Linkers for conjugates and methods of use thereof

Abstract

Provided herein are compounds useful as linkers for conjugates, conjugates comprising these compounds, pharmaceutical compositions thereof, and method of using the compounds, conjugates and pharmaceutical compositions.

Description

Attorney Docket No.: 090322-8001WO01 LINKERS FOR CONJUGATES AND METHODS OF USE THEREOF TECHNICAL FIELD

[0001] The present disclosure generally relates to compounds useful as linkers for conjugates, conjugates comprising these compounds, pharmaceutical compositions thereof, and method of using the compounds, conjugates and pharmaceutical compositions. BACKGROUND OF THE DISCLOSURE

[0002] Antibody-drug conjugates (ADCs) are a potent class of therapeutic constructs advancing the field of cancer therapeutics by allowing targeted delivery of cytotoxic agents to target cells, such as cancer cells. However, future development is increasingly constrained by the intrinsic hydrophobicity of the drug-linkers, increased plasma clearance of ADCs, off-target toxicity due to the nonspecific release of drugs in non-tumorous tissues, reduced efficacy of ADCs due to retro-Michael elimination reaction of the commonly-used maleimide attachment, which ultimately leads to a reduced therapeutic window.

[0003] Therefore, there is a need in the art to design improved linkers for conjugation to therapeutic agents to provide ADCs with improved stability, favorable pharmacokinetics, desirable in vivo activity, and improved safety profile. SUMMARY OF THE DISCLOSURE

[0004] The present disclosure relates to a compound that is useful as linker for conjugation of therapeutic or diagnostic agents and targeting moeity and provides improved stability and safety profile of the conjugates.

[0005] In one aspect, the present disclosure provides a compound of Formula (I):1Attorney Docket No.: 090322-8001WO01 wherein: X is a linking moiety capable of forming a covalent bond with a targeting moiety; W is a polypeptide moiety; L1is an amino acid side chain residue; L2is a spacer moiety; Y is a hydrophilic end group; E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC); T is a therapeutic or a diagnostic agent moiety; n is an integer from 1 to 20; m is an integer from 0 to 5; p is an integer from 1 to 24; a is 0 or 1; and b is 0 or 1.

[0006] In another aspect, the present disclosure provides a compound of Formula (IA), (IB) or (IC):2Attorney Docket No.: 090322-8001WO01 wherein X, W, L1, L2, Y, E, T, n, m, and p are as defined herein.

[0007] In a further aspect, the present disclosure provides a compound of Formula (II):wherein A is a targeting moiety, X’ is a linking moiety connecting to A, r is from 1 to 10, and W, L1, L2, Y, E, T, n, m, p, a, and b are as defined herein.

[0008] In another aspect, the present disclosure provides a compound of Formula (IIA), (IIB), or (IIC):( ), wherein A, X’, W, L1, L2, Y, E, T, n, m, r and p are as defined herein. 3Attorney Docket No.: 090322-8001WO01

[0009] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a compound described herein and a pharmaceutically acceptable carrier.

[0010] In another aspect, the present disclosure provides a method of treating diseases in a subject in need thereof, comprising administering to the subject a therapeutic effective amount of the compound or the pharmaceutical composition provided herein.

[0011] In a further aspect, the present disclosure provides a method of screening an ADC having reduced non-specific internalization into healthy cells, comprising introducing one or more negatively charged group into the linker of the ADC. DESCRIPTION OF DRAWINGS

[0012] Figure 1 shows the SEC and HIC analysis of ADC-1.

[0013] Figure 2 shows the SEC and HIC analysis of ADC-2.

[0014] Figure 3 shows the SEC and HIC analysis of ADC-3.

[0015] Figure 4 shows the SEC and HIC analysis of ADC-4.

[0016] Figure 5 shows the SEC and HIC analysis of ADC-5.

[0017] Figure 6 shows the SEC and HIC analysis of ADC-6.

[0018] Figure 7 shows the SEC and HIC analysis of ADC-7.

[0019] Figures 8A, 8B and 8C show the linker payload stability of ADC-6. Figure 8A: ADC-6 was incubated in the 100x NAC at various time points. Figure 8B: ADC- 6 was incubated in the presence of a final 25 mg / mL HSA at various time points. Figure 8C: ADC-6 was incubated in the presence of 1xPBS, pH 7.4 at various time points.

[0020] Figures 9A, 9B and 9C show the linker payload stability of ADC-7. Figure 9A: ADC-7 was incubated in the 100x NAC at various time points. Figure 9B: ADC- 7 was incubated in the presence of a final 25 mg / mL HSA at various time points. Figure 9C: ADC-7 was incubated in the presence of 1xPBS, pH 7.4 at various time points. 4Attorney Docket No.: 090322-8001WO01

[0021] Figures 10A and 10B show the stability of ADC-5 and Trastuzumab-VC- MMAE measured under stressed condition.

[0022] Figure 11 shows the in vitro cytotoxicity of ADC-1, ADC-2, ADC-3, T-Dxd and Trastuzumab on SKBR3 cells.

[0023] Figure 12 shows the in vitro cytotoxicity of ADC-6, ADC-7 and PSMA antibody on LnCAP cells cells.

[0024] Figure 13 shows the cell-based binding of ADC-1, ADC-2, ADC-3, isotype antibody and Trastuzumab on SKBR3 cells.

[0025] Figure 14 shows the internalization of ADC-1, ADC-2, ADC-3, isotype antibody and Trastuzumab into SKBR3 cells.

[0026] Figure 15 shows the non-specific internalization of ADC-1, T-Dxd, Trastuzumab into human primary corneal epithelial cells.

[0027] Figure 16 shows the in vitro cytotoxicity of ADC-1 and T-Dxd under stressed condition.

[0028] Figure 17 shows the in vitro cytotoxicity of ADC-2 and T-Dxd under stressed condition.

[0029] Figure 18 shows the in vitro cytotoxicity of ADC-3 and T-Dxd under stressed condition.

[0030] Figure 19 shows the HIC-HPLC and SEC profiles of ADC-1, ADC-2, and ADC-3 under stressed condition.

[0031] Figure 20 shows the stability of ADC-1, ADC-2, and ADC-3 in mouse plasma.

[0032] Figure 21 shows the stability of ADC-1, ADC-2, and ADC-3 in human plasma.

[0033] Figure 22 shows the normal PK profile of ADC-1 in rats.

[0034] Figure 23A shows the anti-tumor efficacy of ADC-1 to ADC-5 at 1 mg / kg in NCI-N87 tumor model. 5Attorney Docket No.: 090322-8001WO01

[0035] Figure 23B shows the anti-tumor efficacy of ADC-8 at 2 mg / kg in PA-1 tumor model.

[0036] Figure 24A shows the anti-tumor efficacy of vehicle control as well as ADC- 1 and T-Dxd at a dose of 0.5 mg / kg, 1 mg / kg or 3 mg / kg in in NCI-N87 tumor model.

[0037] Figure 24B shows the body weight change induced by vehicle control as well as ADC-1 and T-Dxd at a dose of 0.5 mg / kg, 1 mg / kg or 3 mg / kg in in NCI-N87 tumor model. DETAILED DESCRIPTION

[0038] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0039] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination.

[0040] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural forms of the same unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to several terms that shall be defined to have the following meanings unless a contrary intention is apparent. DEFINITIONS 6Attorney Docket No.: 090322-8001WO01

[0041] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0042] At various places in the present disclosure, linking substituents are described. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl”, then it is understood that the “alkyl” represents a linking alkylene group.

[0043] When any variable (e.g., Ri) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 Rimoieties, then the group may optionally be substituted with up to two Rimoieties and Riat each occurrence is selected independently from the definition of Ri. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0044] As used herein, a dashat the front or end of a chemical group is used, a matter of convenience, to indicate a point of attachment for a substituent. For example, -OH is attached through the carbon atom; chemical groups may be depicted with or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates a point of attachment of a group. Unless chemically or structurally required, no directionality is indicated or implied by the order in which a chemical group is written or named. As used herein, a solid line coming out of the center of a ring indicates that the point of attachment for a substituent on the ring can be at any ring atom. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds. 7Attorney Docket No.: 090322-8001WO01

[0045] When any variable (e.g., Ri) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 Rimoieties, then the group may optionally be substituted with up to two Rimoieties and Riat each occurrence is selected independently from the definition of Ri. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0046] The term “about”, when used in connection with a numerical value, means that a collection or range of values is included. For example, “about X” includes a range of values that are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numerical value. In one embodiment, the term “about” refers to a range of values which are 5% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 2% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 1% more or less than the specified value.

[0047] Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. A range used herein, unless otherwise specified, includes the two limits of the range. For example, the expressions “n is an integer between 1 and 6” and “n being an integer of 1 to 6” both mean “n being 1, 2, 3, 4, 5, or 6”.

[0048] As used herein, the term “Ci-j” indicates a range of the carbon atoms numbers, wherein i and j are integers and the range of the carbon atoms numbers includes the endpoints (i.e., i and j) and each integer point in between, and wherein j is greater than i. For examples, C1-6 indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms and six carbon atoms. In some embodiments, the term “C1-12” indicates 1 to 12, particularly 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3 or particularly 1 to 2 carbon atoms. In similar manner, the term “m-n membered” ring, wherein m and n are integers and n is greater than m, refers to a ring containing m to n atoms.

[0049] As used herein, the term “alkyl”, whether as part of another term or used independently, refers to a saturated linear or branched-chain hydrocarbon radical, which may be optionally substituted independently with one or more substituents described below. The term “Ci-jalkyl” refers to a linear or branched-chain alkyl 8Attorney Docket No.: 090322-8001WO01 having i to j carbon atoms. For example, alkyl groups contain 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of “C1-6alkyl” include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, s- butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3- methyl-1-butyl, 2-methyl-1-butyl, 2-ethyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2- methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl- 3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0050] As used herein, the term “alkenyl”, whether as part of another term or used independently, refers to linear or branched-chain hydrocarbon radical having at least one carbon-carbon double bond, which may be optionally substituted independently with one or more substituents described herein, and includes radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations. In some embodiments, alkenyl groups contain 2 to 12 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkenyl groups contain 2 carbon atoms. Examples of alkenyl group include, but are not limited to, ethylenyl (or vinyl), propenyl (allyl), butenyl, pentenyl, 1-methyl-2 buten-1-yl, 5-hexenyl, and the like.

[0051] As used herein, the term “alkynyl”, whether as part of another term or used independently, refers to a linear or branched hydrocarbon radical having at least one carbon-carbon triple bond, which may be optionally substituted independently with one or more substituents described herein. In some embodiments, alkenyl groups contain 2 to 12 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkynyl groups contain 2 carbon atoms. Examples of alkynyl group include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

[0052] As used herein, the term “alkoxyl” or “alkoxy”, whether as part of another term or used independently, refers to a radical of the formula -ORawhere Rais an alkyl radical as defined herein. Whenever it appears herein, a numerical range such as “C1-C6alkoxy” or “C1-6alkoxy”, means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkoxy” where no numerical range is designated. In some embodiments, the alkoxy is a C1-109Attorney Docket No.: 090322-8001WO01 alkoxy. In some embodiments, the alkoxy is a C1-6alkoxy. In some embodiments, the alkoxy is a C1-5alkoxy. In some embodiments, the alkoxy is a C1-4alkoxy. In some embodiments, the alkyl is a C1-3alkoxy. In some embodiments, the alkyl is a C1-2alkoxy. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, heteroaryl, and the like.

[0053] As used herein, the term “amino” refers to the group -NRaRb, wherein Raand Rbare independently selected from groups consisting of hydrogen, alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl and each of which may be optionally substituted.

[0054] As used herein, the term “aryl”, whether as part of another term or used independently, refers to monocyclic and polycyclic ring systems having a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 12 ring members. Examples of “aryl” include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more additional rings. In the case of polycyclic ring system, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline). The second ring can also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0055] As used herein, the term “cycloalkyl”, whether as part of another term or used independently, refers to a monovalent non-aromatic, saturated or partially unsaturated monocyclic and polycyclic ring system, in which all the ring atoms are carbon, and which contains at least three ring forming carbon atoms. In some embodiments, the cycloalkyl group may contain 3 to 12 ring forming carbon atoms, 3 to 10 ring forming carbon atoms, 3 to 9 ring forming carbon atoms, 3 to 8 ring forming carbon atoms, 3 to 7 ring forming carbon atoms, 3 to 6 ring forming carbon atoms, 3 to 5 ring forming carbon atoms, 4 to 12 ring forming carbon atoms, 4 to 10 ring forming carbon atoms, 4 to 9 ring forming carbon atoms, 4 to 8 ring forming carbon atoms, 4 to 7 ring forming carbon atoms, 4 to 6 ring forming carbon atoms, 4 to 5 ring forming carbon atoms. The cycloalkyl group may be saturated or partially unsaturated. In some embodiments, the cycloalkyl group may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be a partially 10Attorney Docket No.: 090322-8001WO01 unsaturated cyclic alkyl group that contains at least one double bond or triple bond in its ring system.

[0056] In some embodiments, the cycloalkyl group may be saturated or partially unsaturated monocyclic carbocyclic ring system, examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent- 2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1- cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.

[0057] In some embodiments, the cycloalkyl group may be saturated or partially unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic ring system, which can be arranged as a fused, spiro or bridged ring system. As used herein, the term “fused ring” refers to a ring system having two rings sharing two adjacent atoms, the term “spiro ring” refers to a ring systems having two rings connected through one single common atom, and the term “bridged ring” refers to a ring system with two rings sharing three or more atoms. Examples of fused cycloalkyl include, but are not limited to, naphthyl, benzopyrenyl, anthracenyl, acenaphthenyl, fluorenyl and the like. Examples of spiro cycloalkyl include, but are not limited to, spiro[5.5]undecanyl, spiro-pentadienyl, spiro[3.6]-decanyl, and the like. Examples of bridged cycloalkyl include, but are not limited to bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, bicyclo[3.3.1]nonanyl, bicyclo[3.3.3]undecanyl, and the like.

[0058] As used herein, the term “halo” or “halogen” refers to an atom selected from fluorine (or fluoro), chlorine (or chloro), bromine (or bromo) and iodine (or iodo).

[0059] As used herein, the term “haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

[0060] As used herein, the term “heteroatom” refers to nitrogen, oxygen, sulfur or phosphorus, and includes any oxidized form of nitrogen, sulfur or phosphorus, and any quaternized form of a basic nitrogen.

[0061] “Heterocyclyl”, whether as part of another term or used independently, refers to a 3- to 24-membered partially or fully saturated ring radical comprising 2 to 23 carbon atoms and from 1 to 8 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, silicon, and sulfur. In some embodiments, the 11Attorney Docket No.: 090322-8001WO01 heterocyclyl is fully saturated. In some embodiments, the heterocyclyl is partially unsaturated. In some embodiments, the heterocyclyl comprises one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. In some embodiments, the heterocyclyl comprises one to three heteroatoms selected from nitrogen or oxygen. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic or polycyclic (including but not limited to, bicyclic, tricyclic, or tetracyclic) ring system. The polycyclic ring system may include fused (for example, a heterocyclyl ring fused with a cycloalkyl or another heterocyclyl ring), spiro, or bridged ring systems. The nitrogen, carbon, or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quarternized. Representative heterocyclyls include, but are not limited to, heterocyclyls having from two to fifteen carbon atoms (C2-C15heterocyclyl), from two to ten carbon atoms (C2-C10heterocyclyl), from two to eight carbon atoms (C2-C8heterocyclyl), from two to seven carbon atoms (C2-C7 heterocyclyl), from two to six carbon atoms (C2-C6heterocyclyl), from two to five carbon atoms (C2-C5heterocyclyl), or two to four carbon atoms (C2-C4heterocyclyl). Examples of such heterocyclyl radicals include, but are not limited to, aziridinyl, azetidinyl, oxetanyl, dioxolanyl, dihydrofuryl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, and the like. The term heterocyclyl also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides, and the oligosaccharides. In some embodiments, heterocyclyls have from 2 to 10 carbons in the ring. It is understood that when referring to the number of carbon atoms in a heterocyclyl, the number of carbon atoms in the heterocyclyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocyclyl (i.e. skeletal atoms of the heterocyclyl ring). In some embodiments, the heterocyclyl is a 3- to 8-membered fully saturated heterocyclyl. In some embodiments, the heterocyclyl is a 3- to 7-membered fully saturated heterocyclyl. In some embodiments, the heterocyclyl is a 3- to 6-membered fully saturated heterocyclyl. In some embodiments, the heterocyclyl is a 4- to 6-membered fully saturated heterocyclyl. In some embodiments, the heterocyclyl is a 5- to 6- membered fully saturated heterocyclyl. Unless stated otherwise specifically in the specification, a heterocyclyl may be optionally substituted as described below, for example, with one or more substituents, such as oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, carboxyl, carboxylate, aryl, cycloalkyl, heterocyclyl, heteroaryl, and the like.

[0062] As used herein, the term “hydroxyl” refers to -OH. 12Attorney Docket No.: 090322-8001WO01

[0063] As used herein, the term “-alkyl-cycloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more cycloalkyl radicals, as defined above, and is linked to a parent molecule via the alkyl radical.

[0064] As used herein, the term “-alkyl-aryl” refers to an alkyl radical, as defined above, that is substituted by one or more aryl radicals, as defined above, and is linked to a parent molecule via the alkyl radical.

[0065] As used herein, the term “-alkyl-heterocyclyl” refers to an alkyl radical, as defined above, that is substituted by one or more heterocyclyl radicals, as defined above, and is linked to a parent molecule via the alkyl radical.

[0066] As used herein, the term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the said event or circumstance occurs and instances in which it does not.

[0067] As used herein, the term “partially unsaturated” refers to a radical that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

[0068] As used herein, the term “substituted”, whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and that the substitution results in a stable or chemically feasible compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents may include, but not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halo, haloalkyl, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, thiol, thione, or combinations thereof. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted”, references to chemical moieties herein are understood to include 13Attorney Docket No.: 090322-8001WO01 substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

[0069] As used herein, “amino acid side chain” refers to the additional chemical moiety on the same carbon that bears a primary or secondary amine and a carboxylic acid of an amino acid. As would be appreciated by a person of skill in the art, there are twenty-one “standard” amino acids. Exemplary “standard” amino acids include, without limitation, aspartic acid, glutamic acid, alanine, lysine, serine, tryptophan, tyrosine, proline, arginine, and the like. Other amino acids include, cysteine, selenocysteine, and glycine (e.g., wherein the additional chemical moiety on the same carbon that bears the primary amine and carboxylic acid of glycine is hydrogen). Exemplary amino acid side chains include, without limitation, methyl, sec-butyl, iso- butyl, -CH2CH2SCH3, -CH2-phenyl, iso-propyl, hydroxymethyl, -CH(OH)CH3, -

[0070] As used herein, the term “drug load” refers to the average number of cytotoxic drugs loaded on each targeting moiety in the compound and can also be expressed as the ratio of drug dose to targeting moiety dose. When the targeting moiety in a compound is an antibody, the drug load of can range from 0 to 12 for each antibody (Ab), preferably 1 to 10 cytotoxic drugs (D). In some embodiments, the drug load can be the mean of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA assay, and HPLC characterization were used to identify the average amount of drug for each ADC molecule after coupling reaction.

[0071] As used herein, the term “linker” or “linking moiety” refers to a divalent, trivalent, or multivalent moiety that covalently links, or is capable of covalently linking (e.g., via a reactive group), the targeting moiety to one or more compounds described herein, for instance, therapeutic or diagnostic agent moiety.

[0072] As used herein, the term “residue” refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the term “amino acid residue” refers to the product of an amide coupling or peptide coupling of an amino acid to a suitable coupling partner, wherein, for example, a water molecule is 14Attorney Docket No.: 090322-8001WO01 expelled after the amide or peptide coupling of the amino acid, resulting in the product having the amino acid residue incorporated therein.

[0073] As used herein, the term “targeting moiety” refers to a molecule that recognizes and binds to a cell surface marker or receptor such as, a transmembrane protein, surface immobilized protein, or protoglycan. Examples of targeting moiety includes, but are not limited to antibody, antibody binding fragment, bispecific antibody, immunoglobins or another antibody-based molecule or compound. However, other examples of targeting moieties are known in the art and may be used, such as aptamers, avimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, peptides, small molecules, nanoparticles, or proteins, etc. The targeting moiety, in addition to targeting the compound to a specific cell, tissue or location, may also have certain therapeutic effect such as antiproliferative (cytostatic and / or cytotoxic) activity against a target cell or pathway. The targeting moiety comprises or may be engineered to comprise at least one chemically reactive group such as, -COOR, -SH, amine or a chemically reactive amino acid moiety or side chains such as, for example, tyrosine, histidine, cysteine, or lysine. In some embodiments, a targeting moiety may be a ligand which specifically binds or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population. Following specific binding or complexing of the ligand with its receptor, the cell is permissive for uptake of the ligand or ligand-drug- conjugate, which is then internalized into the cell. The terms “targeting moiety” and “binding moiety” are used synonymously herein.

[0074] As used herein, the term “therapeutic agent” or “drug” refers to a compound, prodrug or payload which provides a physiological response following administration to a biological entity. Exemplary biological responses include, without limitation, increase or decrease in DNA or protein synthesis, upregulation or down-regulation of signalling pathways, and increase or decrease in cell proliferation, and the like. In some embodiments, the therapeutic agent is small molecule drug.

[0075] As used herein, the term “diagnostic agent” refers to a molecule or substance capable of detection, including, but not limited to, fluorescers, chemiluminescers, chromophores, bioluminescent proteins, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, isotopic labels, semiconductor nanoparticles, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like.

[0076] The term “pharmaceutically acceptable salt” refers to a salt of a compound described in the present invention, which is safe and effective when used in a biological entity and has the expected biological activity. The pharmaceutically 15Attorney Docket No.: 090322-8001WO01 acceptable salt includes a salt with base or acid. Non-restrictive examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, magnesium salts, hydrochloride, hydrobromide, hydroiodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate and the like.

[0077] As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, multispecific antibody, or bispecific (bivalent) antibody or a functional portion thereof that binds to a specific antigen. A native intact antibody comprises two heavy chains (H) and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a variable region (VH) and a first, second, and third constant region (CH1, CH2 and CH3, respectively), while each light chain consists of a variable region (VL) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The variable regions of the light and heavy chains are responsible for antigen binding. The variables region in both chains are generally subdivided into three regions of hypervariability called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J.Mol.Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21- 28;342(6252):877-83 (1989); Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. Therefore, each VH and VL comprises of three CDRs and four FRs in the following order (amino acid residues N terminus to C terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to the five major classes based on the amino acid sequence of the constant region of their heavy chain: IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Subclasses of several of the major antibody classes are such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). 16Attorney Docket No.: 090322-8001WO01

[0078] The term “antibody”, as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable, standard technique(s) such as proteolytic digestion or recombinant genetic engineering technique(s) involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein. As used herein, the term “Fab” refers to a monovalent antigen-binding fragment of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond. Fab can be obtained by papain digestion of an antibody at the residues proximal to the N-terminus of the disulfide bond between the heavy chains of the hinge region. As used herein, the term “F(ab’)2” refers to a dimer of Fab’ that comprises two light chains and part of two heavy chains. As used herein, the term “Fc” with regard to an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bond. The Fc portion of the antibody is responsible for various effector functions, but does not function in antigen binding. As used herein, the term “Fv” refers to the smallest fragment of the antibody to bear the complete antigen binding site. A Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. As used herein, the term “single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence. A 17Attorney Docket No.: 090322-8001WO01 “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0079] The antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0080] In some embodiment, the antibody or its antigen binding fragment is chimeric or humanized. As used herein, the term “chimeric” refers to an antibody or antigen-binding fragment that has a portion of heavy and / or light chain derived from one species, and the rest of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region derived from a non-human species, such as from mouse. As used herein, the term “humanized”, with reference to antibody or antigen-binding fragment, refers to the antibody or the antigen-binding fragment comprises CDRs derived from non-human animals (e.g., a rodent, rabbit, dog, goat, horse, or chicken), FR regions derived from human, and when applicable, the constant regions derived from human. In some embodiments, the constant regions from a human antibody are fused to the non-human variable regions. A humanized antibody or antigen-binding fragment is useful as human therapeutics. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, a hamster, or a non-human primate (for example, a monkey (e.g., cynomolgus or rhesus monkey) or an ape (e.g., chimpanzee, gorilla, simian or affen)). In some embodiments, the humanized antibody or antigen-binding fragment is composed of substantially all human sequences except for the CDR sequences which are non-human. In some embodiments, the humanized antibody or antigen-binding fragment is modified to improve the antibody performance, such as binding or binding affinity. For example, one or more amino acid residues in one or more non-human CDRs are altered to reduce potential immunogenicity in human, wherein the altered amino acid residues either are not critical for immunospecific binding or the alterations are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly affected. In some embodiments, the FR regions derived from human may comprise the same amino acid sequence as the human antibody from which it is derived, or it may 18Attorney Docket No.: 090322-8001WO01 comprise some amino acid changes, for example, no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 changes of amino acid. In some embodiments, such change in amino acid could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.

[0081] As used herein, the term “natural amino acid” refers to any one of the common, naturally occurring L-amino acids found in naturally occurring proteins: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamicacid (Glu), asparagine (Asn), glutamine (Gln), cysteine (Cys) and methionine (Met). The amino acid three-letter code and the single-letter code used in the present disclosure are such as J. Boil. Chem. 1968, 243, 3558.

[0082] As used herein, the term “non-natural amino acid” as used herein refers to any amino acid which is not a natural amino acid. This includes, for example, amino acids that comprise α-, β-, ω-, D-, L-amino acyl residues. More generally, the non- natural amino acid comprises a residue of the general formula, wherein the side chain R is other than the amino acid side chains occurring in nature. Exemplary unnatural amino acids, include, but are not limited to, sarcosine (N- methylglycine), citrulline (cit), homocitrulline, β-ureidoalanine, thiocitrulline, hydroxyproline, allothreonine, pipecolic acid (homoproline), α-aminoisobutyric acid, tert-butylglycine, tert-butylalanine, allo-isoleucine, norleucine, α-methylleucine, cyclohexylglycine, β-cyclohexylalanine, β-cyclopentylalanine, α-methylproline, phenylglycine, α-methylphenylalanine and homophenylalanine.

[0083] As used herein, the term “polypeptide”, “protein” or “peptide” can be a single amino acid or a polymer of amino acids. The polypeptide, protein or peptide as described in the present disclosure may contain naturally occurring amino acids and non-naturally occurring amino acids, or analogs and mimetics thereof. The polypeptide, protein or peptide can be obtained by any method well known in the art, for example, but not limited to, by an isolation and a purification from natural materials, a recombinant expression, a chemical synthesis, etc.

[0084] As used herein, the term “bioavailability” refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug or compound administered to a subject. Bioavailability is an absolute term that indicates measurement of both the 19Attorney Docket No.: 090322-8001WO01 time (rate) and total amount (extent) of drug or compound that reaches the general circulation from an administered dosage form.

[0085] As used herein, the term “small molecule” refers to molecules, whether naturally occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Preferred small molecules are biologically active in that they produce a local or systemic effect in animals, such as mammals, for example humans. In certain embodiments, the small molecule is a drug, and the small molecule is referred to as “drug molecule” or “drug” or “therapeutic agent”. In certain embodiments, the drug molecule has MW less than or equal to about 5 kDa. In other embodiments, the drug molecule has MW less than or equal to about 1.5 kDa.

[0086] As used herein the term “cytotoxic” means toxic to cells or a selected cell population (e.g., cancer cells). The toxic effect may result in cell death and / or lysis. In certain instances, the toxic effect may be a sublethal destructive effect on the cell, e.g., slowing, or arresting cell growth. In order to achieve a cytotoxic effect, the drug or prodrug may be selected from a group consisting of a DNA damaging agent, a microtubule disrupting agent, or a cytotoxic protein or polypeptide, amongst others.

[0087] As used herein, the term “tumor antigen” refers to an antigenic substance produced in tumor cells, i.e., it triggers an immune response in the host. Normal proteins in the body are not antigenic because of self-tolerance, a process in which self-reacting cytotoxic T lymphocytes (CTLs) and autoantibody -producing B lymphocytes are culled “centrally” in primary lymphatic tissue (BM) and “peripherally” in secondary lymphatic tissue (mostly thymus for T-cells and spleen / lymph nodes for B cells). Thus, any protein that is not exposed to the immune system triggers an immune response. This may include normal proteins that are well sequestered from the immune system, proteins that are normally produced in extremely small quantities, proteins that are normally produced only in certain stages of development, or proteins whose structure is modified due to mutation.

[0088] As used herein, the term “effective amount” refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, etc. For example, the effective amount of microparticles containing an antigen to be delivered to immunize an individual is the amount that results in an immune response sufficient to prevent infection with an organism having the administered antigen. 20Attorney Docket No.: 090322-8001WO01

[0089] As used herein, “molecular weight” or “MW” of a polymer or polymeric carrier / scaffold or polymer conjugates refers to the weight average molecular weight unless otherwise specified.

[0090] The present disclosure is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C13and C14. Isotopes of hydrogen include H2and H3.

[0091] The present invention is intended to include all isomers of the compound, which refers to and includes, optical isomers, and tautomeric isomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and the optical isomers include isolated optical isomers as well as mixtures of optical isomers including racemic and non-racemic mixtures; where an isomer may be in isolated form or in a mixture with one or more other isomers. COMPOUNDS FOR CONJUGATES

[0092] The present disclosure provides compounds useful as linkers for producing the conjugates described herein. In certain embodiments, the compound can be attached to two or more drugs or active agents and may also include a conjugation moiety useful for conjugation of the drugs or active agents to a targeting moiety (e.g., an antibody). For example, the conjugation moiety in the compound may be conjugated to a targeting moiety, thus indirectly binding the drugs or active agents and the targeting moiety together. The compounds of the present disclosure constitute an effective linker platform for producing ADCs with desirable efficacy, improved stability even under stressed condition, favorable pharmacokinetics and good safety window.

[0093] There is provided a compound of Formula (I):or a pharmaceutically acceptable salt thereof, wherein: 21Attorney Docket No.: 090322-8001WO01 X is a linking moiety capable of forming a covalent bond with a targeting moiety; W is a polypeptide moiety; L1is an amino acid side chain residue; L2is a spacer moiety; Y is a hydrophilic end group; E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC); T is a therapeutic or a diagnostic agent moiety; n is an integer from 1 to 20; m is an integer from 0 to 5; p is an integer from 1 to 24; a is 0 or 1; and b is 0 or 1.

[0094] In one aspect, there is provided a compound of Formula (IA), (IB) or (IC):wherein X, W, L1, L2, Y, E, T, n, m, and p are as defined herein. 22Attorney Docket No.: 090322-8001WO01

[0095] In some embodiments, n is an integer from 1 to 20, from 1 to 19, from 1 to 18, from 1 to 17, from 1 to 16, from 1 to 15, from 1 to 14, from 1 to 13, from 1 to 12, from 1 to 11, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In some embodiments, n is 5. In some embodiments, n is 4. In some embodiments, n is 3. In some embodiments, n is 2.

[0096] In some embodiments, m is an integer from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. In some embodiments, m is 2. In some embodiments, m is 1. In some embodiments, m is 0.

[0097] In some embodiments, p is an integer from 1 to 24, from 1 to 23, from 1 to 22, from 1 to 21, from 1 to 20, from 1 to 19, from 1 to 18, from 1 to 17, from 1 to 16, from 1 to 15, from 1 to 14, from 1 to 13, from 1 to 12, from 1 to 11, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In some embodiments, p is 12. In some embodiments, p is 11. In some embodiments, p is 10.

[0098] In some embodiments, a is 0. In some embodiments, a is 1.

[0099] In some embodiments, b is 0. In some embodiments, b is 1.

[0100] In some embodiments, a is 0 and b is 1. In some embodiments, a is 1 and b is 0.

[0101] In some embodiments,, wherein q is an integer from 1 to 10. In some embodiments, q is an integer from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2. In some embodiments, q is 2. In some embodiments, q is 1.

[0102] In some embodiments, W is selected from a peptide composed of 2-5 amino acids. In some embodiments, each amino acid is independently natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D- α-amino 23Attorney Docket No.: 090322-8001WO01 acids. In some embodiments, the amino acids include but are not limited to alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), tryptophan (Trp), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), Glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), or citrulline (Cit), or a derivative thereof.

[0103] In some embodiments, W is a peptide composed of 2 amino acids. In certain embodiments, W is selected from Gly-Ala, Gly-Gly, Ala-Gly, Glu-Gly, Glu- Ala, Gly-Glu, Asp-Gly, Asp-Ala, Gly-Asp, Cit-Val, Ala-Val, Phe-Lys, Phe-Gly, Lys- Val, Val-Ala, Val-Cit, Ala-Ala, Val-Cys, Val-Gly, Val-Thr, Val-Val, Val-Leu,Val-Ile, Val-Asn or Val-Lys. In certain embodiments, W is Val-Ala, Phe-Gly, or Val-Cit.

[0104] In some embodiments, W is a peptide composed of 3 amino acids. In certain embodiments, W is selected from Glu-Ala-Gly, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Glu-Ala, Asp-Ala-Gly, Asp-Gly-Gly, Gly-Asp-Gly, Gly-Asp-Ala, Gly-Gly-Ala, Gly-Gly-Arg, Gly-Ala-Gly, Gly-F-Gly, Ala-Ala-Gly, Ala-Ala-Ala, Ala-Ala-Asn, Val- Ala-Gly, Val-Cys-Gly, or Val-Lys-Gly.

[0105] In some embodiments, W is a peptide composed of 4 amino acids. In certain embodiments, W is selected from Gly-Gly-Phe-Gly, Gly-Gly Ala-Gly, Gly- Gly-Gly-Gly, Gly-Glu-Gly-Gly, Gly-Glu-Ala-Gly, Gly-Asp-Gly-Gly, Gly-Asp-Ala- Gly, Ala-Ala-Ala-Gly or Glu-Ala-Gly-Gly. In certain embodiments, W is Gly-Gly- Phe-Gly.

[0106] In some embodiments, E is absent. In some embodiments, E is -NHCH2O-. In some embodiments, E is p-aminobenzyloxycarbonyl (PABC).

[0107] In some embodiments, L1is selected from, ,24Attorney Docket No.: 090322-8001WO01

[0108] In some embodiments, L2is selected fromor, wherein the * end indicates the attachment point of L2to L1.

[0109] In some embodiments,

[0111] In some embodiments, Y is selected from -COOH, -OH, -OCH3, -CONH2, - NH2, -N3, -SO3H, -OP(O)(OH)2, -OC(O)OH, -NHCONH2, -SO2NH, or - NH(CH2CH2O)1-5H. In some embodiments, Y is selected from -COOH, -OH, - OCH3, or -CONH2.

[0112] In certain embodiments, the compond is selected from the group consisting of25Attorney Docket No.: 090322-8001WO0126Attorney Docket No.: 090322-8001WO0127Attorney Docket No.: 090322-8001WO0128Attorney Docket No.: 090322-8001WO0129Attorney Docket No.: 090322-8001WO0130Attorney Docket No.: 090322-8001WO0131Attorney Docket No.: 090322-8001WO01. In some embodiments, n is 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. In some embodiments, n is 5. In some embodiments, n is 4. In some embodiments, n is 3. In some embodiments, p is 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. In some embodiments, p is 12. In some embodiments, p is 11. In some embodiments, p is 10. In some embodiments, p is 9. In some embodiments, p is 8.

[0113] In some embodiments, the therapeutic agent used in the conjugate of the present disclosure is a small molecule having a molecular weight not more than about 5 kDa, not more than about 4 kDa, not more than about 3 kDa, not more than about 1.5 kDa or not more than about 1 kDa.

[0114] In some embodiments, the therapeutic agent provided herein is selected from small molecules, protein toxins, enzymes, radionuclides or nanocarriers.

[0115] In some embodiments, the therapeutic agent provided herein is small molecules such as a cytotoxic agent, a cytostatic agent, a protein degrader agent, an immunosuppressive agent, or an imaging agent.

[0116] In some embodiments, the cytotoxic agent is selected from a metal compound, such as a platinum compound (e.g., cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, or tetraplatin) or a gold metal complex; a glycopeptide antibiotic such as bleomycin or pingyangmycin; a DNA topoisomerase inhibitor, such as a topoisomerase I inhibitor (e.g., camptothecin, camptothecin derivatives, camptothecin analogs and non-natural camptothecins, such as irinotecan, SN-38, GI- 147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7- 32Attorney Docket No.: 090322-8001WO01 aminomethyl camptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, or lurtotecan) or a topoisomerase II inhibitor (e.g., actinomycin D, adriamycin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or etoposide); a drug interfering with DNA synthesis, such as methotrexate, 5- fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or narabine; a drugs acting on a structural protein, such as a tubulin inhibitor, a vinblastine alkaloid, a vincristine, vinblastine, paclitaxel, docetaxel or cabazitaxel; a tumor cell signaling pathway inhibitor, such as a serine / threonine kinase inhibitor, a tyrosine kinase inhibitor, a aspartokinase inhibitor or a histidine kinase inhibitor; a proteasome inhibitor; a histone deaceylase inhibitor; a tumor angiogenesis inhibitor; a cyclin inhibitor; a maytansine derivative; a calicheamicin derivative; a auristatin derivative; a pyrrolobenzodiazepine dimers (PBD) derivative; melphalan; mitomycin C; chlorambucil; and other active substances which inhibit the growth of tumor cells, promote the apoptosis or necrosis of tumor cells.

[0117] In some embodiments, the immunosuppressive agent is selected from non- steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocryptine; danazol; dapsone; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, including SOLU-MEDROL® methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); anti-malarial agents such as chloroquine and hydroxychloroquine; sulfasalazine; and leflunomide.

[0118] In some embodiments, the therapeutic agent is an imaging agent (e.g., a fluorophore or a chelator), such as fluorescein, rhodamine, lanthanide phosphors, and their derivatives thereof, or a radioisotope bound to a chelator. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5- FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelators include, but are not limited to, 1,4,7,10- tetraazacyclododecane-N,N’,N”,N’’’-tetraacetic acid (DOTA), 1,4,7- triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 33Attorney Docket No.: 090322-8001WO01 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid) (BAPTA). Examples of a radioisotope or other labels include, but are not limited to,3H,14C,15N,35S,18F,32P,33P,64Cu,68Ga,89Zr,90Y,99Tc,123I,124I,125I,131I,111In,131In,153Sm,186Re,188Re,211At,212Bi, and153Pb.

[0119] In some embodiments, the therapeutic agent provided herein is protein toxins produced from bacteria and plants, including but not limited to Shiga toxin, Shiga-like toxin, Pseudomonas exotoxin, diphtheria toxin and cholera toxin, ricin, modeccin, abrin, volkensin, viscumin and the like.

[0120] In some embodiments, the therapeutic agent provided herein is enzyme, including but not limited to β-glucuronidase, urease, HRPO (horseradish peroxidase), ALP (alkaline phosphatase) and the like.

[0121] In some embodiments, the therapeutic agent provided herein includes lipid- based particles (e.g. liposomes), polymeric nanocarriers (such as polymeric micelles, dendrimers and polymeric nanoparticles), and the like, which are loaded with drugs to be delivered.

[0122] In some embodiments, the therapeutic agent moiety T in the compounds provided herein is of Formula (T-1), Formula (T-2) or Formula (T-3):34Attorney Docket No.: 090322-8001WO01 R1is hydrogen, halogen, alkyl, haloalkyl, -OR1a, -SR1a, -S(O)R1a, or -S(O)2R1a; R2is hydrogen, halogen, alkyl or haloalkyl; each of R3, R6, R7and R10is hydrogen or alkyl; each of R4, R5and R8is independently selected from hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, -alkyl-cycloalkyl, -alkyl-aryl, or -alkyl-heterocyclyl; each R9is independently H, -OH, alkyl, alkoxyl, or cycloalkyl; R11is -[C(R11a)2]2-cycloalkyl, -[C(R11a)2]2-heterocyclyl, or -[C(R11a)2]2-aryl; R12is heterocyclyl or aryl; Z is -O-, -S-, -NH-, or -N(alkyl)-; R13is hydrogen, alkyl, heterocyclyl, or aryl; R1ais hydrogen or alkyl; and each R11ais independently selected from hydrogen, hydroxyl, alkyl, alkoxyl, or cycloalkyl.

[0123] In some embodiments, the therapeutic agent moiety T is selected from:. 35Attorney Docket No.: 090322-8001WO01

[0124] In some embodiments, the diagnostic agent used in the conjugate of the present disclosure can be, for example, a contrast agent (e.g., ultrasound contrast agent, a magnetic resonance imaging (MRI) contrast agent, or a radiocontrast agent), an isotopic label, a fluorescent label, a chemiluminescent label, a bioluminescent label, a paramagnetic ion, an enzyme, or a photoactive agent.

[0125] Exemplary contrast agents include ultrasound contrast agents (e.g. microbubbles), magnetic resonance imaging (MRI) contrast agents (e.g., gadodiamide, gadobenic acid, gadopentetic acid, gadoteridol, gadofosveset, gadoversetamide, gadoxetic acid), and radiocontrast agents, such as for computed tomography (CT), radiography, or fluoroscopy (e.g., diatrizoic acid, metrizoic acid, iodamide, iotalamic acid, ioxitalamic acid, ioglicic acid, acetrizoic acid, iocarmic acid, methiodal, diodone, metrizamide, iohexol, ioxaglic acid, iopamidol, iopromide, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iodoxamic acid, iotroxic acid, ioglycamic acid, adipiodone, iobenzamic acid, iopanoic acid, iocetamic acid, sodium iopodate, tyropanoic acid, and calcium iopodate).

[0126] Exemplary fluorescent labels include fluorescein derivatives, rhodamine derivatives, coumarin derivatives, cyanine derivatives, acridine derivatives, squaraine derivatives, naphthalene derivatives, oxadiazol derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In addition, the fluorescent label may comprise a fluorescent protein, such as, but not limited to, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, rsCherry, and rsCherryRev.

[0127] Exemplary isotopic labels may comprise radioactive isotopes (e.g., gamma- emitters, beta-emitters, and positron-emitters) or non-radioactive isotopes (e.g., stable trace isotopes), such as, but not limited to,3H,2H,14C,32P,15N,13N,110In,111In,177Lu,18F,52Fe,62Cu,64CU,67Cu,67Ga,68Ga,86Y,90Y,89Zr,94mTc,94Tc,99mTc,154Gd,155Gd,156Gd,157Gd,158Gd,15O,186Re,188Re,51M,52mMn,55Co,72As,75Br,76Br,82mRb, and83Sr.

[0128] Exemplary paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), 36Attorney Docket No.: 090322-8001WO01 ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0129] In some embodiments, the compound provided herein is selected from the group consisting of:37Attorney Docket No.: 090322-8001WO0138Attorney Docket No.: 090322-8001WO0139Attorney Docket No.: 090322-8001WO0140Attorney Docket No.: 090322-8001WO0141Attorney Docket No.: 090322-8001WO0142Attorney Docket No.: 090322-8001WO0143Attorney Docket No.: 090322-8001WO0144Attorney Docket No.: 090322-8001WO01. CONJUGATE

[0130] The present disclosure provides a conjugate wherein a targeting moiety is conjugated to one or more compounds of Formula (I), (IA), (IB), or (IC) as described herein.

[0131] In one aspect, there is provided a compound of Formula (II):wherein A is a targeting moiety; X’ is a linking moiety connecting to A; L1is an amino acid side chain residue; L2is a spacer moiety; Y is a hydrophilic end group; E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC); T is a therapeutic or a diagnostic agent moiety; n is an integer from 1 to 20; m is an integer from 0 to 5; p is an integer from 1 to 24; r is from 1 to 10; 45Attorney Docket No.: 090322-8001WO01 a is 0 or 1; and b is 0 or 1.

[0132] In another aspect, there is provided a compound of Formula (IIA), (IIB), or (IIC)):( C), wherein A, X’, W, L1, L2, Y, E, T, n, m, r and p are as defined herein.

[0133] In some embodiments, n is an integer from 1 to 20, from 1 to 19, from 1 to 18, from 1 to 17, from 1 to 16, from 1 to 15, from 1 to 14, from 1 to 13, from 1 to 12, from 1 to 11, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In some embodiments, n is 5. In some embodiments, n is 4. In some embodiments, n is 3. In some embodiments, n is 2.

[0134] In some embodiments, m is an integer from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. In some embodiments, m is 2. In some embodiments, m is 1. In some embodiments, m is 0. 46Attorney Docket No.: 090322-8001WO01

[0135] In some embodiments, p is an integer from 1 to 24, from 1 to 23, from 1 to 22, from 1 to 21, from 1 to 20, from 1 to 19, from 1 to 18, from 1 to 17, from 1 to 16, from 1 to 15, from 1 to 14, from 1 to 13, from 1 to 12, from 1 to 11, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In some embodiments, p is 12. In some embodiments, p is 11. In some embodiments, p is 10.

[0136] In some embodiments, a is 0. In some embodiments, a is 1.

[0137] In some embodiments, b is 0. In some embodiments, b is 1.

[0138] In some embodiments, a is 0 and b is 1. In some embodiments, a is 1 and b is 0.

[0139] In some embodiments,, wherein q is an integer from 1 to 10. In some embodiments, q is an integer from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2. In some embodiments, q is 2. In some embodiments, q is 1.

[0140] In some embodiments, W is selected from a peptide composed of 2-5 amino acids. In some embodiments, each amino acid is independently natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D- α-amino acids. In some embodiments, the amino acids include but are not limited to alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), tryptophan (Trp), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), or citrulline (Cit), or a derivative thereof. 47Attorney Docket No.: 090322-8001WO01

[0141] In some embodiments, W is a peptide composed of 2 amino acids. In certain embodiments, W is selected from Gly-Ala, Gly-Gly, Ala-Gly, Glu-Gly, Glu- Ala, Gly-Glu, Asp-Gly, Asp-Ala, Gly-Asp, Cit-Val, Ala-Val, Phe-Lys, Lys-Val, Val- Ala, Val-Cit, Ala-Ala, Val-Cys, Val-Gly, Val-Thr, Val-Val, Val-Leu,Val-Ile, Val-Asn or Val-Lys. In certain embodiments, W is Val-Ala or Val-Cit.

[0142] In some embodiments, W is a peptide composed of 3 amino acids. In certain embodiments, W is selected from Glu-Ala-Gly, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Glu-Ala, Asp-Ala-Gly, Asp-Gly-Gly, Gly-Asp-Gly, Gly-Asp-Ala, Gly-Gly-Ala, Gly-Gly-Arg, Gly-Ala-Gly, Gly-F-Gly, Ala-Ala-Gly, Ala-Ala-Ala, Ala-Ala-Asn, Val- Ala-Gly, Val-Cys-Gly, or Val-Lys-Gly.

[0143] In some embodiments, W is a peptide composed of 4 amino acids. In certain embodiments, W is selected from Gly-Gly-Phe-Gly, Gly-Gly Ala-Gly, Gly- Gly-Gly-Gly, Gly-Glu-Gly-Gly, Gly-Glu-Ala-Gly, Gly-Asp-Gly-Gly, Gly-Asp-Ala- Gly, Ala-Ala-Ala-Gly or Glu-Ala-Gly-Gly. In certain embodiments, W is Gly-Gly- Phe-Gly.

[0144] In some embodiments, L1is selected from, ,, wherein the * end indicates the attachment point of L1to L2.

[0145] In some embodiments, L2is selected from, wherein the * end indicates the attachment point of L2to L1.

[0146] In some embodiments, L1is48Attorney Docket No.: 090322-8001WO01

[0148] In some embodiments, Y is selected from -COOH, -OH, -OCH3, -CONH2, - NH2, -N3, -SO3H, -OP(O)(OH)2, -OC(O)OH, -NHCONH2, -SO2NH, or - NH(CH2CH2O)1-5H. In some embodiments, Y is selected from -COOH, -OH, - OCH3, or -CONH2.

[0149] In some embodiments, the compound of Formula (II) is selected from:49Attorney Docket No.: 090322-8001WO01.

[0150] In some embodiments, r is from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In some embodiments, r is about 9. In some embodiments, r is about 8. In some embodiments, r is about 7. In some embodiments, r is about 6. In some embodiments, r is about 5. In some embodiments, r is about 4.

[0151] The targeting moiety A directs the linker-therepeutic agent conjugates provided herein to specific tissues, cells, or locations in a cell. The targeting moiety can direct the the linker-therepeutic agent conjugates provided herein in culture or in a whole organism, or both. In each case, the targeting moiety can bind to a ligand that is present on the cell surface of the targeted cell(s) with an effective specificity, affinity, and avidity. In some embodiments, the targeting moiety targets the linker- therepeutic agent conjugates provided herein to a specific tissue such as the liver, kidney, lung, or pancreas. The targeting moiety can target the linker-therepeutic agent conjugates provided herein to a target cell such as a cancer cell, a matrix tissue, a protein associated with cancer such as tumor antigen, or a cell comprising the tumor vasculature.

[0152] In some embodiments, the targeting moiety can target the linker-therepeutic agent conjugates provided herein to a location within the cell, such as the nucleus, the cytoplasm, or the endosome. In certain embodiments, the targeting moiety can enhance cellular binding to receptors, or cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.

[0153] In some embodiments, the targeting moiety comprises natural amino acids that are capable of reacting with a functional group in the linking moiety of the linker- drug conjugate to form a covalent bond. In certain embodiments, the natural amino acid includes cysteine, lysine, tyrosine, aspartic acid and glutamic acid. 50Attorney Docket No.: 090322-8001WO01

[0154] In certain embodiments, the targeting moiety comprises cysteine and the targeting moiety is conjugated to the linker-drug conjugate by a covalent bond via the sulfhydryl group and a functional group of the linking moiety in the linker-drug conjugate.

[0155] In certain embodiments, the targeting moiety comprises lysine and the targeting moiety is conjugated to the linker-drug conjugate by a covalent bond via the amino group and a functional group of the linking moiety in the linker-drug conjugate.

[0156] In some embodiments, the targeting moiety may comprise non-natural amino acids that are capable of reacting with a functional group in the linking moiety of the linker-drug conjugate to form a covalent bond. In certain embodiments, the targeting moiety is conjugated to the linker-drug conjugate by a covalent bond via the amino group and a functional group of the linking moiety in the linker-drug conjugate.

[0157] In some embodiments, the targeting moiety may comprise functional groups that are capable of reacting with a functional group in the linking moiety of the linker- drug conjugate via click reaction to form a covalent bond.

[0158] In some embodiments, the targeting moiety A is selected from a polypeptide, an antibody, an enzyme or a fragment thereof.

[0159] In some embodiments, the targeting moiety A is an antibody. In some embodiments, the targeting moiety A is selected from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody or a fragment thereof. In some embodiments, the targeting moiety A is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, a monoclonal antibody and a polyclonal antibody.

[0160] In some embodiments, the targeting moiety A can be antibodies or fragments thereof specific to cell surface markers, including but not limited to, 5T4, AOC3, ALK, AXL, BCMA, C242, CA-125, CCL11, CCR 5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD44 v6, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD74, CD79-B, CD80, CD125, CD138, CD141, 51Attorney Docket No.: 090322-8001WO01 CD147, CD152, CD 154, CD326, CEA, clumping factor, CTLA-4, CXCR2, EGFR (HER1), ErbB2, ErbB3, EpCAM, EPHA2, EPHB2, EPHB4, FGFR (i.e. FGFR1, FGFR2, FGFR3, FGFR4), FLT3, folate receptor, FAP, GD2, GD3, GPNMB, HGF, HMI.24, ICAM, ICOS-L, IGF-1 receptor, VEGFR1, EphA2, TRPV1, CFTR, CA9, Cripto, c-KIT, c-MET, ACE, APP, adrenergic receptor-beta2, Claudine 3, Mesothelin, MUC1, NaPi2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, RON, ROR1, PD-L1, PD-L2, B7-H3, B7-B4, IL-2 receptor, IL-4 receptor, IL-13 receptor, Trop-2, integrins (including α4, αvβ3, αvβ5, αvβ6, α1β4, α4β1, α4β7, α5β1, α6β4, αIIbβ3intergins), IFN-α, IFN-γ, IgE, IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin (CD62L), MSR1, mucin, MUC1, MUC16, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostatic carcinoma cell, Pseudomonas aeruginosa, rabies, RANKL, respiratory syncytial virus, Rhesus factor, SLAMF7, sphingosine-1-phosphate, STEAP2, TAG- 72, T-cell receptor, tenascin C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL- R2, tumor antigen CTAA16.88, UPK3A, VEGF-A, VEGFR2, vimentin, and the like.

[0161] In some embodiments, the targeting moiety A can be an antibody selected from or derived from an anti-DLL-3 antibody, an anti-MUC16 antibody, an anti- ENPP3 antibody, an anti-TDGF1 antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-Mesothelin antibody, an anti-c-MET antibody, an anti-SLITRK6 antibody, an anti-KIT / CD117 antibody, an anti-STEAP1 antibody, an anti- SLAMF7 / CS1 antibody, an anti-NaPi2B / SLC34A2 antibody, an anti-GPNMB antibody, an anti-HER3 antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti-PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-EphA2 antibody, an anti-FGFR2 antibody, an anti- FGFR3 antibody, an anti-FRα antibody, an anti-CEACAM5 antibody, an anti-GCC antibody, an anti-Integrin Av antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-BCMA antibody, an anti-Tissue Factor antibody, an anti-Foliate Receptor antibody, an anti-ROR1 antibody, an anti-ROR2 antibody, an anti-TROP2 antibody, an anti-EGFR antibody, an anti-HER2 antibody, an anti-HER3 antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD46 antibody, an anti-CD47 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-CD74 antibody, an anti-CD79b antibody, an anti-CD123 antibody, an anti- CD138 antibody, an anti-CD142 antibody, an anti-CD166 antibody, an anti-CD174 antibody, an anti-CD352 antibody, an anti-Claudin6 antibody, an anti-Claudin 18.2 52Attorney Docket No.: 090322-8001WO01 antibody, an anti-GPC3 antibody, an anti-LYPD3 antibody, an anti-BCMA antibody, an anti-CLL1 antibody, an anti-FLT3 antibody, an anti-Nectin-4 antibody, an anti- CD44v9 antibody, an anti-integrin b6 antibody, an anti-CDCP1 antibody, an anti- EpCAM antibody, an anti-PD-L1 antibody, an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-EGFR / HER3 bispecific antibody, an anti-EGFR / c- MET bispecific antibody, an anti-MUC1 / EGFR bispecific antibody, an anti- HER2 / CD63 bispecific antibody, an anti-HER2 / PRLR bispecific antibody, an anti- HER2 / Nectin-4 bispecific antibody, and an anti-HER2 / APLP2 bispecific antibody.

[0162] Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO), adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), aselizumab, atlizumab (tocilizumab, Actemra, RoActemra), atorolimumab, bapineuzumab, basiliximab (Simulect), bavituximab, bectumomab (LYMPHOSCAN), belimumab (BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN), bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab, blinatumomab, brentuximab, briakinumab, canakinumab (ILARIS), cantuzumab, capromab, catumaxomab (REMOVAB), CC49, cedelizumab, certolizumab, cetuximab (ERBITUX), citatuzumab, cixutumumab, clenoliximab, clivatuzumab, conatumumab, CR6261, dacetuzumab, daclizumab (ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab, dorlixizumab, ecromeximab, eculizumab (SOLIRIS), edobacomab, edrecolomab (PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab, elsilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab (REXOMUN), etaracizumab (ABEGRIN), exbivirumab, fanolesomab (NEUTROSPEC), faralimomab, farletuzumab, felvizumab, fezakinumab, figitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab, gantenerumab, gavilimomab, gemtuzumab, girentuximab, glembatumumab, golimumab (SIMPONI), gomiliximab, ibalizumab, ibritumomab, igovomab (INDIMACIS-125), imciromab (MYOSCINT), infliximab (REMICADE), intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, keliximab, labetuzumab (CEA-CIDE), lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (NUMAX), muromonab- CD3 (ORTHOCLONE OKT3), nacolomab, naptumomab, natalizumab (TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM), nofetumomab, ocrelizumab, odulimomab, ofatumumab (ARZERRA), olaratumab, omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX), otelixizumab, pagibaximab, palivizumab (SYNAGIS), panitumumab (VECTIBIX), 53Attorney Docket No.: 090322-8001WO01 panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab (OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO 140, rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), robatumumab, rontalizumab, rovelizumab (LEUKARREST), ruplizumab (ANTOVA), sacituzumab, satumomab pendetide, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab, solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab (LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab (AUREXIS), telimomab, tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA), toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN), tremelimumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab (STELERA), vapaliximab, vedolizumab, veltuzumab, vepalimomab, visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), ziralimumab and zolimomab.

[0163] In some embodiments, the targeting moiety A is an anti-HER2 antibody or an anti-PMSA antibody. In some embodiments, the targeting moiety A is trastuzumab or HJ591.

[0164] The compounds of Formula (I), (IA), (IB), or (IC) as described herein may be conjugated to the targeting moiety (e.g., an antibody) at one or more sites in the targeting moiety. In some embodiments, the resultant conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) (i.e., r in Formula (II), Formula (IIA), Formula (IIB), Formula (IIC)) in the range of from 0.1 to 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In certain embodiments, the conjugates have an average DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugates have an average DAR of 1 to 10. In certain embodiments, the conjugates have an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugates have an average DAR of 5 to 10 (e.g., 8). By average is meant the arithmetic mean.

[0165] In some embodiments, the compound of Formula (II) is selected from: 54Attorney Docket No.: 090322-8001WO01, wherein A is trastuzumab, HJ 591 or hH1L1. SYNTHETIC METHODS

[0166] The therapeutic agent used for the compounds and conjugates provided herein are commercially available or can be prepared in a variety of ways using 55Attorney Docket No.: 090322-8001WO01 commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith, M. B., March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5thedition, John Wiley & Sons: New York, 2001; and Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999, incorporated by reference herein, are useful and recognized reference textbooks of organic synthesis known to those in the art.

[0167] Any available techniques can be used to make the conjugates provided herein or compositions including them, and intermediates and components (e.g., carriers and modifiers) useful for making them.

[0168] The conjugates described herein can be synthesized by coupling the compounds of Formula (I), (IA), (IB), or (IC) as described herein with a targeting moiety, for example, an antibody under standard conjugation conditions (see, e.g., Doronina et al. Nature Biotechnology 2003, 21, 778, which is incorporated herein

[0169] by reference in its entirety).

[0170] When the targeting moiety is an antibody, the antibody may be coupled to the compounds of Formula (I), (IA), (IB), or (IC) provided herein via one or more cysteine or lysine residues of the antibody. The compounds of Formula (I), (IA), (IB), or (IC) provided herein can be coupled to cysteine residues, for example, by subjecting the antibody to a reducing agent, for example, dithiotheritol, to cleave the disulfide bonds of the antibody, purifying the reduced antibody, for example, by gel filtration, and subsequently treating the antibody with a linker-payload containing a suitable reactive moiety, for example, a maleimido group. Suitable solvents include, but are not limited to water, DMA, DMF, and DMSO. Conjugates can be purified using known protein techniques, including, for example, size exclusion chromatography, dialysis, and ultrafiltration / diafiltration.

[0171] Conjugates provided herein, once produced, can be characterized using a variety of assays known to those skilled in the art to determine whether the conjugates have biological activity. For example, the conjugates can be characterized by 56Attorney Docket No.: 090322-8001WO01 conventional assays, including but not limited to those assays described below, to determine the binding activity, binding specificity, cytotoxicity, stability etc. PHARMACEUTICAL COMPOSITION

[0172] For the purposes of administration, in some embodiments, the compounds and conjugates provided herein are administered as a raw chemical or are formulated as pharmaceutical compositions.

[0173] Therefore, in one aspect, the present disclosure provides a pharmaceutical composition comprising one or more compounds or conjugates as disclosed herein and an acceptable carrier. Examples of suitable carriers include, but are not limited to, buffers for maintenance of proper composition pH (e.g., citrate buffers, succinate buffers, acetate buffers, phosphate buffers, lactate buffers, oxalate buffers, and the like), carrier proteins (e.g., human serum albumin), saline, polyols (e.g., trehalose, sucrose, xylitol, sorbitol, and the like), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxolate, and the like), antimicrobials, and antioxidants.

[0174] The administration of the pharmaceutical composition provided herein may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer), intratracheal, intranasal, epidermal, transdermal, oral or parenteral administration including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion or intracranial.

[0175] The pharmaceutical composition provided herein can be formulated into various suitable dosage forms depending on administration routes, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermals, aerosols, powders and sprays.

[0176] The pharmaceutical composition provided herein comprises pharmaceutically effective amount of the compounds or conjugates as disclosed herein. As used herein, the term “pharmaceutically effective amount” refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Pharmaceutically effective amounts for a given situation can be 57Attorney Docket No.: 090322-8001WO01 determined by routine experimentation that is within the skill and judgment of the clinician.

[0177] The pharmaceutical composition provided herein may be administered at a dosage level between about 0.001 mg / kg to about 200 mg / kg of the subject's body weight, between about 0.001 mg / kg to about 150 mg / kg of the subject's body weight, between about 0.001 mg / kg to about 100 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 90 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 80 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 70 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 60 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 50 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 40 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 30 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 20 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 15 mg / kg of the subject's body weight, between about 0.1 mg / kg and about 15 mg / kg of the subject's body weight, between about 0.1 mg / kg and about 20 mg / kg of the subject's body weight, between about 0.1 mg / kg to about 5 mg / kg or about 0.1 mg / kg to about 10 mg / kg of the subject's body weight. In some embodiments, the dosage administered is between about 1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dosage administered is between about 1 mg / kg to about 10 mg / kg of the subject's body weight.

[0178] In some embodiments, the therapeutically effective amount of the compounds or conjugates in the pharmaceutical composition provided herein may be administered on a regular schedule, i.e., daily, weekly, monthly, or yearly basis or on an irregular schedule with varying administration days, weeks, months, etc. Alternatively, the therapeutically effective amount to be administered may vary. In some embodiments, the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses. In some embodiments, the therapeutically effective amount for the first dose is lower than the therapeutically effective amount for one or more of the subsequent doses. Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The number and frequency of dosages corresponding to a completed course of therapy will be determined according to the recommendations of the relevant regulatory bodies and judgment of a health- care practitioner. The therapeutically effective amounts described herein refer to 58Attorney Docket No.: 090322-8001WO01 total amounts administered for a given time period; that is, if more than one different conjugate described herein is administered, the therapeutically effective amounts correspond to the total amount administered. It is understood that the specific dose level for a particular subject depends upon a variety of factors including the activity of the specific conjugate, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, combination with other active agents, and the severity of the particular disease undergoing therapy.

[0179] The compounds, conjugates and pharmaceutical composition provided herein can also be administered in combination with one or more additional therapeutic agents to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects. The one or more additional therapeutic agents can be administered prior to, concurrent with, or after the administration of the compounds, conjugates or pharmaceutical compositions described herein. The additional therapeutic agents may be an anticancer agent, an immunosuppressant agent, and an anti-infectious agent. Examples of anticancer agent include but are not limited to methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5- fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. Examples of immunosuppressant agents include but are not limited to cyclosporine, cyclosporine A, mycophenylate mofetil, sirolimus, tacrolimus, enanercept, prednisone, azathioprine, methotrexate cyclophosphamide, prednisorie, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, and infliximab. Examples of anti-infectious agents include but are not limited to β-lactam antibiotics (such as penicillin G, penicillin V, cloxacilliin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacarnpicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, and ticarcillin), aminoglycosides (such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, and tobramycin), macrolides (such as azithromycin, clarithromycin, erythromycin, lincomycin, and clindamycin), tetracyclines (such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline), quinolones (such as cinoxacin and nalidixic acid), fluoroquinolones (such as ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, and trovafloxicin), and sulfonamides (such as sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethizole, and sulfacetamide). 59Attorney Docket No.: 090322-8001WO01

[0180] The present disclosure also provides pharmaceutical kits comprising one or more containers filled with one or more of the compounds, conjugates and / or compositions of the present disclosure, optionally together with additional therapeutical agents, a device(s) for administering the compounds and / or compositions, and written instructions in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products. The compounds, conjugates or compositions described herein can be packaged as a single dose or for continuous or periodic discontinuous administration. For continuous administration, a package or kit can include the compounds, conjugates or compositions in each dosage unit (e.g., solution or other unit described above or utilized in drug delivery), and optionally instructions for administering the doses daily, weekly, or monthly, for a predetermined length of time or as prescribed. If varying dosing level of the compounds, conjugates or compositions over time is desired, a package or kit may contain a sequence of dosage units which provide the desired variability. METHOD OF TREATMENT OF DISEASE

[0181] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an efficient amount of at least one compound, conjugate or composition provided herein.

[0182] In some embodiments, the compounds, conjugates or compositions provided herein can be administered to treat subjects and / or to modulate the growth of selected cell populations including, for example, cancer. In some embodiments, the particular types of cancers that can be treated with the conjugates provided herein include, but are not limited to: (1) solid tumors, including but not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, head and neck cancer, uterine cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma, multiforme 60Attorney Docket No.: 090322-8001WO01 astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma; (2) blood-borne cancers, including but not limited to leukemia, such as acute lymphoblastic leukemia “ALL”, acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”, acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairy cell leukemia, acute and chronic leukemias, e.g., lymphoblastic myelogenous and lymphocytic myelocytic leukemias; and (3) lymphomas such as Hodgkin's disease, non-Hodgkin's Lymphoma, myeloma such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and polycythemia vera.

[0183] In some embodiments, the compounds, conjugates and compositions provided herein can be administered to treat autoimmune diseases, such as systemic lupus, rheumatoid arthritis, psoriasis, and multiple sclerosis; graft rejections, such as renal transplant rejection, liver transplant rejection, lung transplant rejection, cardiac transplant rejection, and bone marrow transplant rejection; graft versus host disease; viral infections, such as CMV infection, HIV infection, and AIDS; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis, and the like.

[0184] In some embodiments, the compounds, conjugates and compositions provided herein can be administered to treat metabolic diseases, such as glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.

[0185] In a further aspect, the present disclosure provides a method of screening an ADC having reduced non-specific internalization into healthy cells, comprising introducing one or more negatively charged group into the linker of the ADC.

[0186] In some embodiments, the negatively charged group is selected from sulfonate, carboxylate, sulfate, or phosphate. In some embodiments, the negatively charged group is sulfonate or carboxylate.

[0187] Throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific 61Attorney Docket No.: 090322-8001WO01 process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0188] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. EXAMPLES

[0189] For the purpose of illustration, the following examples are included. The Examples provided herein describe the synthesis of the compounds and conjugates disclosed herein as well as intermediates used to prepare the compounds and conjugates. However, it is to be understood that these examples do not limit the present disclosure and are only meant to suggest a method of practicing the present disclosure. Persons skilled in the art will recognize that the chemical reactions described may be readily adapted to prepare a number of other compounds or conjugates of the present disclosure, and alternative methods for preparing the compounds and conjugates of the present disclosure are deemed to be within the scope of the present disclosure. Besides, persons skilled in the art will also understand that individual steps described herein or in the separate batches of a compound may be combined. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the present disclosure. The following description is, therefore, not intended to limit the scope of the present disclosure, but rather is specified by the claims appended hereto. 62Attorney Docket No.: 090322-8001WO01 I. Synthetic of compound containing linker and therapeutic agent moiety (linker- payload) Example 1 Synthesis of linker-exatecan 1 (LP-1)63Attorney Docket No.: 090322-8001WO01

[0191] To a solution of compound 1 (5.0 g, 26.6 mmol, 1.0 eq) in THF / H2O (200 / 50 mL) were added Boc2O (6.4 g, 29.2 mmol, 1.1 eq) and NaHCO3(6.7 g, 79.7 mmol, 3.0 eq) at rt. The reaction mixture was stirred at rt for 16 h. After completion of the reaction (monitored by TLC), the reaction solution was poured into water (100 mL). The solution was adjusted pH = 2-3 with 1N HCl. The mixture was extracted with ethyl acetate (100 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 10% MeOH in DCM as eluent) to afford compound 2 (7.5 g, 98%) as white solid.

[0192] TLC: DCM / MeOH = 10 / 1, Rf (Compound 1) = 0.1, Rf (Compound 2) = 0.4

[0193] Preparation of Compound 4

[0194] To a solution of compound 2 (1.0 g, 3.47 mmol, 1.0 eq) in DMF (10 mL) were added DIEA (1.3 g, 10.4 mmol, 3.0 eq), compound 3 (0.47 g, 3.82 mmol, 1.1 eq) and PyAOP (2.7 g, 5.21 mmol, 1.5 eq) at rt. The reaction mixture was stirred for 16 h at rt, and then poured into water (50 mL). The solution was extracted with ethyl acetate (50 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4and filtered. The filtrate was concentrated under reduced pressure to afford compound 4 (0.8 g, 59%) as white solid.

[0195] TLC: DCM / MeOH = 10 / 1, Rf(Compound 2) = 0.4, Rf(Compound 4) = 0.6, LC-MS: 394.1 [M+H]+.

[0196] Preparation of Compound 664Attorney Docket No.: 090322-8001WO01

[0197] To a solution of compound 4 (0.8 g, 2.04 mmol, 1.0 eq) in DCM (10 mL) were added DIEA (525 mg, 4.07 mmol, 2.0 eq) and compound 5 (451 mg, 2.24 mmol, 1.1 eq) at 0oC. The reaction mixture was stirred at rt for 6 h. After completion of the reaction, the reaction solution was poured into water (30 mL) and extracted with ethyl acetate (30 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 50% EA in PE as eluent) to afford compound 6 (0.8 g, 70%) as yellow solid.

[0198] TLC: PE / EA = 1 / 1, Rf(Compound 4) = 0.2, Rf(Compound 6) = 0.5, LC-MS: 559.5 [M+H]+.

[0199] Preparation of Compound 8

[0200] To a solution of compound 6 (800 mg, 0.36 mmol, 1.0 eq) in DMF (10 mL) was added DIEA (232 mg, 1.80 mmol, 5.0 eq) and compound 7 (762 mg, 0.36 mmol, 1.0 eq) at 0oC. The reaction mixture was stirred at rt for 16 h. After completion of the reaction, the reaction solution was poured into water (30 mL). The solution was extracted with ethyl acetate (30 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 50% EA in PE as eluent) to afford compound 8 (600 mg, 49%) as yellow solid.

[0201] TLC: PE / EA = 1 / 1, Rf(Compound 6) = 0.5, Rf(Compound 8) = 0.3, LC-MS: 855.5 [M+1]+.

[0202] Preparation of Compound 9 65Attorney Docket No.: 090322-8001WO01

[0203] To a solution of compound 8 (1.02 g, 1.19 mmol, 1.0 eq) in DCM (5 mL) was added TFA (2.72 g, 23.8 mmol, 20 eq) at 0oC. The reaction mixture was stirred at rt for 4 h. After completion of the reaction, the reaction solution was adjusted pH = 8 with sat. aq. NaHCO3. The mixture was extracted with DCM (50 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 5% MeOH in DCM as eluent) to afford compound 9 (680 mg, 75%) as yellow oil.

[0204] TLC: DCM / MeOH = 20 / 1, Rf(Compound 8) = 0.4, Rf(Compound 9) = 0.1, LC-MS: 755.2 [M+1]+.

[0205] Preparation of Compound 11

[0206] To a solution of compound 9 (300 mg, 0.39 mmol, 1.0 eq) in DMF (5 mL) were added HATU (227 mg, 0.59 mmol, 1.5 eq), compound 10 (186 mg, 0.43 mmol, 1.1 eq) and DIEA (154 mg, 1.19 mmol, 3.0 eq) at 0oC. The reaction mixture was stirred for 1 h at rt. After completion of the reaction, the reaction solution was poured into water (30 mL) and extracted with ethyl acetate (30 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4,and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 7.5% MeOH in DCM as eluent) to afford compound 11 (400 mg, 86%) as white solid. 66Attorney Docket No.: 090322-8001WO01

[0207] TLC: DCM / MeOH = 10 / 1, Rf(Compound 9) = 0.5, Rf(Compound 11) = 0.6, LC-MS: 1162.3 [M+1]+.

[0208] Preparation of Compound 12

[0209] To a solution of compound 11 (350 mg, 0.30 mmol, 1.0 eq) was added HCOOH (5 mL) at rt. The reaction mixture was stirred at rt for 2 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the solvent to afford compound 12 (201 mg, 60%) as yellow oil.

[0210] TLC: DCM / MeOH = 10 / 1, Rf(Compound 11) = 0.6, Rf(Compound 12) = 0.5, LC-MS: 1106.1 [M+H]+.

[0211] Preparation of Compound 14

[0212] To a solution of compound 12 (180 mg, 0.16 mmol, 1.0 eq) in DMF (2 mL) were added HATU (93 mg, 0.24 mmol, 1.5 eq), compound 13 (121 mg, 0.18 mmol, 1.1 eq) and DIEA (64 mg, 0.49 mmol, 3.0 eq) at 0oC. The reaction mixture was stirred for 1 h at rt. After completion of the reaction, the reaction solution was poured into water (30 mL) and extracted with ethyl acetate (30 mL x 2). The combined organic layer was washed with brine, dried over Na2SO4and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 7.5% MeOH in DCM as eluent) to afford compound 14 (195 mg, 68%) as yellow oil. 67Attorney Docket No.: 090322-8001WO01

[0213] TLC: DCM / MeOH = 10 / 1, Rf(Compound 12) = 0.5, Rf(Compound 14) = 0.6, LC-MS: 1761.6 [M+1]+.

[0214] Preparation of Compound 15

[0215] To a solution of compound 14 (200 mg, 0.11 mmol, 1.0 eq) was added HCOOH (5 mL) at rt. The reaction mixture was stirred at rt for 2 h. After completion of the reaction, the mixture was concentrated under reduced pressure to afford compound 15 (180 mg, 93%) as yellow oil.

[0216] TLC: DCM / MeOH = 10 / 1, Rf(Compound 14) = 0.6, Rf(Compound 15) = 0.5, LC-MS: 1705.0 [M+1]+.

[0217] Preparation of Compound 16

[0218] To a solution of compound 15 (180 mg, 0.10 mmol, 1.0 eq) in DMF (5 mL) was added piperidine (45 mg, 0.52 mmol, 5.0 eq) at rt. The reaction mixture was stirred at rt for 2 h. After completion of the reaction, the mixture was concentrated under reduced pressure to afford compound 16 (120 mg, 76%) as yellow solid.

[0219] TLC: DCM / MeOH = 10 / 1, Rf(Compound 15) = 0.5, Rf(Compound 16) = 0.6, LC-MS: 1483.3 [M+1]+.

[0220] Preparation of Linker-Exatecan 1 (LP-1) 68Attorney Docket No.: 090322-8001WO01

[0221] To a solution of compound 16 (55 mg, 0.04 mmol, 1.0 eq) in DMF (2 mL) were added TEA (8 mg, 0.07 mmol, 2.0 eq) and compound 17 (38 mg, 0.07 mmol, 2.0 eq) at rt. The reaction mixture was stirred at rt for 3 h. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 8.5% MeOH in DCM as eluent) to afford LP-1 (22.7 mg, 32%) as white solid.

[0222] TLC: DCM / MeOH = 10 / 1, Rf(Compound 16) = 0.5, Rf(LP-1) = 0.6, LC- MS: 1881.0 [M+1]+.

[0223] 1H NMR (400 MHz, DMSO) d 12.10 (s, 1 H), 9.90 (s, 1 H), 8.17 – 8.15 (m, 1 H), 8.03 – 7.97 (m, 3 H), 7.95 – 7.93 (m, 1 H), 7.78 – 7.75 (m, 2 H), 7.57 – 7.55 (m, 2 H), 7.33 – 7.31 (m, 2 H), 7.27 (s, 1 H), 6.96 (s, 2 H), 6.48 (s, 1 H), 5.41 (s, 1 H), 5.25 (s, 3 H), 5.04 (s, 2 H), 4.36 – 4.25 (m, 1 H), 4.17 – 4.14 (m, 2 H), 3.57 – 3.55 (m, 7 H), 3.55 – 3.46 (m, 59 H), 3.38 – 3.36 (m, 3 H), 3.18 – 3.17 (m, 2 H), 3.12 – 3.09 (m, 3 H), 2.46 – 2.38 (m, 10 H), 2.26 – 2.15 (m, 4 H), 2.13 – 1.83 (m, 4 H), 2.13 – 1.83 (m, 4 H), 1.27 – 1.25 (m, 3 H), 0.86 – 0.78 (m, 9 H). 69Attorney Docket No.: 090322-8001WO01 Example 2 Synthesis of linker-exatecan 2 (LP-2)

[0225] To a solution of compound 1 (2.0 g, 6.60 mmol, 1.0 eq) in DCM (20 mL) were added HOSu (0.76 g, 6.60 mmol, 1.0 eq), DCC (1.36 g, 6.60 mmol, 1.0 eq) at 0 ℃. The mixture was stirred at room temperature for 16 h. Then the solution was concentrated in vacuo to give compound 2 (2.8 g, crude) as off white solid.

[0226] TLC: PE / EA = 1 / 1, Rf(Compound 1) = 0.1, Rf(Compound 2) = 0.5, LC-MS: 401.1 [M+H]+.

[0227] Preparation of Compound 4 70Attorney Docket No.: 090322-8001WO01

[0228] To a solution of compound 3 (929 mg, 5.49 mmol, 1.0 eq), DIEA (2.1 g, 16.5 mmol, 3.0 eq) in DMF (22 mL) was added compound 2 (2.2 g, crude) at 0 ℃. The mixture was stirred at room temperature overnight. The mixture was purified by reversed phase column (H2O / CAN = 85 / 15) to give compound 4 (1.5 g, 60%) as white solid.

[0229] LC-MS: 452.9 [M-H]-.

[0230] Preparation of Compound 5

[0231] To a solution of compound int-1 (compound 9 in Example 1, 500 mg, 0.66 mmol, 1.0 eq), compound 4 (753 mg, 1.66 mmol, 2.5 eq) and HATU (378 mg, 0.99 mmol, 1.5 eq) in DMF (5 mL) was added DIEA (257 mg, 1.99 mmol, 3.0 eq) at 0 ℃. The mixture was stirred at room temperature for 2 h. The mixture was purified by reversed phase column (H2O / CAN = 65 / 35) to give compound 5 (686 mg, 87%) as light yellow solid.

[0232] TLC: DCM / MeOH = 10 / 1, Rf(nt-1) = 0.3, Rf(Compound 5) = 0.1, LC-MS: 1191.7 [M+H]+.

[0233] Preparation of Compound 671Attorney Docket No.: 090322-8001WO01

[0234] Compound 5 (686 mg, 0.58 mmol, 1.0 eq) in formic acid (5 mL) at room temperature, the mixture was stirred at room temperature for 2 h. The mixture was purified by reversed phase column (H2O / CAN = 50 / 50) to give compound 6 (526 mg, 84%) as light yellow solid.

[0235] LC-MS: 1091.6 [M+H]+.

[0236] Preparation of Compound 7

[0237] To a solution of compound 6 (236 mg, 0.22 mmol, 1.0 eq) in ACN (3 mL) were added sodium bicarbonate (36 mg, 0.4 mmol, 2.0 eq), H2O (3 mL) and Fmoc-Osu (88 mg, 0.26 mmol, 1.2 eq) at room temperature. The mixture was stirred at room temperature for 1 h. The mixture was purified by reversed phase column (H2O / CAN = 75 / 25) to give compound 7 (222 mg, 78%) as off white solid.

[0238] LC-MS: 1313.4 [M+H]+.

[0239] Preparation of Compound 8

[0240] Compound 7 (410 mg, 0.31 mmol, 1.0 eq) was added in formic acid (10 mL) at room temperature, and the mixture was stirred at room temperature for 5 h. The mixture was purified by reversed phase column (H2O / CAN = 55 / 45) to give compound 8 (330 mg, 84%) as yellow solid.

[0241] LC-MS: 1257.2 [M+H]+.

[0242] Preparation of Compound 10 72Attorney Docket No.: 090322-8001WO01

[0243] To a solution of compound 8 (330 mg, 0.26 mmol, 1.0 eq), compound 9 (195 mg, 0.29mmol, 1.1 eq) and HATU (150 mg, 0.39 mmol, 1.5 eq) in DMF (4 mL) was added DIEA (102 mg, 0.79 mmol, 3.0 eq) at 0 ℃. The mixture was stirred at room temperature for 1 h. The mixture was purified by reversed phase column (H2O / CAN = 60 / 40) to give compound 10 (450 mg, 90%) as brown solid.

[0244] LC-MS: 1912.5 [M+H]+.

[0245] Preparation of Compound 11

[0246] Compound 10 (280 mg, 0.15 mmol, 1.0 eq) was added in formic acid (35 mL) at room temperature, and the mixture was stirred at room temperature for 1 h. The mixture was concentrated in vacuo to give compound 11 (270 mg, crude) as brown solid.

[0247] LC-MS: 1856.3 [M+H]+.

[0248] Preparation of Compound 12

[0249] To a solution of compound 11 (270 mg, crude) in DMF (3.5 mL) was added piperidine (62 mg, 0.73 mmol, 5.0 eq) at room temperature. The mixture was stirred at room temperature for 1 h. The mixture was purified by reversed phase column (H2O / CAN = 65 / 35) to give compound 12 (197 mg, 82%) as light yellow solid. 73Attorney Docket No.: 090322-8001WO01

[0250] LC-MS: 1634.4 [M+H]+.

[0251] Preparation of Linker-Exatecan 2 (LP-2)

[0252] To a solution of compound 12 (65 mg, 0.04 mmol, 1.0 eq), TEA (12 mg, 0.12 mmol, 3.0 eq) in DMF (2 mL) was added compound 13 (41 mg, 0.08 mmol, 2.0 eq) at room temperature. The mixture was stirred at room temperature for 4 h. The mixture was purified by reversed phase column (H2O / CAN = 55 / 45) to give LP-2 (24 mg, 30%) as yellow solid.

[0253] LC-MS: 1016.9 [M / 2+H]+.

[0254] 1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.08 – 7.92 (m, 7H), 7.74 (d, J = 11.2 Hz, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 6.96 (s, 2H), 5.42 (s, 2H), 5.26 (s, 3H), 5.09 – 4.97 (m, 2H), 4.63 – 4.62 (m, 1H), 4.26 – 4.17 (m, 2H), 3.95 (t, 1H), 3.63 – 3.53 (m, 9H), 3.46 – 3.43 (m, 60H), 3.17 – 3.09 (m, 6H), 2.97 – 2.95 (m, 1H), 2.82 – 2.80 (m, 1H), 2.41 – 2.38 (m, 3H), 2.34 (s, 5H), 2.30 – 2.26 (m, 2H), 2.17 – 2.07 (m, 5H), 1.86 – 1.66 (m, 4H), 1.28 (d, J = 7.2Hz, 3H), 0.85 – 0.81 (m, 9H) Example 3 Synthesis of linker-exatecan 3 (LP-3)74Attorney Docket No.: 090322-8001WO01

[0255] Preparation of Compound 3

[0256] To a solution of compound 1 (2.0 g, 7.5 mmol, 1.0 eq) in DMF (10 mL) were added compound 2 (1.3 g, 7.5 mmol, 1.0 eq) and DIEA (1.9 g, 15 mmol, 2.0 eq) at rt. The reaction mixture was stirred at rt for 2 h. After completion of the reaction (monitored by LCMS). The mixture was performed to afford compound 3 (2.5 g, crude) without purification.

[0257] LC-MS: 318.8 [M-H]-.

[0258] Preparation of Compound 5

[0259] To a solution of compound 3 (2.5 g, crude) in DMF (10 mL) were added compound 4 (1.3 g, 4.2 mmol, 1.0 eq), DIEA (0.8 g, 6.24 mmol, 1.5 eq) and T3P (50% in DMF) (8.0 g, 12.5 mmol, 3.0 eq) at rt. The reaction mixture was stirred for 3 h at rt. The reaction was monitored by LCMS. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (DCM / MeOH = 92 / 8) to give compound 5 (700 mg, 15% for two steps) as yellow oil.

[0260] LC-MS: 318.8 [M-H]-.

[0261] Preparation of Compound 6

[0262] To a solution of compound 5 (360 mg, 0.57 mmol, 1.0 eq) was added HCOOH (4 mL) at rt. The reaction mixture was stirred at rt for 16 h. The reaction mixture was 75Attorney Docket No.: 090322-8001WO01 monitored by LCMS. After completed, the mixture was purified by reversed phase column (H2O / CAN = 80 / 20) to give compound 6 (240 mg, 74%) as yellow oil.

[0263] LC-MS: 567.9 [M+H]+.

[0264] Preparation of Compound 7

[0265] To a solution of compound 6 (120 mg, 0.22 mmol, 1.0 eq) in DCM (4 mL) were added DCC (65 mg, 0.32 mmol, 1.5 eq) and HOSu (33 mg, 0.28 mmol, 1.3 eq) at rt. The reaction mixture was stirred at rt for 4 h. The reaction was monitored by LCMS. The mixture was purified by reversed phase column (H2O / CAN = 73 / 27) to give compound 7 (60 mg, 43%) as yellow oil.

[0266] LC-MS: 665.0 [M+1]+.

[0267] Preparation of Linker-Exatecan 3 (LP-3)

[0268] To a solution of compound 8 (compound 12 in Example 2, 90 mg, 0.06 mmol, 1.0 eq) in DMF (2 mL) were added compound 7 (60 mg, 0.09 mmol, 1.5 eq) and TEA (12 mg, 0.12 mmol, 2.0 eq) at rt. The reaction mixture was stirred at rt for 2 h. The reaction was monitored by LCMS. The mixture was purified by reversed phase column (H2O / CAN = 78 / 22) to give LP-3 (32.8 mg, 27%) as yellow solid.

[0269] LC-MS: 1016.9 [M / 2+1]+. 76Attorney Docket No.: 090322-8001WO01

[0270] 1H NMR (400 MHz, DMSO) d 9.91 (s, 1H), 8.18 (d, J = 6.8 Hz, 1 H), 8.03 (d, J = 7.0 Hz, 2 H), 7.97 ( d, J = 8 Hz, 1 H), 7.88 - 7.86 (m, 1 H), 7.79 (d, J = 8.4 Hz, 1 H), 7.7 - 7.72 (m, 2 H), 7.56 (d, J = 8 Hz, 2 H), 7.32 (d, J = 6.8 Hz, 2 H), 7.27 (s, 1 H), 6.95 (s, 2 H), 5.41 (s, 2 H), 5.24 (s, 2 H), 5.03 (s, 2 H), 4.35 - 4.32 (m, 1 H), 4.24 - 4.25 (m, 1 H), 4.16 - 4.12 (m, 2 H), 3.57 - 3.30 (m, 64 H), 3.18 - 3.11 (m, 6 H), 2.74 - 2.63 (m, 2 H), 2.40 (s, 2 H), 2.34 (s, 6 H), 2.32 - 2.30 (m, 4 H), 2.92 - 1.85 (m, 4 H), 1.83 - 1.77 (m, 1 H), 1.26 (d, J = 7.2 Hz, 3 H), 0.85 - 0.78 (m, 9 H). Example 4 Synthesis of linker-exatecan 4 (LP-4)

[0271] Preparation of Compound 2

[0272] To a solution of compound 1 (1.0 g, 7.69 mmol, 1.0 eq) in DCM (10 mL) were added DMF (2 d) and (COCl)2(5.85 g, 46.12 mmol, 6.0 eq) at room temperature. The reaction was stirred at room temperature overnight under N2. The reaction was concentrated in vacuo to give compound 2 (950 mg, crude) as brown solid.

[0273] TLC: DCM / MeOH = 30 / 1, Rf(Compound 1) = 0.01, Rf(Compound 2) = 0.6, LC-MS: 149.0 [M+H]+. 77Attorney Docket No.: 090322-8001WO01

[0274] Preparation of Compound 4

[0275] To a solution of compound 3 (683 mg, 4.26 mmol, 1.0 eq) in DCM (10 mL) were added TEA (1.3 g, 12.79 mmol, 3.0 eq) and compound 2 (950 mg, crude) at rt. The reaction was stirred at rt for 1 h. Then water (50 mL) was added and the solution was extracted with DCM (50 mL × 3). The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (100 - 200 silica gel, 5% MeOH in DCM as eluent) to give compound 4 (554 mg, 47%) as yellow solid.

[0276] TLC: DCM / MeOH = 20 / 1, Rf(Compound 2) = 0.85, Rf(Compound 4) = 0.55, LC-MS: 273.0 [M+H]+.

[0277] Preparation of Compound 5

[0278] A solution of compound 4 (554 mg, 2.03 mmol, 1.0 eq) in HCl / EA (10 mL) was stirred at rt for 2 h. The reaction mixture was concentrated in vacuo to give compound 5 (416 mg, 98%) as light yellow solid.

[0279] TLC: DCM / MeOH = 20 / 1, Rf (Compound 4) = 0.55, Rf (Compound 5) = 0.01, LC-MS: 173.1 [M+H]+.

[0280] Preparation of Compound 8 78Attorney Docket No.: 090322-8001WO01

[0281] To a solution of compound 6 (1.8 g, 5.60 mmol, 1.0 eq) in DCM (20 mL) were added DIEA (1.4 g, 11.20 mmol, 2.0 eq) and compound 7 (1.47 g, 7.28 mmol, 1.3 eq) at 0 °C. The reaction was stirred at rt for 2 h. Then water (100 mL) was added and the solution was extracted with DCM (100 mL × 3). The combined organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo The residue was purified by silica gel chromatography (100 - 200 silica gel, 70% EA in DCM as eluent) to give compound 8 (2.2 g, 80%) as colourless oil.

[0282] TLC: PE / EA = 1 / 1, Rf(Compound 7) = 0.7, Rf(Compound 8) = 0.3, LC-MS: 509.0 [M+Na]+.

[0283] Preparation of Compound 9

[0284] To a solution of compound 5 (416 mg, 1.99 mmol, 1.1 eq) in DMF (4 mL) were added DIEA (701 mg, 5.43 mmol, 3.0 eq) and compound 8 (881 mg, 1.81 mmol, 1.0 eq) at 0 °C. The reaction was stirred at rt for 1 h. Then water (100 mL) was added and the solution was extracted with DCM (50 mL × 3). The combined organic phase was washed with saturated NaHCO3 aqueous and brine, dried over sodium sulfate, filtered and concentrated in vacuo The residue was purified by silica gel column chromatography (100 - 200 silica gel, 7% MeOH in DCM as eluent) to give compound 9 (720 mg, 76%) as off white solid.

[0285] TLC: DCM / MeOH = 10 / 1, Rf(Compound 8) = 0.7, Rf(Compound 9) = 0.5, LC-MS: 520.4 [M+H]+.

[0286] Preparation of Compound 10 79Attorney Docket No.: 090322-8001WO01

[0287] A solution of compound 9 (720 mg, 1.38 mmol, 1.0 eq) in HCl / dioxane (10 mL) was stirred at rt for 2 h. The reaction mixture was concentrated in vacuo to give compound 10 (680 mg, crude) as yellow oil.

[0288] TLC: DCM / MeOH = 10 / 1, Rf(Compound 9) = 0.5, Rf(Compound 10) = 0.1, LC-MS: 520.4 [M+H]+.

[0289] Preparation of Compound 11

[0290] To a solution of compound 10 (680 mg, crude) in DCM (10 mL) were added DCC (701 mg, 3.4 mmol) and HOSu (794 mg, 6.90 mmol) at 0 °C. The reaction was stirred at rt overnight. The reaction was filtered and the filtrate was concentrated in vacuo. The residue was purified by reversed phase column (20% ACN in H2O as eluent) to give compound 11 (400 mg, 51% for two steps) as colourless oil.

[0291] TLC: DCM / MeOH = 10 / 1, Rf(Compound 10) = 0.1, Rf(Compound 11) = 0.15, LC-MS: 561.2 [M+H]+.

[0292] Preparation of LP-4

[0293] To a solution of compound 12 (30 mg, 0.018 mmol, 1.0 eq) in DMF (1 mL) were added TEA (5.6 mg, 0.055 mmol, 3.0 eq) and compound 11 (38 mg, 0.068 mmol, 3.7 eq) at rt. The reaction was stirred at rt for 3 h. The residue was purified by reversed phase column (55% ACN in H2O as eluent) to give LP-4 (30 mg, 78%) as yellow solid.

[0294] TLC: DCM / MeOH = 10 / 1.

[0295] Rf(Compound 12) = 0.4, Rf(Compound 11) = 0.15, Rf(LP-4) = 0.45. 80Attorney Docket No.: 090322-8001WO01

[0296] LC-MS: 1040.3 [M+H] / 2+.

[0297] 1H NMR (400 MHz, DMSO-d6) d 9.47 (s, 1H), 8.59 (t, 1H), 8.27 (d, J = 7.6 Hz, 1H), 8.10 – 7.93 (m, 5H), 7.75 (d, J = 10.8 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.28 (s, 1H), 6.96 (d, J = 15.6 Hz, 1H), 6.55 (d, J = 15.6 Hz, 1H), 6.02 (s, 1H), 5.44 (s, 2H) 5.27 (s, 3H), 5.10 – 5.01 (m, 2H), 4.67 – 4.62 (m, 1H), 4.27 – 4.17 (m, 2H), 3.97 (dd, J = 7.2 Hz, 4.8 Hz, 1H), 3.70 (s, 3H), 3.58 – 3.48 (m, 30H), 3.44 – 3.32 (m, 26H), 3.20 – 2.97 (m, 16H), 2.85 – 2.80 (m, 2H), 2.45 – 2.48 (m, 5H), 2.41 (t, 2H), 2.38 – 2.31 (m, 5H), 2.16 – 2.07 (m, 5H), 1.91 – 1.67 (m, 4H), 1.30 (d, J = 7.2 Hz, 3H), 0.87 – 0.81 (m, 9H) Example 5 Synthesis of linker-MMAE 1 (LP-5)81Attorney Docket No.: 090322-8001WO01

[0299] To a solution of compound 1 (2.0 g, 4.87 mmol, 1.0 eq) in DCM / MeOH (30 / 15 mL) were added compound 1 (1.2 g, 9.74 mmol, 2.0 eq) and EEDQ (2.4 g, 9.74 mmol, 2.0 eq) at rt. The reaction mixture was stirred at rt for 24 h. After completion of the reaction (monitored by TLC), the reaction was filtered to afford compound 3 (1.9 g, 75%) as white solid.

[0300] TLC: DCM / MeOH = 10 / 1, Rf(Compound 1) = 0.1, Rf(Compound 3) = 0.6, LC-MS: 516.3 [M+H]+.

[0301] Preparation of Compound 5

[0302] To a solution of compound 3 (1.8 g, 3.49 mmol, 1.0 eq) in DMF (10 mL) were added Py (827 mg, 10.47 mmol, 3.0 eq) and compound 4 (1.4 g, 6.98 mmol, 2.0 eq) at 0oC. The reaction mixture was stirred at rt for 4 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 80% THF in DCM as eluent) to afford compound 5 (0.9 g, 70%) as yellow solid.

[0303] TLC: PE / EA = 1 / 1, Rf(Compound 3) = 0.2, Rf(Compound 5) = 0.5, LC-MS: 681.6 [M+H]+.

[0304] Preparation of Compound 7

[0305] To a solution of compound 5 (900 mg, 1.32 mmol, 1.0 eq) in DMF / Py (10 mL / 2 mL) was added compound 6 (949 mg, 1.32 mmol, 5.0 eq) at 0oC. The reaction mixture was stirred at rt for 16 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 10% MeOH in DCM as eluent) to afford compound 7 (550 mg, 49%) as yellow solid. 82Attorney Docket No.: 090322-8001WO01

[0306] TLC: PE / EA = 1 / 1, Rf(Compound 5) = 0.5, Rf(Compound 7) = 0.1, LC-MS: 1259.8 [M+1]+.

[0307] Preparation of Compound 8

[0308] To a solution of compound 7 (550 mg, 0.43 mmol, 1.0 eq) in DMF (2 mL) was added piperidine (186 mg, 2.18 mmol, 5.0 eq) at rt. The reaction mixture was stirred at rt for 2 h. After completion of the reaction, the solution was concentrated under reduced pressure. The residue was purified by column chromatography (100- 200 silica gel, 10.5% MeOH in DCM as eluent) to afford compound 8 (450 mg, 68%) as yellow oil.

[0309] TLC: DCM / MeOH = 10 / 1, Rf(Compound 7) = 0.6, Rf(Compound 8) = 0.5, LC-MS: 1037.5 [M+1]+.

[0310] Preparation of Compound 10

[0311] To a solution of compound 8 (700 mg, 0.67 mmol, 1.0 eq) in DMF (5 mL) were added HATU (385 mg, 1.01 mmol, 1.5 eq), compound 10 (315 mg, 0.74 mmol, 1.1 eq) and DIEA (435 mg, 3.37 mmol, 5.0 eq) at 0oC. The reaction mixture was stirred for 1 h at rt. After completion of the reaction, the solution was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 11% MeOH in DCM as eluent) to afford compound 10 (720 mg, 73%) as yellow solid.

[0312] TLC: DCM / MeOH = 10 / 1, Rf(Compound 8) = 0.5, Rf(Compound 10) = 0.2

[0313] LC-MS: 1444.9 [M+1]+.

[0314] Preparation of Compound 11 83Attorney Docket No.: 090322-8001WO01

[0315] To a solution of compound 10 (720 mg, 0.49 mmol, 1.0 eq) in DCM (2 mL) was added HCOOH (458 mg, 9.97 mmol, 20 eq) at rt. The reaction mixture was stirred at rt for 2 h. After completion of the reaction, the solution was concentrated under reduced pressure. The residue was purified by reversed phase column (25% ACN in H2O as eluent) to give compound 11 (250 mg, 35%) as white solid.

[0316] TLC: DCM / MeOH = 10 / 1, Rf(Compound 10) = 0.2, Rf(Compound 11) = 0.1

[0317] LC-MS: 1416.8 [M+1]+.

[0318] Preparation of Compound 13

[0319] To a solution of compound 11 (250 mg, 0.17 mmol, 1.0 eq) in DMF (2 mL) were added HATU (100 mg, 0.26 mmol, 1.5 eq), compound 10 (130 mg, 0.19 mmol, 1.1 eq) and DIEA (114 mg, 0.88 mmol, 5.0 eq) at 0oC. The reaction mixture was stirred for 1 h at rt. After completion of the reaction, the solution was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 silica gel, 101% MeOH in DCM as eluent) to afford compound 13 (350 mg, 95%) as yellow solid.

[0320] TLC: DCM / MeOH = 10 / 1, Rf(Compound 11) = 0.1, Rf(Compound 13) = 0.2

[0321] LC-MS: 1037.2 [M+1]+.

[0322] Preparation of Compound 1484Attorney Docket No.: 090322-8001WO01

[0323] To a solution of compound 13 (350 mg, 0.16 mmol, 1.0 eq) in DCM (2 mL) was added HCOOH (155 mg, 3.37 mmol, 20 eq) at rt. The reaction mixture was stirred at rt for 2 h. After completion of the reaction, the solution was concentrated under reduced pressure to afford compound 14 (320 mg, 94%) as yellow oil.

[0324] TLC: DCM / MeOH = 10 / 1, Rf(Compound 13) = 0.2, Rf(Compound 14) = 0.1

[0325] LC-MS: 1037.2 [M+1]+.

[0326] Preparation of Compound 15

[0327] To a solution of compound 14 (320 mg, 0.16 mmol, 1.0 eq) in MeOH (2 mL) was added K2CO3(66 mg, 0.47 mmol, 3.0 eq) at rt. The reaction mixture was stirred at rt for 2 h. The residue was purified by reversed phase column (25% ACN in H2O as eluent) to give compound 15 (200 mg, 80%) as white solid.

[0328] TLC: DCM / MeOH = 10 / 1, Rf(Compound 14) = 0.1, Rf(Compound 15) = 0.05.

[0329] LC-MS: 1766.1 [M+1]+.

[0330] Preparation of linker-MMAE 1 (LP-5)

[0331] To a solution of compound 15 (40 mg, 0.022 mmol, 1.0 eq) in DMF (2 mL) were added TEA (5 mg, 0.045 mmol, 2.0 eq) and compound 16 (23 mg, 0.034 mmol, 85Attorney Docket No.: 090322-8001WO01 1.5 eq) at rt. The reaction mixture was stirred at rt for 4 h. The residue was purified by reversed phase column (30% ACN in H2O as eluent) to give LP-5 (12.9 mg, 24%) as white solid.

[0332] TLC: DCM / MeOH = 10 / 1, Rf(Compound 16) = 0.1, Rf(LP-5) = 0.2, LC-MS: 1158.5 [1 / 2M+1]+

[0333] 1H NMR (400 MHz, DMSO) d 9.87 (s, 1 H), 8.15 – 7.92 (m, 4 H), 7.83 – 7.71 (m, 6 H), 7.54 – 7.26 (m, 8 H), 6.94 (s, 2 H), 5.01 (m, 2 H), 4.46 – 4.14 (m, 8 H), 3.96 – 3.95 (m, 2 H), 3.56 – 3.47 (m, 11 H), 3.46 – 3.36 (m, 42 H), 3.33 – 2.84 (m, 19 H), 2.84 – 2.47 (m, 6 H), 2.47- 2.34 (m, 12 H), 2.34 – 1.26 (m, 16 H), 1.26 – 0.98 (m, 5 H), 0.98 – 0.81 (m, 7 H), 0.81 – 0.73 (m, 26 H). II. Synthesis and characterization of ADC Example 6 General procedure for conjugation of linker-payload having closed- ring maleimide with antibody and purification

[0334] Anti-Her2 antibody Trastuzumab (MCE cat# HY-P9907), humanized PSMA antibody (AhJ591), or anti-Claudin-6 antibody (“hH1L1”, the humanized anti-CLDN6 antibody H1L1 disclosed in CA3078218A1) in 1XPBS buffer (4 mg / mL, pH 7.4) was added with 12 molar excess of TCEP (tris(2-carboxyethyl)phosphine) (Cat# P1021-10). The mixture was incubated at 37 °C for 2 hours at 120 rpm with a benchtop rotating shaker (Serial# 300100524). After that, 10 molar excess of linker-payload (LP-1, LP-2, LP-3 or LP-5) to antibody were added, and the reaction mixture was left at room temperature for 1 hour to reach complete conjugation. At the end of the reaction, 10- fold molar excess of NAC was added to quench the reaction. The conjugation mixture was then purified by PD-10 (cat#: 17-0851-01, Cytiva) and gel filtration by amicon concentrator (Cat#UFC803024, 50 kD cutoff) or FPLC (Superdex 200 Increase 10 / 300 GL, Cytiva) with 1XPBS, pH 7.4 (Cat#21-040-CM) buffer to remove excess small molecules. ADCs were characterized by SEC and HIC. 86Attorney Docket No.: 090322-8001WO01 Example 7 General procedure for conjugation of linker-payload having open- ring maleimide with antibody and purification

[0335] To make ring-opening linker payload ADC, the humanized antibody in 4 mg / mL was buffer exchanged to 50 mM Tris, pH8, 2 mM EDTA, 100 mM NaCI,14 molar excess of TCEP, and 20 molar excess of linker-payload (LP-4) were added simultaneously to the reaction mixture. The reaction was left at RT for overnight (16 hrs). At the end of the reaction, 10-fold molar excess of NAC was added to quench the reaction. The conjugation mixture was then purified by PD-10 (cat#: 17-0851-01, Cytiva) and gel filtration by amicon concentrator (Cat#UFC803024, 50 kD cutoff) or FPLC (Superdex 200 Increase 10 / 300 GL, Cytiva) with 1XPBS, pH 7.4 (Cat#21-040- CM) buffer to remove excess small molecules. ADCs were characterized by SEC and HIC. Example 8 DAR determination

[0336] To determine the drug to antibody ratio (DAR), HIC-HPLC was used. ADC was filtered by 0.2 um filter spinning tube (Costar #8160). 14 μL, 1 mg / mL of ADC in 1Xpbs, pH 7.4 was injected over a TSKgel Butyl-NPR Column, 4.6mmx x 10cm, 2.5µm (TOSOH BIOSCIENCE LLC) running with an Agilent HPLC system (1260 Infinity II Bio Flexible Pump). The ADC was eluted as a reduction of gradient elution concentrations of NH4SO4 in buffer A (25 mM sodium phosphate, 1.5 M NH4SO4, pH 7), to completely buffer B (25mM sodium phosphate; 20% IPA, pH 7) with a flow rate 0.4 mL / min within 20 min. The column was then further washed with buffer B for 20 min and equilibrated with buffer A for continuous running. Example 9 ADC Concentration determination

[0337] To determine the concentration, the extinction coefficient of the linker payload at 280 nm was first measured by lambert's law. The total extinction coefficient of ADC was calculated by the sum of antibody extinction coefficients and the total linker payload extinction coefficient at 280 nm. The ADC concentration was calculated by using the reading at 280 nm from UV-vis spectroscopy divide the total ADC extinction coefficient multiply by 106μM. 87Attorney Docket No.: 090322-8001WO01 Example 10 AnSec

[0338] AnSec was utilized to determine potentially high molecular weight and aggregation species of freshly made ADC conjugates and ADC under stressed conditions. AnSec was run with 90% PBS, 10% acetonitrile, with a flow rate 0.4 ml / min, 30 min.

[0339] The ADCs obtained are listed in Table 1 below. Results of SEC and HIC analysis of the ADCs obtained are shown in Figures 1 to 7. Table 1Example 11 Stability comparison of ADCs obtained from linker-payload with closed and open-ring maleimide

[0340] To determine the stability of open-ring maleimide and closed-ring maleimide ADCs, 2 mg / mL DAR8 ADC-2 (AhJ591-LP2) and ADC-4 (AhJ591-LP4) in 1xPBS, were incubated with 1XPBS, pH 7.4, 25 mg / mL human serum albumin (CAS: 70024- 90-7, sigma), or 100 molar excess NAC (N-Acetyl-L-cysteine, Cat#616-91-1) at 37 °C. At each time point (0 hr, 24 hr, 48 hr, 72 hr, 168 hr, 336 hr), 50 μL were drawn and frozen at -80 °C. At the end of the incubation, all samples were filtered and subjected to HIC-HPLC. Figures 8A-8C and 9A-9C show the linker payload stability of ADC- 6 and ADC-7. As shown in Figures 8A-8C, no apparent cleavage or instability due to retro Michael addition or deconjugation was identified, which demonstrated superior 88Attorney Docket No.: 090322-8001WO01 stability of maleimide linkage. As shown in Figures 9A-9C, no apparent cleavage or instability due to retro Michael addition or deconjugation was identified, which is consistent with the stability of the open ring maleimide. This indicates that the compounds for producing conjugates of the present disclosure provided improved stability for ADCs with maleimide linkage, which would be otherwise instable in known ADCs due to retro-Michael elimination. Without being bound to any specific theory, this is possibly due to the shielding effect of the hydrophilic side chain in the compounds of the present disclosure. Example 12 Stability test under stressed condition

[0341] ADC-5 of the present disclosure and reference ADC (Trastuzumab-VC- MMAE) were stored at 40 °C or -80 °C in PBS buffer at 2 mg / ml for two weeks. Samples were analyzed by AnSec, and the results are shown in Figures 10A-10B. As shown in Figures 10A-10B, ADC-5 is much more stable than Trastuzumab-VC-MMAE under stressed condition. This illustrates that the compounds for producing conjugates of the present disclosure provided improved stability even under stressed condition. Without being bound to any specific theory, this is possibly due to the shielding effect of hydrophilic side chain in the compounds of the present disclosure. III. Biological Assay Example 13 Cell culture

[0342] HER2+ cell lines, NCI-N87, HCC-1954 and SKBR-3, and PSMA+ cell line LnCAP (all from ATCC) were cultured in RPMI1640 (Corning 15-040-CM), Claudin 6+ cell line PA-1 (from ATCC) was cultured in EMEM (ATCC 30-2003), HER2- cell line A549 (from ATCC) was cultured in F-12K Medium (ATCC 30-2004), and Primary Corneal Epithelial Cells (from ATCC) were cultured in Corneal Epithelial Cell Basal Medium (ATCC PCS-700-030) plus one Corneal Epithelial Cell Growth Kit (ATCC PCS-700-040). All media were supplemented with GlutaMAX® (2 mM, ThermoFisher Scientific 3505006), penicillin-streptomycin (100 units / mL, ThermoFisher Scientific 15140122), and 10% fetal bovine serum (ATCC 30-2021). Cells were cultured at 37 °C under 5% CO2and passaged before becoming fully confluent up to 10 passages. All cell lines were periodically tested for mycoplasma contamination. 89Attorney Docket No.: 090322-8001WO01 Example 14 Cell viability assay

[0343] Cells (A549, SKBR-3 or LnCAP) were seeded in a culture-treated black 96- well assay plate (Fisher Scientific, 10285421) (2,000 cells per well in 50 μL culture medium) and incubated at 37 °C under 5% CO2for 24 h. Serially diluted ADC samples (50 μL) were added to each well and the plate was incubated at 37 °C for 120 h, then, incubated at room temperature. CellTiter Glo reagent (Promega, G7572) (100 μL) was added to each well, and incubated the plates for another 30 min. The luminescence of each well was detected using an EnVision Plate Reader. GraphPad Prism software (Version 10.2.3) was used for the calculation of IC50 values. All assays were performed in duplicate. Table 2 and Figure 11 show the results of test ADC samples ADC-1, ADC-2 and ADC-3 provided herein comprising trastuzumab and reference T-Dxd (Enhertu). Table 3 and Figure 12 show the results of test ADC samples ADC-6 and ADC-7 provided herein comprising AhJ591. Table 2Table 3

[0344] As shown in Table 2 and Figure 11, ADC-1, ADC-2 and ADC-3 showed similar potency to T-Dxd on SKBR3 cells. As a negative control, ADC-1 did not show any cytotoxicity on HER2- cell line (A549), indicating the specificity of the ADCs of the present disclosure.

[0345] As shown in Table 3 and Figure 12, ADC-6 and ADC-7 showed potent cytotoxicity on LnCAP cells. PSMA antibody alone did not affect the viability of LnCAP cells. 90Attorney Docket No.: 090322-8001WO01

[0346] These results indicate that the ADCs generated from the compounds of the present disclosure not only had good targeting specificity, but also showed outstanding efficacy. Example 15 Binding and internalization assay

[0347] The binding of naked antibody or ADC was detected using Fluor 488 goat anti- human IgG secondary antibody (Jackson ImmunoResearch 109-545-003). Briefly, SKBR3 cells suspended in FACS buffer (PBS / 0.5% / BSA 2.5 mM EDTA) were incubated with naked Ab or ADC at 4°C for 30 min. Cells were washed three times with FACS buffer and fluorescence-labeled secondary antibody was added and incubated at 4°C for 30 min. The FACS data were collected using a CytoFlex LX (Beckman Coulter Life Sciences). A control sample without primary antibody and another sample with isotype control antibody were used. Mean fluorescence intensity (MFI) was calculated and plotted by GraphPad Prism software.

[0348] For the internalization of naked antibody or ADC in vitro, antibody or ADC was labelled with Fabfluor-pH kits according to the instruction (Human Fabfluor-pH Red Antibody Labeling Reagent, SARTORIUS 4722). SKBR3 cells were plated in 96 well plate and incubated at 37°C for 4 hrs. Serially diluted naked antibody or ADC was added and incubated for 3 hrs. The cells were then resuspended in PBS and the FACS data were collected using a CytoFlex LX. Mean fluorescence intensity (MFI) was calculated and plotted by GraphPad Prism software.

[0349] To test whether the linker helps reduce the non-specific internalization unrelated to the target, human primary corneal epithelial cells (ATCC, Cat# PCS-700- 010 ™) were seeded in 96 well plate and incubated at 37°C overnight. Naked antibody or ADC was added at the final concentration of 1μg / ml. The plate was incubated in Incucyte® S3 Live Cell Analysis System (Sartorius) and monitored for 48 hrs. The fluorescent intensity of the image was calculated and plotted by GraphPad Prism software.

[0350] Binding and internalization results for Trastuzumab or ADCs are shown in Figures 13, 14 and 15. Data showed that the conjugation did not affect the target- related binding and internalization of the antibody (Figures 13 and 14). However, the conjugation did reduce the non-specific internalization unrelated to the target (Figure 15). 91Attorney Docket No.: 090322-8001WO01 Example 16 Cytotoxicity under stressed condition

[0351] ADCs were stored at 40 °C or -80 °C in PBS buffer at 2mg / ml for three weeks. Samples were analyzed by AnSec and HIC. Cytotoxicity assay was performed as above mentioned and T-Dxd stored at 4 °C was used as a positive control. All assays were performed in duplicate.

[0352] ADC-1, ADC-2, and ADC-3 provided herein were also analyzed by HIC- HPLC and SEC.

[0353] Results of the cytotoxicity are shown in Table 4 and Figures 15-17. Results of HIC-HPLC and SEC analysis are shown in Figure 18.

[0354] Table 4

[0355] It can be seen from Table 4 and Figures 15-17 that ADC-1, ADC-2 and ADC- 3 maintained the cytotoxic activity with very minimal changes under 40 °C stressed condition, indicating that the ADCs of the present disclosure were very stable.

[0356] Figure 18 further shows that ADC-1, ADC-2 and ADC-3 maintained stable under stressed condition, suggesting the high stability of the ADCs of the present disclosure. Example 17 Plasma stability assay

[0357] ADCs were incubated at 37 °C in mouse plasma or human plasma at 0.25mg / ml for different time points. Samples were analyzed by LC-MS (Quantall) to test the released exatecan in the serum. GraphPad Prism software was used to plot the stability of the ADCs. Assays were performed in duplicate. Results are shown in Figures 19-20.

[0358] It is shown that ADC-1, ADC-2 and ADC-3 were stable in mouse and human plasma. ADC-2 showed slight payload release in mouse plasma, while all three ADCs 92Attorney Docket No.: 090322-8001WO01 barely released any free payload during the incubation in human plasma. This indicates that the ADCs of the present disclosure exhibited high stability. Example 18 Pharmacokinetic profile in rats

[0359] The test ADC was administered intravenously into groups of 3 male Sprague Dawley rats at 3mg / kg or 10mg / kg. Blood samples were collected at the following timepoints: 0.833, 1, 4, 24, 72, 168, 336, 504 and 672 hour. The plasma concentrations of total Ab and ADC were detected by ELISA. For the total Ab detection, ELISA plates were coated with Goat anti-human IgGs (SouthernBiotech, Cat# C4724-PN24). Detection was performed by using Human Her2-His protein (ACRO, HER2-H5225) and Mouse anti-his tag-HRP (SouthernBiotech, Cat# J1923- P504). For the ADC detection, ELISA plates were coated with mouse anti- DXD&Exatecan monoclonal antibody IgG1 (ACRO, Cat# DXD-M684). Detection was performed by using Goat anti-human IgG-HRP (SouthernBiotech, Cat# 2049-05). The plasma level of free payload was measured by liquid chromatography–tandem mass spectrometry (LC-MS / MS) using a triple quadrupole system.

[0360] The PK profile of ADC-1 was shown in Table 5 and Figure 22.

[0361] Table 5Data is presented as median (range) for Tmax values or mean ± SD for others. 93Attorney Docket No.: 090322-8001WO01 Example 19 Antitumor effect of ADCs in tumor models

[0362] In NCI-N87 tumor model, nude mice were implanted subcutaneously with NCI-N87 cells. When the tumor volume reached 150-2003mm, mice were treated once via intravenous injection of ADCs at a dose of 1mg / kg. As shown in Figure 23A, ADC-1 to ADC-5 showed anti-tumor efficacy.

[0363] In PA-1 tumor model, nude mice were implanted subcutaneously with PA-1 cells. Once the tumor volume reached 100-1503mm, mice were treated once weekly for two doses via intravenous injection of ADCs at a dose of 2mg / kg. As shown in Figure 23B, ADC-8 showed good anti-tumor efficacy. Example 20 Antitumor effect of ADC-1 and T-Dxd in NCI-N87 tumor model

[0364] Nude mice were implanted subcutaneously with NCI-N87 cells. Once the tumor volume reached 150-2003mm, mice were treated via intravenous injection with ADC-1, T-Dxd biosimilar or vehicle control at various doses (0.5 mg / kg, 1 mg / kg and 3 mg / kg). As shown in Figures 24A and 24B, ADC-1 demonstrated superior anti- tumor efficacy compared to T-Dxd, without causing significant bodyweight loss.

[0365] The foregoing description is considered as illustrative only of the principles of the present disclosure. Further, since numerous modifications and changes will be readily apparent to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown as described above. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the invention as defined by the claims that follow.

[0366] The words "comprise", "comprising", "include", "including", and "includes" when used in this specification and in the following claims are intended to specify the presence of stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. 94

Claims

Attorney Docket No.: 090322-8001WO01 WHAT CLAIMED IS:

1. A compound of Formula (I):or a pharmaceutically acceptable salt thereof, wherein: X is a linking moiety capable of forming a covalent bond with a targeting moiety; W is a polypeptide moiety; L1is an amino acid side chain residue; L2is a spacer moiety; Y is a hydrophilic end group; E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC); T is a therapeutic or a diagnostic agent moiety; n is an integer from 1 to 20; m is an integer from 0 to 5; p is an integer from 1 to 24; a is 0 or 1; and b is 0 or 1.

2. The compound of claim 1, wherein the compound is of Formula (IA), (IB) or (IC):1Attorney Docket No.: 090322-8001WO01wherein X, W, L1, L2, Y, E, T, n, m, and p are as defined in claim 1.

3. The compound of claim 1 or 2, wherein X is, or, wherein q is an integer from 1 to 10.

4. The compound of any one of claims 1-3, wherein W is selected from a peptide composed of 2-5 amino acids.

5. The compound of claim 4, wherein W is selected from Gly-Ala, Gly-Gly, Ala- Gly, Glu-Gly, Glu-Ala, Gly-Glu, Asp-Gly, Asp-Ala, Gly-Asp, Cit-Val, Ala-Val, Phe- Lys, Lys-Val, Val-Ala, Val-Cit, Phe-Gly, Ala-Ala, Val-Cys, Val-Gly, Val-Thr, Val- Val, Val-Leu,Val-Ile, Val-Asn or Val-Lys.

6. The compound of claim 4, wherein W is selected from Glu-Ala-Gly, Glu-Gly- Gly, Gly-Glu-Gly, Gly-Glu-Ala, Asp-Ala-Gly, Asp-Gly-Gly, Gly-Asp-Gly, Gly-Asp- Ala, Gly-Gly-Ala, Gly-Gly-Arg, Gly-Ala-Gly, Gly-F-Gly, Ala-Ala-Gly, Ala-Ala-Ala, Ala-Ala-Asn, Val-Ala-Gly, Val-Cys-Gly, or Val-Lys-Gly.

7. The compound of claim 4, wherein W is selected from Gly-Gly-Phe-Gly, Gly- Gly-Ala-Gly, Gly-Gly-Gly-Gly, Gly-Glu-Gly-Gly, Gly-Glu-Ala-Gly, Gly-Asp-Gly- Gly, Gly-Asp-Ala-Gly, Ala-Ala-Ala-Gly or Glu-Ala-Gly-Gly.

8. The compound of claim 4, wherein W is Val-Ala, Val-Cit, or Gly-Gly-Phe-Gly.

9. The compound of any one of claims 1-8, wherein L1is selected from,end indicates the attachment point of L1to L2. 2Attorney Docket No.: 090322-8001WO01 10. The compound of any one of claims 1-9, wherein L2is selected from, wherein the * end indicates the attachment point of L2to L1.

11. The compound of any one of claims 1-10, wherein L1is, and L2is.

12. The compound of any one of claims 1-10, wherein L1is,13. The compound of any one of claims 1-12, wherein Y is selected from -COOH, - OH, -OCH3, or -CONH2.

14. The compound of any one of claims 1-13, wherein n is an integer from 1 to 12.

15. The compound of any one of claims 1-14, wherein p is an integer from 1 to 16.

16. The compound of any one of claims 1-15, wherein m is 0, 1 or 2.

17. The compound of claim 1, selected from the group consisting of:3Attorney Docket No.: 090322-8001WO014Attorney Docket No.: 090322-8001WO015Attorney Docket No.: 090322-8001WO016Attorney Docket No.: 090322-8001WO017Attorney Docket No.: 090322-8001WO018Attorney Docket No.: 090322-8001WO019Attorney Docket No.: 090322-8001WO01.

18. The compound of any one of claims 1-17, wherein T is selected from a cytotoxic agent, a cytostatic agent, a protein degrader agent, a detection agent, a diagnostic agent, a nucleic acid, a radionuclide, a hormone, an immunomodulatory agent, an enzyme, an antibody, a fusion protein, a metal ion or a combination thereof.

19. The compound of claim 18, wherein the cytotoxic agent is an antitubulin agent or a topoisomerase inhibitor.

20. The compound of any one of claims 1-19, wherein T is of Formula (T-1), Formula (T-2) or Formula (T-3): 10Attorney Docket No.: 090322-8001WO01wherein R1is hydrogen, halogen, alkyl, haloalkyl, -OR1a, -SR1a, -S(O)R1a, or -S(O)2R1a; R2is hydrogen, halogen, alkyl or haloalkyl; each of R3, R6, R7and R10is hydrogen or alkyl; each of R4, R5and R8is independently selected from hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, -alkyl-cycloalkyl, -alkyl-aryl, or -alkyl-heterocyclyl; each R9is independently H, -OH, alkyl, alkoxyl, or cycloalkyl; R11is -[C(R11a)2]2-cycloalkyl, -[C(R11a)2]2-heterocyclyl, or -[C(R11a)2]2-aryl; R12is heterocyclyl or aryl; Z is -O-, -S-, -NH-, or -N(alkyl)-; R13is hydrogen, alkyl, heterocyclyl, or aryl; R1ais hydrogen or alkyl; and each R11ais independently selected from hydrogen, hydroxyl, alkyl, alkoxyl, or cycloalkyl.

21. The compound of any one of claims 1-20, wherein T is selected from: 11Attorney Docket No.: 090322-8001WO0112Attorney Docket No.: 090322-8001WO0113Attorney Docket No.: 090322-8001WO0114Attorney Docket No.: 090322-8001WO0115Attorney Docket No.: 090322-8001WO0116Attorney Docket No.: 090322-8001WO0117Attorney Docket No.: 090322-8001WO0118Attorney Docket No.: 090322-8001WO0119Attorney Docket No.: 090322-8001WO01or a pharmaceutically acceptable salt thereof, wherein, A is a targeting moiety; X’ is a linking moiety connecting to A; L1is an amino acid side chain residue; L2is a spacer moiety; Y is a hydrophilic end group; 20Attorney Docket No.: 090322-8001WO01 E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC); T is a therapeutic or a diagnostic agent moiety; n is an integer from 1 to 20; m is an integer from 0 to 5; p is an integer from 1 to 24; r is from 1 to 10; a is 0 or 1; and b is 0 or 1.

24. The compound of claim 23, wherein the compound is of Formula (IIA), (IIB), or (IIC):( C), wherein A, X’, W, L1, L2, Y, E, T, n, m, r and p are as defined in claim 23.

25. The compound of claim 23 or 24, wherein A is selected from a polypeptide, an antibody, an enzyme or a fragment thereof. 21Attorney Docket No.: 090322-8001WO01 26. The compound of claim 25, wherein A is selected from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody or a fragment thereof.

27. The compound of claim 25 or 26, wherein A is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer, a multispecific antibody, a camelized single domain antibody, a nanobody,a domain antibody, a bivalent domain antibody, a monoclonal antibody and a polyclonal antibody.

28. The compound of any one of claims 24-27, wherein A is selected from the group consisting of an anti-DLL-3 antibody, an anti-MUC16 antibody, an anti-ENPP3 antibody, an anti-TDGF1 antibody, an anti-MSLN antibody, an anti-TIM-1 antibody, an anti-LRRC15 antibody, an anti-LIV-1 antibody, an anti-CanAg / AFP antibody, an anti-Mesothelin antibody, an anti-c-MET antibody, an anti-SLITRK6 antibody, an anti-KIT / CD117 antibody, an anti-STEAP1 antibody, an anti-SLAMF7 / CS1 antibody, an anti-NaPi2B / SLC34A2 antibody, an anti-GPNMB antibody, an anti- HER3 antibody, an anti-MUC1 / CD227 antibody, an anti-AXL antibody, an anti- PTK7 / CCK4 antibody, an anti-PRLR antibody, an anti-EFNA4 antibody, an anti-5T4 antibody, an anti-NOTCH3 antibody, an anti-CA6 antibody, an anti-GPR20 antibody, an anti-EphA2 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti- FRα antibody, an anti-CEACAM5 antibody, an anti-GCC antibody, an anti-Integrin α2 antibody, an anti-CAIX antibody, an anti-P-cadherin antibody, an anti-GD3 antibody, an anti-Cadherin 6 antibody, an anti-LAMP1 antibody, an anti-BCMA antibody, an anti-Tissue Factor antibody, an anti-Foliate Receptor antibody, an anti- ROR1 antibody, an anti-ROR2 antibody, an anti-TROP2 antibody, an anti-EGFR antibody, an anti-HER2 antibody, an anti-HER3 antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD46 antibody, an anti-CD47 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-CD74 antibody, an anti-CD79b antibody, an anti-CD123 antibody, an anti-CD138 antibody, an anti- CD142 antibody, an anti-CD166 antibody, an anti-CD174 antibody, an anti-CD352 antibody, an anti-Claudin 6 antibody, an anti-Claudin 18.2 antibody, an anti-GPC3 antibody, an anti-LYPD3 antibody, an anti-BCMA antibody, an anti-CLL1 antibody, an anti-FLT3 antibody, an anti-Nectin-4 antibody, an anti-Tissue Factor antibody, , an anti-CD44v9 antibody, an anti-integrin b6 antibody, an anti-CDCP1 antibody, an anti- EpCAM antibody, an anti-PD-L1 antibody, an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-EGFR / HER3 bispecific antibody, an anti-EGFR / c- MET bispecific antibody, an anti-MUC1 / EGFR bispecific antibody, an anti- HER2 / CD63 bispecific antibody, an anti-HER2 / PRLR bispecific antibody, an anti- HER2 / Nectin-4 bispecific antibody, and an anti-HER2 / APLP2 bispecific antibody.

29. The compound of claim 28, wherein A is an anti-HER2 antibody, anti-PMSA antibody or anti-Claudin-6 antibody. 22Attorney Docket No.: 090322-8001WO01 30. The compound of any one of claims 24-29, whereinwherein q is an integer from 1 to 10, and ** end indicates the attachment point of X’ to A.

31. The compound of any one of claims 24-30, wherein W is selected from a peptide composed of 2-5 amino acids.

32. The compound of claim 31, wherein W is Val-Ala, Val-Cit, Phe-Gly, or Gly-Gly- Phe-Gly.

33. The compound of any one of claims 24-32, wherein L1is, and the * end indicates the attachment point of L1and L2.

34. The compound of any one of claims 24-32, wherein L1is,.

35. The compound of any one of claims 24-33, wherein Y is selected from -COOH, - OH, -OCH3, or -CONH2.

36. The compound of any one of claims 24-35, wherein n is an integer from 1 to 12.

37. The compound of any one of claims 24-36, wherein p is an integer from 1 to 16.

38. The compound of any one of claims 24-37, wherein m is 0, 1 or 2.

39. The compound of any one of claims 24-38, selected from the group consisting of 23Attorney Docket No.: 090322-8001WO01wherein A is trastuzumab, HJ 591 or hH1L1.

40. A pharmaceutical composition comprising one or more of the compounds according to any one of claims 1-39 and an acceptable carrier. 24Attorney Docket No.: 090322-8001WO01 41. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutic effective amount of the compound according to any one of claims 1-39 or the pharmaceutical composition according to claim 40.

42. The method of claim 41, wherein the disease or disorder is cancer, autoimmune disease or metabolic disease.

43. The method of claim 42, wherein the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck cancer, testicular cancer, and esophageal cancer.

44. A method of screening an antibody-drug conjugate having reduced non-specific internalization into healthy cells, comprising introducing one or more negatively charged group into the linker of the antibody-drug conjugate.

45. The method of claim 44, wherein the negatively charged group is sulfonate, carboxylate, sulfate, or phosphate. 25

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