Glucocorticoid receptor agonists and their conjugates

Abstract

The present disclosure describes glucocorticoid agonist compounds for treating diseases or conditions such as autoimmune or inflammatory conditions, their conjugates with binding proteins, their pharmaceutical compositions, and methods and uses.

Description

Background Art

[0001] Autoimmune and inflammatory diseases are immune cell-mediated chronic diseases that often shorten the lifespan through organ damage that mediates the disease, resulting in a high sacrifice to the quality of life of patients. Glucocorticoid receptor agonists (GRAs) and modulators (GRMs) are the mainstay for controlling many autoimmune and inflammatory diseases by reducing immune cell disease activity. However, considering the widespread cell types and tissue expression of the glucocorticoid receptor, as well as the strong activity of the axis on many cellular functions, systemic administration of GRA or GRM results in various unacceptable toxicities, both in terms of the dose and duration-dependent manner (especially harmful bone, cardiovascular, metabolic, and neuropsychiatric toxicities, etc.) even at the low doses required to provide some disease remission.

[0002] Medicinal chemistry attempts to induce effective agonism for diseases that drive immune cells without toxicogenic agonism in other cell types by using systemically delivered small molecule GRAs or GRMs designed to activate subsets of glucocorticoid receptor (GR) cell activity. However, to date, it has been disappointing due to inadequate disease control, still unacceptable toxicity, or both. Considering this, current clinical practice guidelines for many autoimmune and inflammatory diseases limit both the dose and duration of GRA or GRM treatment, despite evidence of reduced loss of disease control. Therefore, alternative strategies are needed for therapies for treating autoimmune or inflammatory conditions.

Summary of the Invention

[0003] In some embodiments, the present invention provides a compound of formula II,

Chemical Formula

Mode for Carrying Out the Invention

[0004] I. Overview The present disclosure describes glucocorticoid agonist compounds for treating diseases or conditions such as autoimmune or inflammatory conditions, their conjugates with binding proteins, their pharmaceutical compositions, and methods and uses.

[0005] II. Definitions "Alkyl" is a straight-chain or branched saturated monovalent hydrocarbon. For example, an alkyl group has 1 to 18 carbon atoms (i.e., C 1-18 alkyl) or 1 to 8 carbon atoms (i.e., C 1-8 alkyl) or 1 to 6 carbon atoms (i.e., C 1-6 alkyl) or 1 to 4 carbon atoms (i.e., C 1-4It can have an alkyl group. Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), and 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), but are not limited thereto. Other alkyl groups include heptyl, octyl, nonyl, decyl, undecyl, dodecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl.

[0006] "Alkylene" refers to a straight-chain or branched saturated aliphatic radical having the indicated number of carbon atoms and linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same or different atoms of the alkylene group. For example, a straight-chain alkylene can be a divalent radical of -(CH2) n- where n is 1, 2, 3, 4, 5, or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene. The alkylene group can be substituted or unsubstituted.

[0007] "Alkenyl" refers to a straight-chain or branched-chain hydrocarbon having at least 2 carbon atoms and at least 1 double bond. Alkenyl can contain any number of carbons such as C2, C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 2-7 , C 2-8 , C 2-9 , C 2-10 , C3, C 3-4 , C 3-5 , C 3-6 , C4, C 4-5 , C 4-6 , C5, C 5-6 , and C6. The alkenyl group can have any suitable number of double bonds including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. The alkenyl group can be substituted or unsubstituted.

[0008] "Alkynyl" refers to any of a straight-chain or branched-chain hydrocarbon having at least 2 carbon atoms and at least 1 triple bond. Alkynyl may contain any number of carbons such as C2, C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 2-7 , C 2-8 , C 2-9 , C 2-10 , C3, C 3-4 , C 3-5 , C 3-6 , C4, C 4-5 , C 4-6 , C5, C 5-6 , and C6. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. The alkynyl group may be substituted or unsubstituted.

[0009] "Alkoxy" refers to an alkyl group having an oxygen atom connecting to the bonding point: alkyl-O-. Regarding the alkyl group, the alkoxy group may have any suitable number of carbon atoms such as C 1-6 . Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexyloxy, and the like. The alkoxy group may be further substituted with various substituents described herein. The alkoxy group may be substituted or unsubstituted.

[0010] "Alkoxyalkyl" refers to an alkoxy group linked to an alkyl group which is linked to the rest of the compound such that the alkyl group is divalent. Alkoxyalkyl is 2-6 (C 2-6 alkoxyalkyl), 2-5 (C 2-5(alkoxyalkyl), 2 - 4 (C 2-4 (alkoxyalkyl), or 2 - 3 (C 2-3 (alkoxyalkyl) and can have any suitable number of carbons such as etc. The number of carbons refers to the total number of carbons in the alkoxy and alkyl groups. For example, C6 alkoxyalkyl refers to ethoxy (C2 alkoxy) linked to butyl (C4 alkyl), and n - propoxy (C3 alkoxy) linked to isopropyl (C3 alkyl). Alkoxy and alkyl are as defined above when the alkyl is divalent, and include methoxymethyl (CH3OCH 2- ), methoxyethyl (CH3OCH2CH 2- ) and others, but are not limited thereto.

[0011] As used herein, "halo" or "halogen" refers to fluoro (-F), chloro (-Cl), bromo (-Br), and iodo (-I).

[0012] As used herein, "haloalkyl" refers to an alkyl as defined herein, wherein one or more hydrogen atoms of the alkyl are independently replaced by the same or different halo substituents. For example, C 1-4 haloalkyl is C 1-4 alkyl in which one or more of the hydrogen atoms of the alkyl are replaced by halo substituents. 1-4 Examples of haloalkyl groups include, but are not limited to, fluoromethyl, fluorochloromethyl, difluoromethyl, difluorochloromethyl, trifluoromethyl, 1,1,1 - trifluoroethyl, and pentafluoroethyl.

[0013] "Haloalkoxy" refers to an alkoxy group in which some or all of the hydrogen atoms are replaced by halogen atoms. Regarding the alkyl group, the haloalkoxy group is C 1-6 ​It can have any suitable number of carbon atoms such as etc. The alkoxy group can be substituted with one, two, three, or more halogens. When all hydrogens are replaced with halogens, for example, fluorine, the compound is oversubstituted, for example, perfluorinated. Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, perfluoroethoxy, etc.

[0014] "Cycloalkyl" refers to a single saturated or partially unsaturated all-carbon ring having 3 to 20 cyclic carbon atoms (i.e., C 3-20 cycloalkyl), for example, 3 to 12 cyclic atoms, for example, 3 to 10 cyclic atoms, or 3 to 8 cyclic atoms, or 3 to 6 cyclic atoms, or 3 to 5 cyclic atoms, or 3 to 4 cyclic atoms. The term "cycloalkyl" also includes multiple fused, saturated, and partially unsaturated all-carbon ring systems (for example, ring systems containing 2, 3, or 4 carbon rings). Thus, cycloalkyl includes bicyclic carbon rings (for example, bicyclic carbon rings having 6 to 12 cyclic carbon atoms such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane), and polycyclic carbon rings (for example, tricyclic and tetracyclic carbon rings having up to 20 cyclic carbon atoms). The rings of a multiple fused ring system can be connected to each other via fused bonds, spiro bonds, and bridging bonds when permitted by valence requirements. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopenta-1-enyl, 1-cyclopenta-2-enyl, 1-cyclopenta-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl.

[0015] As used herein, "heterocyclyl" or "heterocycle" or "heterocycloalkyl" refers to a single saturated or partially unsaturated non-aromatic ring or polycyclic system having at least one heteroatom (i.e., at least one cyclic heteroatom selected from oxygen, nitrogen, and sulfur) within the ring, and the polycyclic system includes at least one non-aromatic ring containing at least one heteroatom. The polycyclic system can also include other aromatic and non-aromatic rings. Unless otherwise specified, a heterocyclyl group has 3 to 20 ring atoms, for example, 3 to 12 ring atoms, for example, 3 to 10 ring atoms, or 3 to 8 ring atoms, or 3 to 6 ring atoms, or 3 to 5 ring atoms, or 4 to 6 ring atoms, or 4 to 5 ring atoms. Thus, the term includes a single saturated or partially unsaturated ring (e.g., 3, 4, 5, 6, or 7-membered ring) having 1 to 6 ring carbon atoms and 1 to 3 ring heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. The heteroatom can optionally be oxidized to form -N(-OH)-, =N(-O - )-, -S(=O)-, or -S(=O)2-. The rings of a polycondensed ring system (e.g., a bicyclic heterocyclyl) system can be connected to each other via fused bonds, spiro bonds, and bridging bonds when permitted by valence requirements. Examples of heterocycles include, but are not limited to, azetidine, aziridine, imidazolidine, morpholine, oxirane (epoxide), oxetane, thietane, piperazine, piperidine, pyrazolidine, piperidine, pyrrolidine, pyrrolidinone, tetrahydrofuran, tetrahydrothiophene, dihydropyridine, tetrahydropyridine, quinuclidine, 2-oxa-6-azaspiro[3.3]heptan-6-yl, 6-oxa-1-azaspiro[3.3]heptan-1-yl, 2-thia-6-azaspiro[3.3]heptan-6-yl, 2,6-diazaspiro[3.3]heptan-2-yl, 2-azabicyclo[3.1.0]hexan-2-yl, 3-azabicyclo[3.1.0]hexanyl, 2-azabicyclo[2.1.1]hexanyl, 2-azabicyclo[2.2.1]heptan-2-yl, 4-azaspiro[2.4]heptanyl, 5-azaspiro[2.4]heptanyl, etc.

[0016] The heterocycloalkyl ring also includes a 9- to 15-membered fused-ring heterocycloalkyl having two, three, or more rings, where at least one ring is an aryl ring and at least one ring is a non-aromatic ring containing at least one heteroatom. Representative fused bicyclic heterocycloalkyls include, as shown in the following structures, indoline (dihydroindole), isoindoline (dihydroisoindole), indazolene (dihydroindazole), benzo[d]imidazole, dihydroquinoline, dihydroisoquinoline, dihydrobenzofuran, dihydroisobenzofuran, benzo[d][1,3]dioxole, dihydrobenzo[b]dioxin, dihydrobenzo[d]oxazole, dihydrobenzo[b]thiophene, dihydroisobenzo[c]thiophene, dihydrobenzo[d]thiazole, dihydrobenzo[c]isothiazole, and benzo[b][1,4]thiazine, but are not limited thereto.

Chemical formula

Chemical formula

[0017] As used herein, "aryl" refers to a single fully carbon aromatic ring or a plurality of fused fully carbon ring systems, at least one of which is aromatic. For example, in some embodiments, an aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Examples of aryl include a phenyl radical. Aryl also includes a plurality of fused ring systems having 9 to 20 carbon atoms, for example, 9 to 16 carbon atoms (e.g., a ring system containing 2, 3, or 4 rings), where at least one ring is aromatic and the other ring may or may not be aromatic (i.e., a carbon ring). Such a plurality of fused ring systems is optionally substituted with one or more (e.g., 1, 2, or 3) oxo groups on any carbon ring moiety of the plurality of fused ring systems. The rings of the plurality of fused ring systems can be connected to each other via fused bonds, spiro bonds, and cross-linking bonds, where permitted by valence requirements. When referring to an aryl having a specific atomic range (e.g., 6- to 10-membered aryl), it should also be understood that the atomic range is for the total ring atoms of the aryl. For example, 6-membered aryl includes phenyl, and 10-membered aryl would include naphthyl and 1,2,3,4-tetrahydronaphthyl. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, etc.

[0018] "Alkylene-aryl" refers to a radical having an alkylene component and an aryl component, and the alkylene component connects the aryl component to the point of attachment. The alkylene component connects to the aryl component and to the point of attachment as defined above. The alkylene component is C 0-6 、C 1-2 、C 1-3 、C 1-4 、C 1-5 、C 1-6 、C 2-3 、C 2-4 、C 2-5 、C 2-6 、C 3-4 、C 3-5 、C 3-6 、C4-5 , C 4-6 , and C 5-6 may contain any number of carbons such as, etc. The aryl component is as defined above. Examples of alkylene-aryl groups include, but are not limited to, benzyl and ethyl-benzene. The alkylene-aryl group may be substituted or unsubstituted.

[0019] As used herein, "heteroaryl" refers to a single aromatic ring having at least one atom other than carbon within the ring, the atom being selected from the group consisting of oxygen, nitrogen, and sulfur, and "heteroaryl" also includes a plurality of fused ring systems having at least one such aromatic ring, which plurality of fused ring systems is further described below. Thus, "heteroaryl" includes a single aromatic ring of 1 to 6 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Sulfur atoms and nitrogen atoms may also be present in oxidized forms, provided that the ring is aromatic. Exemplary heteroaryl ring systems include, but are not limited to, pyridyl, pyrimidinyl, oxazolyl, or furyl. "Heteroaryl" also includes a plurality of fused ring systems (e.g., ring systems containing 2, 3, or 4 rings), and as defined above, a heteroaryl group is fused with one or more rings selected from heteroaryl (e.g., forming 1,8-naphthyridinyl), heterocyclic (e.g., forming 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocyclic (e.g., forming 5,6,7,8-tetrahydroquinolyl), and aryl (e.g., forming indazolyl) to form a plurality of fused ring systems. Thus, heteroaryl (a single aromatic ring or a plurality of fused ring systems) has 1 to 20 carbon atoms and 1 to 6 heteroatoms within the heteroaryl ring. Such a plurality of fused ring systems may optionally be substituted with one or more (e.g., 1, 2, 3, or 4) oxo groups on the carbocyclic or heterocyclic moiety of the fused ring. The rings of the plurality of fused ring systems may be connected to each other via fused bonds, spiro bonds, and bridging bonds, when permitted by valence requirements. It should be understood that the individual rings of the plurality of fused ring systems may be connected to each other in any order. It should be understood that the point of attachment of the heteroaryl or the plurality of fused ring systems of the heteroaryl can be any suitable atom of the heteroaryl or the plurality of fused ring systems of the heteroaryl including carbon atoms and heteroatoms (e.g., nitrogen). When referring to a heteroaryl having a specific atomic range member count (e.g., 5- to 10-membered heteroaryl), the atomic range is for the total ring atoms of the heteroaryl and is understood to include carbon atoms and heteroatoms.For example, 5-membered heteroaryl will include thiazolyl, and 10-membered heteroaryl will include quinolinyl. Exemplary heteroaryls include, but are not limited to, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl benzofuranyl, benzimidazolyl, thianaphthenyl, pyrrololo[2,3-b]pyridinyl, quinazolin-4(3H)-one, and triazolyl.

[0020] "Alkylene-heteroaryl" refers to a radical having an alkylene component and a heteroaryl component, wherein the alkylene component links to the heteroaryl component at the point of attachment. The alkylene component links to the heteroaryl component and at the point of attachment, as defined above. The alkylene component may contain any number of carbons such as C 0-6 、C 1-2 、C 1-3 、C 1-4 、C 1-5 、C 1-6 、C 2-3 、C 2-4 、C 2-5 、C 2-6 、C 3-4 、C 3-5 、C 3-6 、C 4-5 、C 4-6 、and C 5-6 and so forth. The heteroaryl component is as defined above. The alkylene-heteroaryl group may be substituted or unsubstituted.

[0021] A chemical substance having a carbon-carbon double bond or a carbon-nitrogen double bond may exist in the Z-form or E-form (or cis-form or trans-form). Further, some chemical substances may exist in various tautomeric forms. Unless otherwise specified, all Z-forms, E-forms, and tautomeric forms of the compounds of the present disclosure are equally intended to be included.

[0022] A "tautomer" refers to a molecule in which proton transfer is possible from one atom of the molecule to another atom of the same molecule. The compounds presented in this specification exist as tautomers in some embodiments. When tautomerism is possible, a chemical equilibrium state of tautomers exists. The exact ratio of tautomers depends on several factors including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium states are as follows.

Chemical formula

[0023] "The compounds of the present disclosure" include the compounds disclosed in this specification. For example, the compounds of the present disclosure include the compounds of Examples, and the compounds of Formulas I and II.

[0024] The compounds of the present disclosure may, in some cases, exist as diastereomers, enantiomers, or other stereoisomeric forms. The compounds presented in this specification include all forms of diastereomers, enantiomers, and epimers, as well as suitable mixtures thereof. Separation of stereoisomers can be carried out by chromatography, or by forming diastereomers and separating them by recrystallization or chromatography, or by any combination thereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley and Sons, Inc., 1981, which is incorporated herein by reference for the present disclosure). Stereoisomers can also be obtained by stereoselective synthesis.

[0025] As used herein, "composition" is intended to encompass a product containing the specified components in the specified amounts, and any product directly or indirectly obtained from a combination of the specified components in the specified amounts.

[0026] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications and commensurate with a reasonable benefit / risk ratio.

[0027] The terms "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" mean pharmaceutically acceptable materials, compositions, or vehicles such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. "Pharmaceutically acceptable excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, or emulsifying agent that has been approved by the US Food and Drug Administration as acceptable for use in humans or livestock. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0028] The term "salt" or "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counterions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Examples of inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Examples of organic acids that can induce salts include, for example, 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, and salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic bases and organic bases. Examples of inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Examples of organic bases that can induce salts include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Specifically, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine can be mentioned. In some embodiments, the pharmaceutically acceptable base addition salts are selected from ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts.

[0029] As used herein, "binding protein" includes a polypeptide and a binding domain or an antigen-binding fragment thereof that specifically binds to a target or multiple targets. Exemplary binding proteins of the present disclosure include fusion proteins, antibodies (e.g., monoclonal antibodies, bispecific antibodies), antibody constructs, target moieties, or antigen-binding fragments thereof. In some embodiments, the binding protein of the present disclosure includes a binding domain of an antibody or an antigen-binding fragment thereof. In some embodiments, the binding protein or binding polypeptide of the present disclosure (including those for which the term "polypeptide" may be synonymous with the term "protein") includes two or more polypeptides. In some embodiments, the binding protein or polypeptide of the present disclosure includes a complex of two or more polypeptides. In some embodiments, the binding protein or polypeptide further includes a tag, label, bioactive molecule, or any combination thereof. In some embodiments, the binding protein or polypeptide of the present disclosure includes non-natural amino acids.

[0030] In some embodiments, the binding protein or binding polypeptide of the present disclosure comprises or consists of a fragment. As used herein, "polypeptide fragment" means a polypeptide lacking one or more amino acids present in a reference sequence, which may be referred to as an "oligopeptide fragment" or "peptide fragment". In certain embodiments, the polypeptide, oligopeptide, or peptide fragment of the present disclosure comprises a deletion of one or more amino acids present in a reference polypeptide. In further embodiments, the polypeptide, oligopeptide, or peptide fragment of the present disclosure comprises a cleavage of one or more amino acids present in a reference polypeptide. The polypeptide, oligopeptide, or peptide fragment of the present disclosure can comprise a binding domain, antigen, or epitope, e.g., a binding domain, antigen, or epitope present in a reference sequence disclosed herein. The polypeptide fragment of the present disclosure can have at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more of the total number of amino acids of the amino acid sequence of the reference sequence.

[0031] "Treatment", "treating", or "treatment" refers to an intervention that results in any observable beneficial effect of treatment, or any statistically significant success in the treatment or alleviation of a disease or condition, such as alleviation of the signs, symptoms, or progression of a disease or pathological condition. A beneficial effect can be demonstrated, for example, by a reduction in the severity of clinical symptoms of a disease in a subject, a delay or reduction in onset, a reduction in the frequency with which the symptoms of the disease are experienced by the subject, a slower progression of the disease, a reduction in the number of recurrences of the disease, an improvement in the overall health or well-being of the subject, or by other parameters specific to a particular disease.

[0032] A prophylactic treatment that means "preventing" a disease or condition (e.g., tumor formation or proliferation in a subject or patient) is a treatment administered to a subject who does not show signs of the disease or shows only early signs, for the purpose of preventing the onset of pathology or reducing the risk of further progression of the early disease. For example, if an individual at risk of developing an inflammatory or autoimmune condition or having severe symptoms is treated by the methods of the present disclosure and later does not develop an inflammatory or autoimmune condition or have severe symptoms, the disease or the severity of the disease is prevented in that individual for at least a certain period of time. Prophylactic treatment can mean, for example, preventing recurrence or relapse of a disease or condition in a patient previously treated for the disease or condition, by preventing recurrence or relapse of an inflammatory disease or autoimmune disease.

[0033] "Administering" refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, or subcutaneous administration, intrathecal administration, or implantation of a sustained release device, e.g., a mini osmotic pump, into the subject. Administration can be carried out according to a schedule specifying the frequency of administration, the dosage for administration, and other factors.

[0034] As used herein, the terms "parenteral administration" and "administered parenterally" generally mean modes of administration other than enteral and topical administration by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intramedullary, and intrathoracic injections and infusions. As used herein, the terms "intravenous administration" and "administered intravenously" refer to injection or infusion of the conjugate into a vein of the subject. Terms such as "subcutaneous administration", "administer subcutaneously", etc. refer to administration of the conjugate subcutaneously to the subject. For clarity, subcutaneous administration is different from intratumoral injection into a tumor or cancerous lesion located subcutaneously.

[0035] "GR" refers to the glucocorticoid receptor.

[0036] "GR agonist" is a compound that binds to and activates the glucocorticoid receptor.

[0037] III. Compounds In some embodiments, the compounds of the present disclosure are compounds of Formula I,

Chemical formula

[0038] In some embodiments, the compounds of the present disclosure are compounds of formula I,

Chemical formula

Chemical formula

[0039] In certain embodiments, R 200 is -OR 201 and R 201 has one of the following structures.

Chemical formula

[0040] In some embodiments, R 200 is -OR 201 and R 201 has the following structure.

Chemical formula

[0041] In some embodiments, R 200 is -OR 201 and R 201 has the following structure.

Chemical formula

[0042] In some embodiments, R 200 is -OR 201 and R 201 has the following structure.

Chemical formula

[0043] In some embodiments, the compound of the present disclosure is a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein wherein, R 101 、R 102 、R 103 、and R 104 are each independently H or F, R 105 is C 2-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, phenyl, -(C 1-6 alkylene)-phenyl, heteroaryl, -(C 1-6 alkylene)-heteroaryl, C 3-8 cycloalkyl, or heterocyclyl, where alkyl or alkenyl is substituted with 1, 2, or 3 R 107 groups, alkynyl is substituted with 0, 1, 2, or 3 R 108 groups, phenyl is substituted with 2 or 3 R 109 groups, -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 1, 2, or 3 R 110 groups, R 106 is H, or alternatively, R 105 and R 106 combine together to form a C 3-8 cycloalkyl or heterocyclyl, where cycloalkyl or heterocyclyl is substituted with 0, 1, 2, or 3 R 110 groups, each R 107 is independently C 2-6 alkoxyalkyl, C 1-6 haloalkoxy, phenyl, heteroaryl, C 3-8 cycloalkyl, heterocyclyl, or halogen, where phenyl is substituted with 1, 2, or 3 R 111 groups, and heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R 111 groups, each R 108 is independently C 1-6 haloalkoxy, phenyl, heteroaryl, C 3-8Cycloalkyl, heterocyclyl, or halogen, wherein phenyl, heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R 112 and each R is independently C 109 alkyl, C 1-6 alkenyl, C 2-6 alkynyl, C 2-6 alkoxyalkyl, C 2-6 haloalkyl, halogen, -N3, -OR 1-6 , or -N(R 113 )(R 113 ), and alkyl, alkenyl, or alkynyl is substituted with 0 or 1 -S(O)2(C 1-6 alkyl), and each R 110 is independently C 2-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 2-6 alkoxyalkyl, -(C 1-6 alkylene)-OR 114 , -(C 1-6 alkylene)-N(R 114 )(R 1-6 ), C 1-6 haloalkyl, phenyl, -(C 1-6 alkylene)-phenyl, heteroaryl, -(C 115 alkylene)-heteroaryl, halogen, -N3, -OR 115 , -N(R 115 )(CO)R 115 , -N(R 115 )(CO)OR 115 , -N(R 115 )S(O)2R 115 , -(CO)R 115 , -SO2R 115 , or -SO2N(R 115 )(R 116 ), and phenyl, alkylene-phenyl, heteroaryl, or alkylene-heteroaryl is substituted with 0, 1, 2, or 3 R and each R 111 and R 112 is independently C 1-6 alkyl, C1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, halogen, -OR 114 , or -N(R 114 )2, and each R 113 is independently H, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, or phenyl, and haloalkyl is substituted with 0 or 1 N(R 114 )2, each R 114 is independently H or C 1-6 alkyl, each R 116 is -OR 117 , -N(R 117 )2, -N(R 117 )(CO)R 117 , -N(R 117 )(CO)OR 117 , -N(R 117 )S(O)2R 117 , -(CO)R 117 , -SO2R 117 , or -SO2N(R 117 )2, and each R 115 and R 117 are independently H, C 1-6 alkyl, C 1-6 haloalkyl, or phenyl, R 200 is -OR 201 or -N(R 201 )2, and R 201 is H, C 1-6 alkyl, phenyl, or heteroaryl, and phenyl or heteroaryl is substituted with 0, 1, or 2 -OR 202 or -N(R 202 )2, R 202 is H or C 1-6 alkyl, and in each case the heteroaryl is a 5 - 10 membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S The heterocyclyl in each example is a 4- to 10-membered heterocyclyl having 1, 2, or 3 heteroatoms selected from N, O, and S, a compound, or a pharmaceutically acceptable salt thereof.

[0044] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 101 , R 102 , R 103 , and R 104 , one of which is F, and R 101 , R 102 , R 103 , and R 104 , three of which are H. In some embodiments, R 101 , R 102 , R 103 , and R 104 , two of which are F, and R 101 , R 102 , R 103 , and R 104 , two of which are H.

[0045] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 101 is H. In some embodiments, R 101 is F.

[0046] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 102 is H. In some embodiments, R 102 is F.

[0047] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 101 is H and R 102 is F. In certain embodiments, R 101 and R 102 are each F. In some embodiments, R 101 and R 102 are each H. In some embodiments, R 101 is F and R 102 is H.

[0048] In some embodiments of the compound or a pharmaceutically acceptable salt thereof, each R 103 and R 104 is H. In certain embodiments, R 103 and R 104 are each F. In some embodiments, R 103 is F and R 104 is H. In some embodiments, R 103 is H and R 104 is F. In some embodiments, R 103 and R 104 are both H, and R 101 and R 102 are each H. In certain embodiments, R 103 and R 104 are both H, and R 101 and R 102 are each F. In some embodiments, R 103 and R 104 are both H, R 101 is F, and R 102 is H. In certain embodiments, R 103 and R 104 are both H, R 101 is F, and R 102 is H.

[0049] In some embodiments of the compound or a pharmaceutically acceptable salt thereof, R 105 is C 2-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, or C 1-6 haloalkyl, and the alkyl or alkenyl is substituted with 1, 2, or 3 R 107 s, and the alkynyl is substituted with 0, 1, 2, or 3 R 108 s. In some embodiments, R 105 is C 2-6 alkenyl, and the alkenyl is substituted with 1, 2, or 3 R 107 s.

[0050] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is phenyl, -(C 1-6 alkylene)-phenyl, heteroaryl, -(C 1-6 alkylene)-heteroaryl, C 3-8 cycloalkyl, or heterocyclyl, phenyl is substituted with 2 or 3 R 109 , -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 1, 2, or 3 R 110 . In some embodiments, R 105 is phenyl, phenyl is substituted with 2 or 3 R 109 . In some embodiments, R 105 is -(C 1-6 alkylene)-phenyl, heteroaryl, -(C 1-6 alkylene)-heteroaryl, C 3-8 cycloalkyl, or heterocyclyl, -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 1, 2, or 3 R 110 . In some embodiments, R 105 is C 3-8 cycloalkyl or heterocyclyl, cycloalkyl or heterocyclyl is substituted with 1, 2, or 3 R 110 . In some embodiments, R 105 is heteroaryl or -(C 1-6 alkylene)-heteroaryl, heteroaryl or -alkylene heteroaryl is substituted with 1, 2, or 3 R 110 .

[0051] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is heteroaryl, heteroaryl is substituted with 1, 2, or 3 R 110 . In some embodiments, R 105is thienyl, imidazolyl, triazolyl, indolyl, indazolyl, or thienothienyl, which is substituted with 0, 1, 2, or 3 R 110 s. In some embodiments, R 105 is thienyl, which is substituted with 0, 1, 2, or 3 R 110 s. In some embodiments, R 105 is thienyl, which is substituted with 0, 1, or 2 R 110 s.

[0052] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

[0053] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

Chemical formula

[0054] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

Chemical formula

[0055] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

Chemical formula

[0056] In some embodiments, the compound of the present disclosure is a compound of formula II

Chemical formula

[0057] In some embodiments, the compounds of the present disclosure are compounds of Formula II,

Chemical formula

Chemical Structure

[0058] In some embodiments, R 200 is -OR 201 and R 201 has the following structure.

Chemical formula

[0059] In some embodiments, R 200 is -OR 201 and R 201 has the following structure.

Chemical formula

[0060] In some embodiments, R 200 is -OR 201 and R 201 has the following structure.

Chemical formula

[0061] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is C 4-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, or C 1-6 haloalkyl, and the alkyl or alkenyl is substituted with 0, 1, 2, or 3 R 107 groups, and the alkynyl is substituted with 0, 1, 2, or 3 R 108 groups. In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is C 2-6 alkenyl, and the alkenyl is substituted with 0, 1, 2, or 3 R 107 groups.

[0062] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, each R 107 is independently phenyl, heteroaryl, C 3-8is cycloalkyl, heterocyclyl, or halogen, and phenyl, heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, or 2 R 111 .

[0063] In some embodiments of the compound or a pharmaceutically acceptable salt thereof, R 105 is phenyl, -(C 1-6 alkylene)-phenyl, heteroaryl, -(C 1-6 alkylene)-heteroaryl, C 3-8 cycloalkyl, or heterocyclyl, phenyl is substituted with 0, 1, 2, or 3 R 109 , -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R 110 . In some embodiments, R 105 is heteroaryl or -(C 1-6 alkylene)-heteroaryl, and heteroaryl or -alkylene heteroaryl is substituted with 0, 1, 2, or 3 R 110 . In some embodiments, R 105 is thienyl, imidazolyl, triazolyl, indolyl, indazolyl, or thienothienyl, which is substituted with 0, 1, or 2 R 110 . In some embodiments, R 105 is thienyl, which is substituted with 0, 1, 2, or 3 R 110 . In some embodiments, R 105 is thienyl, which is substituted with 0, 1, or 2 R 110 .

[0064] In some embodiments of the compound or a pharmaceutically acceptable salt thereof, each R 110 is independently C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 2-6 alkoxyalkyl, phenyl, -(C 1-6(alkylene)-phenyl, heteroaryl, -(C 1-6 (alkylene)-heteroaryl, heterocyclyl, halogen, -N3, -OR 112 , -N(R 112 )2, -(CO)R 112 , or -S(O)2R 112 wherein phenyl, alkylene-phenyl, heteroaryl, alkylene-heteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R 114 . In some embodiments, each R 110 is independently C 1-3 alkyl or halogen.

[0065] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

[0066] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

Chemical formula

[0067] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

Chemical formula

[0068] In some embodiments of the present compound or a pharmaceutically acceptable salt thereof, R 105 is

Chemical formula

Chemical formula

Chemical formula

[0069] In some embodiments of the compounds of the present disclosure such as the compounds of Formula I or II or pharmaceutically acceptable salts thereof, in each case, the heteroaryl is a 5- to 9-membered heteroaryl having 1 or 2 heteroatoms selected from N, O, and S. In some embodiments, in each case, the heteroaryl is a 5- to 6-membered heteroaryl having 1 or 2 heteroatoms selected from N, O, and S.

[0070] In some embodiments of the compounds of the present disclosure such as the compounds of Formula I or II or pharmaceutically acceptable salts thereof, in each case, the heterocyclyl is a 4- to 9-membered heterocyclyl having 1 or 2 heteroatoms selected from N, O, and S. In some embodiments, in each case, the heterocyclyl is a 4- to 8-membered heterocyclyl having 1 or 2 heteroatoms selected from N, O, and S. In some embodiments, in each case, the heterocyclyl is a 4- to 6-membered heterocyclyl having 1 or 2 heteroatoms selected from N, O, and S.

[0071] In some embodiments of the compounds of the present disclosure or pharmaceutically acceptable salts thereof, the compound has the structure of the compounds described in the examples herein.

[0072] In some embodiments of the compounds of the present disclosure or pharmaceutically acceptable salts thereof, the compound has a structure selected from Table 1.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

[0073] In some embodiments of the compounds of the present disclosure or pharmaceutically acceptable salts thereof, the compound has a structure selected from Table 2.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0074] Characterization Compound 30a 11H NMR (400 MHz, DMSO-d6) δ = 7.63 - 7.59 (m, 1H), 7.27 (d, J = 10.0 Hz, 1H), 6.96 (d, J = 5.2 Hz, 1H), 6.33 - 6.27 (m, 1H), 6.13 (s, 1H), 5.93 (s, 1H), 5.75 - 5.56 (m, 1H), 5.53 (s, 1H), 4.95 (s, 1H), 4.50 (d, J = 19.2 Hz, 1H), 4.28 - 4.13 (m, 2H), 2.74 - 2.56 (m, 1H), 2.36 - 2.28 (m, 1H), 2.27 - 2.17 (m, 1H), 2.09 - 1.99 (m, 1H), 1.74 - 1.64 (m, 3H), 1.55 - 1.41 (m, 4H), 0.85 (s, 3H). MS (ESI) m / z 525.0 [M + H] +

[0075] Compound 30B 1 1H NMR (400 MHz, DMSO-d6) δ = 7.63 - 7.59 (m, 1H), 7.27 (d, J = 10.0 Hz, 1H), 6.96 (d, J = 5.2 Hz, 1H), 6.48 (s, 1H), 6.33 - 6.27 (m, 1H), 6.13 (s, 1H), 5.75 - 5.56 (m, 3H), 5.29 (d, J = 6.0 Hz, 1H), 4.40 - 4.05 (m, 3H), 2.74 - 2.56 (m, 1H), 2.36 - 2.28 (m, 1H), 2.24 - 2.05 (m, 2H), 1.99 - 1.61 (m, 5H), 1.48 (s, 3H), 0.86 (s, 3H). MS (ESI) m / z 525.2 [M + H] +

[0076] Compound 52a 11H NMR (400 MHz, DMSO-d6) δ = 7.64 - 7.57 (m, 1H), 7.29 (d, J = 9.6 Hz, 1H), 6.96 (d, J = 5.2 Hz, 1H), 6.27 - 6.20 (m, 1H), 6.04 (s, 1H), 5.92 (s, 1H), 5.45 (s, 1H), 4.97 - 4.90 (m, 1H), 4.50 (d, J = 19.6 Hz, 1H), 4.22 - 4.14 (m, 3H), 2.69 - 2.54 (m, 2H), 2.48 - 2.42 (m, 1H), 2.40 - 2.31 (m, 1H), 2.22 - 2.10 (m, 1H), 2.07 - 1.99 (m, 1H), 1.90 - 1.80 (m, 1H), 1.70 - 1.60 (m, 3H), 1.49 (s, 3H), 1.44 - 1.33 (m, 1H), 0.86 (s, 3H). MS (ESI) m / z 507.2 [M+H] +

[0077] Compound 52b 1 1H NMR (400 MHz, DMSO-d6) δ = 7.63 - 7.58 (m, 1H), 7.29 (d, J = 10.0 Hz, 1H), 6.97 (d, J = 5.6 Hz, 1H), 6.45 (s, 1H), 6.27 - 6.21 (m, 1H), 6.03 (s, 1H), 5.47 (s, 1H), 5.28 (d, J = 6.8 Hz, 1H), 4.31 (d, J = 19.2 Hz, 1H), 4.22 - 4.15 (m, 1H), 4.07 (d, J = 19.6 Hz, 1H), 2.69 - 2.58 (m, 1H), 2.48 - 2.30 (m, 2H), 2.07 - 1.96 (m, 2H), 1.87 - 1.76 (m, 2H), 1.74 - 1.65 (m, 2H), 1.56 - 1.42 (m, 5H), 0.87 (s, 3H). MS (ESI) m / z 507.2 [M+H] +

[0078] Compound 53a 11H NMR (400 MHz, DMSO-d6) δ = 7.65 - 7.51 (m, 1H), 7.31 (d, J = 9.6 Hz, 1H), 6.96 (d, J = 4.8 Hz, 1H), 6.17 (d, J = 10 Hz, 1H), 5.92 (d, J = 19.6 Hz, 2H), 4.92 (s, 1H), 4.87 - 4.75 (m, 1H), 4.49 (d, J = 18.8 Hz, 1H), 4.32 (s, 1H), 4.16 (d, J = 19.6 Hz, 1H), 2.62 - 2.53 (m, 1H), 2.39 - 2.28 (m, 1H), 2.21 - 1.98 (m, 3H), 1.75 (s, 2H), 1.72 - 1.61 (m, 3H), 1.39 (s, 3H), 1.05 - 0.93 (m, 1H), 0.90 (s, 1H), 0.85 (s, 3H). MS (ESI) m / z 489.0 [M+H] +

[0079] Compound 53b 1 1H NMR (400 MHz, DMSO-d6) δ = 7.63 - 7.57 (m, 1H), 7.31 (d, J = 10.0 Hz, 1H), 6.96 (d, J = 5.2 Hz, 1H), 6.44 (s, 1H), 6.21 - 6.12 (m, 1H), 5.93 (s, 1H), 5.25 (d, J = 6.4 Hz, 1H), 4.90 - 4.68 (m, 1H), 4.38 - 4.27 (m, 3H), 4.06 (d, J = 19.2 Hz, 1H), 2.59 - 2.52 (m, 1H), 2.34 - 2.26 (m, 1H), 2.10 - 1.94 (m, 2H), 1.88 - 1.62 (m, 6H), 1.38 (s, 3H), 1.27 - 1.13 (m, 1H), 1.10 - 1.03 (m, 1H), 0.86 (s, 3H). MS (ESI) m / z 489.2 [M+H] +

[0080] Compound 61a 11H NMR (400 MHz, DMSO-d6) δ = 7.27 (dd, J = 0.8, 10.0 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 3.2 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.88 - 6.82 (m, 2H), 6.75 (d, J = 3.6 Hz, 1H), 6.30 (dd, J = 1.6, 10.0 Hz, 1H), 6.14 (s, 1H), 5.75 (s, 1H), 5.73 - 5.50 (m, 2H), 4.91 (t, J = 2.4 Hz, 1H), 4.49 (d, J = 19.2 Hz, 1H), 4.23 - 4.14 (m, 2H), 4.07 (s, 2H), 2.73 - 2.55 (m, 1H), 2.34 - 2.26 (m, 1H), 2.20 (q, J = 10.0 Hz, 1H), 1.99 (d, J = 13.6 Hz, 1H), 1.72 - 1.57 (m, 3H), 1.49 (s, 3H), 1.47 - 1.39 (m, 1H), 0.84 (s, 3H). MS (ESI) m / z 612.0 [M+H] +

[0081] Compound 61b 1 1H NMR (400 MHz, DMSO-d6) δ = 7.26 (d, J = 11.2 Hz, 1H), 7.21 - 7.15 (m, 1H), 7.04 (d, J = 3.6 Hz, 1H), 6.88 - 6.73 (m, 4H), 6.34 (s, 1H), 6.30 (dd, J = 1.6, 10.0 Hz, 1H), 6.12 (s, 1H), 5.76 - 5.55 (m, 1H), 5.53 (d, J = 3.6 Hz, 1H), 5.26 (d, J = 7.2 Hz, 1H), 4.37 (d, J = 12.8 Hz, 1H), 4.23 - 4.15 (m, 1H), 4.12 - 4.02 (m, 3H), 2.30 - 1.98 (m, 4H), 1.91 - 1.78 (m, 1H), 1.75 - 1.59 (m, 3H), 1.49 (s, 3H), 0.87 (s, 3H). MS (ESI) m / z 612.2 [M+H] +

[0082] Compound 62a 11H NMR (400 MHz, DMSO-d6) δ = 7.29 - 7.23 (m, 2H), 7.20 (br t, J = 7.7 Hz, 1H), 7.05 (d, J = 1.3 Hz, 1H), 6.91 (br s, 1H), 6.87 - 6.78 (m, 2H), 6.30 (dd, J = 1.9, 10.2 Hz, 1H), 6.13 (s, 1H), 5.81 (s, 1H), 5.75 - 5.56 (m, 1H), 5.52 (br d, J = 1.8 Hz, 1H), 4.91 (t, J = 2.6 Hz, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.26 - 4.11 (m, 2H), 3.82 (s, 2H), 2.75 - 2.56 (m, 1H), 2.38 - 2.17 (m, 3H), 2.07 - 1.95 (m, 1H), 1.70 - 1.60 (m, 3H), 1.49 (s, 3H), 1.48 - 1.40 (m, 1H), 0.84 (s, 3H). MS (ESI) m / z 612.1 [M+H] +

[0083] Compound 62b 1 1H NMR (400 MHz, MeOD) δ = 7.48 - 7.39 (m, 1H), 7.32 (br d, J = 10.0 Hz, 2H), 7.21 - 7.11 (m, 2H), 6.98 (br s, 1H), 6.87 (s, 1H), 6.40 (s, 1H), 6.37 - 6.27 (m, 2H), 5.65 - 5.45 (m, 1H), 5.33 (d, J = 6.4 Hz, 1H), 4.39 (d, J = 19.3 Hz, 1H), 4.29 (br d, J = 9.5 Hz, 1H), 4.16 (d, J = 19.3 Hz, 1H), 3.98 (s, 2H), 2.76 - 2.55 (m, 1H), 2.43 - 2.29 (m, 1H), 2.19 - 2.13 (m, 2H), 1.95 - 1.75 (m, 2H), 1.73 - 1.63 (m, 2H), 1.57 (s, 3H), 0.97 (s, 3H). MS (ESI) m / z 612.1 [M+H] +

[0084] Compound 86a 11H NMR (400 MHz, DMSO-d6) δ = 7.36 (d, J = 8.0 Hz, 2H), 7.29 - 7.22 (m, 4H), 7.02 (d, J = 7.6 Hz, 1H), 6.95 - 6.85 (m, 2H), 6.30 (dd, J = 1.6, 10.0 Hz, 1H), 6.13 (s, 1H), 5.71 - 5.60 (m, 2H), 5.46 (s, 1H), 4.95 (d, J = 4.4 Hz, 1H), 4.51 (d, J = 19.2 Hz, 1H), 4.27 - 4.13 (m, 2H), 3.91 (s, 2H), 2.75 - 2.56 (m, 1H), 2.35 - 2.17 (m, 2H), 2.02 (d, J = 13.2 Hz, 1H), 1.76 - 1.63 (m, 3H), 1.54 - 1.45 (m, 4H), 0.86 (s, 3H). MS (ESI) m / z 606.3 [M+H] +

[0085] Compound 86b 1 1H NMR (400 MHz, DMSO-d6) δ = 7.26 (d, J = 10.4 Hz, 1H), 7.23 - 7.16 (m, 5H), 6.89 (d, J = 7.6 Hz, 1H), 6.84 - 6.75 (m, 2H), 6.30 (dd, J = 2.0, 10.2 Hz, 1H), 6.14 - 6.07 (m, 2H), 5.74 - 5.55 (m, 1H), 5.54 - 5.49 (m, 1H), 5.33 (d, J = 7.2 Hz, 1H), 4.26 (d, J = 19.2 Hz, 1H), 4.20 - 4.15 (m, 1H), 4.03 (d, J = 19.2 Hz, 1H), 3.89 (s, 2H), 2.68 - 2.56 (m, 1H), 2.35 - 2.24 (m, 2H), 2.20 - 2.10 (m, 2H), 2.09 - 2.04 (m, 1H), 1.93 - 1.80 (m, 1H), 1.77 - 1.60 (m, 3H), 1.49 (s, 3H), 0.88 (s, 3H). MS (ESI) m / z 606.3 [M+H] +

[0086] Compound 89a 11H NMR (400 MHz, DMSO-d6) δ = 7.29 (d, J = 10.0 Hz, 1H), 7.23 - 7.16 (m, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.92 - 6.81 (m, 3H), 6.75 (d, J = 3.6 Hz, 1H), 6.24 (dd, J = 1.6, 10.0 Hz, 1H), 6.05 (s, 1H), 5.74 (s, 1H), 5.43 (s, 1H), 4.89 (d, J = 3.6 Hz, 1H), 4.49 (d, J = 19.2 Hz, 1H), 4.17 (d, J = 19.2 Hz, 2H), 4.07 (s, 2H), 2.70 - 2.59 (m, 1H), 2.40 - 2.31 (m, 1H), 2.18 - 2.08 (m, 1H), 2.07 - 1.99 (m, 1H), 1.87 - 1.78 (m, 1H), 1.71 - 1.58 (m, 3H), 1.50 (s, 3H), 1.38 - 1.33 (m, 1H), 0.85 (s, 3H). MS (ESI) m / z 594.2 [M+H] +

[0087] Compound 89b 1 1H NMR (400 MHz, DMSO-d6) δ = 7.29 (d, J = 10.0 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 3.6 Hz, 1H), 6.90 - 6.75 (m, 4H), 6.31 (s, 1H), 6.24 (dd, J = 1.6, 10.0 Hz, 1H), 6.04 (s, 1H), 5.45 (d, J = 2.4 Hz, 1H), 5.24 (d, J = 6.8 Hz, 1H), 4.36 (d, J = 19.2 Hz, 1H), 4.18 (d, J = 10.0 Hz, 1H), 4.12 - 4.04 (m, 3H), 2.71 - 2.60 (m, 1H), 2.39 - 2.32 (m, 2H), 2.07 - 1.97 (m, 2H), 1.89 - 1.77 (m, 2H), 1.71 - 1.63 (m, 2H), 1.50 (s, 3H), 0.88 (s, 3H). MS (ESI) m / z 594.3 [M+H] +

[0088] Compound 90a 11H NMR (400 MHz, DMSO-d6) δ = 7.32 (d, J = 10.0 Hz, 1H), 7.26 - 7.19 (m, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.93 - 6.87 (m, 2H), 6.75 (d, J = 3.6 Hz, 1H), 6.19 (dd, J = 1.6, 10.0 Hz, 1H), 5.97 (s, 1H), 5.73 (s, 1H), 4.88 (d, J = 4.4 Hz, 1H), 4.86 - 4.76 (m, 1H), 4.49 (d, J = 19.2 Hz, 1H), 4.29 (d, J = 2.4 Hz, 1H), 4.16 (d, J = 19.6 Hz, 1H), 4.09 (s, 2H), 2.58 - 2.52 (m, 1H), 2.35 - 2.30 (m, 1H), 2.18 - 1.97 (m, 2H), 1.79 - 1.54 (m, 5H), 1.39 (s, 3H), 1.01 - 0.86 (m, 2H), 0.84 (s, 3H). MS (ESI) m / z 576.2 [M+H] +

[0089] Compound 90b 1 1H NMR (400 MHz, DMSO-d6) δ = 7.31 (d, J = 10.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.01 (d, J = 3.2 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.89 - 6.82 (m, 2H), 6.77 (d, J = 3.2 Hz, 1H), 6.30 (s, 1H), 6.17 (d, J = 10.0 Hz, 1H), 5.94 (s, 1H), 5.20 (d, J = 6.8 Hz, 1H), 4.80 (s, 1H), 4.36 (d, J = 19.2 Hz, 1H), 4.29 (s, 1H), 4.10 - 3.99 (m, 3H), 2.50 - 2.40 (m, 1H), 2.12 - 1.95 (m, 2H), 1.87 - 1.59 (m, 5H), 1.38 (s, 3H), 1.27 - 1.09 (m, 1H), 1.05 - 1.00 (m, 1H), 0.86 (s, 3H). MS (ESI) m / z 576.3 [M+H] +

[0090] Compound 91a 11H NMR (400 MHz, DMSO-d6) δ = 7.29 - 7.23 (m, 2H), 7.20 (br t, J = 7.6 Hz, 1H), 7.05 (s, 1H), 6.91 (br s, 1H), 6.87 - 6.78 (m, 2H), 6.30 (dd, J = 2.0, 10.2 Hz, 1H), 6.13 (s, 1H), 5.81 (s, 1H), 5.42 (br d, J = 1.8 Hz, 1H), 4.91 (t, J = 2.6 Hz, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.26 - 4.11 (m, 2H), 3.82 (s, 2H), 2.75 - 2.56 (m, 1H), 2.38 - 2.17 (m, 3H), 2.07 - 1.95 (m, 1H), 1.69 - 1.60 (m, 3H), 1.49 (s, 3H), 1.48 - 1.40 (m, 1H), 0.84 (s, 3H). MS (ESI) m / z 594.1 [M+H] +

[0091] Compound 91b 1 1H NMR (400 MHz, MeOD) δ = 7.44 - 7.37 (m, 2H), 7.29 (d, J = 7.8 Hz, 1H), 7.18 - 7.11 (m, 2H), 6.96 (s, 1H), 6.87 (s, 1H), 6.37 (s, 1H), 6.30 (d, J = 10.4 Hz 1H), 6.10 (s, 1H), 5.32 (d, J = 6.4 Hz, 1H), 4.38 (d, J = 19.2 Hz, 1H), 4.37 - 4.22 (m, 1H), 4.15 (d, J = 19.2 Hz, 1H), 3.99 (s, 2H), 2.81 - 2.70 (m, 1H), 2.68 - 2.51 (m, 1H), 2.49 - 2.40 (m, 1H), 2.28 - 2.10 (m, 2H), 1.99 - 1.52 (m, 5H), 1.63 (s, 3H), 0.97 (s, 3H). MS (ESI) m / z 594.2 [M+H] +

[0092] Compound 92a 11H NMR (400 MHz, DMSO-d6) δ = 7.31 (d, J = 10.0 Hz, 1H), 7.22 (s, 1H), 7.21 - 7.16 (m, 1H), 7.04 (d, J = 1.4 Hz, 1H), 6.90 (br d, J = 4.0 Hz, 1H), 6.87 - 6.79 (m, 2H), 6.17 (dd, J = 2.0, 10.0 Hz, 1H), 5.95 (s, 1H), 5.78 (s, 1H), 4.88 (d, J = 4.8 Hz, 1H), 4.81 (br s, 1H), 4.48 (d, J = 19.2 Hz, 1H), 4.30 (br d, J = 2.8 Hz, 1H), 4.15 (d, J = 19.2 Hz, 1H), 3.82 (s, 2H), 2.60 - 2.52 (m, 1H), 2.37 - 2.28 (m, 1H), 2.18 - 2.01 (m, 2H), 1.76 - 1.71 (m, 2H), 1.70 - 1.65 (m, 2H), 1.64 - 1.59 (m, 1H), 1.39 (s, 3H), 1.05 - 0.86 (m, 2H), 0.85 (s, 3H). MS (ESI) m / z 576.1 [M + H] +

[0093] Compound 92b 1 1H NMR (400 MHz, MeOD) δ = 7.51 - 7.40 (m, 2H), 7.35 (br d, J = 7.2 Hz, 1H), 7.24 - 7.13 (m, 2H), 6.99 (br s, 1H), 6.85 (s, 1H), 6.38 (s, 1H), 6.27 (dd, J = 1.6, 10.0 Hz, 1H), 6.04 (s, 1H), 5.31 (t, J = 3.2 Hz, 1H), 4.44 (br d, J = 2.8 Hz, 1H), 4.41 - 4.34 (m, 1H), 4.17 (d, J = 19.2 Hz, 1H), 4.00 (s, 2H), 2.69 - 2.64 (m, 1H), 2.44 - 2.39 (m, 1H), 2.31 - 2.11 (m, 2H), 2.04 - 1.98 (m, 1H), 1.89 - 1.80 (m, 2H), 1.79 - 1.64 (m, 2H), 1.51 (s, 3H), 1.27 - 1.16 (m, 1H), 1.13 - 1.09 (m, 1H), 0.99 (s, 3H). MS (ESI) m / z 576.2 [M + H] +

[0094] IV. Conjugate In some embodiments, the conjugate of the present disclosure comprises a compound of the present disclosure, a binding protein described herein, and a linker that covalently attaches the compound to the binding protein.

[0095] In some embodiments, the conjugate of the present disclosure has an average ratio of GR agonist to binding protein (referred to herein as drug-to-antibody ratio, or DAR), which ranges from 1 to about 20, 1 to about 10, 1 to about 8, 1 to about 6, 1 to about 5, 1 to about 3, 2 to about 8, 2 to about 6, 2 to about 5, 2 to about 4, about 3 to about 8, about 3 to about 6, or about 3 to about 5, and the drug is a GR agonist of any one of the formulas of the present disclosure. In certain embodiments, the average drug-to-antibody ratio (DAR) of the conjugate of the present disclosure ranges from 1 to about 8, or 2 to about 6, or about 3 to about 5, or about 4. In some embodiments, the average ratio of GR agonist to the binding protein of the conjugate in a pharmaceutical formulation ranges from 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 8, 3 to 6, or 3 to 5, and the drug is a GR agonist of any one of the formulas of the present disclosure. In some embodiments, the average DAR of the conjugate of the present disclosure is 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, or 8 or about 8.

[0096] A. Antibodies and Fusion Proteins In some embodiments of the present disclosure, the binding protein or its conjugate comprises an antibody (e.g., a monoclonal antibody) or a fusion protein that contains a binding domain that specifically binds to a target of interest. In some embodiments, an anti-target antibody or its antigen-binding fragment or fusion protein is conjugated to a compound of the present disclosure, thus forming a conjugate or a binding protein conjugate.

[0097] In some embodiments, the conjugate proteins of the present disclosure include an anti-target fusion protein from a natural or non-natural source sequence. In some embodiments, the antibodies of the present disclosure are recombinant.

[0098] The antibodies of the present disclosure can be derivatized antibodies. For example, derivatized antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cell ligand or other protein.

[0099] Antibodies can be chimeric or humanized. In some embodiments, the antibodies of the present disclosure are chimeric. Chimeric and humanized forms of non-human (e.g., mouse) antibodies can contain sequences derived from non-human immunoglobulins and can be intact (full-length) chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof (e.g., Fv, Fab, Fab’, F(ab’)2, or other target-binding subdomains of an antibody). In some embodiments, the antibodies of the present disclosure are humanized. Generally, a humanized antibody can contain substantially all of at least one, typically two variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions being of human immunoglobulin sequences. A humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) or Fc domain, such as a human immunoglobulin sequence.

[0100] The antibodies of the present disclosure can be human antibodies. As used herein, "human antibody" may include, for example, antibodies having the amino acid sequence of human immunoglobulins, and may include antibodies isolated from a human immunoglobulin library or from transgenic animals for one or more human immunoglobulins, typically not expressing endogenous immunoglobulins. Human antibodies can be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. A fully human antibody that recognizes a selected epitope can be generated using affinity selection. In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to direct the selection of a fully human antibody that recognizes the same epitope.

[0101] The antibody can be of any class, e.g., IgA, IgD, IgE, IgG, and IgM. A particular class can be further divided into isotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins can be α, δ, ε, γ, and μ, respectively. In certain embodiments, the antibodies of the present disclosure include a human IgG1, human IgG2, human IgG3, or human IgG4 heavy chain constant region. The light chain can be either kappa (or κ) or lambda (or λ).

[0102] The antibodies of the present disclosure can be bispecific antibodies or dual variable domain antibodies (DVDs). Bispecific antibodies and DVD antibodies are monoclonal antibodies having binding specificities for at least two different antigens, and are often human or humanized antibodies. In various embodiments, at least one binding domain of the bispecific antibody specifically binds to a target provided by the present disclosure. Similarly, the fusion proteins of the present disclosure can be bispecific and can have two binding domains that bind to two different targets, such as BAFF and APRIL, CD28 and ICOS, or BAFF and ICOS-L. In some embodiments, one of the two binding domains can be derived from an antibody or an antigen-binding domain derived from an antibody.

[0103] In some embodiments, the antibodies or fusion proteins of the present disclosure include an antigen-binding domain and an Fc domain. In various embodiments, an antibody includes two light chain polypeptides (light chains) and two heavy chain polypeptides (heavy chains) that are covalently held together by disulfide bonds. A heavy chain typically includes a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region includes three domains: CH1, CH2, and CH3. The Fc domain is located within the heavy chain CH2 and CH3 domains. Non-limiting exemplary heavy chain constant regions include human IgG1, human IgG2, human IgG3, and human IgG4 constant regions. In some embodiments, the antibodies provided herein include an IgG1 heavy chain constant region. In further embodiments, the antibodies provided herein include an IgG1 heavy chain constant region that includes one or more substitutions that reduce or eliminate effector function. In some embodiments, the antibodies provided herein include an IgG1 heavy chain constant region (e.g., a human IgG1 constant region) that includes the substitutions L117A, L118A, G120A, and / or K205A. In some embodiments, the antibodies provided herein include an IgG1 constant region that includes the substitutions P329G, L234A, L235A, G237A, and / or K322A. Non-limiting exemplary human IgG1 constant region and human IgG1 null constant region amino acid sequences are shown in SEQ ID NOs: 181 and 182. A light chain typically includes a light chain variable region (VL) and a light chain constant region. Non-limiting exemplary light chain constant regions include kappa and lambda constant regions. A non-limiting exemplary human kappa constant region amino acid sequence is shown in SEQ ID NO: 183.

[0104] The antigen recognition region of an antibody variable domain typically includes six complementarity-determining regions (CDRs), or hypervariable regions, present within the frameworks of the heavy-chain variable region and the light-chain variable region at the N-terminals of two heavy chains and two light chains. In some embodiments, the antigen-binding domain includes light-chain complementarity-determining region 1 (LCDR1), light-chain complementarity-determining region 2 (LCDR2), light-chain complementarity-determining region 3 (LCDR3), heavy-chain complementarity-determining region 1 (HCDR1), heavy-chain complementarity-determining region 2 (HCDR2), and heavy-chain complementarity-determining region 3 (HCDR3). In some embodiments, the antibody may be a heavy-chain only antibody, in which case the antigen-binding domain includes HCDR1, HCDR2, and HCDR3, and the antibody lacks a light chain.

[0105] The exemplary CDR sequences of the antibodies disclosed herein, such as anti-BAFF antibodies, anti-LPAM-1 antibodies, anti-CD40 antibodies, anti-CD86 antibodies, anti-ICOS antibodies, anti-ICOSL antibodies, anti-CD28 antibodies, anti-CD80 antibodies, and anti-integrin β7 antibodies, can be determined by one or more methods including Kabat, Chothia, AbM, Contact, IMGT, and AHo. Unless otherwise specified herein, CDR sequences are determined according to the Kabat method. The variable region or CDR numbering as in Kabat, the amino acid numbering as in Kabat, or the CDR sequences determined according to the Kabat method, and variations thereof, refer to the numbering system used for the heavy chain variable region or light chain variable region of the antibody compilation in Kabat et al. ((1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion (residue 52a according to Kabat) after residue 52, and three inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment in the region of sequence homology of the antibody's sequence with the "standard" Kabat-numbered sequence. The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., see Kabat et al., supra).

[0106] The "EU numbering system" or "EU index" is generally used when referring to residues within the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al. (supra)). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0107] Other numbering systems are described, for example, by AbM (see AbM antibody modeling software from Oxford Molecular (see, for example, Antibody Engineering Vol. 2 (Kontermann and Dithel eds., 2d ed. 2010)), Chothia (see Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17), Contact, IMGT (see ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1): 55-77)), and AHon (see Honegger and Plueckthun, 2001, J. Mol. Biol. 309: 657-70), and are well understood by those skilled in the art.

[0108] B. Constant domain and Fc region portion The constant domain of an antibody provides the general framework of the antibody and may not be directly involved in binding to an antigen, but may be involved in various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), ADCP (antibody-dependent cell phagocytosis), CDC (complement-dependent cytotoxicity), and complement fixation, binding to Fc receptors (e.g., CD16, CD32, FcRn), a longer in vivo half-life compared to polypeptides lacking the Fc region, binding to protein A, and in some cases, placental transfer (see Capon et al., Nature 337: 525, 1989).

[0109] As used herein, the term "Fc region constant domain portion" or "Fc region portion" refers to the heavy chain constant region segment of the Fc fragment from an antibody (the "fragment crystallizable" region or Fc region), and can include one or more constant domains such as CH2, CH3, CH4, or any combination thereof. As used herein, the term "Fc domain" refers to a domain from the Fc region portion of an antibody that can specifically bind to an Fc receptor such as an Fcγ receptor or an FcRn receptor. In certain embodiments, the Fc region portion includes the CH2 and CH3 domains of an IgG, IgA, or IgD antibody and any combination thereof, or the CH3 and CH4 domains of an IgM or IgE antibody and any combination thereof.

[0110] The Fc region or domain can interact with different types of FcRs. Different types of FcRs can include, for example, FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcαRI, FcμR, FcεRI, FcεRII, and FcRn. FcRs can be located on the membranes of certain immune cells including, for example, B lymphocytes, natural killer cells, macrophages, neutrophils, follicular dendritic cells, eosinophils, basophils, platelets, and mast cells. When an FcR is involved with an Fc domain, the FcR can initiate functions including, for example, clearance of antigen - antibody complexes via receptor - mediated endocytosis, antibody - dependent cell - mediated cytotoxicity (ADCC), antibody - dependent cell - mediated phagocytosis (ADCP), trogocytosis, trogoptosis, and ligand - induced transmission of signals across the cell membrane, which can lead to secretion, exocytosis, and changes in cell metabolism. FcRs can deliver signals when they are aggregated by antibodies and multivalent antigens at the cell surface. Aggregation of FcRs having immunoreceptor tyrosine - based activation motifs (ITAMs) can successively activate SRC family tyrosine kinases and SYK family tyrosine kinases. ITAMs contain a YXXL sequence (where X = any amino acid) repeated twice adjacent to seven variable residues. SRC and SYK kinases can connect the transduced signals to a common activation pathway.

[0111] In some embodiments, the Fc region portion or its domain can exhibit a reduced binding affinity for one or more Fc receptors such as Fcγ receptors, FcRn receptors, or both Fcγ and FcRn receptors. In some embodiments, the Fc region portion contains an Fc null domain. As used herein, "Fc null domain" refers to a domain that shows little or no binding to any of the Fcγ receptors. In some embodiments, the Fc null domain exhibits at least about a 1000 - fold decrease in binding affinity for Fcγ receptors (e.g., an increase in Kd).

[0112] The Fc region or domain can have one or more, two or more, three or more, or four or more, or up to five amino acid substitutions that reduce the binding of the Fc region portion or its domain to the Fc receptor. In some embodiments, the Fc region portion or its domain exhibits a decrease in binding to FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), or any combination thereof. In some embodiments, the Fc region portion or its domain is IgG1, and one or more substitutions within the Fc region or domain include any one or combination of IgG1 heavy chain mutations corresponding to P329G, E233P, L234V, L234A, L235A, L235E, ΔG236, G237A, E318A, K320A, K322A, A327G, A330S, or P331S according to the EU index of Kabat numbering.

[0113] In some embodiments, the Fc region portion or its domain can include the sequence of an IgG1 isoform modified from the wild-type IgG1 sequence. The modification can include substitutions at more than one amino acid residue, such as five different amino acid residues including L235V / F243L / R292P / Y300L / P396L (referred to as IgG1VLPLL) according to the EU index of Kabat numbering. The modification can include substitutions at more than one amino acid residue, such as two different amino acid residues including S239D / I332E (IgG1DE) according to the EU index of Kabat numbering. The modification can include substitutions at more than one amino acid residue, such as three different amino acid residues including S298A / E333A / K334A (referred to as IgG1AAA) according to the EU index of Kabat numbering. In certain embodiments of the present disclosure, the IgG1 constant region comprises, or consists of, the amino acid sequence of any one of SEQ ID NOs: 181 to 183. In a further embodiment, the antibody of the present disclosure includes a mouse IgG2a heavy chain constant region.

[0114] An antibody or Fc region portion or domain thereof can be modified to acquire or improve at least one constant region-mediated biological effector function, compared to an unmodified antibody or Fc domain, for example, to enhance FcγR interaction. In some embodiments, the modification can increase CD32b binding (and transdelivery in a support, e.g., a PBMC assay), including substitutions at S267L and E329F (also known as the IgG1LF, SELF double mutant) according to the EU index of Kabat numbering. In certain embodiments, the antibodies of the disclosure can comprise an Fc domain, or the fusion proteins of the disclosure can comprise an Fc region portion that binds to FcγRIIA, FcγRIIB, or FcγRIIIA with a higher affinity than the corresponding wild-type Fc domain.

[0115] In some embodiments, the Fc region portion or domain thereof found in the antibodies or fusion proteins of the disclosure can mediate one or more effector functions, lack one or more or all such activities, or have one or more of the effector activities increased by one or more mutations compared to, for example, an unmodified Fc region portion or domain thereof.

[0116] In some embodiments, the IgG Fc domain comprises at least one amino acid substitution that reduces the binding affinity to FcγR1 compared to a wild-type or reference IgG Fc domain. Such modifications can include substitutions at F241 such as F241A, F243 such as F243A, V264 such as V264A, or D265 such as D265A, each according to the EU index of Kabat.

[0117] In some embodiments, the IgG Fc domain comprises at least one amino acid substitution that increases the binding affinity to FcγR1 compared to a wild-type or reference IgG Fc domain. Such modifications can include substitutions at A327 and P329 such as A327Q / P329A according to the EU index of Kabat.

[0118] In some embodiments, the modification comprises one or more amino acid substitutions that reduce the binding affinity of the IgG Fc domain for the FcγRII and FcγRIIIA receptors. The modification can be a substitution of D270, such as D270A, according to the Kabat EU index. The modification can be a substitution of Q295, such as Q295A, according to the Kabat EU index. The modification can be a substitution of A327, such as A237S, according to the Kabat EU index.

[0119] In some embodiments, the modification comprises one or more amino acid substitutions that increase the binding affinity of the IgG Fc domain for the FcγRII and FcγRIIIA receptors. The modification can be a substitution of T256, such as T256A, according to the Kabat EU index. The modification can be a substitution of K290, such as K290A, according to the Kabat EU index.

[0120] In some embodiments, the modification comprises substitution of one or more amino acids in the IgG Fc domain that increases the binding affinity to the FcγRII receptor. The modification can be a substitution of R255, such as R255A, according to the Kabat EU index. The modification can be a substitution of E258, such as E258A, according to the Kabat EU index. The modification can be a substitution of S267, such as S267A, according to the Kabat EU index. The modification can be a substitution of E272, such as E272A, according to the Kabat EU index. The modification can be a substitution of N276, such as N276A, according to the Kabat EU index. The modification can be a substitution of D280, such as D280A, according to the Kabat EU index. The modification can be a substitution of H285, such as H285A, according to the Kabat EU index. The modification can be a substitution of N286, such as N286A, according to the Kabat EU index. The modification can be a substitution of T307, such as T307A, according to the Kabat EU index. The modification can be a substitution of L309, such as L309A, according to the Kabat EU index. The modification can be a substitution of N315, such as N315A, according to the Kabat EU index. The modification can be a substitution of K326, such as K326A, according to the Kabat EU index. The modification can be a substitution of P331, such as P331A, according to the Kabat EU index. The modification can be a substitution of S337, such as S337A, according to the Kabat EU index. The modification can be a substitution of A378, such as A378A, according to the Kabat EU index. The modification can be a substitution of E430, such as E430, according to the Kabat EU index.

[0121] In some embodiments, the modification comprises one or more amino acid substitutions that increase the binding affinity of the IgG Fc domain to the FcγRII receptor and decrease the binding affinity to the FcγRIIIA receptor. The modification can be a substitution of H268, such as H268A, according to the Kabat EU index. The modification can be a substitution of R301, such as R301A, according to the Kabat EU index. The modification can be a substitution of K322, such as K322A, according to the Kabat EU index.

[0122] In some embodiments, the modification comprises one or more amino acid substitutions that decrease the binding affinity of the IgG Fc domain to the FcγRII receptor but do not affect the binding affinity to the FcγRIIIA receptor. The modification can be a substitution of R292, such as R292A, according to the Kabat EU index. The modification can be a substitution of K414, such as K414A, according to the Kabat EU index.

[0123] In some embodiments, the modification comprises one or more amino acid substitutions that decrease the binding affinity of the IgG Fc domain to the FcγRII receptor and increase the binding affinity to the FcγRIIIA receptor. The modification can be a substitution of S298, such as S298A, according to the Kabat EU index. The modification can be substitutions of S239, I332, and A330, such as S239D / I332E / A330L. The modification can be substitutions of S239 and I332, such as S239D / I332E.

[0124] In some embodiments, the modification comprises one or more amino acid substitutions that decrease the binding affinity of the IgG Fc domain to the FcγRIIIA receptor. The modification can be substitutions of F241 and F243, such as F241S / F243S or F241I / F243I, according to the Kabat EU index.

[0125] In some embodiments, the modification comprises one or more amino acid substitutions that reduce the binding affinity of the IgG Fc domain to the FcγRIIIA receptor and do not affect the binding affinity to the FcγRII receptor. The modification can be a substitution of S239, such as S239A, according to the Kabat EU index. The modification can be a substitution of E269, such as E269A, according to the Kabat EU index. The modification can be a substitution of E293, such as E293A, according to the Kabat EU index. The modification can be a substitution of Y296, such as Y296F, according to the Kabat EU index. The modification can be a substitution of V303, such as V303A, according to the Kabat EU index. The modification can be a substitution of A327, such as A327G, according to the Kabat EU index. The modification can be a substitution of K338, such as K338A, according to the Kabat EU index. The modification can be a substitution of D376, such as D376A, according to the Kabat EU index.

[0126] In some embodiments, the modification comprises one or more amino acid substitutions that increase the binding affinity of the IgG Fc domain to the FcγRIIIA receptor and do not affect the binding affinity to the FcγRII receptor. The modification can be a substitution of E333, such as E333A, according to the Kabat EU index. The modification can be a substitution of K334, such as K334A, according to the Kabat EU index. The modification can be a substitution of A339, such as A339T, according to the Kabat EU index. The modification can be substitutions of S239 and I332, such as S239D / I332E.

[0127] In some embodiments, the modification comprises one or more amino acid substitutions that increase the binding affinity of the IgG Fc domain to the FcγRIIIA receptor. The modification can be a substitution of L235, F243, R292, Y300, and P396, such as L235V / F243L / R292P / Y300L / P396L (IgG1VLPLL), according to the Kabat EU index. The modification can be a substitution of S298, E333, and K334, such as S298A / E333A / K334A, according to the Kabat EU index. The modification can be a substitution of K246, such as K246F, according to the Kabat EU index.

[0128] Other substitutions within the IgG Fc domain that affect its interaction with one or more Fcγ receptors are disclosed in U.S. Patent Nos. 7,317,091 and 8,969,526, which are incorporated herein by reference.

[0129] In some embodiments, the IgG Fc domain comprises at least one amino acid substitution that reduces the binding affinity to FcRn as compared to the wild-type or reference IgG Fc domain. The modification can include a substitution at H435, such as H435A, according to the Kabat EU index. The modification can include a substitution at I253, such as I253A, according to the Kabat EU index. The modification can include a substitution at H310, such as H310A, according to the Kabat EU index. The modification can include substitutions at I253, H310, and H435, such as I253A / H310A / H435A, according to the Kabat EU index.

[0130] The modification can include a substitution of one amino acid residue that increases the binding affinity of the IgG Fc domain for FcRn as compared to the wild-type or reference IgG Fc domain. The modification can include a substitution at V308, such as V308P, according to the Kabat EU index. The modification can include a substitution at M428, such as M428L, according to the Kabat EU index. The modification can include a substitution at N434, such as N434A or N434H according to the Kabat EU index. The modification can include substitutions at T250 and M428, such as T250Q and M428L, according to the Kabat EU index. The modification can include substitutions at M428 and N434, such as M428L and N434S, N434A, or N434H, according to the Kabat EU index. The modification can include substitutions at M252, S254, and T256, such as M252Y / S254T / T256E, according to the Kabat EU index. The modification can include a substitution of one or more amino acids selected from P257L, P257N, P257I, V279E, V279Q, V279Y, A281S, E283F, V284E, L306Y, T307V, V308F, Q311V, D376V, and N434H. Other substitutions within the IgG Fc domain that affect its interaction with FcRn are disclosed in U.S. Patent No. 9,803,023, the disclosure of which is incorporated herein by reference.

[0131] In some embodiments, the antibody is a human IgG2 antibody that includes an IgG2 Fc region. In some embodiments, the heavy chain of the human IgG2 antibody can be mutated to cysteine at positions 127, 232, or 233. In some embodiments, the light chain of the human IgG2 antibody can be mutated to cysteine at position 214. The mutations in the heavy and light chains of the human IgG2 antibody can be from a cysteine residue to a serine residue.

[0132] C. Target As used herein, "target" refers to a molecule for the purpose of enabling specific delivery of a conjugate of the present disclosure to a location or tissue where the target is located. Exemplary targets of the present disclosure include one or more targets selected from B cell activating factor (BAFF), BAFF receptor (BAFF-R), proliferation-inducing ligand (APRIL), transmembrane activator and CAML interactor (TACI), Peyer's patch-specific homing receptor (LPAM-1), B cell maturation antigen (BCMA), CD40, CD40 ligand (CD40L), T lymphocyte activation antigen CD86 (CD86), cytotoxic T lymphocyte protein 4 (CTLA4), tyrosine kinase-type cell surface receptor HER2 (HER2), inducible T cell co-stimulator (ICOS), ICOS ligand (ICOSL), T cell-specific surface glycoprotein CD28 (CD28), T lymphocyte activation antigen CD80 (CD80), integrin β7, integrin α4, mucosal addressin cell adhesion molecule 1 (MADCAM), tumor necrosis factor α (TNFα), and tumor necrosis factor receptor 2 (TNF-R2). In some embodiments, the target is selected from the group consisting of cluster of differentiation 40 (CD40, tumor necrosis factor receptor superfamily 5 (TNFSF5)), CD40 ligand (CD40L, CD154), T lymphocyte activation antigen CD86 (CD86), cytotoxic T lymphocyte protein 4 (CTLA4), inducible T cell co-stimulator (ICOS), ICOS ligand (ICOSL), T cell-specific surface glycoprotein CD28 (CD28), T lymphocyte activation antigen CD80 (CD80), integrin β7, integrin α4, mucosal addressin cell adhesion molecule 1 (MADCAM), tumor necrosis factor α (TNFα), tumor necrosis factor receptor 2 (TNF-R2), killer cell lectin-like receptor G1 (KLRG1), B cell activating factor (BAFF), BAFF receptor (BAFFR), transmembrane activator and CAML interaction (TACI), Peyer's patch-specific homing receptor (LPAM-1), B cell maturation antigen (BCMA), and proliferation-inducing ligand (APRIL).In some embodiments, the target of the present disclosure comprises, or consists of, a number of amino acids sufficient to be specifically bound by a binding domain, such as having at least 5, 6, 7, 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids of any of the target sequences provided herein.

[0133] In some embodiments, the binding proteins of the present disclosure include an anti-target antibody, an anti-target fusion protein, or an antigen-binding fragment thereof. Exemplary anti-target binding proteins include belimumab, tabalumab, rozanolixizumab (also known as AMG-570, two tandem copies of a BAFF-binding peptide fused to the C-terminus of the anti-ICOSL mAb heavy chain), blisibimod (also known as AMG-623, a BAFF-binding domain fused to the N-terminus of hIgG1), atacicept (TACI ectodomain fused to hIgG1 Fc), briobacept (extracellular ligand-binding portion of BAFF-R fused to hIgG1 Fc), tibulizumab (an anti-IL-17 scFv derived from ixekizumab fused to anti-BAFF tabalumab via a Gly-rich linker), ALPN-303 (a high-affinity variant form of TACI fused to hIgG), ianamumab (also known as VAY736), vedolizumab, etrolizumab, brazikumab (also known as 4D11, ASKP-1240, ASKP1240), dacetuzumab, ziloverumab, isakizumab (also known as CFZ-533, NVP-CFZ533, OM11-62MF), lucatumumab, mitazalimab, ravulizumab (also known as ABBV-323), serclidemab, sotigalimab, vanalizumab, BI 655064, rupatuzumab, tralokinumab, dupilumab pegol (also known as CDP7657), retifanlimab (also known as BMS-986004, a domain antibody targeting CD40L fused to IgG1 Fc from the C-terminus), dazodalibep, abatacept (CTLA4 fused to hIgG1 Fc), belatacept (high-affinity CTLA4 fused to hIgG1 Fc, CTLA4-Ig (ASP2408), CTLA4-Ig (ASP2409), arglifyrmab, feralizumab, izalizumab (a bispecific hybrid mAb and scFv targeting ICOS and PD-1), boptelimumab, akdacortcept (also known as ALPN-101, an ICOS-L variant fused to null huFc), rulizumab (also known as BMS-931699 or lh-239-891 (D70C)), abrilumab, natalizumab, ontamalizumab, adalimumab, etanercept, golimumab, infliximab, certolizumab pegol, civerlimab (also known as VIS-649), and one or more of BION-1301.

[0134] In some embodiments, the conjugate protein conjugate of the present disclosure comprises a compound of the present disclosure linked to a BAFF-binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to BAFF. Such anti-BAFF binding proteins of the present disclosure, and their GR agonist conjugates, can specifically bind to BAFF-expressing cells. In certain embodiments, the anti-BAFF binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human BAFF (e.g., see www.uniprot.org / uniprot / Q9Y275, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In certain other embodiments, the fusion protein conjugate of the present disclosure comprises human TACI or an extracellular portion thereof (e.g., see www.uniprot.org / uniprot / O14836, the sequence of which is incorporated herein by reference in its entirety). In further embodiments, the anti-BAFF binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-BAFF antibody (e.g., belimumab or tabalumab) or an anti-BAFF fusion protein (e.g., ALPN-303, a high-affinity variant form of the TACI ectodomain fused to human IgG).

[0135] In another embodiment, the binding domain of the targeting protein also binds to a non-cell-bound soluble target and confers a reduction in inflammation and / or autoimmunity (e.g., soluble BAFF, APRIL, TNFα).

[0136] As background, the BAFF receptor (BAFF-R) is expressed on B lymphocytes and, when activated by BAFF alone, results in increased B cell survival and autoimmune signaling via NF-κB. When the anti-BAFF binding protein conjugate of the present disclosure binds to BAFF, the conjugate blocks this signaling pathway and effectively blocks BAFF-R by binding to both membrane-bound and soluble BAFF. Thus, one result of administration of the anti-BAFF conjugate of the present disclosure is to inhibit B cell activation via BAFF-R. At the same time, myeloid cells internalize the anti-BAFF conjugate via macropinocytosis, Fc receptor-mediated uptake, or both. Once internalized by myeloid cells, the anti-BAFF conjugate releases its GR or GM agonist payload within the myeloid cells, which alters cell surface and soluble molecule production (e.g., cytokines and chemokines) that affect the immune activation of nearby cells such as T cells. In addition, the anti-BAFF conjugate of the present disclosure can also secondarily inhibit T cells and other immune cells adjacent to dendritic cells (or myeloid cells) due to bystander activity (i.e., release of the payload by dendritic cells that subsequently inhibits GR signaling in T cells). In some embodiments, the conjugates of the present disclosure comprise an anti-BAFF binding protein, such as belimumab or tabalumab or antigen-binding domains thereof, that reduces pathogenic B cell activity, and a payload that delivers disease-suppressing GR agonism to myeloid cells and other cell types (e.g., plasmacytoid dendritic cells or T cells).

[0137] In some embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist that binds to an anti-BAFF antibody, and the anti-BAFF antibody comprises the heavy chain CDR amino acid sequences of CDR1 (VH-CDR1), VH-CDR2, and VH-CDR3 from belimumab and the light chain CDR amino acid sequences of CDR1 (VL-CDR1), VL-CDR2, and VL-CDR3 from belimumab. In certain embodiments, the conjugate of the present disclosure comprises a heavy chain variable (VH) region comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belimumab VH region amino acid sequence, and a light chain variable (VL) region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belimumab VL region amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belimumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belimumab light chain amino acid sequence.

[0138] In some embodiments, the conjugate of the present disclosure comprises an anti-BAFF antibody, and the anti-BAFF antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from tabalumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from tabalumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tabalumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tabalumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tabalumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tabalumab light chain amino acid sequence.

[0139] In a further embodiment, the conjugate of the present disclosure comprises a compound of the present disclosure linked to a bispecific antibody construct consisting of an antibody specific for BAFF and a binding domain specific for IL-17, the binding domain specific for IL-17 being linked to the anti-BAFF antibody heavy chain via a peptide spacer, and the peptide spacer linking the binding domains specific for IL-17 and the anti-ICOSL antibody comprising from about 5 to about 15 amino acids (preferably 14 amino acids), and the binding domain specific for IL-17 being linked via a second peptide spacer comprising from about 10 to about 30 amino acids (preferably 20 amino acids) and comprising the VH and VL regions from ixekizumab. In any of these conjugate embodiments, the BAFF-binding peptide comprises an anti-BAFF binding domain from tabalumab. In certain embodiments, the conjugate of the present disclosure comprises a GR agonist linked to a fusion protein comprising a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the chimeric anti-CD20 antibody heavy chain amino acid sequence and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the chimeric anti-CD20 antibody light chain amino acid sequence.

[0140] In some embodiments, the conjugate of the present disclosure comprises a GR agonist linked to an anti-BAFF binding protein, and the binding protein comprises a BAFF binding peptide that specifically binds to BAFF. In further embodiments, the conjugate of the present disclosure comprises a GR agonist linked to a bispecific antibody construct consisting of a BAFF binding peptide and an antibody specific for inducible costimulatory ligand (ICOSL), and one or more BAFF binding peptides are linked to the anti-ICOSL antibody heavy chain via a peptide spacer. The peptide spacer linking the BAFF binding peptide and the anti-ICOSL antibody comprises from about 5 to about 15 amino acids, and two or more BAFF binding peptides are linked via a second peptide spacer comprising from about 15 to about 35 amino acids. In any of these conjugate embodiments, the BAFF binding peptide comprises a BAFF binding peptide from Rhizobius sp. alpha. In certain embodiments, the conjugate of the present disclosure comprises a GR agonist linked to a fusion protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Rhizobius sp. alpha.

[0141] In some embodiments, the conjugate of the present disclosure comprises an anti-BAFF fusion protein comprising one or both of the Blisibimod BAFF binding peptide amino acid sequences fused to the Fc region moiety. In certain embodiments, the conjugate of the present disclosure comprises an anti-BAFF fusion protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Blisibimod amino acid sequence.

[0142] In some embodiments, the conjugate of the present disclosure comprises a compound of the present disclosure linked to an anti-BAFF binding protein, and the binding protein comprises a TACI ectodomain or a portion thereof that specifically binds to BAFF. In further embodiments, the conjugate of the present disclosure comprises a GR agonist linked to a bispecific binding fusion protein comprising a TACI ectodomain or a portion thereof that specifically binds to BAFF and APRIL. In certain embodiments, the conjugate of the present disclosure comprises a GR agonist linked to a fusion protein consisting of a TACI ectodomain and an Fc region portion, and the TACI ectodomain is optionally fused to the Fc region portion via a peptide spacer consisting of about 5 to about 15 amino acids. In any of these conjugate embodiments, the BAFF-binding peptide or the BAFF and APRIL-binding peptide comprises a BAFF or BAFF and APRIL-binding peptide from atacicept or ALPN-303. In some embodiments, the conjugate of the present disclosure comprises a GR agonist linked to a fusion protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the atacicept amino acid sequence or the ALPN-303 amino acid sequence.

[0143] In some embodiments, the conjugate of the present disclosure comprises a compound of the present disclosure linked to an anti-BAFF binding protein, and the binding protein comprises a BAFF-R ectodomain or a portion thereof that specifically binds to BAFF. In further embodiments, the conjugate of the present disclosure comprises a compound of the present disclosure linked to a fusion protein consisting of a BAFF-R ectodomain and an Fc region portion such as a human IgG1 Fc region, and the TACI ectodomain is optionally fused to the Fc region portion via a peptide spacer consisting of about 5 to about 15 amino acids. In certain embodiments, the anti-BAFF fusion protein comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the briobacept amino acid sequence.

[0144] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a compound of the present disclosure linked to a BAFF-R binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to BAFF-R. Such anti-BAFF-R binding proteins of the present disclosure and their GR agonist conjugates can specifically bind to BAFF-R expressing cells. In certain embodiments, the anti-BAFF-R binding protein of the present disclosure, or its conjugate, specifically binds to human BAFF-R (e.g., see www.uniprot.org / uniprot / Q96RJ3, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-BAFF-R binding protein of the present disclosure, or its conjugate, comprises an anti-BAFF-R antibody (e.g., belimumab) or an anti-BAFF-R fusion protein.

[0145] In some embodiments, the conjugate of the present disclosure comprises an anti-BAFF antibody, and the anti-BAFF-R antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from ianalumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from ianalumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ianalumab VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ianalumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ianalumab heavy chain amino acid sequence, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ianalumab light chain amino acid sequence.

[0146] In certain embodiments, the binding protein conjugate of the present disclosure comprises a compound of the present disclosure linked to a TACI binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to TACI. Such anti-TACI binding domains and conjugates of the present disclosure can specifically bind to TACI-expressing cells. In certain embodiments, the anti-TACI binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human TACI (see, e.g., Uniprot#O14836, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-TACI binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-TACI antibody or anti-TACI fusion protein.

[0147] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist linked to an α4 integrin or α4β7 integrin heterodimer binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to α4 integrin or α4β7 integrin. Such anti-α4 integrin or anti-α4β7 integrin binding domains and conjugates of the present disclosure can specifically bind to α4 integrin or α4β7 integrin-expressing cells. In certain embodiments, the anti-α4β7 integrin binding protein or its GR agonist conjugate of the present disclosure specifically binds to human LPAM-1 (see, e.g., Uniprot#P26010, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In certain embodiments, the anti-α4 integrin binding protein or its GR agonist conjugate of the present disclosure specifically binds to human α4 integrin (see, e.g., Uniprot#P13612, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-LPAM-1 binding protein, or its GR agonist conjugate of the present disclosure, comprises an anti-LPAM-1 antibody (e.g., vedolizumab or natalizumab) or an anti-LPAM-1 fusion protein.

[0148] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist linked to the anti-α4 subunit of an α4β7 integrin heterodimer antibody, and the anti-α4β7 integrin antibody comprises the heavy chain amino acid sequences of CDR1 (VH-CDR1), VH-CDR2, and VH-CDR3 from abrilumab, and the amino acid sequences of (VL-CDR1), VL-CDR2, and VL-CDR3 light chain CDR1 from abrilumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the abrilumab VH region amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the abrilumab VL region amino acid sequence, of an anti-α4β7 integrin antibody. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the abrilumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the abrilumab light chain amino acid sequence, of an anti-α4β7 integrin antibody.

[0149] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist linked to an anti-α4 integrin antibody, and the anti-α4β7 integrin antibody comprises the heavy chain amino acid sequences of CDR1 (VH-CDR1), VH-CDR2, and VH-CDR3 from natalizumab and the amino acid sequences of the light chain CDR1 of (VL-CDR1), VL-CDR2, and VL-CDR3 from natalizumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the natalizumab VH region amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the natalizumab VL region amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the natalizumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the natalizumab light chain amino acid sequence.

[0150] In some embodiments, the anti-integrin α4 binding domain of the present disclosure specifically binds to an epitope of integrin α4 comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids of at least one amino acid of the isoforms shown in Table A (www.uniprot.org / uniprot / P13612).

[0151] In certain embodiments, the anti-integrin α4 antibody, or antigen-binding fragment thereof, of the present disclosure specifically binds to an epitope of integrin α4 comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids of at least one isoform shown in Table A (www.uniprot.org / uniprot / P13612). In certain embodiments, the anti-integrin α4 fusion protein of the present disclosure specifically binds to an epitope of integrin α4 comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids of at least one amino acid of the isoform shown in Table A (www.uniprot.org / uniprot / P13612).

[0152] In some embodiments, the epitope of the present disclosure is continuous. In some embodiments, the epitope of the present disclosure is discontinuous. In some embodiments, the epitope of the present disclosure is conformational.

[0153] Further information regarding integrin α4 can be found at www.uniprot.org / uniprot / P13612, the contents of which are incorporated by reference in their entirety.

[0154] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist linked to an LPAM-1 binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to LPAM-1. Such anti-LPAM-1 binding domains and conjugates of the present disclosure can specifically bind to LPAM-1 expressing cells. In certain embodiments, the anti-LPAM-1 binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human LPAM-1 (e.g., see www.uniprot.org / uniprot / P26010, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-LPAM-1 binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-LPAM-1 antibody (e.g., vedolizumab or etrolizumab) or an anti-LPAM-1 fusion protein.

[0155] As background, LPAM-1 is most highly expressed on subsets of lymphocytes and mediates the adhesion of lymphocytes to mucosal high endothelial venules and homing to the gastrointestinal tract through its interaction with MADCAM-1. Lymphocytes expressing LPAM-1 internalize the anti-LPAM-1 GR agonist conjugate by endosomal uptake, and upon internalization, the anti-LPAM-1 GR agonist conjugate can release the GR agonist payload within the lymphocyte. In some embodiments, the conjugate of the present disclosure comprises a binding protein that directly modifies the activation of pathogenic T cells, and the conjugate payload delivers GR agonism to pathogenic T cells. In some embodiments, the binding protein that directly modifies the activation of pathogenic T cells comprises a binding domain specific for LPAM-1.

[0156] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist linked to an anti-LPAM-1 antibody, and the anti-LPAM-1 antibody comprises the heavy chain amino acid sequence of CDR1 (VH-CDR1), VH-CDR2, and VH-CDR3 from vedolizumab, and the amino acid sequences of light chain CDR1 (VL-CDR1), VL-CDR2, and VL-CDR3 from vedolizumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH region amino acid sequence of vedolizumab, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL region amino acid sequence of vedolizumab, and an anti-LPAM1 antibody having the same. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain amino acid sequence of vedolizumab, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the light chain amino acid sequence of vedolizumab, and an anti-LPAM-1 antibody having the same.

[0157] In some embodiments, the conjugates of the present disclosure comprise an anti-LPAM-1 antibody or an anti-β7 subunit of the α4β7 or αEβ7 integrin heterodimer, and the anti-LPAM-1 or anti-β7 subunit antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from etrolizumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from etrolizumab. In certain embodiments, the conjugates of the present disclosure comprise a VH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the etrolizumab VH amino acid sequence and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the etrolizumab VL amino acid sequence, wherein the anti-LPAM-1 or anti-β7 subunit antibody comprises the VH and VL region. In other embodiments, the conjugates of the present disclosure comprise a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the etrolizumab heavy chain amino acid sequence and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the etrolizumab light chain amino acid sequence, wherein the anti-LPAM-1 or anti-β7 subunit antibody comprises the heavy chain and the light chain.

[0158] In certain embodiments, the conjugate proteins of the present disclosure comprise a GR agonist linked to a BCMA-binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to BCMA. Such anti-BCMA binding domains and conjugates of the present disclosure can specifically bind to BCMA-expressing cells. In certain embodiments, the anti-BCMA binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human BCMA (e.g., see www.uniprot.org / uniprot / Q02223, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-BCMA binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-BCMA antibody or an anti-BCMA fusion protein.

[0159] In some embodiments, the conjugate of the present disclosure comprises an anti-BCMA antibody, and the anti-BCMA antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from belantamab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from belantamab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belantamab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belantamab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises an anti-BCMA antibody having a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belantamab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belantamab light chain amino acid sequence.

[0160] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a compound of the present disclosure linked to a CD40 binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to CD40. Such anti-CD40 binding domains and conjugates of the present disclosure can specifically bind to CD40-expressing cells (e.g., antigen-presenting cells including B cells). In certain embodiments, the anti-CD40 binding protein of the present disclosure, or a conjugate thereof, specifically binds to human CD40 (e.g., see www.uniprot.org / uniprot / P25942, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-CD40 binding protein of the present disclosure, or a conjugate thereof, comprises an anti-CD40 antibody or an anti-CD40 fusion protein.

[0161] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from blisibimod, and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from blisibimod. In certain embodiments, the conjugate of the present disclosure comprises a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the blisibimod VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the blisibimod VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the blisibimod heavy chain amino acid sequence, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the blisibimod light chain amino acid sequence.

[0162] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from dacetuzumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from dacetuzumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dacetuzumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dacetuzumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dacetuzumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dacetuzumab light chain amino acid sequence.

[0163] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from ziloverumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from ziloverumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ziloverumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ziloverumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ziloverumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ziloverumab light chain amino acid sequence.

[0164] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from isakizumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from isakizumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the isakizumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the isakizumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the isakizumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the isakizumab light chain amino acid sequence.

[0165] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from lucatumumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from lucatumumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the lucatumumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the lucatumumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the lucatumumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the lucatumumab light chain amino acid sequence.

[0166] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from mitazalimab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from mitazalimab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the mitazalimab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the mitazalimab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the mitazalimab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the mitazalimab light chain amino acid sequence.

[0167] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from ravulizumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from ravulizumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ravulizumab VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ravulizumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ravulizumab heavy chain amino acid sequence, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ravulizumab light chain amino acid sequence.

[0168] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from certolizumab pegol and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from certolizumab pegol. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab pegol VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab pegol VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab pegol heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab pegol light chain amino acid sequence.

[0169] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from sotigalimab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from sotigalimab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sotigalimab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sotigalimab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sotigalimab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sotigalimab light chain amino acid sequence.

[0170] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40 antibody, and the anti-CD40 antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from baliximab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from baliximab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the baliximab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the baliximab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the baliximab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the baliximab light chain amino acid sequence.

[0171] In certain embodiments, the binding protein conjugate of the present disclosure comprises a GR agonist linked to a CD40L binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to CD40L. Such anti-CD40L binding domains and conjugates of the present disclosure are capable of specifically binding to CD40L-expressing cells. In certain embodiments, the anti-CD40L binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human CD40L (e.g., see www.uniprot.org / uniprot / P25942, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-CD40L binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-CD40L antibody or anti-CD40L fusion protein.

[0172] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40L antibody, and the anti-CD40L antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from rhuplizumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from rhuplizumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the rhuplizumab VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the rhuplizumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the rhuplizumab heavy chain amino acid sequence, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the rhuplizumab light chain amino acid sequence.

[0173] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40L antibody, and the anti-CD40L antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from tocilizumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from tocilizumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tocilizumab VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tocilizumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tocilizumab heavy chain amino acid sequence, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the tocilizumab light chain amino acid sequence.

[0174] In some embodiments, the conjugates of the present disclosure include an anti-CD40L binding domain, and the anti-CD40L binding domain includes the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from daratumumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from daratumumab. In certain embodiments, the conjugates of the present disclosure include a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the daratumumab VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the daratumumab VL amino acid sequence, and the anti-CD40L binding domain, optionally, the conjugates of the present disclosure include a pegylated anti-CD40L binding domain. In further embodiments, the conjugates of the present disclosure include a pegylated anti-CD40L binding domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the daratumumab pegol.

[0175] In some embodiments, the conjugates of the present disclosure include an anti-CD40L fusion protein, and the fusion protein includes an anti-CD40L binding domain having the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from rituximab fused to an Fc region moiety. In certain embodiments, the conjugates of the present disclosure include an anti-CD40L fusion protein having a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the rituximab VH amino acid sequence fused to an Fc region moiety.

[0176] In some embodiments, the conjugate of the present disclosure comprises an anti-CD40L fusion protein comprising one or both of the dazodalibep anti-CD40L binding peptide amino acid sequences fused to human serum albumin (HSA), wherein the dazodalibep anti-CD40L binding peptide is linked to HSA via a peptide spacer of about 5 to about 20 amino acids (preferably 10 amino acids), and the anti-CD40L binding peptide is linked via a second peptide spacer comprising about 5 to about 30 amino acids (preferably 20 amino acids). In certain embodiments, the conjugate of the present disclosure comprises an anti-CD40L fusion protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the dazodalibep amino acid sequence.

[0177] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist linked to a CD86-binding protein or a CD80-binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to CD86, CD80, or both. Such CD86 or CD80 binding domains and conjugates of the present disclosure can specifically bind to cells that express CD86, CD80, or both. In certain embodiments, the binding protein or its GR agonist conjugate of the present disclosure specifically binds to human CD86 (e.g., see www.uniprot.org / uniprot / P42081, the sequence of which is incorporated herein by reference in its entirety), human CD80 (e.g., see www.uniprot.org / uniprot / P33681, the sequence of which is incorporated herein by reference in its entirety), both human CD86 and human CD80, or an epitope thereof. In further embodiments, the fusion protein conjugate of the present disclosure comprises human CTLA4 or an extracellular portion thereof (e.g., see www.uniprot.org / uniprot / P16410, the sequence of which is incorporated herein by reference in its entirety). In further embodiments, the CD86 or CD80 binding protein, or its GR agonist conjugate, of the present disclosure comprises an anti-CD86 antibody, an anti-CD80 antibody, or a CTLA4 fusion protein (e.g., abatacept, belatacept, ASP2408, or ASP2409).

[0178] As background, when a CTLA4 fusion protein conjugate of the present disclosure (e.g., a CTLA4-Ig conjugate) binds to CD80 or CD86 (the receptor for both CD28 and CTLA4), the conjugate blocks the CD28 signaling pathway and effectively blocks T cell activation. Thus, one result of administration of the CTLA4 fusion protein conjugate of the present disclosure is to inhibit T cell activation via binding of CD80 or CD86. At the same time, APCs expressing CD80 or CD86 internalize the CTLA4 fusion protein conjugate by endosomal uptake, and upon internalization, the GR agonist payload is released within the APC. In some embodiments, these two actions not only occur simultaneously within T cells and APCs, but also provide a resulting synergistic combination for treating inflammatory or autoimmune conditions. In some embodiments, the conjugates of the present disclosure include a binding protein that directly modulates T cell activation, and the conjugate GR agonist payload delivers immunosuppressive GR agonism to antigen-presenting cells, including myeloid antigen-presenting cells, via ligand binding or macropinocytosis. In certain embodiments, the binding protein that directly modulates T cell activation includes a binding domain specific for CD80, CD86, or both.

[0179] In some embodiments, the conjugates of the present disclosure include an anti-CD86 or anti-CD80 fusion protein comprising a CTLA ectodomain fused to an Fc region moiety. In certain embodiments, the conjugates of the present disclosure have a CTLA ectodomain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CTLA ectodomain amino acid sequence from belatacept, abatacept, ASP2408, or ASP2409. In further embodiments, the conjugates of the present disclosure include an anti-CD86 or anti-CD80 fusion protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the belatacept, abatacept, ASP2408, or ASP2409 amino acid sequence.

[0180] In certain embodiments, the binding protein conjugates of the present disclosure include a GR agonist linked to an anti-CTLA4 binding protein, wherein the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to CTLA4. Such anti-CTLA4 binding domains and conjugates of the present disclosure can specifically bind to CTLA4-expressing cells. In certain embodiments, the anti-CTLA4 binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human CTLA4 (e.g., see www.uniprot.org / uniprot / P16410, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-CTLA4 binding protein of the present disclosure, or its GR agonist conjugate, includes an agonist anti-CTLA4 antibody or an agonist anti-CTLA4 fusion protein.

[0181] As background, CTLA4 is expressed on activated T lymphocytes and Treg T cells. Binding by an agonist anti-CTLA4 antibody conjugate results in decreased T cell activation and increased Treg immunosuppression by activating the CTLA4 signaling pathway and through internalization and delivery of GR agonism to T cells. In some embodiments, the conjugates of the present disclosure include a binding protein that directly modifies activation and immunosuppression of Treg cells, and the conjugate GR agonist payload delivers immunosuppressive GR agonism to macropinocytic myeloid cells. In certain embodiments, the binding protein that directly modifies activation and immunosuppression of activated T cells and Treg T cells includes a binding domain specific for CTLA4.

[0182] In certain embodiments, the binding protein conjugates of the present disclosure include a GR agonist linked to an ICOS binding protein, and the binding protein includes a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to ICOS. Such anti-ICOS binding domains and conjugates of the present disclosure can specifically bind to ICOS-expressing cells. In certain embodiments, the anti-ICOS binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human ICOS (e.g., see www.uniprot.org / uniprot / Q9Y6W8, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-ICOS binding protein of the present disclosure, or its GR agonist conjugate, includes an agonist anti-ICOS antibody or an agonist anti-ICOS fusion protein.

[0183] As background, ICOS is expressed on T lymphocytes (T cells). ICOS ligand is expressed on B cells, macrophages, and dendritic cells. When binding to ICOS-L, ICOS mediates T cell proliferation and cytokine secretion. The anti-ICOS GR agonist conjugate of the present disclosure blocks this signaling pathway, effectively blocking both T cell proliferation and cytokine secretion, and at the same time can deliver immunosuppressive GR agonism into ICOS-expressing cells (T cells). In some embodiments, the conjugate of the present disclosure includes a binding protein that directly modifies the activation of pathogenic T cells, and the conjugate payload delivers GR agonism to pathogenic T cells. In some embodiments, the binding protein that directly modifies the activation of pathogenic T cells includes a binding domain specific for ICOS.

[0184] In some embodiments, the conjugate of the present disclosure includes an anti-ICOS antibody, and the anti-ICOS antibody includes the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from avelumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from avelumab. In certain embodiments, the conjugate of the present disclosure includes a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the avelumab VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the avelumab VL amino acid sequence, and includes an anti-ICOS antibody. In other embodiments, the conjugate of the present disclosure includes a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the avelumab heavy chain amino acid sequence, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the avelumab light chain amino acid sequence, and includes an anti-ICOS antibody.

[0185] In some embodiments, the conjugate of the present disclosure comprises an anti-ICOS antibody, and the anti-ICOS antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from feralizumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from feralizumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the feralizumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the feralizumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the feralizumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the feralizumab light chain amino acid sequence.

[0186] In some embodiments, the conjugate of the present disclosure comprises an anti-ICOS antibody, and the anti-ICOS antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from boptelimumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from boptelimumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the boptelimumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the boptelimumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the boptelimumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the boptelimumab light chain amino acid sequence.

[0187] In a further embodiment, the conjugate of the present disclosure comprises a GR agonist linked to a bispecific antibody construct consisting of an antibody specific for ICOS and a binding domain specific for PD-1, wherein the binding domain specific for PD-1 is optionally linked via a peptide spacer of about 5 to about 30 amino acids, and the binding domain specific for PD-1 comprises a scFv comprising VH and VL regions linked via a peptide spacer comprising about 10 to about 30 amino acids. In certain embodiments, the conjugate of the present disclosure comprises a GR agonist linked to a fusion protein comprising a heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the isralimab heavy chain amino acid sequence and a light chain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the isralimab light chain amino acid sequence.

[0188] In some embodiments, the conjugate of the present disclosure comprises an anti-ICOS or anti-CD28 fusion protein comprising an ICOS-L fragment fused to a null Fc region moiety. In certain embodiments, the conjugate of the present disclosure comprises an ICOS-L fragment having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ICOS-L fragment amino acid sequence from atacicept. In a further embodiment, the conjugate of the present disclosure comprises an anti-ICOS or anti-CD28 fusion protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the atacicept amino acid sequence.

[0189] In certain embodiments, the conjugate proteins of the present disclosure comprise a GR agonist linked to an ICOS-L binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to ICOS-L. Such anti-ICOS-L binding domains and conjugates of the present disclosure can specifically bind to ICOS-L expressing cells. In certain embodiments, the anti-ICOS-L binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human ICOS-L (e.g., see www.uniprot.org / uniprot / O75144, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof.

[0190] In further embodiments, the anti-ICOS-L binding protein of the present disclosure, or its GR agonist conjugate, comprises an agonist anti-ICOS-L antibody or an agonist anti-ICOS-L fusion protein.

[0191] As background, the anti-ICOS-L conjugate or ICOS fusion protein conjugate of the present disclosure can block the ICOS signaling pathway, effectively block both T cell proliferation and cytokine secretion, and at the same time deliver immunosuppressive GR agonism into ICOS-L expressing cells (e.g., B cells, macrophages, or dendritic cells), providing a synergistic combination of activities for treating inflammatory or autoimmune conditions.

[0192] In certain embodiments, the conjugate proteins of the present disclosure comprise a GR agonist conjugated to a CD28 binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to CD28. Such anti-CD28 binding domains and conjugates of the present disclosure can specifically bind to CD28-expressing cells. In certain embodiments, the anti-CD28 binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human CD28 (e.g., see www.uniprot.org / uniprot / P10747, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-CD28 binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-CD28 antibody or anti-CD28 fusion protein. In some embodiments, the conjugate of the present disclosure comprises a binding protein that directly modulates the activation of pathogenic T cells, and the conjugate payload delivers GR agonism to pathogenic T cells. In some embodiments, the binding protein that directly modulates the activation of pathogenic T cells comprises a binding domain specific for CD28.

[0193] In some embodiments, the conjugate of the present disclosure comprises an anti-CD28 domain antibody, and the domain antibody comprises an anti-CD28 binding domain having the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from ririluzumab, and optionally, the anti-CD28 domain antibody is pegylated. In certain embodiments, the conjugate of the present disclosure optionally comprises an anti-CD28 domain antibody having a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the pegylated (ririluzumab pegol) ririluzumab VL amino acid sequence.

[0194] In certain embodiments, the conjugate proteins of the present disclosure include a GR agonist linked to a MADCAM-binding protein, and the binding protein includes a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to MADCAM. Such anti-MADCAM binding domains and conjugates of the present disclosure can specifically bind to MADCAM-expressing cells. In certain embodiments, the anti-MADCAM binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human MADCAM (e.g., see www.uniprot.org / uniprot / Q13477, the sequence of which is hereby incorporated by reference in its entirety) or an epitope thereof. In further embodiments, the anti-MADCAM binding protein of the present disclosure, or its GR agonist conjugate, includes an anti-MADCAM antibody or an anti-MADCAM fusion protein.

[0195] In some embodiments, the conjugate of the present disclosure comprises an anti-MADCAM antibody, and the anti-MADCAM antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from ontamalimab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from ontamalimab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ontamalimab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ontamalimab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ontamalimab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ontamalimab light chain amino acid sequence.

[0196] In certain embodiments, the binding protein conjugate of the present disclosure comprises a GR agonist linked to a TNFα binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to TNFα. Such anti-TNFα binding domains and conjugates of the present disclosure can specifically bind to TNFα-expressing cells. In certain embodiments, the anti-TNFα binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human TNFα (e.g., see www.uniprot.org / uniprot / P01375, the sequence of which is hereby incorporated by reference in its entirety) or an epitope thereof. In further embodiments, the anti-TNFα binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-TNFα antibody or anti-TNFα fusion protein.

[0197] In some embodiments, the conjugate of the present disclosure comprises an anti-TNFα antibody, and the anti-TNFα antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from adalimumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from adalimumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the adalimumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the adalimumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the adalimumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the adalimumab light chain amino acid sequence.

[0198] In some embodiments, the conjugate of the present disclosure comprises an anti-TNFα antibody, and the anti-TNFα antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from infliximab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from infliximab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the infliximab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the infliximab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the infliximab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the infliximab light chain amino acid sequence.

[0199] In some embodiments, the conjugate of the present disclosure comprises an anti-TNFα antibody, and the anti-TNFα antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from golimumab and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from golimumab. In certain embodiments, the conjugate of the present disclosure comprises a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the golimumab VH amino acid sequence, and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the golimumab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the golimumab heavy chain amino acid sequence, and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the golimumab light chain amino acid sequence.

[0200] In some embodiments, the conjugates of the present disclosure comprise an anti-TNFα antibody, and optionally a pegylated anti-TNFα antibody, which comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from certolizumab, and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from certolizumab. In certain embodiments, the conjugates of the present disclosure comprise an anti-TNFα antibody that is optionally pegylated and has a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab VL amino acid sequence. In other embodiments, the conjugates of the present disclosure comprise an anti-TNFα antibody that is optionally pegylated and has a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the certolizumab light chain amino acid sequence (certolizumab pegol).

[0201] In some embodiments, the conjugates of the present disclosure comprise an anti-TNFα fusion protein comprising 1 to 6 etanercept TNFα-binding peptide amino acid sequences fused to the Fc region moiety. In certain embodiments, each of the 1 to 6 etanercept TNFα-binding peptides is a TNFR ectodomain. In certain embodiments, the conjugates of the present disclosure comprise an anti-TNFα fusion protein having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the etanercept amino acid sequence.

[0202] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist linked to a TNFR2 binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to TNFR2. Such anti-TNFR2 binding domains and conjugates of the present disclosure can specifically bind to TNFR2-expressing cells. In certain embodiments, the anti-TNFR2 binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human TNFR2 (e.g., see www.uniprot.org / uniprot / P20333, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-TNFR2 binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-TNFR2 antibody or an anti-TNFR2 fusion protein.

[0203] In certain embodiments, the conjugate protein conjugate of the present disclosure comprises a GR agonist bound to an APRIL binding protein, and the binding protein comprises a binding domain, antibody, antibody construct, fusion protein, or targeting moiety that specifically binds to APRIL. Such anti-APRIL binding domains and conjugates of the present disclosure can specifically bind to APRIL-expressing cells. In certain embodiments, the anti-APRIL binding protein of the present disclosure, or its GR agonist conjugate, specifically binds to human APRIL (e.g., see www.uniprot.org / uniprot / O75888, the sequence of which is incorporated herein by reference in its entirety) or an epitope thereof. In further embodiments, the anti-APRIL binding protein of the present disclosure, or its GR agonist conjugate, comprises an anti-APRIL antibody or an anti-APRIL fusion protein.

[0204] In some embodiments, the conjugate of the present disclosure comprises an anti-APRIL antibody, and the anti-APRIL antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from sibeprentamab, and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from sibeprentamab. In certain embodiments, the conjugate of the present disclosure comprises a VH region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sibeprentamab VH amino acid sequence, and a VL region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sibeprentamab VL amino acid sequence. In other embodiments, the conjugate of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sibeprentamab heavy chain amino acid sequence, and a light chain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sibeprentamab light chain amino acid sequence.

[0205] In some embodiments, the conjugate of the present disclosure comprises an anti-APRIL antibody, and the anti-APRIL antibody comprises the heavy chain CDR amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 from BION-1301 and the light chain CDR amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 from BION-1301. In certain embodiments, the conjugate of the present disclosure comprises a VH region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the BION-1301 VH amino acid sequence and a VL region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the BION-1301 VL amino acid sequence, and an anti-APRIL antibody. In other embodiments, the conjugate of the present disclosure comprises a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the BION-1301 heavy chain amino acid sequence and a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the BION-1301 light chain amino acid sequence, and an anti-APRIL antibody.

[0206] In any of the foregoing embodiments, the binding protein or its GR agonist conjugate of the present disclosure specifically binds to an epitope having a continuous amino acid sequence or a discontinuous amino acid sequence. In any of the foregoing embodiments, the binding protein or its conjugate of the present disclosure specifically binds to a conformational epitope.

[0207] D. Nucleic Acids, Vectors, and Host Cells The present disclosure provides an isolated nucleic acid encoding a fusion protein of the present disclosure (e.g., an anti-target fusion protein provided herein) or an antibody (e.g., an anti-target antibody provided herein), or a target or antigen-binding fragment thereof. In some embodiments, the nucleic acid encoding the fusion protein or antibody of the present disclosure, or an antigen-binding fragment thereof, is codon-optimized to enhance or maximize expression in a particular cell type (e.g., Scholten et al., Clin. Immunol. 119:135-145, 2006). As used throughout the present disclosure, a "codon-optimized" polynucleotide is a heterologous polypeptide having codons modified with silent mutations corresponding to the abundance of host cell tRNA levels.

[0208] In some embodiments, the nucleic acid molecule encodes a fusion protein of the present disclosure (e.g., an anti-target fusion protein provided herein) or an antibody (e.g., an anti-target antibody provided herein), or a target or antigen-binding fragment thereof (e.g., an antibody heavy and light chains, or an antibody-binding domain comprising a V H binding region and a V L binding region), and two or more chains or regions are separated by a cleavage site. In further embodiments, the cleavage site is a self-cleaving amino acid sequence comprising a 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or any combination thereof (e.g., see Kim et al., PLOS One 6:e18556, 2011, the 2A nucleic acid and amino acid sequences of which are incorporated herein by reference in their entirety). In some embodiments, a nucleic acid molecule is provided that comprises a nucleotide sequence encoding the heavy chain or variable region thereof of a fusion protein or antibody (e.g., an anti-target antibody provided herein). In some embodiments, a nucleic acid molecule is provided that comprises a nucleotide sequence encoding the light chain or variable region thereof of a fusion protein or antibody (e.g., an anti-target antibody provided herein).

[0209] In certain embodiments, provided is a nucleic acid encoding a fusion protein (e.g., an anti-target fusion protein provided herein) or an antibody (e.g., an anti-target antibody provided herein) of the present disclosure, or an expression construct comprising its target or antigen-binding fragment. In some embodiments, the nucleic acid can be operably linked to an expression control sequence. As used herein, an “expression construct” refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences that can affect the expression of the nucleic acid molecule in a suitable host. The expression construct can be present in a vector (e.g., a bacterial vector, a viral vector) or can be integrated into the genome. The term “operably linked” refers to the association of two or more nucleic acids on a single polynucleotide fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it can affect the expression of the coding sequence (i.e., if the coding sequence is under the transcriptional control of the promoter). The term “expression control sequence” (also referred to as a regulatory sequence) refers to a nucleic acid sequence that affects the expression and processing of the coding sequence to which it is operably linked. For example, expression control sequences can include transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and optionally sequences that enhance protein secretion.

[0210] In some embodiments, a fusion protein (e.g., an anti-target fusion protein provided herein) or an antibody (e.g., an anti-target antibody provided herein), or a nucleic acid or expression construct encoding its target or antigen-binding fragment, is present within a vector. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. The vector can include chromosomal, extrachromosomal, semisynthetic or synthetic nucleic acids, for example, it may be a plasmid, cosmid, virus, RNA vector, or linear or circular DNA or RNA molecule. Exemplary vectors are those capable of autonomous replication (episomal vectors), or those capable of expressing the nucleic acids linked thereto (expression vectors).

[0211] Exemplary viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, ortho-myxoviruses (e.g., influenza virus), negative-strand RNA viruses such as rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian type C, B-type viruses, D-type viruses, HTLV-BLV group, lentiviruses, spumaviruses (Coffin, J.M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B.N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). In some embodiments, the vector is a plasmid. In some other embodiments, the vector is a viral vector. In some such embodiments, the viral vector is a lentiviral vector or a γ-retroviral vector.

[0212] In some embodiments, the present disclosure provides an isolated host cell comprising a nucleic acid, expression construct, or vector encoding a fusion protein of the present disclosure (e.g., an anti-target fusion protein provided herein) or an antibody (e.g., an anti-target antibody provided herein), or a target or antigen-binding fragment thereof. As used herein, the term "host" refers to a cell or microorganism targeted for genetic recombination with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., a fusion protein or its target-binding domain, an antibody or its antigen-binding fragment). In some embodiments, the host cell may optionally already have or be modified to include other genetic recombinations (e.g., incorporation of a detectable marker) that confer desired properties related or unrelated to the biosynthesis of a heterologous or exogenous protein. Multiple heterologous or exogenous nucleic acid molecules can be introduced into the host cell as separate nucleic acid molecules, as multiple individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or as any combination thereof. When two or more exogenous nucleic acid molecules are introduced into the host cell, it is understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., in a single vector), by separate vectors, and integrated into the host chromosome at a single site or multiple sites. The number of heterologous nucleic acid molecules or protein activities referred to refers to the number of coding nucleic acid molecules or protein activities, not the number of separate nucleic acid molecules introduced into the host cell.

[0213] E. Linkers and Linker-Payloads Compounds of the present disclosure, such as compounds of Formula I or II, can be linked to a linker, for example, a linker containing a peptide or a cleavable linker. In some embodiments, the linker can also be linked to a binding domain (e.g., a fusion protein), an antibody, an antibody construct, or a polypeptide comprising a targeting moiety that specifically binds to a target, thereby forming a conjugate comprising the polypeptide and the compound. The linker of the conjugate should not affect the binding of the conjugate to its cognate binding partner, such as an antigen, of the active moiety of the conjugate, for example, an antigen-binding domain, an Fc region or domain, a target-binding domain, an antibody, a targeting moiety, etc. to the target. The conjugate can contain multiple linkers, each linking one or more compounds. The multiple linkers can be the same or different linkers contained on a single conjugate or separate conjugates.

[0214] In some embodiments of the present disclosure, the linker connects one or more compounds of the present disclosure to a polypeptide comprising a target binding domain (e.g., a fusion protein, or an antibody or antigen-binding fragment thereof) by forming a covalent bond to the compound at one position and a covalent bond to the polypeptide comprising the binding domain at another position. The covalent bond can be formed by a reaction between a functional group on the linker and functional groups on the compound and on the polypeptide comprising the binding domain. As used herein, the term "linker" refers to (i) the unbound form of a linker that can comprise a functional group capable of covalently bonding the linker to a compound and a functional group capable of covalently bonding the linker to a polypeptide comprising a binding domain or a binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof), (ii) the partially bound form of a linker that can comprise a functional group capable of covalently bonding the linker to a polypeptide comprising a binding domain or a binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof) and capable of covalently bonding to a compound, or vice versa, and (iii) the fully bound form of a linker that can covalently bond to both a GR agonist and a polypeptide comprising a binding domain or a binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof). In some embodiments, the functional group on the linker and the covalent bond formed between the linker and the polypeptide comprising the binding domain can be specifically exemplified as Rx and Rx’, respectively.

[0215] The linker can be short, flexible, rigid, cleavable, non-cleavable, hydrophilic, or hydrophobic. The linker can contain segments having different properties, such as flexible segments or rigid segments. The linker may be chemically stable to the extracellular environment, e.g., may be chemically stable in the bloodstream, or may contain bonds that are unstable or selectively unstable. The linker can contain bonds designed to be cleaved or broken specifically or non-specifically intracellularly, or otherwise degraded. A cleavable linker can be sensitive to an enzyme. A cleavable linker can be cleaved by an enzyme such as a protease.

[0216] Cleavable linkers can include valine-citrulline (Val-Cit) peptides, valine-alanine (Val-Ala) peptides, phenylalanine-lysine (Phe-Lys), or other peptides, such as peptides that form protease recognition and cleavage sites. Such peptide-containing linkers can contain a pentafluorophenyl group. Peptide-containing linkers can include a succinimide or maleimide group. Peptide-containing linkers can include a para-aminobenzoic acid (PABA) group. Peptide-containing linkers can include an aminobenzyloxycarbonyl (PABC) group. Peptide-containing linkers can include a PABA or PABC group and a pentafluorophenyl group. Peptide-containing linkers can include a PABA or PABC group and a succinimide group. Peptide-containing linkers can include a PABA or PABC group and a maleimide group.

[0217] Non-cleavable linkers are generally protease-insensitive and insensitive to intracellular processes. Non-cleavable linkers can include a maleimide group. Non-cleavable linkers can include a succinimide group. Non-cleavable linkers can be maleimide-alkyl-C(O)-linkers. Non-cleavable linkers can be maleimidocaproyl linkers. Maleimidocaproyl linkers can be N-maleimidomethylcyclohexane-1-carboxylate. Maleimidocaproyl linkers can include a succinimide group. Maleimidocaproyl linkers can include a pentafluorophenyl group.

[0218] The linker can be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. The linker can be a maleimide-PEG4 linker. The linker can be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. The linker can be a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules. The linker can contain maleimide(s) linked to a polyethylene glycol molecule, and the polyethylene glycol can enable more flexibility of the linker or can be used to lengthen the linker.

[0219] The linker can be a (maleimidocaproyl)-(valine-alanine)-(para-aminobenzyloxycarbonyl) linker. The linker can be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonyl) linker. The linker can be a (maleimidocaproyl)-(phenylalanine-lysine)-(para-aminobenzyloxycarbonyl) linker. The linker can be a linker suitable for binding to engineered cysteine (THIOMAB). The THIOMAB linker can be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonyl)-linker.

[0220] The linker can also contain segments of alkylene, alkenylene, alkynylene, polyether, polyester, polyamide, polyamino acid, peptide, polypeptide, cleavable peptide, or aminobenzyl-carbamate. The linker can contain maleimide at one end and N-hydroxysuccinimidyl ester at the other end. The linker can contain N-terminal amine acetylation and lysine having a valine-citrulline, valine-alanine, or phenylalanine-lysine cleavage site. The linker can be a link made by microbial transglutaminase, and the link can be made between an amine-containing portion and a portion engineered to contain glutamine as a result of an enzyme that catalyzes the formation of a bond between the acyl group of the glutamine side chain and the primary amine of the lysine chain. The linker can contain a reactive primary amine. The linker can be a sortase A linker. The sortase A linker can be made by a sortase A enzyme that fuses an LPXTG recognition motif to an N-terminal GGG motif to regenerate a native amide bond. Thus, the linker produced can link a portion bound to the LPXTG recognition motif and a portion bound to the N-terminal GGG motif. The linker can be a junction that occurs between a non-natural amino acid on one portion and an oxime bond formed by modifying a ketone group with an alkoxyamine on another portion. The portion can be part of a conjugate. The portion can be part of an antibody. The portion can be part of a GR agonist. The portion can be part of a binding domain. The linker can be unsubstituted or can be substituted, for example, with a substituent. Substituents can include, for example, hydroxyl group, amino group, nitro group, cyano group, azide group, carboxyl group, carboxyl aldehyde group, imine group, alkyl group, alkenyl group, alkynyl group, alkoxy group, acyl group, acyloxy group, amide group, and ester group.

[0221] In some embodiments of the conjugates of the present disclosure, the compound, or a salt, stereoisomer, solvate or prodrug thereof, is herein designated as L or L 3It is linked to a polypeptide comprising a target binding domain (e.g., a fusion protein, or an antibody or antigen-binding fragment thereof) via a linker(s) also referred to as such. As used herein, L can be selected from any of the linker moieties discussed herein. The linker that conjugates a compound or a salt thereof to the polypeptide of the conjugate's binding domain can be short, long, hydrophobic, hydrophilic, flexible, or rigid, or each can independently be composed of segments having one or more of the above-mentioned properties such that the linker can include segments having different properties. The linker can be multivalent to covalently bind more than one compound or salt to a single site on the polypeptide comprising the binding domain or fragment thereof, or monovalent to covalently bind a single compound or salt to a single site on the binding domain or fragment thereof.

[0222] The linker can be multivalent to covalently bind more than one compound of the present disclosure to a single site on the binding domain (e.g., a fusion protein, or an antibody or antigen-binding fragment thereof), or monovalent to covalently bind a single compound to a single site on the binding domain or fragment thereof.

[0223] In some embodiments of the compounds of the present disclosure, such as compounds of Formula I or II, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, the compound may further comprise a linker (L) that provides a linker-payload. The linker may covalently attach at any position on the compound, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, as valence permits. For example, the linker may attach to a nitrogen atom, such as an amine, or an oxygen atom, such as a hydroxyl of the compound, sulfur, such as a thiol, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof. The linker may comprise an electrophilic group, such as a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamine, unnatural amino acid residue, or glutamic acid residue, that is capable of reacting to form a covalent bond with a reactive moiety, such as a reactive moiety of a binding protein of the present disclosure. In some embodiments, the compound, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, may covalently attach to a binding domain, such as an antibody, antibody construct, or targeting moiety, via the linker.

[0224] In a conjugate, a compound of the present disclosure, such as a compound of Formula I or II, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, is attached to an antibody, antibody construct, or targeting moiety via a polypeptide (e.g., a fusion protein) comprising a binding domain, e.g., a linker(s) (also referred to herein as L), which may be selected from any of the linker moieties discussed herein. The linker that links the compound or salt to a polypeptide of a binding domain, such as an antibody, antibody construct, or targeting moiety of a conjugate, may be short, long, hydrophobic, hydrophilic, flexible, or rigid, or each may independently be composed of segments having one or more of the above-described properties such that the linker may comprise segments having different properties. The linker may be multivalent so as to covalently attach more than one compound or salt to a single site on a polypeptide comprising a binding domain, such as an antibody, antibody construct, or targeting moiety, or may be monovalent so as to covalently attach a single compound or salt to a single site on a polypeptide comprising a binding domain, such as an antibody, antibody construct, or targeting moiety.

[0225] The linker (L) of the present disclosure may have from about 10 to about 500 atoms in the linker, such as from about 10 to about 400 atoms, such as from about 10 to about 300 atoms in the linker. In some embodiments, the linker of the present disclosure has from about 30 to about 400 atoms in the linker, such as from about 30 to about 300 atoms.

[0226] The linker of the present disclosure may form a conjugate by covalently linking a compound of the present disclosure, such as a compound of Formula I or II, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, to a binding protein of the present disclosure via a covalent bond between the linker, the binding protein of the present disclosure, and the GR agonist compound.

[0227] When used throughout this disclosure, the expression "linker" refers to: (i) a linker in unconjugated form that includes a functional group capable of covalently bonding the linker to a compound of the disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and a functional group capable of covalently bonding the linker to a binding protein of the disclosure; (ii) a linker in a partially conjugated form that can covalently bond to a polypeptide and includes a functional group capable of covalently bonding to at least one compound of the disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof; and (iii) a linker in a fully conjugated form that covalently bonds to both a compound of the disclosure, or a pharmaceutically acceptable isomer, racemate, hydrate, solvate, isotope, or salt thereof, and a binding protein of the disclosure. Some embodiments relate to conjugates formed by contacting a binding protein of the disclosure that binds a cell surface receptor or antigen expressed on a target cell with a linker compound of the disclosure under conditions such that the linker compound covalently bonds to the binding protein of the disclosure. Further embodiments relate to methods of making conjugates formed by contacting a linker compound under conditions such that the linker compound covalently bonds to a binding protein of the disclosure.

[0228] In some embodiments, a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, is covalently attached to a linker (L) to form a linker-payload (L-P or "linker payload"). The linker can be covalently attached to any position of the compound, as long as the valence permits. The linker can include an electrophilic group that can react to form a covalent bond with a portion of a binding protein of the present disclosure, such as, for example, lysine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamine, unnatural amino acid residues, or glutamic acid residues. In some embodiments, a linker-payload comprising a GR agonist compound or a salt of a GR agonist compound and linker L is covalently attached to a binding protein of the present disclosure via the linker.

[0229] In some embodiments, a linker-payload comprising a compound of the present disclosure or a salt thereof, and linker L, is covalently attached to an antibody via L. In further embodiments, a linker-payload comprising a compound of the present disclosure or a salt thereof, and linker L, is covalently attached to an antigen-binding fragment of an antibody via L. In still further embodiments, a linker-payload comprising a compound of the present disclosure or a salt thereof, and linker L, is covalently attached to a fusion protein via L. In some embodiments, for a linker-payload comprising a compound of Formula I or II, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, L is a non-cleavable linker. Alternatively, in some embodiments, for a linker-payload comprising a compound of Formula I or II, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, L is a cleavable linker, such as a linker cleavable by lysosomal enzymes. In some embodiments, the polypeptide can further include a second antigen or target-binding domain.

[0230] In certain embodiments, the GR agonist compounds of the present disclosure are covalently bound to an antibody. In further embodiments, the GR agonist compounds of the present disclosure are covalently bound to an antigen-binding fragment of an antibody. In other embodiments, the compounds of the present disclosure are covalently bound to a fusion protein. In some embodiments, the binding proteins of the present disclosure further comprise a second target-binding domain.

[0231] 1. Category I linker Exemplary multivalent linkers that can be used to link the compounds of the present disclosure to polypeptides comprising a target-binding domain, such as an antibody construct, are described. For example, Fleximer® linker technology has the potential to enable high-DAR conjugates with favorable physicochemical properties. As shown below, Fleximer® linker technology is based on incorporating a drug molecule into a solubilizing polyacetal backbone via a sequence of ester linkages.

Chemical formula

[0232] In some embodiments, the moieties, constructs, or conjugates of the present disclosure are symbolized by

Chemical formula

[0233] By way of non-limiting example, some cleavable and non-cleavable linkers that can be included in the conjugate are described below in addition to any others of the present disclosure.

[0234] Sulfamide linkers can be used to link many compounds of the present disclosure to antibody constructs. Examples of sulfamide linkers of the present disclosure include, for example, U.S. Patent Publication No. 2019 / 0038765, the linker of which is incorporated herein by reference.

[0235] Cleavable linkers can be cleavable in vitro, in vivo, or both. Cleavable linkers can include chemically or enzymatically labile or degradable bonds. Cleavable linkers can rely on intracellular processes to release Category A-K compounds, such as cytoplasmic reduction, exposure to acidic conditions within lysosomes, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers can incorporate one or more chemical bonds that are chemically or enzymatically cleavable while the remainder of the linker is non-cleavable.

[0236] In some embodiments, L is a linker containing a reactive moiety. In some embodiments, for a linker payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, -L is represented by the following formula.

Chemical formula

[0237] In some embodiments, -L is represented by the following formula, [Chemical formula] In the formula, each R 30 is independently selected from optionally substituted C1-C6 alkyl and optionally substituted phenyl, and RX is a reactive moiety. RX may contain a leaving group. RX may be a maleimide. L may further covalently bind to the binding protein of the present disclosure. In some embodiments, -L- is represented by the following formula, [Chemical formula] In the formula, RX * is a bond, a succinimide moiety, or a hydrolyzed succinimide moiety bound to a residue of the binding protein of the present disclosure. In the formula, on RX* [Chemical formula] represents a binding point to a residue of the polypeptide, and each R 30 is independently selected from optionally substituted C1-C6 alkyl and optionally substituted phenyl.

[0238] In some embodiments, for the linker-payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, L comprises a methylene carbamate unit.

[0239] In some embodiments, for the linker-payload (L-P) comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and the linker L-RX * L-P is part of the conjugate, and RX * comprises a hydrolyzed succinimide moiety and binds to a cysteine residue of a polypeptide comprising a binding domain.

[0240] By way of example and not limitation, some cleavable and non-cleavable linkers that may be included in the conjugate are described below in addition to any other of the present disclosure.

[0241] The linker may contain a chemically labile group such as a hydrazone or disulfide group. Linkers containing chemically labile groups can take advantage of the different properties between plasma and some cytoplasmic compartments. Intracellular conditions that facilitate the release of the compounds of the present disclosure for hydrazones containing a linker, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, may be the acidic environment of endosomes and lysosomes, while linkers containing disulfides can be reduced in the cytosol, which may contain a high thiol concentration, such as glutathione. The plasma stability of linkers containing chemically labile groups can be increased by introducing steric hindrance using substituents near the chemically labile group.

[0242] Groups that are labile to acids, such as hydrazones, can remain intact during systemic circulation in the neutral pH environment (pH 7.3 - 7.5) of the blood, and when this conjugate is internalized into the mildly acidic endosome (pH 5.0 - 6.5) and lysosome (pH 4.5 - 5.0) compartments of the cell, it can undergo hydrolysis and release the compounds of the present disclosure. This pH-dependent release mechanism may be associated with non-specific release of the drug. To enhance the stability of the hydrazone group of the linker, the linker can be changed by chemical modification, such as substitution, and tuning allows for minimizing losses in circulation and achieving more efficient release within the lysosome.

[0243] In some embodiments, for a linker payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and linker L, -L comprises a hydrazone moiety. For example, L is

Chemical formula

[0244] The hydrazone-containing linker may contain additional cleavage sites such as a cleavage site labile to additional acids or an enzymatically labile cleavage site. Conjugates containing exemplary hydrazone-containing linkers can include, for example, the following structures,

Chemical formula

[0245] Other acid-labile groups that can be included in the linker include cis-aconityl-containing linkers. Cis-aconityl chemistry can use a carboxylic acid juxtaposed to an amide bond to accelerate amide hydrolysis under acidic conditions.

[0246] Cleavable linkers can also include disulfide groups. Disulfides may be thermodynamically stable at physiological pH and designed to release the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof upon internalization within cells, and the cytosol may provide a significantly more reducing environment compared to the extracellular environment. Cleavage of the disulfide bond may require the presence of a cytoplasmic thiol cofactor such as (reduced) glutathione (GSH), such that the disulfide-containing linker is reasonably stable in circulation and can selectively release the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, in the cytosolic compartment. Intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to the preferential cleavage of disulfide bonds within cells. GSH can be present in cells in a concentration range of 0.5 - 10 mM, compared to a significantly lower concentration of GSH or cysteine, the most abundant low molecular weight thiol in circulation, at approximately 5 μM. Tumor cells, where irregular blood flow can cause hypoxia, can result in enhanced activity of reductases and thus further increase glutathione concentrations. The in vivo stability of disulfide-containing linkers can be improved by chemical modification of the linker, such as the use of steric hindrance adjacent to the disulfide bond.

[0247] Conjugates comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and an exemplary disulfide-containing linker may comprise the following structures,

Chemical formula

[0248] Another type of linker that can be used is a linker that is specifically cleaved by an enzyme. For example, the linker can be cleaved by a lysosomal enzyme. Such a linker may be peptide-based or may contain a peptide region that can act as a substrate for an enzyme. Peptide-based linkers can be more stable in plasma and the extracellular environment than chemically labile linkers.

[0249] Peptide bonds can have very low activity in blood due to endogenous inhibitors and the blood pH value being unfavorably high compared to lysosomes, so they can have good serum stability. Release of the compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, from the binding protein of the present disclosure can occur due to the action of lysosomal proteases, such as cathepsin and plasmin. These proteases can be present at high levels in certain tumor tissues. The linker can be cleavable by lysosomal enzymes. Lysosomal enzymes can be, for example, cathepsin B, β-glucuronidase, or β-galactosidase.

[0250] The cleavable peptide can be selected from tetrapeptides such as Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, or dipeptides such as Val-Cit, Val-Ala, and Phe-Lys. Dipeptides can have lower hydrophobicity compared to longer peptides.

[0251] A variety of peptide-based cleavable linkers can be used with the disclosed conjugate proteins to form conjugates of the disclosed compounds, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof.

[0252] Enzymatically cleavable linkers can include self-immolative spacers for spatially separating the disclosed compounds, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof from the enzyme cleavage site. A direct bond of the disclosed compounds, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof to a peptide linker can result in proteolytic release of the disclosed compounds, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, or amino acid adducts of the disclosed compounds, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, thereby impairing their activity. The use of self-immolative spacers can enable the release of the unmodified, fully active compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof upon hydrolysis of the amide bond.

[0253] One self-immolative spacer can be a bifunctional para-aminobenzyl alcohol (PABA) group that can be linked to a peptide via an amino group to form an amide bond, while the amine-containing compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, can be linked to the benzyl hydroxyl group of the linker via a carbamate functional group (to obtain p-amidobenzyl carbamate, PABC). The resulting prodrug is activated upon protease-mediated cleavage, resulting in a 1,6-elimination reaction and releasing the unmodified compound of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, carbon dioxide, and the remainder of the linker group.

[0254] In some embodiments, a compound of categories A - K, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, is attached to an LI - type linker, which may contain a reactive group capable of forming a covalent bond with a reactive group (e.g., - SH or NH2) on the binding protein of the present disclosure, or the linker may already be covalently bound to the binding protein of the present disclosure. In some embodiments, the linker has the following structure,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0255] The following scheme shows the fragmentation of p - amidobenzyl carbamate and the release of the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof,

Chemical formula

[0256] In some other embodiments, D is bonded to the L1 - type linker via an S atom on D. In such embodiments, the following scheme shows the fragmentation of p - amidobenzyl carbamate and the release of the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof,

Chemical formula

[0257] In some embodiments, for the linker - payload comprising the compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, the linker (i.e., L) is represented by the following formula,

Chemical formula

Chemical formula

[0258] In some embodiments, for a linker payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, -L is represented by the following formula,

Chemical formula

Chemical formula

[0259] In some embodiments, for a linker payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, -L is represented by the following formula,

Chemical formula

[0260] In some embodiments, for a GR agonist compound, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, in a linker-payload comprising, L-P is part of the conjugate, and -L is represented by the following formula,

Chemical formula

Chemical formula

[0261] In a further embodiment, L-P is part of the conjugate, and -L- is represented by the following formula,

Chemical formula

Chemical formula

Chemical formula

[0262] In some embodiments, L-P is part of the conjugate, and -L- is represented by the following formula

Chemical formula

Chemical formula

[0263] In some embodiments, -L is [Chemical formula] .

[0264] This heterocyclic variant of the self - sacrificial group can also be used.

[0265] The enzymatically cleavable linker can be a β - glucuronic acid - based linker. The facile release of the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, can be achieved by cleavage of the β - glucuronide glycoside bond by the lysosomal enzyme β - glucuronidase. This enzyme can be abundantly present within lysosomes and can be overexpressed in some tumor types, but can have low extracellular enzyme activity. The β - glucuronic acid - based linker can be used to avoid the tendency of the polypeptide conjugates of the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, to aggregate due to the hydrophilic nature of the β - glucuronide. In some embodiments, the β - glucuronic acid - based linker can conjugate the binding protein of the present disclosure to the hydrophobic compound of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof.

[0266] The following scheme shows the release from the conjugates of the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, and the compounds of the present disclosure containing a (D)β - glucuronic acid - based linker, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, [Chemical formula] wherein Ab represents the binding protein of the present disclosure.

[0267] Various cleavable β-glucuronate-based linkers are described that are useful for conjugating drugs such as auristatin, camptothecin analogs, doxorubicin analogs, CBI minor groove binders, and psinverine to antibodies. These β-glucuronate-based linkers can be used in conjugates. In some embodiments, the enzymatically cleavable linker is a β-galactoside-based linker. β-Galactosides are abundant within lysosomes but have low extracellular enzyme activity.

[0268] In addition, a compound of the present disclosure containing a phenol group, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, can covalently bond to a linker via a phenolic oxygen. One such linker depends on a methodology in which a diamino-ethane "Space Link" is used in combination with a conventional "PABO" type self-sacrificing group to deliver phenol.

[0269] The cleavable linker may contain a non-cleavable moiety or segment, or the cleavable segment or moiety may be contained within another non-cleavable linker to make it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers may contain cleavable groups within the polymer backbone. For example, a polyethylene glycol or polymer linker may contain one or more cleavable groups such as disulfide, hydrazone, or dipeptide.

[0270] Other cleavable linkages that may be included in the linker can include an ester bond formed by the reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group on any one of the compounds of categories A - K, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and such ester groups can hydrolyze under physiological conditions to release any one of the compounds of categories A - K, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof. Cleavable linkages by hydrolysis can include a carbonate bond, an imine bond resulting from the reaction of an amine and an aldehyde, a phosphate ester bond formed by reacting an alcohol with a phosphate group, an acetal bond which is the reaction product of an aldehyde and an alcohol, an orthoester bond which is the reaction product of a formate and an alcohol, and a phosphoramidite group including the end of a polymer, and an oligonucleotide bond formed by a 5'-hydroxyl group of an oligonucleotide.

[0271] The linker can contain an enzymatically cleavable peptide, for example, a linker containing the following structural formulas (LI - CIIIa), (LI - CIIIb), (LI - CIIIc), or (LI - CIIId),

Chemical formula

[0272] In some embodiments, the peptide can be selected to contain natural amino acids, non-natural amino acids, or any combination thereof. In some embodiments, the peptide can be a tripeptide or a dipeptide. In certain embodiments, the dipeptide is an L-amino acid, such as Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit, or salts thereof.

[0273] Exemplary embodiments of the linker according to Structural Formula (LI-CIIIa) are illustrated below (as illustrated, the linker contains a reactive group suitable for covalently bonding the linker to the binding protein of the present disclosure).

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0274] Exemplary embodiments of the linker according to Structural Formula (CIIIb), (CIIIc), or (CIIId) that can be included in the conjugate can include the linkers illustrated below (as illustrated, the linker contains a reactive group suitable for covalently bonding the linker to a fusion protein, or a polypeptide containing a binding domain such as an antibody or antigen-binding fragment thereof).

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0275] The linker includes an enzymatically cleavable sugar moiety, for example, a linker including the following structural formulas (CIVa), (CIVb), (CIVc), (CIVd), or (CIVe), [Chemistry] or a salt thereof, wherein q is 0 or 1, r is 0 or 1, and X 1 is CH2, O, or NH, [Chemistry] represents the bonding point of the linker (L) to the compound of the present disclosure, or its pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt, and * represents the bonding point to the remaining part of the linker.

[0276] Exemplary embodiments of the linker according to structural formula (CIVa) that may be included in an antibody construct conjugate of a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, can include the linkers exemplified below (as exemplified, the linker includes a group suitable for covalently bonding the linker to a binding protein of the present disclosure),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0277] Exemplary embodiments of the linker according to structural formula (CIVb) that may be included in the conjugate include the linkers exemplified below (as exemplified, the linker includes a group suitable for covalently bonding the linker to a binding protein of the present disclosure),

Chemical formula

Chemical formula

Chemical formula

[0278] Exemplary embodiments of the linker according to structural formula (CIVc) that may be included in the conjugate include the linkers exemplified below (as exemplified, the linker includes groups suitable for covalently bonding the linker to the conjugate protein of the present disclosure),

Chem.

Chem.

Chem.

Chem.

[0279] Exemplary embodiments of the linker according to structural formula (CIVd) that may be included in the conjugate include the linkers exemplified below (as exemplified, the linker includes groups suitable for covalently bonding the linker to the conjugate protein of the present disclosure),

Chem.

Chem.

[0280] Exemplary embodiments of the linker according to structural formula (CIVe) that may be included in the conjugate include the linkers exemplified below (as exemplified, the linker includes groups suitable for covalently bonding the linker to the conjugate protein of the present disclosure),

Chem.

[0281] Cleavable linkers can offer certain advantages, although linkers including conjugates need not be cleavable. For non-cleavable linkers, payload compound release may not depend on the different properties between plasma and some cytoplasmic compartments. Release of the payload compound may occur after internalizing the conjugate via antigen-mediated endocytosis and delivering it to the lysosomal compartment, where the binding protein of the present disclosure can be degraded to the amino acid level by intracellular proteolytic degradation. This process can release the payload compound derivative (the metabolite of the conjugate containing the non-cleavable linker heterocyclic compound) formed by the payload compound, the linker, and the amino acid residue(s) to which the linker is covalently bound. The payload compound derivative from the conjugate with a non-cleavable linker may be more hydrophilic and have lower membrane permeability, which can result in less bystander effect and non-specific toxicity compared to the conjugate with a cleavable linker. The conjugate with a non-cleavable linker can have higher stability in circulation than the conjugate with a cleavable linker. The non-cleavable linker may include an alkylene chain, or may be a polymer based on, for example, a polyalkylene glycol polymer, an amide polymer, or may include segments of an alkylene chain, a polyalkylene glycol, or an amide polymer. The linker may contain a polyethylene glycol segment having 1 to 6 ethylene glycol units.

[0282] The linker may be non-cleavable in vivo and can be, for example, a linker-payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and linker L, where -L is of the following formula:

Chemical formula

Chemical formula

[0283] In some embodiments, for the linker-payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and linker L, -L is represented by the following formula:

Chemical formula

Chemical formula

[0284] In some embodiments, for the linker-payload comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and linker L, -L is represented by the following formula:

Chemical formula

Chemical formula

[0285] In some embodiments, for the conjugate comprising a compound of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, linker L, and the binding protein of the present disclosure, -L- is represented by the following formula,

Chemical formula

Chemical formula

Chemical formula

[0286] Exemplary embodiments of the linker according to structural formulas (CVa) to (Ve) that can be included in the conjugate include the linkers exemplified below (as exemplified, the linker includes a group suitable for covalently bonding the linker to the binding protein of the present disclosure,

Chemical formula

Chemical formula

[0287] The linking groups used to attach a linker to the binding protein of the present disclosure can be substantially electrophilic and include, for example, maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, halocarboxylates, acid halides, alkyls, and benzyl halides such as haloacetamides. There are also new technologies regarding "self-stabilizing" maleimides and "crosslinking disulfides" that can be used with the GR agonist compounds of the present disclosure. Examples of cysteine-based linkers are provided in PCT Patent Application Publication No. 2020 / 092385, which is incorporated herein by reference.

[0288] Maleimide groups are frequently used in the preparation of conjugates due to their specificity for reacting with the thiol groups of cysteine groups of the binding proteins of the present disclosure, for example. The reaction between the thiol group of the binding protein of the present disclosure and a drug having a linker (linker-payload) containing a maleimide group begins according to the following scheme.

Chemical formula

[0289] A reverse reaction leading to the elimination of maleimide from the thio-substituted succinimide can also occur. This reverse reaction is undesirable because the maleimide group may then react with another available thiol group, such as another protein in the body having available cysteine. Thus, the reverse reaction can interfere with the specificity of the conjugate. One way to prevent the reverse reaction is to incorporate a basic group into the linking group shown in the above scheme. Without wishing to be bound by theory, the presence of the basic group can increase the nucleophilicity of nearby water molecules and promote the ring-opening hydrolysis of the succinimide group. The hydrolyzed form of the linking group is resistant to deconjugation in the presence of plasma proteins. So-called "self-stabilizing" linkers provide conjugates with improved stability. A representative schematic is shown below.

Chemical formula

[0290] The hydrolysis reaction schematically represented above can occur at either of the carbonyl groups of the succinimide group. Thus, as shown below, two possible isomers can occur.

Chemical formula

[0291] The identity of the base, as well as the distance between the base and the maleimide group, can be modified, for example, by improving the specificity and stability of the conjugate, to adjust the hydrolysis rate of the thio-substituted succinimide group and optimize the delivery of the conjugate to the target.

[0292] Before conjugating to the binding protein of the present disclosure, a base suitable for inclusion in a linker, for example, any L having a maleimide group, can promote the hydrolysis of nearby succinimide groups formed after conjugating the binding protein of the present disclosure to the linker. The base can be, for example, an amine (e.g., -N(R 26 )(R 27 ), where R 26 and R 27 are independently selected from H and C 1-6 alkyl), a nitrogen-containing heterocycle (e.g., a 3- to 12-membered heterocyclic ring containing one or more nitrogen atoms and optionally one or more double bonds), an amidine, a guanidine, and a carbocyclic or heterocyclic ring substituted with one or more amine groups (e.g., optionally containing a heteroatom such as a nitrogen atom and having the form -N(R 26 )(R 27 ), where R 26 and R 27 are independently selected from H or C 1-6 alkyl), and a 3- to 12-membered aromatic or non-aromatic ring substituted with one or more amines). The basic unit can be separated from the maleimide group, for example, by an alkylene chain in the form of -(CH2) m- , where m is an integer from 0 to 10. The alkylene chain can optionally be substituted with other functional groups of the present disclosure.

[0293] The linker (L) having a maleimide group is -C(O)R, =O, -CN, -NO2, -CX3, -X, -C(O)OR, -C(O)NR2, -C(O)R, -C(O)X, -SO2R, -SO2OR, -SO2NHR, -SO2NR2, -PO3R2, -P(O)(CH3)NHR, -NO, -NR3 + , -CR=CR2, and -C≡CR and the like may contain an electron-withdrawing group, and each R is independently selected from H and C 1-6 alkyl, and each X is independently selected from F, Br, Cl, and I. The self-stabilizing linker may also include a group optionally substituted with an electron-withdrawing group such as aryl, for example, phenyl, or heteroaryl, for example, those of the present disclosure.

[0294] Examples of self-stabilizing linkers are provided, for example, in US Patent Application Publication No. 2013 / 0309256, the linker of which is incorporated herein by reference. It will be understood that self-stabilizing linkers useful in combination with the compounds of the present disclosure may be equivalently described as linkers containing unsubstituted maleimide, linkers containing thio-substituted succinimide, or linkers containing hydrolyzed ring-opened thio-substituted succinimide.

[0295] In some embodiments, for the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, and linker payloads containing linker L, -L contains a self-stabilizing moiety. For example, L may be selected from the following.

Chemical formula

[0296] In the scheme provided above, the bottom structure may be referred to as (maleimide)-DPR-Val-Cit-PAB, where DPR refers to diaminopropionic acid, Val refers to valine, Cit refers to citrulline, and PAB refers to para-aminobenzylcarbonyl.

Chemical formula

[0297] A method for crosslinking a pair of sulfhydryl groups derived from the reduction of a native hinge disulfide bond is disclosed and shown in the following schematic. The advantage of this methodology is the ability to synthesize homogeneous conjugates by complete reduction of IgG (obtaining four pairs of sulfhydryls from the interchain disulfides where the DAR can range from 1 to 8), followed by reaction with four equivalents of an alkylating agent. Conjugates containing "crosslinked disulfides" are also claimed to have increased stability.

Chem.

[0298] Similarly, as shown below, maleimide derivatives capable of crosslinking a pair of sulfhydryl groups have been developed.

Chem.

[0299] The linker L of the present disclosure has the following structural formula (CVIa), (CVIb), or (CVIc),

Chem.

[0300] Exemplary embodiments of the linker according to structural formulas (CVIa) and (CVIb) that can be included in the linker-payload and conjugate structures of the present disclosure include the linkers exemplified below (as exemplified, the linker includes a group suitable for covalently bonding the linker to an antibody construct),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0301] Exemplary embodiments of the linker according to structural formula (CVIc) that can be included in the linker-payload and conjugate structures of the present disclosure include the linkers exemplified below (as exemplified, the linker includes a group suitable for covalently bonding the linker to the binding protein of the present disclosure),

Chemical formula

Chemical formula

Chemical formula

[0302] Several exemplary linkers (L) are described in the following paragraphs. In some embodiments of the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, the linker attachment is at the nitrogen of the compound, the conjugation is at the cysteine residue of the antibody or targeting moiety, and -L is represented by the formula shown in Table 3 below. [Table 3-1] [Table 3-2] Wherein, [Chemical Formula] represents the attachment to the nitrogen of the compound or salt of the present disclosure, and L 4 is the C-terminus of the peptide, and L 5 is selected from a bond, alkylene, and heteroalkylene, and L 5 is optionally substituted with one or more groups independently selected from R 30 , and R 30 is independently, at each occurrence, halogen, -OH, -CN, -O-alkyl, -SH, =O, =S, -S(O)2OH, -NH2, -NO2, and C 1-10 alkyl, C 2-10 alkenyl, and C 2-10 alkynyl, each of which is independently optionally substituted, at each occurrence, with one or more substituents selected from halogen, -OH, -CN, -O-alkyl, -SH, =O, =S, -S(O)2OH, -NH2, and -NO2, and RX represents a reactive moiety. The reactive moiety can be selected from, for example, an electrophilic group, such as an α,β-unsaturated carbonyl such as maleimide, and a leaving group. In certain embodiments, RX of any one of linkers L1 to L11 is maleimide. In certain further embodiments, RX is [Chemical Formula] , wherein RX *is a bond, succinimide moiety, or hydrolyzed succinimide moiety attached to a cysteine residue of an antibody, antibody construct, or targeting moiety, wherein on RX*

Chem.

[0303] In some embodiments of the compounds of the present disclosure, or pharmaceutically acceptable isomers, tautomers, racemates, hydrates, solvates, isotopes, or salts thereof, the linker attachment is to a nitrogen of the compounds of the present disclosure, or a pharmaceutically acceptable isomer, tautomer, racemate, hydrate, solvate, isotope, or salt thereof, and the conjugation is to a lysine residue of the conjugated protein of the present disclosure. In certain embodiments, -L is represented by the formula shown in Table 4 below.

Table 4

Chem.

Chem.

Chem.

[0304] As is known to those skilled in the art, the linker selected for a particular conjugate can be affected by various factors, including the binding site to the binding protein of the present disclosure, lysine, cysteine, or other amino acid residues, the structural constraints of the drug pharmacophore, and the lipophilicity of the drug. The specific linker selected for the conjugate should strive to balance these different factors for the combination of the polypeptide containing the binding domain (e.g., a fusion protein, or an antibody or its antigen-binding fragment) and the drug.

[0305] For example, it has been observed that cytotoxic conjugates result in the killing of bystander antigen-negative cells present in the vicinity of antigen-positive tumor cells. The mechanism of the bystander effect by cytotoxic conjugates has shown that metabolites formed during the intracellular processing of the conjugate can play a role. The neutral cytotoxic metabolites generated by the metabolism of the conjugate in antigen-positive cells appear to play a role in the killing of bystander cells, while charged metabolites can prevent diffusion across the membrane into or out of the medium, and thus cannot affect the killing of cells via the bystander effect. In some embodiments, the linker is selected to attenuate the bystander effect caused by the cellular metabolites of the conjugate. In further embodiments, the linker is selected to increase the bystander effect.

[0306] The properties of the linker, or linker-payload, can also affect conjugate aggregation under conditions of use or storage. Generally, conjugates reported in the literature contain about 3 to 4 drug molecules per antibody molecule. Attempts to obtain higher drug-to-antibody ratios (“DARs”) often fail, especially when both the drug and the linker are hydrophobic due to conjugate aggregation. In many cases, a DAR greater than 3 to 4 can be beneficial as a means of increasing potency. When the payload compound is in fact more hydrophobic, especially when it is desirable for the DAR to exceed 3 to 4, it may be desirable to select a relatively hydrophilic linker as a means of reducing conjugate aggregation. Thus, in some embodiments, the linker incorporates a chemical moiety that reduces conjugate aggregation during storage or use. The linker can incorporate a charged group, or a polar or hydrophilic group such as a group that is charged at physiological pH to reduce conjugate aggregation. For example, the linker can incorporate a charged group that is deprotonated at physiological pH, such as a carboxylate, or protonated, such as an amine.

[0307] In preferred embodiments, conjugate aggregation during storage or use is less than about 40% as determined by size exclusion chromatography (SEC). In certain embodiments, conjugate aggregation during storage or use is less than about 35%, such as less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, or even less, as determined by size exclusion chromatography (SEC).

[0308] 2. Linkers of Category II Other linkers useful in various embodiments include linkers of Category II. In certain embodiments, the conjugate comprises a binding protein of the present disclosure and the linker is a Category II linker that links a GR agonist to a spacer that does not affect the binding of the polypeptide containing the segment, such as the active portion of the conjugate, i.e., the antigen-binding domain, or the release of the drug compound.

[0309] In another embodiment, the conjugate comprises a Category II linker and is represented by formula (LII-1), or

Chemical formula

[0310] In another embodiment of LII-1, a is an integer from 1 to 4, b is an integer from 1 to 10, and m is 0. In yet another embodiment, a is an integer from 1 to 4, m is 0, n is 0, and R 3 and R 3’ are each hydrogen. In some embodiments, the linker L is Category II represented by formula (LII-2),

Chemical formula

Chemical formula

Chemical formula

[0311] In some embodiments, W is an L II self-sacrificing moiety segment in a linker of the L group. In some embodiments, W is a group of its own and is selected from the following,

Chemical formula

Chemical formula

Chemical formula

[0312] In other embodiments, W is selected from the following.

Chemical formula

[0313] In some embodiments, W is the following.

Chemical formula

[0314] In a further embodiment, Z in linker LII-2 comprises a peptide that can be enzymatically cleaved. In an even further embodiment, Z is a cathepsin-cleavable group of the peptide. In some embodiments, Z is a two-amino acid peptide selected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys, and Lys-Phe. In one embodiment, Z is Val-Ala or Ala-Val. In certain embodiments, U and U' are modifiable conjugates in linker LII-2. In some embodiments of linker LII-2, U and U' are independently absent or selected from the following,

Chemical formula

Chemical formula

Chemical formula

[0315] In one embodiment, U' is absent and U is represented by the following formula (LII-3).

Chemical formula

[0316] In some embodiments, Q is a substitutable conjugate in linker LII-2. In other embodiments, Q is a heterobifunctional group or RG that can bind to Ab by chemical or enzyme-mediated conjugation. In a further embodiment, Q is selected from the following,

Chemical formula

Chemical formula

[0317] In some embodiments, a GR agonist of categories A - K, or a pharmaceutically acceptable salt thereof, is linked to the binding protein of the present disclosure via linker LII-4, [Chemical formula] wherein t is 1, W is absent or is a self-sacrificing group, and Z is absent or is a peptide of two amino acids.

[0318] In another embodiment, the conjugate of the present disclosure is represented by formula (LII-5), [Chemical formula] or a pharmaceutically acceptable salt thereof, wherein, a is an integer from 1 to 20, b is an integer from 1 to 20, k is 0, 1, 2, or 3, m is 0, 1, 2, 3, or 4, D-NH- is a compound of the present invention, one H is replaced by a covalent bond to a -C(=O)X- group, R 2 is selected from H, C1-C4 alkyl, and -(CH2CH2O) s -CH3, s is an integer from 1 to 10, R 4 is selected from hydrogen and any natural amino acid side chain, R 5 is selected from C1-C4 alkyl and O-C1-C4 alkyl, L is a cleavable linker, and Ab is the binding protein of the present disclosure.

[0319] In another embodiment, the linker of category II is represented by formula (LII-6), wherein, [Chemistry] [Chemistry] is a bonding point to the carbonyl group, [Chemistry] is a bonding point to Ab, W is a self - sacrificial moiety, Z is either absent or a peptide of 2 - 5 amino acids, U and U’ are independently either absent or a spacer, and Q is a heterobifunctional group.

[0320] In another embodiment, the conjugate of the present disclosure is represented by formula (LII - 7), or [Chemistry] or a pharmaceutically acceptable salt thereof, wherein D - NH - is a compound of categories A - K, and one H is replaced by a covalent bond to the - C(=O)X - group.

[0321] In one embodiment, the LII - 2 type is represented by formulas (LII - 8 - LII - 10), [Chemistry] wherein [Chemistry] is a bonding point to the amino group in the compound of the present invention.

[0322] In one embodiment, the term “self-sacrificing moiety” or “self-sacrificing group” refers to a moiety, functional group, or functional group that undergoes an electron cascade resulting in the release of the moiety, functional group, or molecule to which it is attached. In some embodiments, the self-sacrificing group comprises one or more groups that can undergo 1,4-elimination, 1,6-elimination, 1,8-elimination, 1,6-cycloelimination, 1,5-cycloelimination, 1,3-cycloelimination, intramolecular 5-exo-trig cyclization, or 6-exo-trig cyclization. In some embodiments, the self-sacrificing group can be any of the groups disclosed in PCT Publications WO2018 / 200812 and WO2018 / 100558, the portions of which are hereby incorporated by reference in their entirety.

[0323] In one embodiment, the “Z” group of the Category II linker is absent or is a peptide of 2 to 5 amino acids. In some embodiments, the peptide is the cleavage site of the linker, thereby facilitating the release of the drug upon exposure to an intracellular protease such as a lysosomal enzyme (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Examples of peptides having two amino acids include alanine-alanine (ala-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides having three amino acids include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The above amino acid combinations can also exist in the reverse order (i.e., cit-val).

[0324] The peptides of the present disclosure may contain natural or non-natural amino acid residues. The term "natural amino acid" refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. "Non-natural amino acids" include homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), beta-alanine, L- or D-naphthalanine, ornithine ("Orn"), and the like. The peptides can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-related proteases, cathepsin B, C, and D, or plasmin proteases.

[0325] Amino acids also include the D-forms of natural and non-natural amino acids. "D-" indicates an amino acid having a "D" (right-handed) configuration, in contrast to the configuration in naturally occurring ("L-") amino acids. Natural and non-natural amino acids can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

[0326] The "U" and "U'" groups in the Category II linkers are independently either absent or are spacers. As used herein, the term "spacer" refers to a chemical moiety that functions as a connector. In the present disclosure, a spacer can connect the binding protein of the present disclosure to a heterobifunctional group, or can connect a heterobifunctional group to a peptide "Z", or, if "Z" is absent, can connect to the "W" group. Non-limiting exemplary spacers include -NH-, -S-, -O-, -NHC(=O)CH2CH2-, -S(=O)2-CH2CH2-, -C(=O)NHNH-, -C(=O)O-, -C(=O)NH-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2=CH2-, -C°C-, -CH=N-O-, polyethylene glycol (PEG),

Chemical Structure

[0327] In the compounds having a linker of Category II, when "U" is present, it can be a branched group substituted with 1 to 10 "-C(O)-W-Z-" groups. In some embodiments, "U" is substituted with 1 to 5 "-C(O)-W-Z-" groups. In some embodiments, "U" is substituted with 1 or 2 "-C(O)-W-Z-" groups. In some embodiments, "U" is substituted with 1 "-C(O)-W-Z-" group. In some embodiments, the spacer can be any of those disclosed in PCT Publications WO2018 / 200812, WO2018 / 100558, which are hereby incorporated by reference in their entirety.

[0328] The group "Q" as defined for compounds having a linker of Category II is a heterobifunctional or reactive group (RG). In the present disclosure, the term "heterobifunctional group" refers to the chemical moiety that connects the linker of which it is a part to the binding protein of the present disclosure. See, for example, WO2017 / 191579. A heterobifunctional group is characterized by having different reactive groups at both ends of this chemical moiety. The heterobifunctional group may be directly bonded to "Ab" or, alternatively, may be connected via the linker "U". The bond to "Ab" can be achieved by chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves a controlled reaction of accessible amino acid residues on the surface of a polypeptide containing a binding domain having a reactive handle on "Q" or "U". Examples of chemical conjugation include lysine amide coupling, cysteine coupling, and coupling via non-natural amino acids incorporated by genetic engineering, where a non-natural amino acid residue having the desired reactive handle is attached to "Ab". In enzymatic conjugation, the enzyme mediates the coupling of the linker to accessible amino residues on the binding protein of the present disclosure. Examples of enzymatic conjugation include transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation can also be used sequentially. For example, enzymatic conjugation can also be used to attach a unique reactive handle on "Ab" for use in subsequent chemical conjugation. In some embodiments, the heterobifunctional group can be any of those disclosed in PCT publications WO2018 / 200812, WO2018 / 100558, which are hereby incorporated by reference in their entirety.

[0329] 3. Linker of Category III In some other aspects, the present disclosure relates to conjugates comprising a compound of categories A - K linked to a binding protein of the present disclosure via a category III linker, the conjugate having the following formula (LIII - I),

Chemical formula

Chemical formula

[0330] In certain embodiments, the present disclosure relates to a binding protein suitable for forming conjugates of compounds of categories A - K that include a linker of category III represented by formula (LIII - 3) or (LIII - 4),

Chemical formula

Chemical formula

[0331] In one embodiment, the peptide moiety of the Category III linker contains from 3 to about 10 amino acids, such as about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acids.

[0332] In one embodiment, the hydrophilic group contains the following.

Chemical formula

[0333] In another embodiment, the hydrophilic group contains the following.

Chemical formula

[0334] In some embodiments, the aminopolyalcohol is

Chemical formula

[0335] In some embodiments, the hydrophilic group is

Chemical formula

[0336] In certain embodiments, the hydrophilic group includes polyethylene glycol, for example, polyethylene glycol having about 6 to about 24 PEG subunits. In some embodiments, the hydrophilic group includes polyethylene glycol having about 6 to about 12 PEG subunits.

[0337] In some embodiments, the hydrophilic group includes polyethylene glycol having about 8 to about 12 PEG subunits.

[0338] In a further embodiment, when L 3 is present, it includes -X-C 1-10 alkylene-C(Q)-, where X is directly connected to L M and X is CH2, O, or NR5, and R5 is hydrogen, C1-6 alkyl, C6- 10 aryl, C3-8 cycloalkyl, COOH, or COO-C 1-6It is alkyl.

[0339] In some embodiments, L 3 , when present, is -NR5-(CH2)v-C(O)- or -CH2-(CH2)v-C(O)-NR5-(CH2)v-C(O)-, and each v is, independently, an integer from 1 to 10 (for example, each v is, independently, an integer from 1 to 6, or from 2 to 4, or 2). In some embodiments, LIII is -NH-(CH2)2-C(O)- or -(CH2)2-C(0)-NH-(CH2)2-C(O)-.

[0340] In some embodiments, a4 is 1, 2, or 3. In some embodiments, d 13 is an integer from about 1 to about 6. In some embodiments, d 13 is an integer from about 1 to about 4. In some embodiments, d 13 is an integer from about 4 to about 6. In some embodiments, d 13 is an integer from about 2 to about 4. In some embodiments, d 13 is an integer from about 1 to about 2. In some embodiments, d 13 is 2. In some embodiments, each W p , when present, is independently

Chemical formula

[0341] In some embodiments, R1K is halo or RC(O)O−, and R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0342] In one embodiment, R 1A is

Chemical formula

[0343] In some embodiments, each W P is independently

Chemical formula

[0344] In some embodiments, W P is as follows.

Chemical formula

[0345] In some embodiments, when W P is

Chemical formula

Chemical formula

[0346] In some embodiments, W P is [Chemical formula] wherein, one of Xa and Xb is H and the other is a maleimide blocking moiety. In some embodiments, a maleimide blocking compound (i.e., a compound capable of reacting with maleimide to convert it to succinimide) can be used, for example, to quench the reaction between a linker-drug moiety and PERM (e.g., engineered cysteine of PERM), and the maleimide blocking moiety refers to a chemical moiety that binds to succinimide upon conversion. In some embodiments, the maleimide blocking moiety is a moiety capable of covalently binding to one of two olefin carbon atoms when the maleimide group reacts with a thiol-containing compound of formula (LIII-7), (LIII-7) R90-(CH2) (LIII-7) R90-(CH2) d -SH wherein, R 90 is NHR 91 , OH, COOR 93 , CH(NHR 91 )COOR 93 , or a substituted phenyl group, R 93 is hydrogen or C1-4 alkyl, R 91 is hydrogen, CH3, or CH2CO, d is an integer from 1 to 3.

[0347] In some embodiments, the maleimide blocking compound can be cysteine, N-acetylcysteine, cysteine methyl ester, N-methylcysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH2SH), benzylthiol optionally substituted with one or more hydrophilic substituents on phenyl, or 1-aminopropane-1-thiol. In some embodiments, one or more hydrophilic substituents on phenyl include OH, SH, methoxy, ethoxy, COOH, CHO, COC 1-4 alkyl, F, cyano, SO3H, PO3H, etc.

[0348] In some embodiments, the maleimide blocking group is -S-(CH2) d -R 90 wherein R 90 is OH, COOH, or CH(NHR 91 )COOR 93 and R 93 is hydrogen or CH3, R 91 is hydrogen or CH3CO, and d is 1 or 2.

[0349] In one embodiment, the maleimide blocking group is -S-CH2-CH(NH2)COOH.

[0350] In one embodiment, the spacer unit M p is a group by itself.

[0351] In some embodiments, M p , when present, is -(Z4)-[(Z5)-(Z6)] z where Z4 is connected to L p ' or L p , Z6 is connected to L M , z is 1, 2, or 3, Z 4 is

Chemical formula

[0352] In some embodiments, Z4 is as follows.

Chemical formula

[0353] In other embodiments, Z4 is as follows, [Chemical formula] wherein b1 is 1 or 4.

[0354] In some embodiments, Z4 is as follows. [Chemical formula]

[0355] In other embodiments, Z4 is [Chemical formula] and, for example, b1 is 1.

[0356] In other embodiments, Z4 is [Chemical formula] and, for example, in the formula, bi is 0.

[0357] In some embodiments, Z4 is [Chemical formula] as follows.

[0358] In other embodiments, Z4 is [Chemical formula] as follows.

[0359] In some embodiments, b1 is 0. In some embodiments, one of R 66 is O and the other is NH.

[0360] In some embodiments, Z4 is as follows.

Chem.

[0361] In some embodiments, Z4 is

Chem.

[0362] In some embodiments, each Z5 is independently a polyalkylene glycol (PAO) including, but not limited to, polymers of lower alkylene oxides (such as polymers of propylene oxide, polypropylene glycol, polyethylene glycol (PEG), polyoxyethylenated polyols, copolymers thereof, and block copolymers thereof). In some embodiments, the polyalkylene glycol is polyethylene glycol (PEG) including polydisperse PEG, monodisperse PEG, and separate PEGs. In some embodiments, polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is purified from the heterogeneous mixture and thus provides a single chain length and molecular weight. In some embodiments, the PEG units are separate PEGs. In some embodiments, the separate PEGs provide single molecules having a defined and specified chain length. In some embodiments, the PEG is mPEG.

[0363] As used herein, when referring to PEG units, the subunit refers to a polyethylene glycol subunit having the following formula.

Chem.

[0364] In some such embodiments, the PEG units include multiple PEG subunits.

[0365] In some embodiments, when z is 2 or 3, at least one Z5 is a polyalkylene glycol (PAO), for example, a PEG unit.

[0366] In some embodiments, when z is 2, at least one Z5 is a polyalkylene glycol (PAO), for example, a PEG unit.

[0367] In some embodiments, when z is 3, at least one Z is a polyalkylene glycol (PAO), for example, a PEG unit.

[0368] In some embodiments, the PEG unit contains 1 to 6 subunits. In some embodiments, the PEG unit contains 1 to 4 subunits. In some embodiments, the PEG unit contains 1 to 3 subunits. In some embodiments, the PEG unit contains 1 subunit. In some embodiments, the PEG unit contains 2 subunits. In some embodiments, the PEG unit contains 3 subunits. In some embodiments, the PEG unit contains 4 subunits. In some embodiments, the PEG unit contains 5 subunits. In some embodiments, the PEG unit contains 6 subunits.

[0369] In some embodiments, the PEG unit contains one or more PEG subunits linked together by a PEG linking unit. In some embodiments, the PEG linking unit connecting one or more chains repeating the CH2CH2O-subunit is Z6. In some embodiments, Z6 is -C1-10 alkyl-R3-, -C 2-10 alkyl-NH-, -C2-10 alkyl-C(O)-, -C2-10 alkyl-O-, or -C1-10 alkyl-S, wherein R3 is -C())-NR5-, or -NR5-C(O)-.

[0370] In some embodiments, the PEG linking unit is -C1-10 alkyl -C(O)-NH- or -C1-10 alkyl -NH-C(O)-. In some embodiments, the PEG linking unit is -C1-10 alkyl -C(0)-NH-. In some embodiments, the PEG linking unit is -C1-10 alkyl -NH-C(O)-.

[0371] In some embodiments, the PEG linking unit is -(CH2)2 -C(O)-NH-.

[0372] In some embodiments, each Z5 is absent.

[0373] In some embodiments, when z is 2 or 3, at least one Z5 is absent.

[0374] In some embodiments, when z is 2, at least one Z5 is absent. In some embodiments, when z is 3, at least one Z5 is absent.

[0375] In some embodiments, each Z5 is -(CH2-CH2-O-)2-.

[0376] In some embodiments, when z is 2 or 3, at least one Z5 is -(CH2-CH2-0-)2-. In some embodiments, when z is 2, at least one Z5 is ---(CH2-CH2-0-)2-. In some embodiments, when z is 3, at least one Z5 is --(CH2-CH2-O-)2-.

[0377] In some embodiments, each Z5 is independently R 57 -R 17 and in some embodiments, each Z5 is independently R 17 , NHR 17 , OR 17 , or SR 17 .

[0378] In some embodiments, when z is 2 or 3, at least one Z5 is R 57 -R 17 (e.g., R17, NHR17, OR 17 , or SR:-).

[0379] In some embodiments, when z is 2, at least one Z5 is R 57 -R 17 (e.g., R17, NHR17, OR17, or SR17). In some embodiments, when z is 3, at least one Z5 is R57-R17 (e.g., R17, NHR17, OR17, or SR17).

[0380] In some embodiments, each Z6 is absent.

[0381] In some embodiments, when z is 2 or 3, at least one Z6 is absent.

[0382] In some embodiments, when z is 2, at least one Z6 is absent. In some embodiments, when z is 3, at least one Z6 is absent.

[0383] In some embodiments, at least one of Z5 and Z6 is not absent.

[0384] In some embodiments, each Z6 is independently -C 1-10 alkyl-R3-, -C 1-10 alkyl-NH-, -C 1-10 alkyl-C(O)-, -C 1-10 alkyl-O-, -C 1-10 alkyl-S-, or -(C 1-10 alkyl-R3)g1-C 1-10 alkyl-C(O)-.

[0385] In some embodiments, g1 is an integer from 1 to 4.

[0386] In some embodiments, when z is 2 or 3, at least one Z6 is -C 1-10 alkyl-R3-, -C 1-10 alkyl-NH-, -C 1-10 alkyl-C(O)-, -C 1-10 alkyl-O-, -C 1-10 alkyl-S-, or -(C 1-10 alkyl-R3)g1-C 1-10 alkyl-C(O)-. In some embodiments, g1 is an integer from 1 to 4.

[0387] In some embodiments, each Z6 is independently - C 2-10 alkyl-C(O)-(e.g., -(-(CH2)2C(O)-)).

[0388] In some embodiments, at least one Z6 is -C 2-10 alkyl-C(O)-(e.g., --(-(CH2)2C(O)-)).

[0389] In some embodiments, each Z6 is independently -C 2-10 alkyl - R3-C 2-10 alkyl-C(O)-(e.g., -(CH2)2-C(O)NH-(CH2)2-C(O)-).

[0390] In some embodiments, at least one Z6 is -C 2-10 alkyl-R3-C 2-10 alkyl-C(O)-(e.g., -(CH2)2-C(O)NH-(CH2)2-C(O)-).

[0391] In some embodiments, each Z6 is independently -(C 2-10 alkyl-R3) g1 -C 2-10 alkyl-C(O)-(e.g., -(CH2)2-C(O)NH-(CH2)2-NHC(O)-(CH2)-C(O)-).

[0392] In some embodiments, at least one Z6 is -(C 2-10 alkyl-R3) g1 -C 2-10 alkyl-C( ’ O)-(e.g., -(CH2)2-C(O)NH-(CH2)2-NHC(O)-(CH2)-C(O)-) or -(CH2)2-NH-C(O)-(CH2)2-C(O)-NH-(CH2)-C(O)-).

[0393] In some embodiments, each Z6 is independently -(CH2)2-NH-C(O)-(CH2)2-C(O)-NH-(CH2)-C(O)-).

[0394] In some embodiments, -[(Z5)-(Z6)]z- does not necessarily exist.

[0395] In some embodiments, -[(Z5)-(Z6)]z- is a bond.

[0396] In some embodiments, -[(Z5)-(Z6)]z- is -(CH2CH2O)2-(CH2)2-C(O)-.

[0397] In some embodiments, -[(Z5)-(Z6)]z- is -(CH2CH2O)2-(CH2)2-C(O)-NH-(CH2CH2O)2-.

[0398] In some embodiments, -[(Z5)-(Z6)]z- is -(CH2CH2O)2-(CH2)2-C(O)-NH-(CH2)-C(O).

[0399] In some embodiments, M p when present,[[]]

Chemical formula

Chemical formula

[0400] In some embodiments, M p , when present, is (1)

Chemical formula

Chemical formula

[0401] In one embodiment, L M is a bond and a2 is 1.

[0402] In some embodiments, a2 is 2 and L M is

Chemical formula

Chemical formula

[0403] In some embodiments, a2 is 2, and L M is

Chemical formula

[0404] In some embodiments, a2 is 3, and L M is

Chemical formula

Chemical formula

Chemical formula

[0405] In some embodiments, a2 is 3, and L M is

Chemical formula

[0406] In one embodiment, the peptide moiety M A is a moiety of itself.

[0407] In other embodiments, M A comprises a peptide moiety containing at least about 5 amino acids.

[0408] In one embodiment, M A comprises a peptide moiety containing at most about 16 amino acids.

[0409] In some embodiments, M A comprises a peptide moiety containing about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 amino acids.

[0410] In some embodiments, M A comprises a peptide moiety containing at most about 10 amino acids.

[0411] In some embodiments, M A comprises a peptide moiety containing about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acids.

[0412] In some embodiments, M A comprises a peptide moiety comprising from about 3 to about 10 amino acids selected from glycine, serine, glutamic acid, aspartic acid, lysine, cysteine, their stereoisomers (e.g., isoglutamic acid or isoaspartic acid), and combinations thereof.

[0413] In some embodiments, M A comprises a peptide moiety comprising at least 4 glycines and at least 1 serine.

[0414] In some embodiments, M A comprises a peptide moiety comprising at least 4 glycines and at least 1 glutamic acid.

[0415] In some embodiments, M A comprises a peptide moiety comprising at least 4 glycines, at least 1 serine, and at least 1 glutamic acid.

[0416] In some embodiments, the peptide moiety

Chemical formula

[0417] In some embodiments, the peptide moiety comprises (glycine)-(serine), wherein the peptide moiety is via glycine, L 3Combine (if present), or L M (if L 3 is not present) and combine, The peptide moiety binds, via serine, to T 1 (if present), The peptide moiety binds, via serine, to L D (if present).

[0418] In some embodiments, the peptide moiety [Chemical formula] includes, wherein * represents a bond to L 3 (if present), or a bond to L M (if L 3 is not present), ** represents a bond to T 1 (if present), or a bond to -OH (if T 1 is not present), *** represents a bond to L D (if present), or a bond to -H (if L° is not present).

[0419] In some embodiments, the peptide moiety includes (glycine)4-(serine), and the peptide moiety binds, via one of the glycines, to L 3 (if present), or binds to L M (if L 3 is not present), The peptide moiety binds, via serine, to T 1 (if present), The peptide moiety binds, via serine, to L D (if present).

[0420] In some embodiments, the peptide moiety [Chemical formula] includes, wherein * represents a bond to L3 indicates the bond to (if present), or L M (L 3 if not present) indicates the bond to, ** is T 1 (if present) indicates the bond to, or -OH (T 1 if not present) indicates the bond to, *** is L D (if present) indicates the bond to, or -H (L D if not present) indicates the bond to.

[0421] In some embodiments, the peptide moiety comprises (serine)-(glycine)4, wherein the peptide moiety binds via serine to L 3 (if present), or L M (L 3 if not present) binds to, the peptide moiety binds via one of the glycines to T 1 (if present), and the peptide moiety binds via serine to L D (if present).

[0422] In some embodiments, the peptide moiety is

Chemical Formula

[0423] In some embodiments, the peptide moiety is [Chemical formula] comprising, wherein * represents a bond to L 3 (if present), or I. M (L 3 if not present), represents a bond to ** represents a bond to T 1 (if present), or a bond to -OH (T 1 if not present), represents a bond to *** represents a bond to L D (if present), or a bond to -H (L D if not present), represents a bond to.

[0424] In some embodiments, the peptide moiety is (-alanine)-(glycine) 1-4 -(serine), wherein the peptide moiety binds to L 3 (if present) via β-alanine, or binds to L M (L 3 if not present), the peptide moiety binds to T 1 (if present) via serine, the peptide moiety binds to L D (if present) via serine.

[0425] In some embodiments, the peptide moiety is [Chemical formula] comprising, wherein * represents a bond to L 3 (if present), or a bond to L M (L 3 if not present), represents a bond to ** represents a bond to T 1 (if present), or a bond to -OH (T 1 if not present), represents a bond to *** represents a bond to L DIndicates the bond to (if present), or -H (when L D is not present) indicates the bond to.

[0426] In some embodiments, the peptide moiety comprises (P-alanine)-(glycine)4-(serine), the peptide moiety binds to L 3 (if present) via β-alanine, or L M (when L 3 is not present) binds, the peptide moiety binds to T via serine 1 (if present), the peptide moiety binds to L via serine D (if present).

[0427] In some embodiments, the peptide moiety

Chemical Formula

[0428] In some embodiments, the peptide moiety comprises (glycine) 1-4 -(glutamic acid), the peptide moiety binds to L via one of the glycines 3 (if present), or L M (when L 3 is not present) binds, the peptide moiety binds to T via glutamic acid1 binds, if present, The peptide moiety binds, via glutamic acid, to L D if present.

[0429] In some embodiments, the peptide moiety is (glycine) 1-4 - containing glutamic acid, and the peptide moiety binds, via glutamic acid, to L 3 if present, or to L M (when L 3 is absent). The peptide moiety binds, via glycine, to T 1 if present. The peptide moiety binds, via glutamic acid, to L D if present.

[0430] In some embodiments, the peptide moiety

Chemical Formula

[0431] In some embodiments, the peptide moiety includes (glycine)-(glutamic acid), the peptide moiety binds, via glycine, to L if present, or to L M (when L 3 is absent), the peptide moiety binds, via glutamic acid, to T 1(if present) binds, and the peptide moiety binds via glutamic acid to L D (if present).

[0432] In some embodiments, the peptide moiety

Chemical formula

[0433] In some embodiments, the peptide moiety includes (glycine)4-(glutamic acid), and the peptide moiety binds via one of the glycines to L 3 (if present), or binds to L M (when L 3 is absent), and the peptide moiety binds via glutamic acid to T 1 (if present), and the peptide moiety binds via glutamic acid to L D (if present).

[0434] In some embodiments, the peptide moiety

Chemical formula

[0435] In some embodiments, the peptide moiety comprises (glutamic acid)-(glycine)4, the peptide moiety binds to L 3 (if present) via glutamic acid or binds to L M (when L 3 is not present), the peptide moiety binds to T 1 (if present) via one of the glycines, the peptide moiety binds to L D (if present) via glutamic acid.

[0436] In some embodiments, the peptide moiety

Chemical formula

[0437] In some embodiments, the peptide moiety

Chemical formula

[0438] In some embodiments, the peptide moiety comprises (p-alanine)-(glycine) 1-4 -(glutamic acid), the peptide moiety binds to L 3 (when present) via β-alanine, or binds to L M (where L 3 is absent), the peptide moiety binds to T 1 (when present) via glutamic acid, and the peptide moiety binds to L D (when present) via glutamic acid.

[0439] In some embodiments, the peptide moiety

Chemical Formula

[0440] In some embodiments, the peptide moiety comprises (β-alanine)-(glycine)4-(glutamic acid), and the peptide moiety binds, via β-alanine, to L 3 (when present), or binds to L M (when L 3 is not present). The peptide moiety binds, via glutamic acid, to T 1 (when present), and the peptide moiety binds, via glutamic acid, to L D (when present).

[0441] In some embodiments, the peptide moiety [Chemical formula] and includes, wherein * indicates a bond to L 3 (when present), or indicates a bond to L M (when L 3 is not present), ** indicates a bond to T 1 (when present), or indicates a bond to -OH (when T 1 is not present), *** indicates a bond to L D (when present), or indicates a bond to -H (when L D is not present).

[0442] Linker L III In some embodiments of L D each occurrence of L A is independently a divalent linker moiety connecting D to M

[0443] and includes at least one cleavable bond such that when the bond is cleaved, D is released in an active form for its intended therapeutic effect. D In some embodiments, L D is a component of a releasable assembly unit. In other embodiments, L

[0444] In some embodiments, L D comprises one cleavable linkage.

[0445] In some embodiments, L D comprises multiple cleavage sites or linkages.

[0446] In some embodiments, the functional groups for forming cleavable linkages may include, for example, sulfhydryl groups for forming disulfide linkages, aldehyde, ketone, or hydrazine groups for forming hydrazone linkages, hydroxylamine groups for forming oxime linkages, carboxylic acid or ammo groups for forming peptide linkages, carboxylic acid or hydroxy groups for forming ester linkages, and sugars for forming glycoside linkages. In some embodiments, L D comprises disulfide linkages cleavable by disulfide exchange, acid-labile linkages cleavable at acidic pH, or linkages cleavable by hydrolases (e.g., peptidases, esterases, and glucuronidases).

[0447] In some embodiments, L D comprises a carbamate linkage (i.e., -O-C(O)-NR-, where R is H or alkyl, etc.).

[0448] In some embodiments, the structure and sequence of the cleavable linkage in I D can be such that the linkage is cleaved by the action of an enzyme present at the target site. In other embodiments, the cleavable linkage can be cleaved by other mechanisms.

[0449] In some embodiments, the structure and sequence of the cleavable linkage in L D can be such that the linkage is cleaved by the action of an enzyme present at the target site. In other embodiments, the cleavable linkage can be cleaved by other mechanisms.

[0450] In some embodiments, the cleavable linkage(s) can be enzymatically cleaved by one or more enzymes, including tumor-related proteases, to release the linker-payload, and the conjugate, or an intermediate thereof, or the backbone of the present disclosure can be protonated in vivo upon release to provide the linker-payload.

[0451] In some embodiments, L D can comprise one or more amino acids. In some embodiments, for example, each amino acid in L D can be natural or unnatural, or D- or L-isomers, provided that a cleavable linkage is present. In some embodiments, L D comprises α, β, or γ amino acids, which can be natural or unnatural. In some embodiments, L D comprises from 1 to 12 (e.g., from 1 to 6, or from 1 to 4, or from 1 to 3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acids in a contiguous sequence.

[0452] In some embodiments, L D can comprise only natural amino acids. In some embodiments, L° can comprise only unnatural amino acids. In some embodiments, L D can comprise natural amino acids linked to unnatural amino acids. In some embodiments, L D can comprise natural amino acids linked to D-isomers of natural amino acids. In some embodiments, L D comprises dipeptides such as -Val-Cit-, -Phe-Lys-, or -Val-Ala-.

[0453] In some embodiments, L D comprises single peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units.

[0454] In some embodiments, L D comprises a peptide (e.g., 1 to 12 amino acids) conjugated directly to the payload. In some such embodiments, the peptide is a single amino acid or a dipeptide. In some such embodiments, the peptide is a single amino acid. In some such embodiments, the peptide is a dipeptide.

[0455] In some embodiments, L D each amino acid of is independently selected from alanine, β-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, amino alkanoic acid, amino alkynoic acid, amino aikandioic acid, amino benzoic acid, amino heterocycloalkanoic acid, heterocyclocarboxylic acid, citrulline, statine, diamino aikanoic acid, and derivatives thereof.

[0456] In some embodiments, each amino acid is independently selected from alanine, b-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, citrulline, and selenocysteine.

[0457] In some embodiments, each amino acid is independently selected from the group consisting of alanine, b-alanine, arginine, asparti...

Claims

1. A compound of formula II, 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein, In the formula, R １０５ is C ４－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C １－６ haloalkyl, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, C ３－８ cycloalkyl, or heterocyclyl, wherein the alkyl or alkenyl is substituted with 0, 1, 2, or 3 R １０７ s, the alkynyl is substituted with 0, 1, 2, or 3 R １０８ s, the phenyl is substituted with 0, 1, 2, or 3 R １０９ s, and the -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１０ s. R １０６ is H, or Alternatively, or alternatively, R １０５ and R １０６ are combined together to form C ３－８ cycloalkyl or heterocyclyl, wherein the cycloalkyl is substituted with 1, 2, or 3 R １１０ and the heterocyclyl is substituted with 0, 1, 2, or 3 R １１０ and Each R １０７ and R １０８ is independently C １－６ alkyl, C ２－６ alkoxyalkyl, C １－６ haloalkoxy, phenyl, heteroaryl, C ３－８ cycloalkyl, heterocyclyl, or halogen, wherein said phenyl, heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１１ groups, Each R １０９ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, -(C １－６ alkylene)-OR １１２ , -(C １－６ alkylene)-N(R １１２ ) ２ , C １－６ haloalkyl, C １－６ haloalkoxy, phenyl, -(C ２－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, -(C １－６ alkylene)-heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ wherein the alkynyl or haloalkoxy is substituted with 0, 1, 2, or 3 R １１３ and the phenyl, -alkylenephenyl, heteroaryl, -alkylenheteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ . Each R １１０ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, -(C １－６ alkylene)-OR １１２ , -(C １－６ alkylene)-N(R １１２ ) ２ , C １－６ haloalkyl, C １－６ haloalkoxy, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, -(C １－６ alkylene)-heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ and the alkynyl or haloalkoxy is substituted with 0, 1, 2, or 3 R １１３ , and the phenyl, -alkylenephenyl, heteroaryl, -alkyleneheteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ . Each R １１１ is independently C １－６ alkyl, C １－６ alkoxy, C １－６ haloalkyl, C １－６ haloalkoxy, or halogen, and Each R １１２ is independently H, C １－６ alkyl, C １－６ haloalkyl, or phenyl, and Each R １１３ is independently —S(O) ２ (C １－６ alkyl) or —N(R １１５ ) ２ and Each R １１４ is independently C １－６ alkyl, -(C １－６ alkyl)-OR １１６ , -(C １－６ alkyl)-N(R １１６ ) ２ , -OR １１６ , -N(R １１６ ) ２ , -N(R １１６ )(CO)R １１６ , -N(R １１６ )(CO)OR １１６ , -N(R １１６ )(O)S ２ R １１６ , -C(O)R １１６ , -S(O) ２ R １１６ , -S(O) ２ N(R １１６ ) ２ , or R ３００ ; Each R １１５ and R １１６ are each, independently, H, C １－６ alkyl, C １－６ haloalkyl, or R ３００ is R ２００ is -OR ２０１ or -N(R ２０１ ) ２ and R ２０１ is H, C １－６ alkyl, phenyl, or heteroaryl, wherein the phenyl or heteroaryl is substituted with 0, 1, or 2 —OR ２０２ —N(R ２０２ ) ２ or R ３００ and is substituted R ２０２ is H or C １－６ and is alkyl, R ３００ has one of the following structures, 【Chemical 2】 In the formula, R ３００ａ is H or C １－６ alkyl, R ３００ｂ is C １－６ alkyl or C １－６ alkoxy, and R ３００ｃ is H, C １－６ alkyl, -CH ２ OH, or C １－６ is alkoxy, R ３００ｄ is H or C １－６ alkyl, R ３００ｅ is H or C １－６ and is alkyl, In each case, said heteroaryl is a 5- to 10-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S, In each case, said heterocyclyl is a 4- to 10-membered heterocyclyl having 1, 2, or 3 heteroatoms selected from N, O, and S, a compound, or a pharmaceutically acceptable salt thereof.

2. R １０５ is C ４－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, or C １－６ haloalkyl, wherein the alkyl or alkenyl is substituted with 0, 1, 2, or 3 R １０７ and the alkynyl is substituted with 0, 1, 2, or 3 R １０８ The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

3. R １０５ is C ２－６ alkenyl, and the alkenyl is substituted with 0, 1, 2, or 3 R １０７ The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. Each R １０７ is independently phenyl, heteroaryl, C ３－８ cycloalkyl, heterocyclyl, or halogen, and the phenyl, heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, or 2 R １１１ The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

5. R １０５ is phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, C ３－８ cycloalkyl, or heterocyclyl, wherein said phenyl is substituted with 0, 1, 2, or 3 R １０９ and said -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１０ The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

6. R １０５ is heteroaryl or -(C １－６ alkylene)-heteroaryl, and the heteroaryl or -alkylene-heteroaryl is substituted with 0, 1, 2 or 3 R １１０ The compound according to claim 1 or 5, or a pharmaceutically acceptable salt thereof.

7. R １０５ is phenyl, imidazolyl, triazolyl, indolyl, indazolyl, or thienothienyl, and 0, 1, or 2 R １１０ substituted compounds according to claim 1, 5, or 6, or a pharmaceutically acceptable salt thereof.

8. Each R １１０ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ and the phenyl, alkylene-phenyl, heteroaryl, alkylene-heteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ s, a compound according to any one of claims 1 and 5 to 7, or a pharmaceutically acceptable salt thereof.

9. Each R １１０ is independently C １－３ alkyl or halogen, the compound according to any one of claims 1 and 5 to 8, or a pharmaceutically acceptable salt thereof.

10. R １０５ is 【Chemical Formula 3】 In the formula, Each X １ａ , X ２ａ , X ３ａ , and X ４ａ is, independently, CH or N, R １１０ is CH ３ , CH ２ F, CHF ２ or CF ３ and R １１４ is -NH(CO)CH ３ , -NHS(O) ２ CH ３ , or R ３００ and R １１６ is CH ３ , CH ２ F, CHF ２ , CF ３ or R ３００ and R １１８ is H or R ３００ The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein

11. R １０５ is 【Chemical Formula 4-1】 【Chemical Formula 4-2】 【Chemical Formula 4-3】 The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein

12. A compound of formula I, 【Chemical Formula 5】 or a pharmaceutically acceptable salt thereof, comprising, In the formula, R １０１ 、R １０２ 、R １０３ 、and R １０４ are each independently H or F, R １０５ is C ２－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C １－６ haloalkyl, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, C ３－８ cycloalkyl, or heterocyclyl, wherein said alkyl or alkenyl is substituted with 1, 2, or 3 R １０７ groups, said alkynyl is substituted with 0, 1, 2, or 3 R １０８ groups, said phenyl is substituted with 2 or 3 R １０９ groups, and said -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 1, 2, or 3 R １１０ groups, R １０６ is H, or Alternatively, instead, R １０５ and R １０６ are combined together to form C ３－８ cycloalkyl or heterocyclyl, said cycloalkyl or heterocyclyl being substituted with 0, 1, 2, or 3 R １１０ groups, Each R １０７ is independently C ２－６ alkoxyalkyl, C １－６ haloalkoxy, phenyl, heteroaryl, C ３－８ cycloalkyl, heterocyclyl, or halogen, wherein said phenyl is substituted with 1, 2, or 3 R １１１ and said heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１１ and Each R １０８ is independently C １－６ haloalkoxy, phenyl, heteroaryl, C ３－８ cycloalkyl, heterocyclyl, or halogen, wherein said phenyl, heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１２ groups, Each R １０９ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, C １－６ haloalkyl, halogen, -N ３ , -OR １１３ , or -N(R １１３ ), ２ and the alkyl, alkenyl, or alkynyl is substituted with 0 or 1 -S(O) ２ (C １－６ alkyl). Each R １１０ is independently C ２－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, -(C １－６ alkylene)-OR １１４ , -(C １－６ alkylene)-N(R １１４ ) ２ , C １－６ haloalkyl, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, halogen, -N ３ , -OR １１５ , -N(R １１５ ) ２ , -N(R １１５ )(CO)R １１５ , -N(R １１５ )(CO)OR １１５ , -N(R １１５ )(SO) ２ R １１５ , -(CO)R １１５ , -SO ２ R １１５ , or -SO ２ N(R １１５ ) ２ and the phenyl, alkylene-phenyl, heteroaryl, or alkylene-heteroaryl is substituted with 0, 1, 2, or 3 R １１６ s, Each R １１１ and R １１２ is independently C １－６ alkyl, C １－６ alkoxy, C １－６ haloalkyl, C １－６ haloalkoxy, halogen, -OR １１４ or -N(R １１４ ) ２ wherein Each R １１３ is independently H, C １－６ alkyl, C １－６ alkoxy, C １－６ haloalkyl, or phenyl, and the haloalkyl is substituted with 0 or 1 N(R １１４ ), ２ and Each R １１４ is independently H or C １－６ alkyl, Each R １１６ is -OR １１７ , -N(R １１７ ), ２ , -N(R １１７ )(CO)R １１７ , -N(R １１７ )(CO)OR １１７ , -N(R １１７ )(SO) ２ R １１７ , -(CO)R １１７ , -SO ２ R １１７ , -SO ２ N(R １１７ ), ２ or R ３００ and Each R １１５ and R １１７ are each independently H, C １－６ alkyl, C １－６ haloalkyl, phenyl, or R ３００ is R ２００ is -OR ２０１ or -N(R ２０１ ) ２ and R ２０１ is H, C １－６ alkyl, phenyl, or heteroaryl, wherein said phenyl or heteroaryl is substituted with 0, 1, or 2 of -OR ２０２ , -N(R ２０２ ), ２ or R ３００ and is substituted R ２０２ is H or C １－６ and is alkyl, R ３００ has one of the following structures, 【Chemical Formula 6】 In the formula, R ３００ａ is H or C １－６ is alkyl, R ３００ｂ is C １－６ alkyl or C １－６ alkoxy, and R ３００ｃ is H, C １－６ alkyl, -CH ２ OH, or C １－６ alkoxy, and R ３００ｄ is H or C １－６ is alkyl, R ３００ｅ is H or C １－６ is alkyl, In each case, said heteroaryl is a 5- to 10-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S, In each case, said heterocyclyl is a 4- to 10-membered heterocyclyl having 1, 2, or 3 heteroatoms selected from N, O, and S, a compound, or a pharmaceutically acceptable salt thereof.

13. R １０５ is C ２－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, or C １－６ haloalkyl, wherein the alkyl or alkenyl is substituted with 1, 2, or 3 R １０７ and the alkynyl is substituted with 0, 1, 2, or 3 R １０８ , the compound according to claim 12, or a pharmaceutically acceptable salt thereof.

14. R １０５ is C ２－６ alkenyl, and the alkenyl is substituted with 1, 2, or 3 R １０７ groups, the compound according to claim 12 or 13, or a pharmaceutically acceptable salt thereof.

15. R １０５ is phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, C ３－８ cycloalkyl, or heterocyclyl, wherein the phenyl is substituted with 2 or 3 R １０９ s, and the -(alkylene)-phenyl, heteroaryl, -(alkylene)-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 1, 2, or 3 R １１０ s, the compound according to claim 12, or a pharmaceutically acceptable salt thereof.

16. R １０５ is phenyl, and the phenyl is substituted with two or three Rs １０９ The compound according to claim 12 or 15, or a pharmaceutically acceptable salt thereof.

17. R １０５ is -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, C ３－８ cycloalkyl, or heterocyclyl, wherein said -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 1, 2, or 3 R １１０ groups, the compound according to claim 12 or 15, or a pharmaceutically acceptable salt thereof.

18. R １０５ is C ３－８ cycloalkyl or heterocyclyl, said cycloalkyl or heterocyclyl being substituted with 1, 2, or 3 R １１０ groups, the compound according to claim 12 or 15, or a pharmaceutically acceptable salt thereof.

19. R １０５ is heteroaryl or -(C １－６ alkylene)-heteroaryl, and the heteroaryl or -alkylene-heteroaryl is substituted with 1, 2 or 3 R １１０ The compound according to claim 12 or 15, or a pharmaceutically acceptable salt thereof.

20. R １０５ is 【Chemical Formula 7】 wherein, In the formula, Each X １ａ , X ２ａ , X ３ａ , and X ４ａ is, independently, CH or N, R １１０ is CH ３ , CH ２ F, CHF ２ or CF ３ and R １１６ is -NH(CO)CH ３ , -NH S(O) ２ CH ３ , or R ３００ is, R １１７ is CH ３ 、CH ２ F, CHF ２ 、CF ３ 、or R ３００ and is R １１８ is H or R ３００ The compound according to any one of claims 12 to 19, or a pharmaceutically acceptable salt thereof, wherein

21. R １０５ is 【Chemical Formula 8-1】 【Chemical Formula 8-2】 The compound according to any one of claims 12 to 20, or a pharmaceutically acceptable salt thereof, wherein

22. R １０３ and R １０４ each is H, the compound according to any one of claims 12 to 21.

23. R １０１ and R １０２ is each H, the compound according to any one of claims 12 to 22.

24. R １０１ and R １０２ each is F, the compound according to any one of claims 12 to 22.

25. R １０１ is F, and R １０２ is H, the compound according to any one of claims 12 to 22.

26. R １０１ is F and R １０２ is H, the compound according to any one of claims 12 to 22.

27. The compound, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure selected from Table 1.

28. (a) A compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, and (b) A binding protein comprising a binding domain capable of specifically binding to a target(s), wherein the target is selected from the group consisting of CD40, CD40 ligand, T lymphocyte activation antigen CD86 (CD86), cytotoxic T lymphocyte protein 4 (CTLA4), inducible T cell co-stimulator (ICOS), ICOS ligand (ICOSL), T cell-specific surface glycoprotein CD28 (CD28), T lymphocyte activation antigen CD80 (CD80), integrin β7, integrin α4, mucosal addressin cell adhesion molecule 1 (MADCAM), tumor necrosis factor α (TNFα), tumor necrosis factor receptor 2 (TNF-R2), killer cell lectin-like receptor G1 (KLRG1), B cell-activating factor (BAFF), BAFF receptor (BAFFR), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), gut-homing receptor (LPAM-1), B cell maturation antigen (BCMA), and proliferation-inducing ligand (APRIL), a binding protein, and (c) A linker that covalently attaches the compound to the binding protein, a conjugate. **Claim 29** The compound has a structure of formula II-1a or II-Ib, 【Chemical Formula 9】 or a pharmaceutically acceptable salt thereof, wherein, R １０５ is C ４－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C １－６ haloalkyl, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, C ３－８ cycloalkyl, or heterocyclyl, wherein said alkyl or alkenyl is substituted with 0, 1, 2, or 3 R １０７ groups, said alkynyl is substituted with 0, 1, 2, or 3 R １０８ groups, said phenyl is substituted with 0, 1, 2, or 3 R １０９ groups, and said -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１０ groups, R １０６ is H, or Alternatively, instead, R １０５ and R １０６ are combined together to form C ３－８ cycloalkyl or heterocyclyl, wherein the cycloalkyl is substituted with 1, 2, or 3 R １１０ groups, and the heterocyclyl is substituted with 0, 1, 2, or 3 R １１０ groups. Each R １０７ and R １０８ is independently C １－６ alkyl, C ２－６ alkoxyalkyl, C １－６ haloalkoxy, phenyl, heteroaryl, C ３－８ cycloalkyl, heterocyclyl, or halogen, wherein said phenyl, heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１１ groups, Each R １０９ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, -(C １－６ alkylene)-OR １１２ , -(C １－６ alkylene)-N(R １１２ ) ２ , C １－６ haloalkyl, C １－６ haloalkoxy, phenyl, -(C ２－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, -(C １－６ alkylene)-heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ and the alkynyl or haloalkoxy is substituted with 0, 1, 2, or 3 R １１３ and the phenyl, -alkylenephenyl, heteroaryl, -alkyleneheteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ . Each R １１０ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, -(C １－６ alkylene)-OR １１２ , -(C １－６ alkylene)-N(R １１２ ) ２ , C １－６ haloalkyl, C １－６ haloalkoxy, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, -(C １－６ alkylene)-heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ and the alkynyl or haloalkoxy is substituted with 0, 1, 2, or 3 R １１３ and the phenyl, -alkylenephenyl, heteroaryl, -alkylenheteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ . Each R １１１ is independently C １－６ alkyl, C １－６ alkoxy, C １－６ haloalkyl, C １－６ haloalkoxy, or halogen, and Each R １１２ is independently H, C １－６ alkyl, C １－６ haloalkyl, or phenyl, and Each R １１３ is independently —S(O) ２ (C １－６ alkyl) or —N(R １１５ ) ２ and Each R １１４ is independently C １－６ alkyl, -(C １－６ alkyl)-OR １１６ ,-(C １－６ alkyl)-N(R １１６ ) ２ ,-OR １１６ ,-N(R １１６ ) ２ ,-N(R １１６ )(CO)R １１６ ,-N(R １１６ )(CO)OR １１６ ,-N(R １１６ )(SO) ２ R １１６ ,-C(O)R １１６ ,-SO ２ R １１６ ,-SO ２ N(R １１６ ) ２ or R ３００ and Each R １１５ and R １１６ is, in each occurrence, independently H, C １－６ alkyl, C １－６ haloalkyl, or R ３００ is, X ２００ is -O- or -NH-, covalently bonded to the linker, R ３００ has one of the following structures, 【Chemical 10】 wherein, R ３００ａ is H or C １－６ and alkyl, R ３００ｂ is C １－６ alkyl or C １－６ alkoxy, and R ３００ｃ is H, C １－６ alkyl, -CH ２ OH, or C １－６ alkoxy, and R ３００ｄ is H or C １－６ is alkyl, R ３００ｅ is H or C １－６ alkyl, and in each case, the heteroaryl is a 5- to 10-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S, in each case, the heterocyclyl is a 4- to 10-membered heterocyclyl having 1, 2, or 3 heteroatoms selected from N, O, and S, the conjugate according to claim 28. **Claim 30** The compound has a structure of formula II-3a or II-3b, 【Chemical 11】 or a pharmaceutically acceptable salt thereof, wherein, R １０５ is C ４－６ alkylene, C ２－６ alkenylene, C ２－６ alkynylene, C １－６ haloalkylene, phenylene, -(C １－６ alkylene)-phenylene, heteroarylene, -(C １－６ alkylene)-heteroarylene, C ３－８ cycloalkylene, or heterocyclylene, wherein the alkylene or alkenylene is substituted with 0, 1, 2, or 3 R １０７ groups, the alkynylene is substituted with 0, 1, 2, or 3 R １０８ groups, the phenylene is substituted with 0, 1, 2, or 3 R １０９ groups, and the -alkylene-phenylene, heteroarylene, -alkylene-heteroarylene, cycloalkylene, or heterocyclylene is substituted with 0, 1, 2, or 3 R １１０ groups, R １０６ is H, or Alternatively, or alternatively, R １０５ and R １０６ are combined together to form C ３－８ cycloalkylene or heterocyclylene, wherein the cycloalkylene is substituted with 1, 2, or 3 R １１０ and the heterocyclylene is substituted with 0, 1, 2, or 3 R １１０ and R １０５ is covalently bonded to the linker, Each R １０７ and R １０８ is independently C １－６ alkyl, C ２－６ alkoxyalkyl, C １－６ haloalkoxy, phenyl, heteroaryl, C ３－８ cycloalkyl, heterocyclyl, or halogen, wherein said phenyl, heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１１ groups, Each R １０９ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, -(C １－６ alkylene)-OR １１２ , -(C １－６ alkylene)-N(R １１２ ) ２ , C １－６ haloalkyl, C １－６ haloalkoxy, phenyl, -(C ２－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, -(C １－６ alkylene)-heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ wherein the alkynyl or haloalkoxy is substituted with 0, 1, 2, or 3 R １１３ and the phenyl, -alkylene phenyl, heteroaryl, -alkylene heteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ . Each R １１０ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, -(C １－６ alkylene)-OR １１２ , -(C １－６ alkylene)-N(R １１２ ) ２ , C １－６ haloalkyl, C １－６ haloalkoxy, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, -(C １－６ alkylene)-heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ wherein the alkynyl or haloalkoxy is substituted with 0, 1, 2, or 3 R １１３ and the phenyl, -alkylenephenyl, heteroaryl, -alkylenheteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ . Each R １１１ is independently C １－６ alkyl, C １－６ alkoxy, C １－６ haloalkyl, C １－６ haloalkoxy, or halogen, and Each R １１２ is independently H, C １－６ alkyl, C １－６ haloalkyl, or phenyl, and Each R １１３ is independently -S(O) ２ (C １－６ alkyl) or -N(R １１５ ) ２ and Each R １１４ independently is C １－６ alkyl, -(C １－６ alkyl)-OR １１６ -(C １－６ alkyl)-N(R １１６ ) ２ -OR １１６ -N(R １１６ ) ２ -N(R １１６ )(CO)R １１６ -N(R １１６ )(CO)OR １１６ -N(R １１６ )(SO) ２ R １１６ -C(O)R １１６ -SO ２ R １１６ -SO ２ N(R １１６ ) ２ or R ３００ and Each R １１５ and R １１６ is, in each occurrence, independently H, C １－６ alkyl, C １－６ haloalkyl, or R ３００ is, R ２０３ is H or R ３００ and R ３００ has one of the following structures, 【Chemical Formula 12】 wherein, R ３００ａ is H or C １－６ and is alkyl, R ３００ｂ is C １－６ alkyl or C １－６ alkoxy, and R ３００ｃ is H, C １－６ alkyl, -CH ２ OH, or C １－６ alkoxy, and R ３００ｄ is H or C １－６ is alkyl, R ３００ｅ is H or C １－６ is alkyl, in each case, the heteroaryl is a 5- to 10-membered heteroaryl having 1, 2, or 3 heteroatoms selected from N, O, and S, in each case, the heterocyclyl is a 4- to 10-membered heterocyclyl having 1, 2, or 3 heteroatoms selected from N, O, and S, the conjugate according to claim 28. **Claim 31** R １０５ is phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, C ３－８ cycloalkyl, or heterocyclyl, wherein said phenyl is substituted with 0, 1, 2, or 3 R １０９ and said -alkylene-phenyl, heteroaryl, -alkylene-heteroaryl, cycloalkyl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１０ The conjugate according to claim 29, wherein **Claim 32** R １０５ is heteroaryl or -(C １－６ alkylene)-heteroaryl, and the heteroaryl or -alkylene-heteroaryl is substituted with 0, 1, 2, or 3 R １１０ as defined in claim 29, or a pharmaceutically acceptable salt thereof. **Claim 33** R １０５ is phenyl, imidazolyl, triazolyl, indolyl, indazolyl, or thienothienyl, and 0, 1, or 2 R １１０ substituted, the conjugate according to claim 29, or a pharmaceutically acceptable salt thereof. **Claim 34** Each R １１０ is independently C １－６ alkyl, C ２－６ alkenyl, C ２－６ alkynyl, C ２－６ alkoxyalkyl, phenyl, -(C １－６ alkylene)-phenyl, heteroaryl, -(C １－６ alkylene)-heteroaryl, heterocyclyl, halogen, -N ３ , -OR １１２ , -N(R １１２ ) ２ , -(CO)R １１２ , or -S(O) ２ R １１２ wherein the phenyl, alkylene-phenyl, heteroaryl, alkylene-heteroaryl, or heterocyclyl is substituted with 0, 1, 2, or 3 R １１４ s, the conjugate according to any one of claims 31 to 33, or a pharmaceutically acceptable salt thereof. **Claim 35** Each R １１０ is independently C １－３ alkyl or halogen, the conjugate according to any one of claims 31 to 34, or a pharmaceutically acceptable salt thereof. **Claim 36** R １０５ is 【Chemical Formula 13】 and wherein, Each X １ａ , X ２ａ , X ３ａ , and X ４ａ is independently CH or N, R １１０ is CH ３ CH ２ F, CHF ２ or CF ３ and R １１４ is -NH(CO)CH ３ , -NHS(O) ２ CH ３ , or R ３００ and R １１６ is CH ３ , CH ２ F, CHF ２ , CF ３ or R ３００ and R １１８ wherein R is H or R ３００ The conjugate according to claim 29, or a pharmaceutically acceptable salt thereof. **Claim 37** R １０５ is 【Chemical Formula 14-1】 【Chemical Formula 14-2】 【Chemical Formula 14-3】 is, the conjugate according to claim 29, or a pharmaceutically acceptable salt thereof. **Claim 38** R ２０３ The conjugate according to claim 30, wherein R is H. **Claim 39** R ２０３ wherein R ３００ is as defined in claim 30, the conjugate according to claim 30. **Claim 40** The conjugate according to any one of claims 29 to 39, wherein the target is CD40.

41. The conjugate according to claim 40, wherein the binding protein is an anti-CD40 antibody.

42. The conjugate according to any one of claims 29 to 41, wherein the linker contains a succinimide group.

43. The conjugate according to any one of claims 29 to 42, wherein the linker contains a hydrophilic element.

44. The conjugate according to claim 43, wherein the hydrophilic element contains polyethylene glycol, polysarcosine, cyclodextrin, c-glycoside, or a combination thereof.

45. A pharmaceutical composition comprising the compound according to any one of claims 1 to 27, or a pharmaceutically acceptable salt thereof, or the conjugate according to any one of claims 28 to 44, and a pharmaceutically acceptable excipient.

46. The pharmaceutical composition according to claim 45, for use in a method of treating or preventing an autoimmune or inflammatory condition in a subject.

47. A method of treating or preventing an autoimmune or inflammatory condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the conjugate according to any one of claims 28 to 44 or the pharmaceutical composition according to claim 45.

48. Use of an effective amount of the conjugate according to any one of claims 28 to 44 or the pharmaceutical composition according to claim 45 in the manufacture of a medicament in a method of treating or preventing an autoimmune or inflammatory condition in a subject.

49. An effective amount of the conjugate according to any one of claims 28 to 44, or the pharmaceutical composition according to claim 45, for use in treating or preventing an autoimmune or inflammatory condition in a subject.

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