm.

[0379] (9H-fluorene-9-yl)methyl N-[(1S)-4-(carbamoylamino)-1-{[4-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamate (17)

[0380] To a solution of compound 16 (0.20 g, 0.40 mmol) in anhydrous THF (30 mL), 1.5 g of 4A molecular sieve and Fmoc-Cit-OPFP (CAS: 1356537-03-5, 0.34 g, 0.60 mmol) were added, and the mixture was stirred at room temperature for half an hour. Trimethylphosphine (1 M in THF, 0.80 mL, 0.80 mmol) was added to the mixture, and the reaction mixture was stirred at room temperature for 1 hour until compound 16 was completely consumed, as monitored by LCMS. The resulting mixture was filtered, and the solution was concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography (ammonium bicarbonate aqueous solution (10 mM) in 0–70% acetonitrile) to give compound 17 (50 mg, 15% yield) as a white solid. ESI m / z: 848.3 (M + H) + (Very weak).

[0381] {4-[(2S)-2-amino-5-(carbamoylamino)pentamido]phenyl}methyl N-({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]) 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxymethyl)carbamate (18)

[0382] Diethylamine (43 mg, 0.59 mmol) was added to a solution of compound 17 (50 mg, 59 μmol) in DMF (2 mL), and the reaction mixture was stirred at room temperature for 2 hours until Fmoc was completely removed, as monitored by LCMS. The resulting mixture was directly separated by reversed-phase rapid chromatography (0–70% acetonitrile in aqueous ammonium bicarbonate solution (10 mM)) to give compound 18 (25 mg, 68% yield) as a white solid. ESI m / z: 626.3 (M + H) + .

[0383] tert-Butyl(4S)-4-{[(2S)-1-[(2,5-dioxopyrrolidone-1-yl)oxy]-3-methyl-1-oxobutane-2-yl]carbamoyl}-4-[1-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butyrate (19)

[0384] To H-Glu(O) t A solution of Bu)-Val-OH (29 g, 56 mmol) in DMF (200 mL) was added to a solution of Fmoc-PEG4-OSu (31 g, 53 mmol) in DCM (200 mL) and DIPEA (7.2 g, 56 mmol, 9.7 mL), and the reaction mixture was stirred at room temperature for 2 hours, as monitored by LCMS. Volatile substances were then removed under vacuum. The residue was diluted with water (100 mL), washed with MTBE (80 mL × 3), and acidified to pH 5 with citric acid. The mixture was extracted with ethyl acetate (120 mL × 2), and the combined organic solutions were washed with brine (60 mL × 2), dried over anhydrous sodium sulfate, and concentrated under vacuum to give Fmoc-PEG4-OSu (O) as a yellow oil. t Bu)-Val-OH (33 g). ESI m / z: 772 (M+ H) + .

[0385] Add DIC (6.5 g, 51 mmol) and the aforementioned Fmoc-PEG4-Glu (O) to a solution of HOSu (7.9 g, 68 mmol) in DMF (150 mL) and DCM (150 mL). tBu)-Val-OH (33 g). The reaction mixture was stirred at room temperature for 12 hours, as monitored by TLC and LCMS. The resulting mixture was filtered, and the filtrate was concentrated under vacuum. The residue was diluted with water (200 mL) and extracted with ethyl acetate (150 mL x 3). The combined organic solutions were washed with brine (150 mL × 3), dried over anhydrous sodium sulfate, and concentrated under vacuum to give compound 19 (14 g) as a yellow oil. ESI m / z 870 (M + H) + . 1 H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz, 2H), 7.64-7.58 (m, 2H), 7.42-7.37 (m, 2H), 7.34-7.28 (m, 2H), 4.86-4.76 (m, 1H), 4.56-4.34 (m, 3H), 4.28-4.18 (m, 1H), 3.76-3.73 (m, 3H), 3.66-3.56 (m, 13H), 3.39(br d, J = 4.9 Hz, 1H), 2.81 (br s, 6H), 2.50-2.46 (m, 2H), 2.40-2.32 (m,2H), 2.14-2.04 (m 1H), 1.97-1.81 (m, 4H), 1.44 (s, 9H), 1.05 (s, 3H), 1.04(s, 3H) ppm.

[0386] (4S)-4-{[(2S)-1-[(2,5-dioxopyrrolidone-1-yl)oxy]-3-methyl-1-oxobutane-2-yl]carbamoyl}-4-[1-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]butyric acid (20)

[0387] To compound 19 (Fmoc-PEG4-Glu(O) tA solution of Bu-Val-OSu (0.10 g, 0.12 mmol) in ethyl acetate (2 mL) was mixed with a solution of HCl in ethyl acetate (4 N, 4 mL, 16 mmol), and the reaction mixture was stirred at 0 °C for 4 h, as monitored by LCMS. Volatile substances were removed under vacuum, and the residue was purified by reversed-phase rapid chromatography (0.02% formic acid in 0-50% acetonitrile) to give compound 20 (Fmoc-PEG4-Glu-Ala-OSu) as a white solid (32 mg, 33% yield). ESI m / z: 813.2 (M + H) + .

[0388] Alternative methods (amplification methods): TFA (0.72 g, 0.47 mL, 6.3 mmol) was added to a solution of compound 19 (0.14 g, 0.16 mmol, obtained as above) in DCM (0.5 mL), and the reaction mixture was stirred at room temperature for 1 h, as monitored by LCMS. Volatiles were removed under vacuum, and the residue was milled in MTBE (10 mL × 2). A grayish-white precipitate was collected by centrifugation to give compound 20 (0.13 g, yield of H-Glu(O)) as a grayish-white solid. t 22% of Bu)-Val-OH). ESI m / z: 813 (M +H) + .

[0389] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-[1-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecanyl-15-amido]butyric acid (21)

[0390] DIPEA (16 mg, 0.12 mmol) and compound 20 (32 mg, 40 μmol) were added to a solution of compound 18 (25 mg, 40 μmol) in DMF (3 mL), and the reaction mixture was stirred at room temperature for 2 h, as monitored by LCMS. The resulting mixture was purified by reversed-phase rapid chromatography (0–70% acetonitrile in aqueous ammonium bicarbonate solution (10 mM)) to give compound 21 (25 mg, 47% yield) as a white solid. ESI m / z: 698.3 (M / 2 + H) + .

[0391] (4S)-4-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl]oxymethyl]carbamoyl]oxymethyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]butyric acid (LP5A)

[0392] Diethylamine (8.0 mg, 0.11 mmol) was added to a solution of compound 21 (15 mg, 11 μmol) in DMF (2 mL), and the reaction mixture was stirred at room temperature for 2 hours until Fmoc was completely removed, as monitored by LCMS. The resulting mixture was directly separated by preparative HPLC (10%–95% acetonitrile in aqueous ammonium bicarbonate solution (10 mM)) to give LP5A (2.2 mg, 18% yield) as a white solid. ESI m / z: 1101 (M + H) + .

[0393] LP5: (4S)-4-[1-(4-{2-azatricyclic[10.4.0.0]] 4 , 9[Hexadecane-1(12),4(9),5,7,13,15-hexen-10-yn-2-yl}-4-oxobutamido)-3,6,9,12-tetraoxapentadecan-15-amido]-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl]oxymethyl]carbamoyl]oxymethyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]butyric acid (LP5)

[0394] Except for LP5A, the junction-load LP5 (2.7 mg, 18% yield) was obtained as a white solid following a similar procedure to LP3A to LP3. ESI m / z: 695.0 (M / 2 + H) + .

[0395] LP51 and LP52 (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy]methyl)carbamoyl]oxy]methyl]phenyl]carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamitoyl]-3,6,9,12-tetraoxapentadecanyl-15-amido]butyric acid (LP51)

[0396] Except for the reaction of LP5A with COT-OSu, a linker-loaded LP51 (1.0 mg, 16% yield) was obtained as a white solid following a similar procedure to that from LP3A to LP3. ESI m / z: 632.3 (M / 2 + H) + .

[0397] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(1-{2-[4-(6-methyl-1,2,4,5-tetraazin-3-yl)phenyl]acetamido}-3,6,9,12-tetraoxapentadecanyl-15-amido)butyric acid (LP52)

[0398] Except for the reaction of LP5A with MeTz-PhAc-NHS (CAS: 1644644-96-1), a linker-loaded LP52 (0.9 mg, 14% yield) was obtained as a white solid following a similar procedure to that from LP3A to LP3. ESI m / z: 657.3 (M / 2 + H) + .

[0399] LP6 The synthesis of LP6 is described in Scheme 7 below.

[0400] Scheme 7. Synthesis of LP6. LP6 is disclosed as SEQ ID NO:292.

[0401]

[0402] 2-(cyclooctyl-2-yne-1-oxy)-N-{({[({[({[1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]methyl]carbamoyl]methyl]carbamoyl]methyl]carbamoyl]methyl]carbamoyl]methyl]acetamide (LP6). LP6 is disclosed as SEQ ID NO:292.

[0403]

[0404] HATU (38 mg, 0.10 mmol) and DIPEA (26 mg, 0.20 mmol) were added to a solution of COT-Gly3-OH (CAS: 2504011-15-6, synthesized according to WO2022015656, 24 mg, 66 μmol) in DMF (3 mL), and the mixture was stirred at room temperature for 10 min, followed by the addition of ProDHT (25 mg, 66 μmol). The reaction mixture was stirred at room temperature for 4 h, as monitored by LCMS. The resulting mixture was purified by preparative HPLC (TFA aqueous solution (0.01%) in 5%–95% acetonitrile) to give LP6 (7.2 mg, 15% yield) as a white solid. ESI m / z (M LP4 – M DHT + H) + , 734 (M +Na) + . 1 H NMR (400 MHz, DMSO d6 ) δ 8.49 (t, J = 6.5 Hz, 1H), 8.20 (t, J = 5.7 Hz,1H), 8.18-8.09 (m, 2H), 7.83 (t, J = 5.7 Hz, 1H), 4.65-4.51 (m, 1H), 4.51-4.49 (m, 1H), 4.35-4.29 (m, 1H), 4.00-3.66 (m, 10H), 2.50-2.05 (m, 8H), 1.98-1.60 (m, 8H), 1.66-1.08 (m, 15H), 0.97 (s, 3H), 0.90-0.83 (m, 2H), 0.68 (s, 3H) ppm.

[0405] LP7 The synthesis of LP7 is described in Scheme 8 below.

[0406] Scheme 8. Synthesis of LP7

[0407] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]} 2 , 7 .011 , 15 [Heptadecanyl-14-yl]oxy}methyl)carbamoyl]methyl}carbamoyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]-4-(3-{2-[2-(3-{3-[2-({2-[2-(2-{[(1S)-1-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}methyl)carbamoyl]methyl}carbamoyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl]-2-methylpropyl]carbamoyl]-3-carboxypropyl]carbamoyl]ethoxy)ethoxy]ethyl}carbamoyl)ethoxy]-2-[3-(2-{2-[2-(cyclooctyl-2-yn-1-yloxy)acetamyl]ethoxy}ethoxy)propamido]propoxy}propamido)ethoxy]ethoxy}propamido)butyric acid (LP7)

[0408] ProDHT (60 mg, 0.16 mmol) and HOBt (10 mg, 74 μmol) were added to a solution of compound 22 (0.18 g, 82 μmol) in DMF (5 mL), and the reaction mixture was stirred at room temperature for 4 h, as monitored by LCMS. The resulting mixture was directly separated by reversed-phase rapid chromatography (10%–95% acetonitrile in ammonium bicarbonate aqueous solution (10 mM)) to give LP7 (20 mg, 9% yield) as a white solid. ESI m / z: 1331.7 (M / 2 + H) + . 1 H NMR (400MHz, DMSO d6 ) δ 10.0 (s, 2H), 8.55 (t, J = 6.4 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.93 (t, J = 6.4 Hz, 2H), 7.75 (d, J= 7.2 Hz,2H), 7.60-7.55 (m, 4H), 7.41 (t, J = 6.4 Hz, 2H), 7.29-7.27 (m, 4H), 6.00-5.98 (m, 2H), 5.40 (s, 4H), 5.00 (s, 4H), 4.60-4.50 (m, 2H), 4.45-4.35 (m,2H), 4.30-4.20 (m, 5H), 4.20-4.15 (m, 2H), 3.80-3.75 (m, 1H), 3.75-3.50 (m,11H), 3.55 (br, 10H), 3.50-3.45 (m, 6H), 3.45-3.40 (m, 10H), 3.10 (br, 6H),3.00-2.90 (m, 3H), 2.25-2.15 (m, 16H), 1.80-1.60 (m, 19H), 1.50-1.45 (m,20H), 1.45-1.40 (m, 20H), 0.95-0.90 (m, 16H), 0.85 (s, 6H), 0.60-0.55 (m, 9H)ppm.

[0409] LP41, LP42, LP43 and LP44 The synthesis of LP41, LP42, LP43 and LP44 is described in Scheme 9 below.

[0410] Scheme 9. Synthesis of LP41, LP42, LP43 and LP44

[0411] LP41, LP42, LP43 and LP44 tert-Butyl N-[(1S)-5-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}pentyl]carbamate (23a)

[0412] A solution of Boc-Lys(Fmoc)-OH (1.0 g, 2.1 mmol) in DMF (10 mL) was added with (4-aminophenyl)methanol (0.26 g, 2.1 mmol), HATU (1.2 g, 3.2 mmol), and 2,6-dimethylpyridine (0.68 g, 6.3 mmol), and the mixture was stirred at room temperature for 3 hours. The resulting mixture was purified by reversed-phase rapid chromatography (TFA aqueous solution (0.1%) in 0–95% acetonitrile) to give compound 23a (1.0 g, 82% yield) as a white solid. ESI m / z: 574.3 (M+H) + .

[0413] {4-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-6-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)hexamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl carbonate (24a)

[0414] DMAP (1.2 g, 10 mmol) was added to a solution of DHT (0.50 g, 1.7 mmol) in DCM (10 mL). After cooling the mixture to 0 °C, triphosgene (0.15 g, 0.85 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, and 23a (1.0 g, 3.4 mmol) was added. The mixture was stirred at room temperature for 2 hours. The resulting mixture was concentrated under vacuum, and the residue was purified by rapid chromatography (5%–95% acetonitrile in TFA aqueous solution (0.1%)) to give 24a (0.50 g, 50% yield) as a white solid. ESI m / z: 890.2 (M + H) + .

[0415] {4-[(2S)-2-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutamido]-6-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)hexamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15Heptadecan-14-yl carbonate (25a)

[0416] TFA (1.0 mL) was added to a solution of compound 24a (0.50 g, 1.7 mmol) in DCM (8 mL), and the reaction mixture was stirred at room temperature for 3 hours. The resulting mixture was concentrated under vacuum, and the residue was dissolved in DMF (5 mL). Boc-valine (0.11 g, 0.5 mmol), HATU (0.29 g, 0.75 mmol), and DIPEA (0.19 g, 1.5 mmol) were added to the solution, and the reaction mixture was stirred at room temperature for 3 hours. The resulting mixture was purified by reversed-phase rapid chromatography (TFA aqueous solution (0.1%) in 0–95% acetonitrile) to give compound 25a (0.30 g, 54% yield) as a white solid. ESI m / z: 990.2 (M + H) + .

[0417] {4-[(2S)-2-[(2S)-2-(1-{[(tert-butoxy)carbonyl]amino}-3,6,9,12-tetraoxapentadecan-15-amido)-3-methylbutamido]-6-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)hexamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl carbonate (26a)

[0418] In addition to using N Compound 26a, 0.20 g (58% yield), was obtained as a white solid, except that the -Boc-PEG4- acid replaced Boc-valine. The procedure was similar to that for 25a. ESI m / z: 1237.6 (M + H) + .

[0419] {4-[(2S)-2-[(2S)-2-(1-amino-3,6,9,12-tetraoxopentadecane-15-amido)-3-methylbutyramido]-6-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)hexamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .011 , 15 Heptadecan-14-yl carbonate (27)

[0420] TFA (1.0 mL) was added to a solution of 26a (0.20 g, 0.16 mmol) in DCM (8 mL), and the mixture was stirred at room temperature for 3 hours. The resulting mixture was concentrated under vacuum, and the residue was purified by reversed-phase rapid chromatography (TFA aqueous solution (0.1%) in 5%–95% acetonitrile) to give compound 27 (0.14 g, 77% yield) as a white solid. ESI m / z: 1137.5 (M + H) + .

[0421] {4-[(2S)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamyl]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutamido]-6-({[(9H-fluorene-9-yl)methoxy]carbonyl}amino)hexamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl carbonate (28a)

[0422] Add COT-OSu (34 mg, 0.12 mmol) and DIPEA (46 mg, 0.36 mmol) to a solution of 27 (0.14 g, 0.12 mmol) in DMF (2 mL), and stir the reaction mixture at room temperature for 3 hours. Separate the resulting mixture directly by reversed-phase rapid chromatography (TFA aqueous solution (0.1%) in 5%–95% acetonitrile) to give 28a (90 mg, 57% yield) as a white solid. ESI m / z: 1301.7 (M + H) + .

[0423] {4-[(2S)-6-amino-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamyl]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutamido]hexamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .011 , 15 Heptadecan-14-yl carbonate (LP41)

[0424] To a solution of 28a (90 mg, 68 μmol) in DMF (2 mL), piperidine (0.2 mL) was added, and the reaction mixture was stirred at room temperature for 3 hours. The mixture was directly separated by reversed-phase rapid chromatography (TFA aqueous solution (0.1%) in 5%–95% acetonitrile) to give LP41 (40 mg, 54% yield) as a white solid. ESI m / z: 1079.5 (M + H) + .

[0425] {4-[(2S)-6-amino-2-[(2S)-2-{1-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)acetamyl]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutamido]hexamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl carbonate (LP42)

[0426] Except for the use of AMAS (CAS: 55750-61-3) instead of COT-OSu, the linker-load LP42 (4.5 mg, 20% yield) was obtained as a white solid following a procedure similar to that for LP41. ESI m / z: 1051.6 (M + H) + .

[0427] {4-[(2S)-2-[(2S)-2-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-3-methylbutyramido]-5-(carbamoylamino)pentamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl carbonate (LP44)

[0428] In addition to N-Starting with Boc-Cit-OH, compounds 23b, 24b, 25b, 26b and LP44 were obtained following a similar procedure to compound 27.

[0429] LP44 (20 mg, 15% gross yield): ESI m / z: 943.5 (M + H) + .

[0430] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-oxy)acetamyl]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutamido]pentamido]phenyl}methyl(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 Heptadecan-14-yl carbonate (LP43)

[0431] Except that LP44 was used instead of compound 27, the linker-loaded LP43 (2.5 mg, 60% yield) was obtained as a white solid following a similar procedure to that for LP41. ESI m / z: 1107.7 (M + H) + .

[0432] LP61 and LP61A The synthesis of LP61 is described in Scheme 10 below.

[0433] Scheme 10. Synthesis of LP61

[0434] LP61 and LP61A (9H-fluorene-9-yl)methyl N-[(1S)-1-{[(1S)-1-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy}methyl)carbamoyl]methyl}carbamoyl]-5-(dipropylamino)pentyl]carbamoyl}-2-methylpropyl]carbamate (29)

[0435] To a solution of compound 28 (synthesized according to WO2023143208, 33 mg, 60 μmol) in DMF (3 mL), HATU (38 mg, 0.10 mmol) and DIPEA (26 mg, 0.20 mmol) were added, and the mixture was stirred at room temperature for 10 min, followed by the addition of ProDHT (19 mg, 50 μmol). The reaction mixture was stirred at room temperature for 4 h, as monitored by LCMS. The resulting mixture was purified by preparative HPLC (TFA aqueous solution (0.01%) in 5%–95% acetonitrile) to give compound 29 (10 mg, 22% yield) as a white solid. ESI m / z 910.6 (M + H) + .

[0436] 1-Amino-N-[(1S)-1-{[(1S)-1-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0] 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]-5-(dipropylamino)pentyl]carbamoyl]-2-methylpropyl]-3,6,9,12-tetraoxapentadecanyl-15-amide (LP61A)

[0437] Except for the use of compound 29 instead of Fmoc-vcPAB-ProDHT, the linker-loaded LP61A (6.0 mg, 58% yield) was obtained as a white solid following a similar procedure to LP2A. ESI m / z: 935.7 (M + H) + .

[0438] 1-[2-(cyclooctyl-2-yn-1-yloxy)acetamyl]-N-[(1S)-1-{[(1S)-1-({[({[(1S,2S,7S,10R,11S,14S,15S)-2,15-dimethyl-5-oxotetracyclo[8.7.0.0]] 2 , 7 .0 11 , 15 [Heptadecanyl-14-yl]oxy]methyl]carbamoyl]methyl]carbamoyl]-5-(dipropylamino)pentyl]carbamoyl]-2-methylpropyl]-3,6,9,12-tetraoxapentadecanyl-15-amide (LP61)

[0439] Except for replacing compound 27 with LP61A, following a similar procedure to 28a, a linker-loaded LP61 (0.5 mg, 21% yield) was obtained as a white solid. ESI m / z: 1100.0 (M + H) + .

[0440] Example 10: Preparation of Antibody-Drug Conjugates (ADCs) The general procedure for preparing site-specific conjugates is disclosed below and shown in Figure 8.

[0441] ADC conjugation uses non-glycosylated human antibody IgG (IgG1, IgG4, etc.) containing N297Q or N297D mutations. Conjugation is performed via a two-step method. Figure 8A The results of the fusion with structure and MS-DAR values ​​are summarized in Table 5.

[0442] Table 5. List of Antibodies, Azide-functionalized Antibodies, and ADCs

[0443] Table 6: Exemplary Antibody-Connectors and ADCs Selected According to This Disclosure

[0444] Step 1: The handle-functionalized amine is site-specifically conjugated with the antibody to generate an antibody conjugate containing 1-4 handles per antibody.

[0445] Non-glycosylated human antibody IgG containing N297Q or N297D mutations was mixed with ≥100 molar equivalents of unbranched or branched handpiece amine (AL) in BupH buffer (pH 7.4). The resulting solution was then mixed with transglutaminase (350 U / mL; 1 U mTG / mg antibody, SLCK1576, Sigma; or 25 U / mL; 1 U mTG / mg antibody, Zedira, Darmstadt, Germany; or 10 U / mL; 0.06 mg mTG / mg antibody, Modernist Pantry-ACTIVA TI containing maltodextrin from Ajinomoto, Japan) to achieve a final antibody concentration of 0.5–20 mg / mL. The reaction mixture was incubated at 25–37 °C for 24 hours with gentle shaking, while monitoring by ESI-MS. After completion, excess amine and mTG were removed by size exclusion chromatography (SEC) or protein A column chromatography. The conjugates were characterized by UV-Vis, SEC, and ESI-MS.

[0446] Step 2: Click reaction between the handle-functionalized antibody (Ab-handle) and the linker payload (LP) in Table 3 to generate a site-specific ADC.

[0447] Handle-functionalized antibody (Ab-(AL)) in PBS (pH 7.4) n The linker-load (LP) was dissolved in an organic solvent such as DMSO or DMA (10 mg / mL) to make the final reaction mixture contain 5%–15% organic solvent (v / v) at 1–20 mg / mL. The mixture was incubated at 25–37 °C for 1–48 h with gentle shaking. The reaction was monitored by ESI-MS. After completion, excess LP and organic solvent were removed by desalting with a BupH (pH 7.4) column, and protein aggregates (if present) were removed by size exclusion chromatography (SEC). The purified conjugate Ab-(AL-LP) was then... n ADC or Ab-(AL-(LP)2)4ADC was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. The monomer purity of the conjugate was >95% by SEC.

[0448] All ADCs were purified by SEC using an ÄKTA instrument from Cytiva, employing a 16 / 600 Superdex® 200 column, eluted with DPBS at a flow rate of 1.5 mL / min, pH 7.4. The DAR values ​​of the ADCs were measured by ESI-MS. A 4 x LP mass increase from Ab-[AL]4 was observed, correlated with a 4DAR ADC.

[0449] Detailed joining procedure The following example illustrates a representative 4DAR ADC from Method I. A non-glycosylated anti-CACNG1 human IgG antibody containing the N297Q mutation was mixed with 100 molar equivalents of azido-dPEG3-amine (AL1, MW 218.26 g / mol). The resulting solution was mixed with microbial transglutaminase (350 U / mL; 1 U mTG per mg antibody, Sigma), resulting in a final antibody concentration of 3.4 mg / mL. The reaction mixture was incubated at 32°C for one hour with gentle shaking, monitored by ESI-MS. After completion, excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS. The antibody with the azido linker attached resulted in an 811 Da mass increase compared to the mAb, indicating a conjugation of 4 ALs with an antibody having 4 azido handles (Ab-(AL)4).

[0450] The site-specific antibody azide conjugate (6.9 mg / mL) in PBS (pH 7.0) was mixed with 6 molar equivalents of linker-load (LP1) in 2 mM DMSO to make the reaction mixture contain 12% organic solvent (v / v), and the solution was incubated at 37°C for 4 hours with gentle shaking. The reaction was monitored by ESI-MS. After completion, excess linker-load and protein aggregates were removed by size exclusion chromatography (SEC). The purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. The monomer purity of the conjugate was 99.8% by SEC. The antibody attaching the drug resulted in a mass increase of 5264 Da in the DAR4 conjugate. The monomer purity of the conjugate was >99% by SEC.

[0451] Table 7: Selection criteria as follows Figure 8B The ADC prepared in one step as shown

[0452] Interchain disulfide bond entanglement Antibody (2 mg / ml) in 50 mM HEPES, 2 mM EDTA (pH 7.4) was treated with 1 mM dithiothreitol or 5 mM TCEP for 90 min at 37 °C. After Amicon filtration (30 K), maleimide linker payload derivatives (1.0–2.0 equivalents / SH group of cysteine ​​residues, LP33 or LP42) in DMSO or DMA (8 mM solution) were added to the reduced antibody. The reaction was confirmed by LC-MS after 1 h. The conjugates were purified by size exclusion chromatography using PBS (1X) pH 7.4 under isocratic buffer elution conditions or by hydrophobic interaction chromatography using 3 M NaCl in 50 mM sodium phosphate as binding buffer (pH 7.4) and 50 mM sodium phosphate with 15%–20% IPA as elution buffer (pH 7.4) under gradient elution conditions. The concentrated fractions were exchanged for buffer in pH 7.4 PBS (1x) through a 30 K Amicon filter, then prepared with 5% (v / v) glycerol and aseptically filtered. Protein and linker payload concentrations were determined by UV spectroscopy. Size exclusion HPLC confirmed that all conjugates used were >95% monomers, and RP-HPLC confirmed the presence of <0.5% unconjugated linker payloads.

[0453] It is known in the art that the 5-membered succinimide ring formed during the conjugation process exists in equilibrium between a closed ring and two open ring isoforms through hydrolysis under neutral or basic conditions.

[0454] Table 8: Selection criteria as follows Figure 8C The ADC prepared by the Cys-maleimide procedure shown

[0455] Example 11: Characterization of an Exemplary ADC The ESI-MS spectra of the selected ADC and Ab handle are shown in Figures 9-11.

[0456] Example 11A: SDS-PAGE for ADC integrity and purity analysis In one method, SDS-PAGE running conditions included non-reduced and reduced samples (1–2 μg) and Precision Plus protein dual-color standards (Bio-rad, 500 μl, Cat# 1610374), loaded per channel into Novex 4%–20% Tris-Glycine-free gels (1.0 mm × 10 wells), and run at 180 V, 300 mA for 80 minutes. Non-reduced samples were prepared using NuPAGE® LDS sample buffer (4×) (Thermo Fisher Scientific, Cat# 1887691), and reduced samples were prepared using SDS sample buffer (4×) containing 10% sample reducing agent (10X) (Thermo Fisher Scientific, Cat# 1769410).

[0457] The molecular weights of antibodies and ADCs on SDS-PAGE were determined under both reducing and non-reducing conditions. Under non-reducing conditions, the mass shift may not be significant due to the relatively small percentage change in mass. However, the mass of the heavy chain increases from naked antibodies to azido-functionalized antibodies, and further increases to ADC conjugates.

[0458] Example 11B: Size exclusion chromatography (SEC) for ADC analysis and purification Size exclusion chromatography (SEC) was performed to determine the purity of the antibody-drug conjugates. SEC analysis was conducted using a Thermo UltiMate™ 3000 instrument, run on an XBridge protein BEH SEC column (Waters, 200 Å, 3.5 μm, 7.8 mm × 300 mm), with each sample (30–40 μg, 20 μL) treated with PBS pH 7.4 and 15% 2-propanol at a flow rate of 0.5 mL / min, and monitored at λ280 nm using a Thermo DAD-3000 RS rapid separation diode array detector.

[0459] The ADC was purified by size exclusion chromatography (SEC) and concentrated by ultracentrifugation. To separate the antibody-drug conjugate from the reaction mixture, preparative SEC purification was performed using an ÄKTA instrument from GE Healthcare on a Superdex® 200 increase 10 / 300 GL (1.0 × 30 cm) column, eluted with BupH at pH 7.4 at a flow rate of 0.6 mL / min, and monitored at λ280 nm. To concentrate the product, an Amicon® Ultra-4 centrifuge filter (Ultracel-10K) was used in an Allegra x-12r centrifuge, and the solution was stirred after each concentration to avoid excessive aggregation.

[0460] Example 11C: Experimental Procedure for Assaying CACNG1 Androgen Reporter The CACNG1 androgen reporter assay is described below, including the relevant cell lines, proteins, reagents, and instrument type and model. CACNG1 is the γ1 subunit of the dihydropyridine receptor specifically expressed in skeletal muscle. Therefore, antibodies generated against CACNG1 can be used to specifically deliver conjugated therapeutic payloads to skeletal muscle to enhance therapeutic efficacy in muscle and reduce off-target toxicity. Conjugation of CACNG1 antibodies to dihydrotestosterone (DHT), a potent metabolite of testosterone, can allow androgen receptor signaling in muscle, leading to increased muscle mass and function. Here, CACNG1 antibodies conjugated to linkers with DHT payloads were tested in androgen receptor (AR) reporter cell lines to determine whether these antibody conjugates can specifically activate AR in CACNG1-expressing cells in vitro.

[0461] To evaluate signaling via AR, LNCaP cells were transfected with lentivirus (Qiagen; ARE.Luc Cignal Lenti) to generate a stable cell line expressing the AR-luciferase reporter (AR.Luc). A subset of these selected cells were transduced to express human CACNG1 and further selected; this cell line was named hCACNG1.AR.Luc.

[0462] For bioassays, AR.Luc or hCACNG1.AR.Luc cells were seeded at 5,000 cells / well in OptiMEM and 0.5% carbon-peeled FBS in PDL-coated 96-well plates. Cells were then incubated for 24, 48, or 72 hours with a CACNG1 antibody conjugated to DHT via a linker, an allotype control antibody, or DHT alone. All antibodies were conjugated to DHT at a drug-to-antibody ratio (DAR) of ~4. After the corresponding time points, cells were lysed and incubated with One-GLO buffer, and luminescence was read on an Envision plate reader. Relative luminescence units (RLU) were plotted against logarithmic concentration in mol / L and adjusted for DAR.

[0463] Results Summary and Conclusions Unconjugated DHT activated AR in both the AR.Luc and hCACNG1.AR.Luc cell lines, while DHT conjugated to the isotype control antibody Ab 1 did not activate AR in either of these cell lines. Several CACNG1 antibody-DHT conjugates activated AR only in the hCACNG1.AR.Luc cell line and not in the AR.Luc cell line. Although the efficacy and potency of AR activation by CACNG1 antibody-DHT conjugates were lower than that of unconjugated DHT at 24 hours post-treatment, these conjugates maintained AR activation at 48 and 72 hours, while unconjugated AR signaling was significantly reduced at these time points. Overall, these data indicate that DHT conjugation to CACNG1 antibodies allows for specific activation of AR in hCACNG1-expressing cells, and that DHT conjugated to CACNG1 antibodies maintains sustained AR signaling in hCACNG1-expressing cells in vitro for several days.

[0464] Example 11D: Cell-free assay of DHT-AR binding affinity In AR measurements conducted via Thermo Fisher Scientific's SelectScreen™ Profiling Service, DHT exhibited an IC50 of 1.43 nM. 50 .

[0465] AR (androgen receptor) antagonist screening, activated by R1881 (metribolone or methyltrienolone, an AR agonist). AR-UAS-bla GripTite™ 293 cells were thawed and prepared for agonist screening as described above (see Example 5). 4 µL of cyproterone acetate (control antagonist starting concentration, 3,160 nM) or a 10X serial dilution of the compound was added to the appropriate wells of the poly-D-lysine assay plate. 32 µL of cell suspension was added to the wells, and the plates were pre-incubated in a humidified incubator at 37°C / 5% CO2 for 30 min with the compound and control antagonist titrant. 4 µL of the predetermined EC80 concentration of 10X control agonist R1881 was added to the wells containing the control antagonist or compound. The plates were incubated in a humidified incubator at 37°C / 5% CO2 for 16–24 h. 8 µL of 1 µM substrate loading solution was added to each well, and the plates were incubated at room temperature for 2 h. The plates were read on a fluorescent plate reader. The results on the screen are as shown. Figure 12 As shown.

[0466] Example 11E: Plasma stability of ACACNG1 DHT-ADC To determine the plasma stability of CACNG1-DHT ncADCs carrying DHT payloads against hCACNG1 Ab 5-L3 and [L2], the ncADCs were incubated in vitro with plasmas from different species, and the drug-to-antibody ratio (DAR) was evaluated.

[0467] The ncADC solution was spiked into pooled mouse, rat, cynomolgus monkey, or IgG-depleted human plasma (Bipoint) to a final concentration of 50 μg / mL, and then incubated at 37 °C on a ThermoMixer C (Eppendorf, Cat#2231000574). 100 μL aliquots were collected at 0, 24, 48, 72, and 168 hours and immediately frozen at -80 °C until analysis.

[0468] For DAR analysis, ncADC was purified from plasma samples using an immunoaffinity capture process via a KingFisher Apex automated processor (Thermo Scientific, Cat#5400930). First, biotinylated anti-human Fc antibody (a Regeneron-generated reagent) was immobilized on Dynabeads M280 streptavidin beads (Invitrogen, Cat#60210). Each plasma sample containing ncADC was gently mixed with 0.5 mg of the beads (Regeneron-generated reagent-coupled beads) at 950 rpm at room temperature with gentle shaking for 1.5 h. The beads were then washed three times with 500 μL HBS-EP pH 7.4 buffer (GE Healthcare, Cat#BR100188), once with 500 μL water, and once with 500 μL 10% acetonitrile in water (VWR Chemicals, Cat#BDH83640.100E). After washing, the ncADC was eluted by incubating the beads in 70 μL of 1% formic acid in a 30:70 acetonitrile:water (v / v) solution at room temperature for 15 min. The 50 μL of eluted sample was further reduced by adding 50 μL of 10 mM TCEP (Sigma, Cat 646547-10X1ML) and incubated in ThermoMixer C at 37 °C for 20 min.

[0469] The reduced ncADC sample was injected into a 1x50 mm 1.7 μm BEH300 C4 column (Waters, Cat#186005589) for separation and then detected by a Synapt G2-Si mass spectrometer (Waters, Milford, MA). The flow rate was 80 µL / min (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile). The ncADC was eluted by an HPLC gradient between 2.0 and 6.5 min, corresponding to 25%–40% of mobile phase B. The acquired spectra were deconvolved using MaxEnt1 software with the following parameters: mass range: 20–60 kDa; m / z Range: 800-2500 Da; Resolution: 1.0 Da / channel; Full width at half maximum (FWHM): 0.7 Da; Minimum intensity ratio: 33%; Maximum number of iterations: 12.

[0470] Results summary and conclusions: After incubation for 168 hours in rat, cynomolgus monkey, or IgG-depleted human plasma, no significant changes in DAR were observed in either ncADC. Approximately 65% ​​and 15% DAR loss, respectively, were observed in mouse plasma for anti-hCACNG1 Ab 5-L2 and anti-hCACNG1 Ab 5-L3.

[0471] The list data is summarized in Table 9 below. Figures 13-17 Presented in the middle. Figure 16 The following are shown as relative light units (RLU; y-axis) after 24 hours of incubation with LNCaP cell lines modified to express luciferase upon androgen receptor activation: dihydrotestosterone (DHT) alone (unconjugated DHT), anti-hCACNG1 antibodies (anti-hCACNG1 Ab6, anti-hCACNG1 Ab7, anti-hCACNG1 Ab8, anti-hCACNG1 Ab9, anti-hCACNG1 Ab10 or anti-hCACNG1 Ab5) conjugated to DHT (L2) via VC-PAB linkers; or anti-FelD isotype control antibody (isotype control Ab1) conjugated to DHT (L2) via VC-PAB linkers. Figure 17 The following are shown: LNCaP cell lines expressing CACNG1 and modified to also express luciferase upon androgen receptor activation (hCACNG1.AR.Luc) androgen receptor (AR) activation levels, expressed in relative light units (RLU; y-axis), after incubation at different concentrations (Log[concentration(M)]; x-axis) for 24, 48, or 72 hours: dihydrotestosterone (DHT) alone (unconjugated DHT), anti-hCACNG1 antibodies conjugated to DHT (L2) via VC-PAB linkers (anti-hCACNG1 Ab 6, anti-hCACNG1 Ab 7, anti-hCACNG1 Ab 8, anti-hCACNG1 Ab 9, anti-hCACNG1 Ab 10, or anti-hCACNG1 Ab 5); or anti-FelD isotype control antibodies conjugated to DHT (L2) via VC-PAB linkers (isotype control Ab 1).

[0472] Table 9. DAR values ​​of anti-hCACNG1 Ab 5-L2 and anti-hCACNG1 Ab 5-L3 in plasma after incubation

[0473] * * * Since various changes can be made to the subject matter described above without departing from the scope and spirit of the invention, it is intended that all subject matter contained in the above description or defined in the appended claims be construed as descriptive and illustrative of the invention. Many modifications and variations of the invention are possible in light of the above teachings. Therefore, this description is intended to cover all such alternatives, modifications, and variations falling within the scope of the appended claims.

[0474] All patents, applications, publications, test methods, documents and other materials cited herein are incorporated herein by reference in their entirety as if they were physically present in this specification.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, comprising an antigen-binding protein that specifically binds to the human calcium voltage-gated channel helper subunit γ1 (hCACNG1), wherein the antigen-binding protein is conjugated directly or via a linker to at least one therapeutic agent, wherein the antibody-drug conjugate has a structure according to formula (I): A – [L – P] y (I), Where A is an antigen-binding protein; L is absent or is a connector; P is a therapeutic agent, and y is an integer from 1 to 8.

2. The antibody-drug conjugate according to claim 1, wherein the antigen-binding protein is an anti-hCACNG1 antibody or its antigen-binding fragment, comprising an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 4-6-8-12-AAS-16, SEQ ID NO: 20-22-24-26-28-ATS-32, SEQ ID NO: 36-38-40-44-KAS-48, SEQ ID NO: 52-54-56-60-GAS-64; SEQ ID NO: 68-70-72-76-AAS-80; SEQ ID NO: 84-86-88-92-AAS-96; SEQ ID NO: 100-102-104-108-AAS-112; SEQ ID NO: 116-118-120-124-GA-128; SEQ ID NO: 132-134-136-140-GAS-144; SEQ ID NO: 148-150-152-156-RN-160; SEQ ID NO: 164-166-168-172-DNN-176; SEQ ID NO: 180-182-186-188-GAS-192; SEQ ID NO: 36-297-299-303-YNS-305; and SEQ ID NO: 316-318-152-324-RNN-326.

3. The antibody-drug conjugate according to any one of claims 1-2, wherein the anti-hCACNG1 antibody or its antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair having at least 90% sequence identity with an 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, SEQ ID NO: 178 / 186, SEQ ID NO: 294 / 301, and SEQ ID NO: 314 / 322.

4. The antibody-drug conjugate according to any one of claims 1-3, wherein the therapeutic agent is dihydrotestosterone (DHT) or its prodrug or derivative.

5. The antibody-drug conjugate according to any one of claims 1-3, wherein the therapeutic agent is selected from the group consisting of: having a structure DHT and with selectivity and The structure of the group composed of proDHT, R1 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R2 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R3 is selected from the following groups: H and C 1-3 Alkyl, and R4 is selected from the following groups: H and C 1-3 alkyl.

6. The antibody-drug conjugate according to any one of claims 1-3, wherein n is 2 or 4.

7. The antibody-drug conjugate according to any one of claims 1-6, wherein the linker has the following structure: -L1-B-L2-, where: L1 is the first linker unit covalently attached to the antigen-binding protein; B is a unit that either does not exist or is an adduct containing at least one group B', wherein the group B' is selected from -N3, , ; ;and Where Q is C or N; L2 is absent or is a second connector unit covalently attached to unit B via at least one group B', wherein the groups B' and B'' form the at least one adduct. The condition is that when B is absent, L2 is also absent and L1 is covalently attached to the therapeutic agent, and when L2 is present, L2 is covalently attached to the therapeutic agent.

8. The antibody-drug conjugate according to claim 7, wherein L1 comprises C 1-6 Alkyl, phenyl, -NH-, -C(O)-, -(CH2) u -NH-C(O)-、-(CH2) u -C(O)-NH-、-(CH2-CH2-O) v -、-(CH2) u -(O-CH2-CH2) v -C(O)-NH-, a peptide unit containing 2 to 4 amino acids, or a combination thereof; each of which may optionally be substituted by one or more of -S-, -S(O2)-, -C(O)-, -C(O2)- and CO2H; wherein the subscripts u and v are independent integers from 1 to 8.

9. The antibody-drug conjugate according to claim 8, wherein L1 is .

10. The antibody-drug conjugate according to any one of claims 7-9, wherein B has a structure selected from the group consisting of: , , , and , where Q is C or N.

11. The antibody-drug conjugate according to any one of claims 7-10, wherein the L2 has the following structure: -SP1-AA-SP2- (L2), where: SP1 is absent or is the first spacer unit; AA either does not exist or contains peptide units of 2 to 4 amino acids; SP2 is absent or is a second spacer unit covalently attached to the therapeutic agent.

12. The antibody-drug conjugate of claim 11, wherein SP1 is absent or selected from the group consisting of: , , , , C 1-6 Alkyl group, -(CH2-CH2-O) v -、-(CH2-CH2-O) v -(CH2) u -、-(CH2-CH2-O) v -(CH2) u -C(O), -O-CH2-C(O)-NH, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -C(O)-NH, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -、-NH-(CH2) u -C(O)-、-NH-(CH2-CH2-O) v -、-NH-(CH2-CH2-O) v -C(O)-、-NH-(CH2-CH2-O) v -(CH2) u -、-NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof; where the subscripts u and v are independent integers from 1 to 8.

13. The antibody-drug conjugate according to claim 11 or claim 12, wherein the AA is a peptide unit comprising 2 to 4 amino acids selected from the group consisting of glycine, valine, phenylalanine, proline, glutamic acid, lysine, N,N-dipropyllysine, phenylalanine, and citrulline, and combinations thereof.

14. The antibody-drug conjugate according to claim 13, wherein the AA is valine-citrulline, valine-alanine, valine-lysine, valine-N,N-dipropyllysine, phenylalanine-lysine, glycine-glycine-glycine (GGG), glycine-glycine-glycine-glycine (GGGG (SEQ ID NO:289)), glycine-glycine-phenylalanine (GGF), glycine-glycine-phenylalanine-glycine (GGFG (SEQ ID NO:290)), L-glutamic acid-valine-citrulline (GGFG, G ... L EVC) and D-glutamic acid-valine-citrulline ( D EVC).

15. The antibody-drug conjugate according to any one of claims 11-14, wherein SP2 is absent or selected from the group consisting of: , , , , and their combinations.

16. The antibody-drug conjugate according to any one of claims 1-15, wherein the linker-therapeutic agent (LP) has a structure selected from the group consisting of:

17. The antibody-drug conjugate according to any one of claims 1-16, wherein the linker is attached to the side chain of the glutamine residue of the antigen-binding protein.

18. The antibody-drug conjugate of claim 17, wherein the glutamine residue is naturally present in the CH2 or CH3 domain of the antigen-binding protein.

19. The antibody-drug conjugate of claim 17, wherein the glutamine residue is introduced into the antigen-binding protein by modifying one or more amino acids.

20. The antibody-drug conjugate according to any one of claims 1-19, wherein the antibody-drug conjugate has a structure selected from the group consisting of: Where Ab is the antigen-binding protein, and n is an integer from 1 to 4.

21. Compounds according to formula (L2-P) or (L2′-P): B”-SP1-AA-SP2-P (L2-P), H2N-SP1-AA-SP2-P) p (L2'-P), Or its pharmaceutically acceptable salt, wherein: B" is selected from the following groups: -N3, , , , and ; SP1 either does not exist or is selected as the first spacer unit of the following group: and C 1-6 Alkyl group, -(CH2-CH2-O) v -、-(CH2-CH2-O) v -(CH2) u -、-(CH2-CH2-O) v -(CH2) u -C(O), -O-CH2-C(O)-NH, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -, -O-CH2-C(O)-NH-(CH2-CH2-O) v -(CH2) u -C(O)-NH, -NH-, -C(O)-, -NH-C(O)-, -NH-(CH2) u -、-NH-(CH2) u -C(O)-、-NH-(CH2-CH2-O) v -、-NH-(CH2-CH2-O) v -C(O)-、-NH-(CH2-CH2-O) v -(CH2) u -、-NH-(CH2-CH2-O) v -(CH2) u -C(O)-, -(CH2) u -NH-C(O)-, -NH-(CH2) u -NH-C(O)-, -NH-(CH2) u -C(O)-NH-, or combinations thereof; where the subscripts u and v are independent integers from 1 to 8; AA either does not exist or contains peptide units of 2 to 4 amino acids; SP2 either does not exist or is selected from the following groups of second spacer units: , , , , and their combinations and P is a therapeutic agent selected from the group consisting of: having a structure DHT and with selectivity and The structure of the group composed of proDHT, R1 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R2 is selected from the following groups: H, C 1-6 Alkyl group, (CH2) 0-6 -OH, (CH2) 0-6 -NH2、(CH2) 0-6 -NH-C 1-3 Alkyl groups and (CH2) 0-6 -N-(C 1-3 Alkyl)2; R3 is selected from the following groups: H and C 1-3 Alkyl, and R4 is selected from the following groups: H and C 1-3 alkyl.

22. The compound according to claim 21, having a structure selected from the group consisting of:

23. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, comprising an antigen-binding protein that specifically binds to the human calcium voltage-gated channel helper subunit γ1 (hCACNG1), said antigen-binding protein conjugating to a linker-payload selected from the group consisting of: , and .

24. A composition comprising a group of antibody-drug conjugates according to any one of claims 1-20 and 23, said composition having a drug-antibody ratio (DAR) of about 0.5 to about 8.

0.

25. The composition according to claim 24, wherein the composition has a DAR of about 1.0 to about 2.

5.

26. The composition according to claim 25, wherein the composition has a DAR of about 2.

0.

27. The composition according to claim 24, wherein the composition has a DAR of about 3.0 to about 4.

5.

28. The composition according to claim 27, wherein the composition has a DAR of about 4.

0.

29. A pharmaceutical composition comprising an antibody-drug conjugate as described in any one of claims 1-20 and 23, as well as a diluent, a carrier, and / or an excipient.

30. A pharmaceutical dosage form comprising the antibody-drug conjugate of any one of claims 1-20 and 23 or the pharmaceutical composition of claim 29.

31. A method of treating a condition in a subject with a corresponding need, wherein the method comprises administering to the subject an antibody-drug conjugate of any one of claims 1-20 and 23, a pharmaceutical composition of claim 29, or a pharmaceutical dosage form of claim 30.

32. The method of claim 31, wherein the condition is selected from the group consisting of: muscle atrophy and hereditary muscle diseases.

33. The method of claim 32, wherein the condition is selected from the group consisting of: adult spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), anoctaminopathy, autoimmune neuropathy, Becker muscular dystrophy, Bethlem myopathy, calcium dystrophy, pituitary muscular dystrophy, central nervous system disease, peroneal muscular dystrophy (CMT), congenital fibrous type dysplasia (CFTD), congenital muscular dystrophy, congenital myasthenia gravis (CMS), congenital myopathy, congenital myotonic dystrophy, dermatomyositis (DM), neuromuscular junction disorder, distal myopathy with rimmed vacuoles (DMRV), Dichenne muscular dystrophy (DMD), Emory-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), facioscapulohumeral muscular dystrophy (FSPD), progressive ossification of muscle (FOP), glycogen storage disease type V (GSD). V), GNE myopathy (GNEM), hereditary inclusion body myopathy (HIBM), hereditary inclusion body myopathy type 2 (HIBM2), hereditary motor and sensory neuropathy (HMSN), hereditary neuropathies, inclusion body myositis (IBM), infantile progressive spinal muscular atrophy, inflammatory neuropathies, infectious myelitis, intermediate spinal muscular atrophy, juvenile dermatomyositis, juvenile spinal muscular atrophy, Landouzy-Dejerine syndrome, limb-girdle muscular dystrophy (LGMD), McCardell's disease, merosin deficiency congenital muscular dystrophy, metabolic myopathy, minicore myopathy. Multiaxial hollow myopathy, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myofibril myopathy, myositis, progressive ossifying myositis (MOP), myotonic dystrophy, myotube and other central nucleus myopathy, rod body myopathy, mid-field myopathy, oculopharyngeal muscular dystrophy (OPMD), periodic paralysis, polymyositis (PM) and dermatomyositis (DM), quadriceps-preserving myopathy (QSM), myoglucanopathy, sarcopenia, SEPN1-related myopathy, spinal muscular atrophy (SMA), Steinert's disease, Ulrich's congenital muscular dystrophy, VCP disease, and age-related progressive loss of muscle mass and strength.

34. A method for selectively delivering a compound into cells, wherein the compound is an antibody-drug conjugate according to any one of claims 1-20 and 23.

35. A method for selectively targeting antigens on a cell surface with a compound, wherein the compound is any one of claims 1-20 and 23.

36. The method of claim 35, wherein the cell is a mammalian cell.

37. The method of claim 36, wherein the cell is a human cell.

38. The method according to any one of claims 35-37, wherein the cell is a muscle cell.

39. The method according to any one of claims 31-38, wherein the antibody-drug conjugate is administered intramuscularly, intravenously, or subcutaneously.

40. A method for internalizing a compound into muscle fibers, the method comprising contacting the muscle fibers with an antibody-drug conjugate according to any one of claims 1-20 and 23.

41. A method for preparing a compound having the structure according to formula (A) or a pharmaceutically acceptable salt thereof: Ab-(L1-B-L2-P) n (A), in: Ab is an antigen-binding protein according to any one of claims 1-14; L1 is the first linker that covalently binds to the side chain of the glutamine residue of Ab; B is the portion containing triazole; L2 is the second linker that covalently binds to the therapeutic agent P; P is a therapeutic agent selected from the group consisting of DHT and proDHT, and n is an integer from 1 to 8, wherein the method includes the following steps: a) In the presence of transglutaminase, contact the A containing at least one glutamine residue with at least one compound L1-B'; b) Contact the product of step a) with one or more equivalent amounts of compound B”-L2-P, wherein group B” is covalently attached to group B’. One of the groups B' and B” is selected from -N3 and ; and the other of the groups B' and B” is selected from , and Where Z is C or N; and c) The resulting compound of formula (A) was isolated.

42. The method of claim 41, wherein A has glutamine residues at positions 295 (Q295) and 297 (N297Q).

43. The method of claim 41, wherein the L1-B' has a structure .

44. The method of claim 41, wherein the compound B”-L2-P has a structure selected from the group consisting of:

45. The method of claim 41, wherein the compound of formula (A) has a structure selected from the group consisting of:

46. ​​A method for preparing a compound having the structure according to formula (I): A – [L – P] y (I), Where A is an antigen-binding protein; L stands for connector; P is a therapeutic agent selected from the group consisting of DHT and proDHT, and y is an integer from 1 to 8, and the method includes the following steps: a) In the presence of transglutaminase, contacting A, which contains at least one glutamine residue, with at least one compound LP, wherein said compound LP has at least one terminal amine moiety, and b) The resulting compound of formula (I) after separation.

47. The method of claim 46, wherein the compound LP has a structure selected from the group consisting of:

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