Polymer-drug conjugates

Abstract

The present invention discloses polymer scaffolds useful for conjugating to targeting moieties to form targeting moiety-polymer-drug conjugates. Also disclosed are targeting moiety-polymer-drug conjugates prepared from the polymer scaffolds. Also disclosed are compositions containing the conjugates, methods for preparing them, and methods for treating various disorders using the conjugates or compositions thereof.

Description

[Technical Field]

[0001] The present disclosure relates generally to compounds, conjugates comprising the compounds, pharmaceutical compositions thereof, and methods of treating diseases or disorders using the conjugates or pharmaceutical compositions thereof. [Background technology]

[0002] Traditionally, therapeutic agents, primarily small molecules, are delivered to the body via oral / GI absorption or systemic injection, followed by delivery to the site of action via the blood circulation. However, many challenges remain. For example, many therapeutic agents typically undergo partial degradation or accumulation in non-target tissues, or both, before reaching their desired target in the body, limiting or reducing their efficacy and therapeutic effectiveness.

[0003] Therefore, there is a need to deliver therapeutic agents intact to specific target sites in the body through a system that stabilizes the drug and controls the movement of the therapeutic agent in vivo, thereby achieving maximum cytotoxicity of the therapeutic agent. Summary of the Invention

[0004] The present disclosure relates to polymer scaffold delivery systems that exhibit high drug loading and strong binding to target antigens, thereby efficiently delivering and releasing drugs to target sites. The present disclosure also relates to polymer scaffolds that are useful for conjugating targeting moieties to obtain polymer scaffold delivery systems.

[0005] In one aspect, the present disclosure provides a polymer scaffold of formula (I) useful for conjugating a targeting moiety: [ka] where: the polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with said targeting moiety. pis a linking moiety comprising M a L to B a It is a stretcher that connects to the part, B a is the functional group W attached to the -NH- moiety M is a branched portion including each G 1 is independent, L P is a functional group that connects the Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are functional groups that can be independently converted to a charged state, n is an integer from 0 to 1000, m is an integer from 0 to 1000, p is an integer from 0 to 1000, and q is an integer from 0 to 1000.

[0006] In another aspect, the present disclosure provides a polymer scaffold of formula (II): [ka] where: the polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with said targeting moiety. p is a linking moiety comprising M a L to B a It is a stretcher that connects to the part, B a is the functional group W attached to the -NH- moiety M is a branched portion including each G 2 are functional groups that can be independently converted to a charged state, each G 3independently comprise a functional group capable of reacting with a reactive group on the drug release mechanism to connect the drug release mechanism to the linear polyglycerol; n is an integer from 0 to 1000, m is an integer from 0 to 1000, p is an integer from 0 to 1000, and q is an integer from 0 to 1000.

[0007] In another aspect, the present disclosure provides a polymer scaffold of formula (IIa), (IIb), (IIA) or (IIB): [ka] [ka] to provide.

[0008] In another aspect, the present disclosure provides a polymer scaffold of formula (IId), (IIe), (IIf), (IIg) or (IIh): [ka] [ka] to provide.

[0009] In another aspect, the present disclosure provides a polymer scaffold of formula (III): [ka] where: the polymer scaffold comprises linear polyglycerol; PBRM is the targeting part, Each L a independently, the targeting moiety is M a is a bivalent moiety that connects to Each M a is independent, L a B a It is a stretcher that connects to the part, B ais the functional group W attached to the -NH- moiety M is a branched portion including each G 1 is independent, L P is a functional group that connects the Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are functional groups that can be independently converted to a charged state, n is an integer from 0 to 1000, m is an integer from 0 to 1000, p is an integer from 0 to 1000; q is an integer from 0 to 1000, and s is an integer from 1 to 8.

[0010] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a polymer scaffold or conjugate described herein and a pharmaceutically acceptable carrier.

[0011] In another aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polymer scaffold or conjugate described herein, or a pharmaceutical composition provided herein. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the in vitro cytotoxicity of ADC PLAD-042 and ADC PLAD-043 to KG1α cells.

[0013] [Figure 2] 1 shows the in vitro cytotoxicity of ADC PLAD-047 and ADC PLAD-053 to NCI-H358 cells (A), MDA-MB-231 cells (B), and MCF-7 cells (C).

[0014] [Figure 3] FIG. 1 shows the in vitro cytotoxicity of ADC PLAD-072 to IGROV1, OVCAR3, KB, JEG3, and SKOV3 cells.

[0015] [Figure 4] FIG. 1 shows cell-based binding of mAb B854, ADC PLAD-072, and hIgG1 to SKOV3 cells.

[0016] [Figure 5] FIG. 1 shows the internalization of mAb B854 and ADC PLAD-072 into SKOV-3 cells.

[0017] [Figure 6] FIG. 1 shows the in vitro cytotoxicity of ADC PLAD-085 to IGROV1, OVCAR3, JEG3, and SKOV3 cells.

[0018] [Figure 7] FIG. 1 shows the in vitro cytotoxicity of ADC PLAD-114 to IGROV1, OVCAR3, JEG3, and SKOV3 cells.

[0019] [Figure 8] FIG. 1 shows cell-based binding of B854, ADC PLAD-114, and hIgG1 to SKOV3 cells.

[0020] [Figure 9] FIG. 1 shows the internalization of mAb B854 and ADC PLAD-114 into SKOV-3 cells.

[0021] [Figure 10] FIG. 1 shows the in vitro cytotoxicity of ADC PLAD-119 to OVCAR3 cells.

[0022] [Figure 11] FIG. 1 shows cell-based binding of A149-32, ADC PLAD-119, and hIgG1 to OVCAR3 cells.

[0023] [Figure 12] FIG. 1 shows the internalization of A149-32 and the ADC PLAD-119 into OVCAR3 cells.

[0024] [Figure 13] 1 shows the in vitro cytotoxicity of ADC PLAD-134 and ADC PLAD-135 to OVCAR3 cells (A) and HEK293 overexpressing cells (B).

[0025] [Figure 14] FIG. 1 shows cell-based binding of A149-32, ADC PLAD-134, ADC PLAD-135, and hIgG1 to OVCAR3 cells.

[0026] [Figure 15] FIG. 1 shows the internalization of A149-32, ADC PLAD-134, and ADC PLAD-135 into OVCAR3 cells.

[0027] [Figure 16] 1 shows the in vitro cytotoxicity of PLAD-141 and PLAD-142 to OVCAR3 cells (A), OV90 cells (B), and HEK293 overexpressing cells (C).

[0028] [Figure 17] FIG. 1 shows cell-based binding of A149-32, ADC PLAD-141, ADC PLAD-142, and hIgG1 to OVCAR3 cells.

[0029] [Figure 18]FIG. 1 shows the internalization of A149-32, ADC PLAD-141, and ADC PLAD-142 into OVCAR3 cells. DETAILED DESCRIPTION OF THE INVENTION

[0030] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying structures and formulas. While the present invention will be described in connection with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the present invention is intended to cover all variations, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, and may use them in practicing 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, including but not limited to defined terms, term usage, described techniques, etc., differs or contradicts with this application, this application controls.

[0031] It will be 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, while for brevity, are described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0032] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. In this specification and the following claims, reference will be made to a number of terms, which will be defined to have the following meanings unless a contrary intention is apparent. definition

[0033] The definitions of specific functional groups and chemical terms are described in more detail below.For the purpose of this disclosure, chemical elements are identified according to the Periodic Table of Elements, CAS edition, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein.In addition, the general principles of organic chemistry and 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, 5th edition, John Wiley & Sons, Inc. New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc. New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0034] At various points in this disclosure, linking substituents are described. Where a structure expressly requires a linking group, it is understood that the Markush variable listed for that group is the linking group. For example, if a structure requires a linking group and the Markush group definition for that variable lists "alkyl," it is understood that "alkyl" represents a linking alkylene group.

[0035] When any variable (e.g., R) occurs more than once 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 to 2 R moieties, that group may be optionally substituted with up to two R moieties, where R is independently selected from the definitions of R at each occurrence. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0036] As used herein, a dash "-", used for convenience before or at the end of a chemical group, indicates the point of attachment of the substituent. For example, -OH is attached through a carbon atom, and chemical groups can be drawn with or without one or more dashes without losing their normal meaning. A wavy line drawn through a straight line in a structure indicates the point of attachment of the group. No directionality is indicated or implied by the order in which chemical groups are written or named, unless chemically or structurally required. As used herein, a solid line emanating from the center of a ring indicates that the point of attachment of a substituent on the ring can be any ring atom. When substituents are listed without indicating the intervening atoms through which such substituents are attached to the remainder of the compound of a given formula, such substituents can be attached through any atom in such formula. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0037] When any variable (e.g., R) occurs more than once 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 to 2 R moieties, that group may be optionally substituted with up to two R moieties, where R is independently selected from the definitions of R at each occurrence. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0038] When used in connection with a numerical value, the term "about" is meant to include a group or range of values. For example, "about X," where X is a numerical value, includes a range of values ​​that are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X. In one embodiment, the term "about" refers to a range of values ​​that are 5% above or below a particular value. In another embodiment, the term "about" refers to a range of values ​​that are 2% above or below a particular value. In another embodiment, the term "about" refers to a range of values ​​that are 1% above or below a particular value.

[0039] The recitation of ranges of values ​​is intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. Ranges used herein include the two endpoints of the range unless otherwise specified. For example, the expressions "n is an integer between 1 and 6" and "n is an integer from 1 to 6" both mean "x is 1, 2, 3, 4, 5, or 6."

[0040] As used herein, the term "Ci-j" refers to a range where i and j are integers, the range of carbon atoms is inclusive of the endpoints (e.g., i and j) and each integer point between them, and j is greater than i. For example, C1-6 refers to a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms. In some embodiments, the term "C1-12" refers to 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 a similar manner, the term "m- to n-membered" ring refers to a ring containing m to n atoms, where m and n are integers and n is greater than m.

[0041] As used herein, the term "aliphatic" includes both saturated and unsaturated, straight-chain (i.e., unbranched) or branched aliphatic hydrocarbons, optionally substituted with one or more functional groups. As will be understood by those skilled in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, and alkynyl moieties.

[0042] As used herein, the term "alkyl," whether used as part of another term or independently, refers to a saturated straight- or branched-chain hydrocarbon radical, which may be optionally substituted independently with one or more substituents described below. The term "Ci-j alkyl" refers to a straight- or branched-chain alkyl having i to j carbon atoms. For example, the alkyl group contains 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 "Ci_6 alkyl" 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.

[0043] As used herein, the term "alkenyl," whether used as part of another term or independently, refers to a straight- or branched-chain hydrocarbon radical having at least one carbon-carbon double bond, which may be optionally substituted with one or more substituents described herein, and may include radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. In some embodiments, alkenyl groups contain 2-12 carbon atoms. In some embodiments, alkenyl groups contain 2-11 carbon atoms. In some embodiments, alkenyl groups contain 2-11 carbon atoms, 2-10 carbon atoms, 2-9 carbon atoms, 2-8 carbon atoms, 2-7 carbon atoms, 2-6 carbon atoms, 2-5 carbon atoms, 2-4 carbon atoms, 2-3 carbon atoms, and in some embodiments, alkenyl groups contain 2 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethylenyl (ie, vinyl), propenyl (allyl), butenyl, pentenyl, 1-methyl-2 buten-1-yl, 5-hexenyl, and the like.

[0044] As used herein, the term "alkynyl," whether used as part of another term or independently, refers to a linear or branched hydrocarbon radical having at least one carbon-carbon triple bond, and 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 groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

[0045] As used herein, the term "amino" refers to -NR a R b where R refers to the group a and R b is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl, each of which may be optionally substituted.

[0046] As used herein, the term "aryl," whether used as part of another term or independently, refers to monocyclic and polycyclic ring systems having a total of 5 to 20 ring members, wherein at least one ring of the system is aromatic and each ring of 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. The term "aryl," as used herein, also includes groups in which an aromatic ring is fused to one or more additional rings. In polycyclic systems, only one of the rings need be aromatic (e.g., 2,3-dihydroindole), although all rings may be aromatic (e.g., quinoline). The second ring may also be fused or bridged. Examples of polycyclic aryls include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0047] As used herein, the term "cycloalkyl," whether used as part of another term or independently, refers to monovalent non-aromatic, saturated or partially unsaturated, monocyclic and polycyclic ring systems in which all ring atoms are carbon and which contain at least three ring-forming carbon atoms. In some embodiments, cycloalkyl groups can 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, or 4 to 5 ring-forming carbon atoms. Cycloalkyl groups can be saturated or partially unsaturated. In some embodiments, cycloalkyl groups can be saturated cyclic alkyl groups. In some embodiments, cycloalkyl groups can be partially unsaturated cyclic alkyl groups containing at least one double or triple bond in their ring system.

[0048] In some embodiments, a cycloalkyl group can be a 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.

[0049] In some embodiments, cycloalkyl groups can be saturated or partially unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic ring systems, which can be arranged as fused, spiro, or bridged ring systems. As used herein, the term "fused ring" refers to a ring system having two rings that share two adjacent atoms, the term "spiro ring" refers to a ring system in which two rings are connected through a single common atom, and the term "bridged ring" refers to a ring system in which two rings share three or more atoms. Examples of fused carbocyclic compounds include, but are not limited to, naphthyl, benzopyrenyl, anthracenyl, acenaphthenyl, fluorenyl, and the like. Examples of spirocarbocyclic compounds include, but are not limited to, spiro[5.5]undecanyl, spiro-pentadienyl, spiro[3.6]-decanyl, and the like. Examples of bridged carbocyclic compounds 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.

[0050] 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).

[0051] 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.

[0052] As used herein, the term "heteroaliphatic" refers to an aliphatic moiety in which one or more carbon atoms in the backbone are replaced with heteroatoms. Thus, a heteroaliphatic group refers to an aliphatic chain containing, for example, one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of carbon atoms. The heteroaliphatic moiety can be branched or straight-chain unbranched. As will be understood by those skilled in the art, "heteroaliphatic," as used herein, is intended to include, but is not limited to, heteroalkyl, heteroalkenyl, and heteroalkynyl moieties. In certain embodiments, a heteroaliphatic moiety is one in which one or more hydrogen atoms are replaced with an aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, F, Cl, Br, I, -NO, -CN, -CF, -CHCF, -CHC, or the like. 12 , -CH2OH, -CH2CH2OH, -CH2NH2, -CH2SO2CH3 or -GR G1 and wherein G is substituted by being independently substituted with one or more moieties including, but not limited to, —O—, —S—, —NR G2 -, -C(=O)-, -S(=O)-, -SO2-, -C(=O)O-, -C(=O)NR G2 -, -OC(=O)-, -NR G2 C(=O)-, -OC(=O)O-, -OC(=O)NR G2 -, -NR G2 C(=O)O-, -NR G2 C(=O)NR G2 -, -C(=S)-, -C(=S)S-, -SC(=S)-, -SC(=S)S-, -C(=NR G2 )-, -C(=NR G2 )O-, -C(=NR G2 )NR G3 -, -OC(=NR G2 )-, -NR G2 C(=NR G3 )-, -NR G2 SO2-, -NRG2 SO2NR G3 -, or -SO2NR G2 - (where, in each occurrence, R G1 , R G2 and R G3 are independently including, but not limited to, hydrogen, halogen, or an optionally substituted aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety. Additional examples of generally applicable substituents are exemplified by the specific embodiments shown in the examples described herein.

[0053] As used herein, the term "heteroalkyl" refers to an alkyl in which at least one carbon atom is replaced with a heteroatom selected from N, O, or S. A heteroalkyl can be a carbon radical or a heteroatom radical (i.e., the heteroatom can occur at the center or end of the radical), and can be optionally substituted independently with one or more substituents described herein. The term "heteroalkyl" encompasses alkoxyl and heteroalkoxy radicals.

[0054] As used herein, the term "heteroalkenyl" refers to an alkenyl in which at least one carbon atom is replaced with a heteroatom selected from N, O, or S. A heteroalkenyl can be a carbon radical or a heteroatom radical (i.e., the heteroatom can occur at the center or end of the radical), and can be optionally substituted independently with one or more substituents described herein.

[0055] As used herein, the term "heteroalkynyl" refers to an alkynyl in which at least one carbon atom is replaced with a heteroatom selected from N, O, or S. The heteroalkynyl can be a carbon radical or a heteroatom radical (i.e., the heteroatom can occur at the center or at the end of the radical), and can be optionally substituted independently with one or more substituents described herein.

[0056] As used herein, the term "heteroaryl," whether used as part of another term or independently, refers to an aryl group having one or more heteroatoms in addition to carbon atoms. Heteroaryl groups can be monocyclic. Examples of monocyclic heteroaryls include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl, and pteridinyl. Heteroaryl groups also include polycyclic groups in which a heteroaromatic ring is fused to one or more aryl, heteroaryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of polycyclic heteroaryls include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0057] As used herein, the term "heterocycloalkyl" refers to a saturated or partially unsaturated cycloalkyl group, in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, wherein one or more ring atoms are optionally independently substituted with one or more substituents. In some embodiments, a heterocycloalkyl is a saturated heterocycloalkyl. In some embodiments, a heterocycloalkyl is a partially unsaturated heterocycloalkyl having one or more double bonds in its ring system. In some embodiments, a heterocycloalkyl can contain any oxidized form of carbon, nitrogen, or sulfur, and any quaternized form of a basic nitrogen. A heterocycloalkyl radical can be carbon- or nitrogen-linked, where possible. In some embodiments, a heterocycle is carbon-linked. In some embodiments, a heterocycle is nitrogen-linked. For example, a pyrrole-derived group can be pyrrol-1-yl (nitrogen-linked) or pyrrol-3-yl (carbon-linked). Additionally, the imidazole-derived group can be imidazol-1-yl (nitrogen-linked) or imidazol-3-yl (carbon-linked).

[0058] Heterocycloalkyl groups can be monocyclic. Examples of monocyclic heterocycloalkyls include, but are not limited to, oxetanyl, 1,1-dioxothietanylpyrrolidyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydrothienyl, azetidinyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperazinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazonyl, pyrrolidinyl, triazinonyl, and the like.

[0059] Heterocycloalkyl groups can be polycyclic, and include fused, spiro, and bridged ring systems. Fused heterocycloalkyl groups include radicals in which the heterocycloalkyl radical is a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Examples of fused heterocycloalkyls include, but are not limited to, phenyl- or pyridinyl-fused rings, such as quinolinyl, isoquinolinyl, quinoxalinyl, quinolidinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[1,2-a]pyridinyl, furo[3,4-d]pyrimidinyl, pyrrolo[3,4-d]pyrimidinyl, dihydrofuro[3,4-b]pyridinyl groups, and the like. Examples of spiroheterocycloalkyl include, but are not limited to, spiropyranyl, spirooxazinyl, 5-aza-spiro[2.4]heptanyl, 6-aza-spiro[2.5]octanyl, 6-aza-spiro[3.4]octanyl, 2-oxa-6-aza-spiro[3.3]heptanyl, 2-oxa-6-aza-spiro[3.4]octanyl, 6-aza-spiro[3.5]nonanyl, 7-aza-spiro[3.5]nonanyl, 1-oxa-7-aza-spiro[3.5]nonanyl, 3,8-dioxa-1-azaspiro[4.5]dec-1-enyl, and the like. Examples of bridged heterocycloalkyls include, but are not limited to, 3-aza-bicyclo[3.1.0]hexanyl, 8-aza-bicyclo[3.2.1]octanyl, 1-aza-bicyclo[2.2.2]octanyl, 2-aza-bicyclo[2.2.1]heptanyl, 1,4-diazabicyclo[2.2.2]octanyl, and the like.

[0060] As used herein, the term "hydroxyl" refers to --OH.

[0061] As used herein, the term "leaving group" refers to a molecular fragment that departs with an electron pair in anisotropic bond decomposition. Leaving groups can be anions or neutral molecules. Leaving groups include, but are not limited to, halides, e.g., Cl. - , Br - and I - , sulfonic acid esters, such as paratoluenesulfonate ("tosylate", TsO - ) and RC(O)O, where R is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety - Includes:

[0062] 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 said event or circumstance occurs and instances where it does not occur.

[0063] As used herein, the term "partially unsaturated" refers to a radical that contains at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

[0064] As used herein, the term "protecting group" means that a specific functional moiety, such as O, S, or N, is temporarily blocked so that a reaction in a multifunctional compound can be selectively carried out at another reactive site. In some embodiments, the protecting group reacts selectively in good yield, resulting in a protected substrate that is stable for the anticipated reaction. The protecting group must be selectively removed in good yield with a reagent that is readily available, preferably non-toxic, and does not attack other functional groups. The protecting group forms an easily separable derivative (more preferably, without creating a new asymmetric center). The protecting group has minimal additional functionality to avoid further reaction sites. As described herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. For example, in some embodiments, certain exemplary oxygen protecting groups may be utilized. These oxygen-based protecting groups include, but are not limited to, methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM (p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, and TBDPS (t-butyldiphenylsilyl ether), esther (e.g., ethoxymethyl ether), ethoxymethyl ether), ethoxymethyl ether, ... Examples of suitable protecting groups include esters (e.g., formates, acetates, benzoates (Bz), trifluoroacetates, and dichloroacetates), carbonates, cyclic acetals, and ketals. In some other embodiments, nitrogen protecting groups are utilized. Nitrogen protecting groups and methods of protection and deprotection are known in the art. Nitrogen protecting groups include, but are not limited to, carbamates (methyl, ethyl, and substituted ethyl carbamates (e.g., Troc), amides, cyclic imide derivatives, N-alkyl and N-aryl amines, imine derivatives, and enamine derivatives. In yet other embodiments, certain exemplary sulfur protecting groups may be utilized.Sulfur protecting groups include, but are not limited to, the oxygen protecting groups described above, as well as aliphatic carboxylic acids (e.g., acrylic acid), maleimide, vinylsulfonyl, and optionally substituted maleic acid. While certain other exemplary protecting groups are detailed herein, it is understood that the present invention is not intended to be limited to these protecting groups, and a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the present invention. Additionally, a variety of protecting groups are described in "Protective Groups in Organic Synthesis," 3rd Edition, Greene, TW and Wuts, PG, John Wiley & Sons (eds.), New York: 1999, the entire contents of which are incorporated herein by reference.

[0065] As used herein, the term "leaving group" refers to a molecular fragment that departs with an electron pair in anisotropic bond decomposition. Leaving groups can be anions or neutral molecules. Leaving groups include, but are not limited to, halides, e.g., Cl. - , Br - and I - , sulfonic acid esters, such as paratoluenesulfonate ("tosylate", TsO - ) and RC(O)O, where R is hydrogen, aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety - Includes:

[0066] As used herein, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogen atoms of the targeted moiety have been replaced with a suitable substituent. "Substituted" or "substituted with" is understood to include, with the express proviso that such substitution is in accordance with the allowed valence of the substituted atom and that the substitution results in a stable or chemically feasible compound, e.g., a compound that does not undergo unintended transformation by rearrangement, cyclization, elimination, or the like. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when multiple positions in any given structure may be substituted with multiple substituents selected from a specified group, the substituents may be the same or different at each position. Substituents may include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azide, 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. Those skilled in the art will understand that, where appropriate, the substituents themselves may also be substituted. Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, references to an "aryl" group or moiety unconditionally include both substituted and unsubstituted variants.

[0067] 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, a surface-immobilized protein, or a protoglycan. Examples of targeting moieties include, but are not limited to, antibodies or fragments thereof, lipocalin, proteins, peptides, or peptidomimetics. In addition to targeting the polymer scaffold to a specific cell, tissue, or location, the targeting moiety may also have a specific therapeutic effect, such as antiproliferative (cytostatic and / or cytotoxic) activity against the target cell or pathway. The targeting moiety may contain or be engineered to contain at least one chemically reactive group, such as -COOR, -SH, amine, or a chemically reactive amino acid moiety or side chain, such as tyrosine, histidine, cysteine, or lysine. In some embodiments, the targeting moiety may be a ligand that specifically binds to 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 complexation of the ligand to its receptor, the cell becomes permissive for uptake of the ligand or ligand-drug conjugate, which is then internalized within the cell. As used herein, a ligand that "specifically binds to or complexes with" or "targets" a cell surface molecule binds preferentially to the cell surface molecule via intermolecular forces.

[0068] As used herein, the term "ligand" refers to a variety of chemical or biological molecules that can have specific binding affinity to a selected target, where the selected target can be, for example, a cell surface receptor, a cell surface antigen, a cell, a tissue, an organ, etc. In some embodiments, a ligand can specifically bind to a protein or marker expressed on the surface of a target cell. In some embodiments, the ligands of the present disclosure specifically bind to cell surface proteins or markers. -6 ~10 -11 In some embodiments, the ligands of the present disclosure bind to cell surface proteins or markers with an affinity of at least 10 M (Kd value). -7, at least 10 -8 and at least 10 -9 In some embodiments, the ligands of the present disclosure bind to cell surface proteins or markers with an affinity of 10 M (Kd value). -6 Less than 10 -7 Less than and 10 -8 The ligands of the present disclosure bind with an affinity of less than M (Kd value). In some embodiments, the ligands of the present disclosure bind to a cell surface protein or marker with a specific affinity, where specific affinity refers to the affinity of the ligand for the target cell surface protein or marker, which is at least 2, 3, 4, 5, 6, 8, 10, 20, 50, 100 or more times higher than for a non-target cell surface protein or marker. In some embodiments, the expression of the cell surface protein or marker of the present disclosure in target cells (e.g., cancer cells) is significantly higher than that in normal cells. As used herein, the term "significantly" refers to a statistically significant difference or a notable difference that can be recognized by one of ordinary skill in the art.

[0069] As used herein, the term "targeting moiety" refers to a molecule, complex, or aggregate that specifically or selectively binds to target molecules, cells, particles, tissues, or aggregates.Examples of targeting moieties include, but are not limited to, antibodies, antibody-binding fragments, bispecific antibodies, immunoglobulins, or other antibody-based molecules or compounds.However, other examples of targeting moieties are known in the art and can be used, such as aptamers, avimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, peptides, small molecules, nanoparticles, or proteins.The terms "targeting moiety" and "binding moiety" are used interchangeably herein.

[0070] As used herein, the term "drug" refers to a compound (e.g., an active ingredient) that is biologically active and produces a desired physiological effect following administration to a subject in need thereof.

[0071] 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 natural intact antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a variable region (VH) and first, second, and third constant regions (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 variable regions of both chains are generally subdivided into three hypervariable regions called complementarity-determining regions (CDRs) (the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3). The extent of the CDRs for the antibodies and antigen-binding fragments disclosed herein may be defined or specified according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., December 5, 186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. December 21-28, 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between adjacent segments known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. Thus, each VH and VL consists of three CDRs and four FRs, in the following order (amino acid residues N- to C-terminus): FR1, CDR1, FR2, CDR2, FR3, DR3, 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 five major classes based on the amino acid sequence of the constant regions of their heavy chains: IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some subclasses of the major antibody classes are, for example, 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).

[0072] As used herein, the term "Fab" with respect to an antibody refers to a monovalent antigen-binding fragment of an antibody consisting of a variable region and one light chain (both variable and constant regions) linked by a disulfide bond to the first constant region of one heavy chain. Fab can be obtained by papain digestion of an antibody at residues proximal to the N-terminus of the disulfide bond between the hinge regions of the heavy chains.

[0073] As used herein, the term "Fab" refers to a Fab fragment that can be obtained by pepsin digestion of an antibody at a residue proximal to the C-terminus of the disulfide bond between the hinge regions of the heavy chains, and thus contains a portion of the hinge region that differs from Fab in a small number of residues in the hinge region (including one or more cysteines).

[0074] As used herein, the term "F(ab')2" refers to a dimer of Fab' comprising two light chains and portions of two heavy chains.

[0075] As used herein, the term "Fc" in reference to an antibody refers to the portion of an antibody that consists of the second and third constant regions of a first heavy chain and is linked to the second and third constant regions of a second heavy chain via disulfide bonds. The Fc region of IgG and IgM contains three heavy chain constant regions (the second, third, and fourth heavy chain constant regions in each chain). This can be obtained by papain digestion of an antibody. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not function in antigen binding.

[0076] As used herein, the term "Fv" in reference to an antibody refers to the smallest antibody fragment that retains a complete antigen-binding site. An Fv fragment consists of the variable region of one light chain bound to the variable region of one heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of one light chain and the variable region of one heavy chain is a disulfide bond.

[0077] As used herein, "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 each other directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). An "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions with a linker. An "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions connected to each other via a linker. H -V L dimerizes with the V moiety and forms a single V H is the other part of V L V cooperates with V to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L The term "scFv dimer" may be a bivalent diabody or a bivalent ScFv (BsFv) comprising a V H1 and V L1 cooperated, V H2 and V L2V cooperates with each other, with each cooperated pair having a different antigen specificity. L1 -V H2 V attached to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0078] As used herein, the term "single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv attached to the Fc region of an antibody.

[0079] As used herein, the term "camelized single domain antibody", "heavy chain antibody", "nanobody" or "HCAb" refers to a camelized single domain antibody consisting of two V HIt refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. December 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. June;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally obtained from camelids (camels, dromedaries, and llamas). Although they lack light chains, camelized antibodies have authentic antigen-binding repertoires (Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae: a case of evolutionary innovation," Immunogenetics. April;54(1):39-47 (2002); Nguyen VK. et al., Immunology. May;109(1):93-101 (2003)). The variable domains of heavy-chain antibodies (VHH domains) represent the smallest known antigen-binding units produced by adaptive immune responses (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8 Epub 2007 June 15 (2007)). "Diabodies" include small antibody fragments with two antigen-binding sites, where the fragments are V L V connected to the domain H Domain Included (V H -V L or V L -V H ) (See, e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). Two domains on the same chain cannot pair because the linker is too short, so the domains are forced to pair with complementary domains on another chain, thereby generating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes).

[0080] As used herein, the term "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some embodiments, two or more V H The domains are covalently linked with peptide linkers to form bivalent or multivalent domain antibodies. H The domains may target the same or different antigens.

[0081] As used herein, the term "(dsFv)2" refers to an antigen-binding fragment consisting of three peptide chains: two V connected by a peptide linker and separated by disulfide bridges. L Two V's bonded to the moiety H portion.

[0082] As used herein, the term "bispecific ds diabody" refers to a V L1 -V H2 (linked by a peptide linker) to V H1 and V L1 V is linked via a disulfide bridge between H1 -V L2 (also linked by a peptide linker).

[0083] As used herein, the term "bispecific dsFv" or "dsFv-dsFv" refers to an antigen-binding fragment consisting of three peptide chains, i.e., the heavy chains are each connected by a peptide linker (e.g., a long flexible linker), V L1 and V L2 V binds to and pairs with V via a disulfide bridge. H1 -V H2 Each of the disulfide-paired heavy and light chains has a different antigen specificity.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof is chimeric or humanized.

[0085] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the remaining heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0086] As used herein, the term "humanized," with respect to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that comprises CDRs derived from a non-human animal (e.g., a rodent, rabbit, dog, goat, horse, or chicken), human-derived FR regions, and, where applicable, human-derived constant regions. In some embodiments, constant regions from a human antibody are fused to non-human variable regions. Humanized antibodies or antigen-binding fragments are useful as human therapeutics. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, hamster, or a non-human primate (e.g., a monkey (e.g., a cynomolgus monkey or a rhesus monkey) or an ape (e.g., a chimpanzee, gorilla, monkey, or affen). In some embodiments, a humanized antibody or antigen-binding fragment is composed of substantially all human sequences, except for CDR sequences, which are non-human. In some embodiments, a humanized antibody or antigen-binding fragment is modified to improve antibody performance, e.g., binding or binding affinity. For example, by modifying one or more amino acids in one or more non-human CDRs. In some embodiments, amino acid residues are modified to reduce potential immunogenicity in humans, where the modified amino acid residues are not critical for immunospecific binding or the modifications are conservative changes such that binding of the humanized antibody to the antigen is not significantly affected. In some embodiments, the human-derived FR region may contain an amino acid sequence identical to that of the human antibody from which it is derived, or may contain some amino acid changes, e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes. In some embodiments, such amino acid changes may occur only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0087] 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), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), cysteine ​​(Cys), and methionine (Met). As used herein, the term "unnatural amino acid" refers to any amino acid that is not a natural amino acid. This includes, for example, amino acids that contain α-, β-, ω-, D-, or L-aminoacyl residues. More generally, unnatural amino acids can be represented by the general formula: [ka] wherein the side chain R is different from a naturally occurring amino acid side chain. 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.

[0088] As used herein, the terms "polypeptide," "protein," or "peptide" may refer to a single amino acid or a polymer of amino acids. Polypeptides, proteins, or peptides, as described in this disclosure, may include naturally occurring and non-naturally occurring amino acids, or analogs and mimetics thereof. Polypeptides, proteins, or peptides may be obtained by any method well known in the art, including, but not limited to, separation and purification from natural sources, recombinant expression, chemical synthesis, etc.

[0089] As used herein, the term "biocompatible" refers to a compound that exerts minimal disruptive or host reaction effects while in contact with body fluids or living cells or tissues.Therefore, biocompatible groups, as used herein, refer to aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moieties, which fall within the definition of the term biocompatible as defined above and herein.As used herein, the term "biocompatible" also refers to a compound that exhibits minimal interaction with recognition proteins, such as naturally occurring antibodies, cellular proteins, cells, and other components of biological systems, unless such interaction is specifically desired.Therefore, substances and functional groups, such as drugs and prodrugs, that are specifically intended to cause the above-mentioned minimal interaction are considered to be biocompatible. In some embodiments, compounds are "biocompatible" if, when added to normal cells in vitro at concentrations similar to their intended systemic concentrations in vivo, they cause 1% or less cell death within a time period equal to the compound's half-life in vivo (e.g., the time required for 50% of the administered compound to be removed / cleared in vivo), and if their administration in vivo induces minimal and medically acceptable inflammation, bodily responses to foreign substances, immunotoxicity, chemotoxicity, and / or other such adverse effects. As used herein, the term "normal cells" refers to cells that are not intended to be destroyed or otherwise significantly affected by the test compound.

[0090] As used herein, a "biodegradable" polymer is a polymer that is susceptible to biological processes in vivo. As used herein, a "biodegradable" compound or moiety, when taken up by a cell, can be decomposed by lysosomes or other chemical mechanisms or hydrolysis into components that the cell can reuse or dispose of without significant toxic effects to the cell. The term "biocleavale" as used herein has the same meaning as "biodegradable." The degradation fragments preferably induce little or no organ or cellular overload, pathological processes caused by such overload, or other adverse effects in vivo. Examples of biodegradation processes include enzymatic and non-enzymatic hydrolysis, oxidation, and reduction. Suitable conditions for non-enzymatic hydrolysis of the biodegradable protein-polymer-drug conjugates described herein (or their components, such as biodegradable polymer carriers and linkers between the carriers and antibodies or drug molecules) include, for example, exposure of the biodegradable conjugate to water at the temperature and pH of the lysosomal intracellular compartment. The biodegradation of some protein-polymer-drug conjugates (or their components, e.g., biodegradable polymer carriers and linkers between the carrier and antibodies or drug molecules) can also be enhanced extracellularly, e.g., in low-pH regions of an animal's body, such as in areas of inflammation, activated macrophages, or in close proximity to other cells that release degradation-promoting factors. In certain embodiments, the effective size of the polymer carrier at a pH of about 7.5 does not detectably change over 1-7 days and remains within 50% of the original polymer size for at least several weeks. On the other hand, at a pH of about 5, the polymer carrier preferably degrades detectably over 1-5 days and is completely converted to low-molecular-weight fragments within a time frame of 2 weeks to several months. The integrity of the polymer in such tests can be measured, for example, by size-exclusion HPLC. While more rapid degradation may be preferred in some cases, it may generally be more desirable for the polymer to degrade intracellularly at a rate that does not exceed the rate at which the polymer fragments can be metabolized or excreted by the cell.In certain embodiments, the polymers and polymer biodegradation by-products are biocompatible.

[0091] 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 time (rate) and the total amount (extent) of drug or compound that reaches the general circulation from an administered dosage form.

[0092] As used herein, the term "drug release mechanism" refers to a linking moiety that is biocleavable / biodegradable under intracellular conditions, such that cleavage of the linking moiety releases the drug into the intracellular environment. In some embodiments, the linking moiety is hydrolytically unstable, i.e., susceptible to hydrolysis at a specific pH, in water or aqueous solutions, including, for example, body fluids such as blood. In some embodiments, the linking moiety is enzymatically unstable, i.e., degradable by one or more enzymes. In some embodiments, the linking moiety is photolabile, making it useful on body surfaces and in many body cavities accessible to light. In some embodiments, the linking moiety is biocleavable under reducing conditions, under which the activity of the drug is not affected.

[0093] As used herein, the term "therapeutic agent" or "drug" refers to a compound (e.g., an active ingredient) that is biologically active and produces a desired physiological effect following administration to a subject in need thereof. In some embodiments, the therapeutic agent is a small molecule drug.

[0094] As used herein, the term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially created (e.g., via chemical synthesis). Preferred small molecules are biologically active in producing a local or systemic effect in an animal, e.g., a mammal, e.g., a human. In certain embodiments, the small molecule is a drug, and the small molecule is referred to as a "drug molecule" or "drug" or "therapeutic agent." In certain embodiments, the drug molecule has a MW of about 5 kDa or less. In other embodiments, the drug molecule has a MW of about 1.5 kDa or less.

[0095] Classes of drug molecules that may be used in the present disclosure include, but are not limited to, anti-cancer agents, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, antivirals, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, anti-parasitic and / or anti-protozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotics, antiemetics, and imaging agents.

[0096] In certain embodiments, polymers may be used as therapeutic agents in the present disclosure. Examples of suitable polymers include, but are not limited to, amino acid-based molecules such as peptides, polypeptides, enzymes, antibodies, immunoglobulins, or functional fragments thereof, among others.

[0097] In some embodiments, the therapeutic agents used in the present disclosure are therapeutic agents that have antiproliferative (cytostatic and / or cytotoxic) activity against target cells or pathways. The drugs may have chemically reactive groups, such as -COOH, primary amines, secondary amines -NHR, -OH, -SH, -C(O)H, -C(O)R, -C(O)NHR', -C(S)OH, -S(O)OR', -P(O)OR', -CN, -NC, or -ONO, where R is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, and R' is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl moiety.

[0098] As used herein, the term "cytotoxic" means toxic to a cell or a selected cell population (e.g., cancer cells). A toxic effect can result in cell death and / or lysis. In certain cases, a toxic effect can be a sublethal destructive effect on a cell, such as slowing or stopping cell growth. To achieve a cytotoxic effect, the drug or prodrug can be selected from the group consisting of a DNA damaging agent, a microtubule disrupting agent, or a cytotoxic protein or polypeptide, among others.

[0099] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen. In some embodiments, the antibodies or antigen-binding fragments provided herein exhibit a binding affinity (K) of about 0.01 nM to about 100 nM, about 0.1 nM to about 100 nM, 0.01 nM to about 10 nM, about 0.1 nM to about 10 nM, 0.01 nM to about 1 nM, about 0.1 nM to about 1 nM, or about 0.01 nM to about 0.1 nM for H7.4. D ) specifically binds to the target antigen. As used herein, K D is the ratio of the dissociation rate to the association rate (k off / k on ) and can be determined using surface plasmon resonance using an instrument such as a Biacore.

[0100] As used herein, the term "tumor antigen" refers to an antigenic substance produced in tumor cells, i.e., tumor antigens induce an immune response in the host. Normal proteins in the body are not antigenic due to self-tolerance, a process in which autoreactive cytotoxic T lymphocytes (CTLs) and autoantibody-producing B lymphocytes are culled "centrally" in primary lymphoid tissues (BM) and "peripherally" in secondary lymphoid tissues (mostly the thymus for T cells and the spleen / lymph nodes for B cells). Therefore, any protein that is not exposed to the immune system can trigger an immune response. This may include normal proteins that are well isolated from the immune system, proteins that are normally produced in very small amounts, proteins that are normally produced only at certain stages of development, or proteins whose structure is altered due to mutation.

[0101] As used herein, the term "effective amount" refers to the amount necessary to elicit a desired biological response. As will be understood by those skilled in the art, the effective amount of a drug or device can vary depending on factors such as the desired biological endpoint, the drug being delivered, the composition of the encapsulating matrix, the target tissue, and other factors. For example, an effective amount of antigen-containing microparticles delivered to immunize an individual is an amount that generates an immune response sufficient to prevent infection with the administered antigen-bearing organism.

[0102] As used herein, the "molecular weight" or "MW" of a polymer or polymeric carrier / scaffold or polymeric conjugate refers to the weight average molecular weight, unless otherwise specified.

[0103] The present disclosure is intended to include all isotopes of atoms present in the present compounds. Isotopes include 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 C 13 and C 14 Includes:

[0104] The present invention is intended to include all isomers of the compounds, which refers to and includes optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include separated optical isomers and mixtures of optical isomers, including racemic and non-racemic mixtures, where an isomer may be in separated form or in mixture with one or more other isomers. polymer support

[0105] The conjugates of the present disclosure are useful for biomedical applications, such as drug delivery and tissue engineering, and the polymeric carriers used in the conjugates of the present disclosure are biocompatible and biodegradable. In some embodiments, the polymeric carrier is a soluble polymer, nanoparticle, gel, liposome, micelle, suture, implant, etc.

[0106] In some embodiments, the polymeric carrier is from about 400 to about 3,000,000 Da, e.g., from about 1,000 to about 2,000,000 Da, from about 1,000 to about 1,000,000 Da, from about 1,000 to about 900,000 Da, from about 1,000 to about 800,000 Da, from about 1,000 to about 700,000 Da, from about 1,000 to about 600,000 Da, from about 1,000 to about 500,000 Da, from about 1,000 to about 400,000 Da, from about 1,000 to about 300,000 Da, from about 1,000 to about 200,000 Da, or from about 1,000 to about 100,000 Da. The weight average molecular weight Mw may be about 1,000 to about 90,000 Da, about 1,000 to about 80,000 Da, about 1,000 to about 70,000 Da, about 1,000 to about 60,000 Da, about 1,000 to about 50,000 Da, about 1,000 to about 40,000 Da, about 1,000 to about 30,000 Da, about 1,000 to about 20,000 Da, about 1,000 to about 10,000 Da, about 2,000 to about 10,000 Da, about 3,000 to about 10,000 Da, about 4,000 to about 10,000 Da, or about 5,000 to about 10,000 Da.

[0107] In some embodiments, the polymeric carrier used in the present disclosure is polyglycerol. In certain embodiments, the polymeric carrier used in the present disclosure is linear polyglycerol. In certain embodiments, the linear polyglycerol has a molecular weight of about 400 to about 3,000,000 Da, e.g., about 1,000 to about 2,000,000 Da, about 1,000 to about 1,000,000 Da, about 1,000 to about 900,000 Da, about 1,000 to about 800,000 Da, about 1,000 to about 700,000 Da, about 1,000 to about 600,000 Da, about 1,000 to about 500,000 Da, about 1,000 to about 400,000 Da, about 1,000 to about 300,000 Da, about 1,000 to about 200,000 Da, or about 1,000 to about 100,000 Da. The weight average molecular weight Mw may be about 1,000 Da, about 1,000 to about 90,000 Da, about 1,000 to about 80,000 Da, about 1,000 to about 70,000 Da, about 1,000 to about 60,000 Da, about 1,000 to about 50,000 Da, about 1,000 to about 40,000 Da, about 1,000 to about 30,000 Da, about 1,000 to about 20,000 Da, about 1,000 to about 10,000 Da, about 2,000 to about 10,000 Da, about 3,000 to about 10,000 Da, about 4,000 to about 10,000 Da, or about 5,000 to about 10,000 Da. therapeutic agent

[0108] In some embodiments, the therapeutic agent used in the conjugates of the present disclosure is a small molecule having a molecular weight of about 5 kDa or less, about 4 kDa or less, about 3 kDa or less, about 1.5 kDa or less, or about 1 kDa or less.

[0109] In some embodiments, the therapeutic agent has an IC 50 It has.

[0110] IC above approximately 1 μM 50Some therapeutic agents having an IC of greater than about 1 μM are unsuitable for conjugation with a targeting moiety using art-recognized conjugation techniques. Without wishing to be bound by theory, such therapeutic agents cannot be conjugated to sufficient copies (i.e., more than 8) of the drug using art-recognized techniques without reducing the pharmacokinetics and physiochemical properties of the conjugate, and have insufficient potency for use in moiety-drug conjugate targeting using conventional techniques. However, using the conjugation strategies described herein, sufficiently high loading of these relatively low-potency drugs can be achieved, thereby obtaining high therapeutic agent loading while maintaining desirable pharmacokinetics and physiochemical properties. Thus, in some embodiments, an IC of greater than about 1 μM can be achieved. 50 Therapeutic agents having the formula:

[0111] Small molecule therapeutic agents (e.g., antiproliferative (cytotoxic and cytostatic) agents that can be linked to polymeric carriers) used in the present disclosure include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PI3K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperone inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / Hedgehog signaling inhibitors, and RNA polymerase inhibitors.

[0112] Broad spectrum cytotoxins include, but are not limited to, DNA binding, intercalating or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, topoisomerase I inhibitors and protein synthesis inhibitors.

[0113] Exemplary DNA-binding, intercalating or alkylating drugs include CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, nemorubicin and its derivatives, PNU-159682), bisnapthalimide compounds such as elinafide (LU79553) and its analogs, alkylating agents such as calicheamicin, dactinomycin, mitromycines, pyrrolobenzodiazepines, and the like. Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin A, duocarmycin C1, duocarmycin C2, duocarmycin B1, duocarmycin B2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, seco-adozelesin, and related analogs and prodrug forms, examples of which are described in U.S. Patent Nos. 5,475,092, 5,595,499, 5,846,545, 6,534,660, 6,586,618, 6,756,397, and 7,049,316. ​​Doxorubicin and its analogs include those described in U.S. Patent No. 6,630,579. Calicheamicins include, for example, enediynes, such as esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116. Duocarmycins include those described in U.S. Patent Nos. 5,070,092, 5,101,038, 5,187,186, 6,548,530, 6,660,742, and 7,553,816(B2) and Li et al., Tet Letts., 50:2932-2935 (2009).

[0114] Pyrrolobenzodiazepines (PBDs) and analogs thereof include those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000) and Antonow and Thurston, Chem Rev., 2815-2864 (2010).

[0115] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel, maytansinoids, auristatins and their analogs, vinca alkaloid derivatives, epothilones, and cryptophycins.

[0116] Exemplary maytansinoids or maytansinoid analogs include maytansinol and maytansinol analogs, such as maytansine or DM-1 and DM-4, ​​as described in U.S. Patent Nos. 5,208,020, 5,416,064, 6,333,410, 6,441,163, 6,716,821, RE39,151, and 7,276,497. In certain embodiments, the cytotoxic agent is a maytansinoid, another group of antitubulin agents (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131), a maytansinoid, or a maytansinoid analog. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Suitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219, 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929, 4,331,598, 4,361,650, 4,362,663, 4,364,866, 4,450,254, 4,322,348, 4,371,533, 6,333,410, 5,475,092, 5,585,499, and 5,846,545.

[0117] Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F, auristatin F phenylenediamine (AFP), auristatin F HPA, and dolastatin. Suitable auristatins also are described in U.S. Patent Application Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248, PCT Application Publication Nos. WO09 / 117531, WO2005 / 081711, WO04 / 010957, WO02 / 088172, and WO01 / 24763, and U.S. Patent Nos. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,124,431, 6,034,065, 5,780,588, 5,790,591, 5,800,592, 5,800,593, 5,800,594, 5,800,595, 5,800,596, 5,800,597, 5,800,598, 5,800,59 ... Nos. 67,237, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, the entire disclosures of which are incorporated herein by reference.

[0118] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). Suitable vinca alkaloids that may be used in the present disclosure are also disclosed in U.S. Patent Application Publication Nos. 2002 / 0103136 and 2010 / 0305149, and U.S. Patent No. 7,303,749 (B1), the entire contents of which are incorporated herein by reference.

[0119] Exemplary epothilone compounds include epothilones A, B, C, D, E and F, and derivatives thereof. Suitable epothilone compounds and their derivatives are described, for example, in U.S. Pat. Nos. 6,956,036, 6,989,450, 6,121,029, 6,117,659, 6,096,757, 6,043,372, 5,969,145, and 5,886,026, as well as WO97 / 19086, WO98 / 08849, WO98 / 22461, WO98 / 25929, WO98 / 38192, WO99 / 01124, WO99 / 02514, WO99 / 03848, WO99 / 07692, WO99 / 27890, and WO99 / 28324, the entire disclosures of which are incorporated herein by reference.

[0120] Exemplary cryptophycin compounds are described in US Pat. Nos. 6,680,311 and 6,747,021.

[0121] Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin (PARAPLATIN®), oxaliplatin (ELOXATINE®), iproplatin, ormaplatin, and tetraplatin.

[0122] Still other classes of compounds or compounds with these or other cytotoxic modes of action can be selected, including, for example, mitomycin C, mitomycin A, daunorubicin, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, aminopterin, bleomycin, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol, pyrrolobenzodiazepine (PBD) polyamides and dimers thereof. Other suitable cytotoxic agents include, for example, puromycin, topotecan, rhizoxin, echinomycin, combretastatin, netropsin, estramustine, cryptophycin, cemadotin, discodermolide, eleutherobin, and mitoxantrone.

[0123] Exemplary topoisomerase I inhibitors include camptothecin, camptothecin derivatives, camptothecin analogs, and unnatural camptothecins, such as CPT-11 (irinotecan), SN-38, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, rubitecan, gimatecan, karenitecin, ciratecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan, and 539625. Other camptothecin compounds that can be used in the present invention include, for example, those described in J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30:1774 (1987).

[0124] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, and MetAP2 inhibitors.Exemplary VGFR and PDGFR inhibitors include sorafenib (Nexavar), sunitinib (Sutent) and vatalanib.Exemplary MetAP2 inhibitors include fumagillol analogs, which refer to any compound that contains the fumagillin core structure, including fumagillamine, that inhibits the ability of MetAP-2 to remove NH2-terminal methionine from proteins, as described in Rodeschini et al., J.Org.Chem., 69, 357-373, 2004 and Liu et al., Science 282, 1324-1327, 1998. Non-limiting examples of "fumagillol analogs" are disclosed in J. Org. Chem., 69, 357, 2004; J. Org. Chem., 70, 6870, 2005; European Patent Application No. 0 354 787; J. Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg. Med. Chem., 14, 91, 2004; Tet. Lett., 40, 4797, 1999; WO 99 / 61432, U.S. Patent Nos. 6,603,812, 5,789,405, 5,767,293, 6,566,541, and 6,207,704.

[0125] Exemplary cell cycle progression inhibitors include CDK inhibitors such as BMS-387032 and PD0332991, Rho kinase inhibitors such as GSK429286, checkpoint kinase inhibitors such as AZD7762, Aurora kinase inhibitors such as AZD1152, MLN8054 and MLN8237, PLK inhibitors such as BI2536, BI6727 (vorasertib), GSK461364, ON-01910 (Estybon), and KSP inhibitors such as SB743921, SB715992 (ispinesib), MK-0731, AZD8477, AZ3146 and ARRY-520.

[0126] Exemplary PI3K / m-TOR / AKT signaling pathway inhibitors include phosphoinositide 3-kinase (PI3K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors, and PDK-1 inhibitors.

[0127] Exemplary PI3 kinase inhibitors are disclosed in U.S. Pat. No. 6,608,053 and include BEZ235, BGT226, BKM120, CAL101, CAL263, demethoxyviridine, GDC-0941, GSK615, IC87114, LY294002, Palomid529, perifosine, PI-103, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, wortmannin, XL147, and XL765.

[0128] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0129] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.

[0130] Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,994 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, RO5126766 and RO4987655, PD0325901, AZD6244, AZD8330 and GDC-0973.

[0131] Exemplary B-raf inhibitors include CDC-0879, PLX-4032 and SB590885.

[0132] Exemplary B p38 MAPK inhibitors include BIRB796, LY2228820 and SB202190.

[0133] Receptor tyrosine kinases (RTKs) are cell surface receptors that are often associated with signal transduction pathways that stimulate the uncontrolled growth of cancer cells and angiogenesis. Many RTKs have been identified that are overexpressed or mutated, leading to constitutive receptor activation, including but not limited to VEGFR, EGFR, FGFR, PDGFR, EphR, and RET receptor family receptors. Exemplary specific RTK targets include ErbB2, FLT-3, c-Kit, and c-Met.

[0134] Exemplary ErbB2 receptor (EGFR family) inhibitors include, but are not limited to, AEE788 (NVP-AEE788), BIBW2992 (afatinib), lapatinib, erlotinib (Tarceva), and gefitinib (Iressa).

[0135] Exemplary RTK inhibitors that target more than one signaling pathway (multiple-targeted kinase inhibitors) include AP24534 (ponatinib), which targets FGFR, FLT-3, VEGFR-PDGFR, and Bcr-Abl receptors; ABT-869 (linifanib), which targets FLT-3 and VEGFR-PDGFR receptors; AZD2171, which targets VEGFR-PDGFR, Flt-1, and VEGF receptors; CHR-258 (dovitinib), which targets VEGFR-PDGFR, FGFR, Flt-3, and c-Kit receptors; sunitinib (sutent), which targets VEGFR, PDGFR, KIT, FLT-3, and CSF-IR; and sorafenib (nexavar) and vatalanib, which target intracellular serine / threonine kinases in the VEGFR, PDGFR, and Raf / Mek / Erk pathways.

[0136] Exemplary protein chaperone inhibitors include HSP90 inhibitors. Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922, and KW-2478.

[0137] Exemplary HDAC inhibitors include belinostat (PXD101), CUDC-101, droxinostat, ITF2357 (divinostat, gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, dacinostat), LBH-589 (panobinostat), MC1568, MGCD0103 (mosetinostat), MS-275 (entinostat), PCI-24781, pyroxamide (NSC696085), SB939, trichostatin A, and vorinostat (SAHA).

[0138] Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (veliparib), AG014699, CEP 9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0139] Exemplary Wnt / Hedgehog signaling pathway inhibitors include vismodegib (RG3616 / GDC-0449), cyclopamine (11-deoxojervine) (a Hedgehog pathway inhibitor), and XAV-939 (a Wnt pathway inhibitor).

[0140] Exemplary RNA polymerase inhibitors include amatoxins, including α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanulin, amanuric acid, amaninamide, amanine, and proamanulin.

[0141] Exemplary protein synthesis inhibitors include trichothecene compounds.

[0142] In some embodiments, the therapeutic agent of the present disclosure is a topoisomerase inhibitor (such as, for example, a non-natural camptothecin compound), a vinca alkaloid, a kinase inhibitor (such as, for example, a PI3 kinase inhibitor (GDC-0941 and PI-103)), a MEK inhibitor, a KSP inhibitor, an RNA polymerase inhibitor, a protein synthesis inhibitor, a PARP inhibitor, docetaxel, paclitaxel, doxorubicin, a duocarmycin, an auristatin, a dolastatin, a calicheamicin, topotecan, SN38, camptothecin, exatecan, nemorubicin and its derivatives, PNU-159682, CC1065, elinafide, a trichothecene, a pyrrolobenzodiazepine, a maytansinoid, a DNA-binding drug, or a platinum compound, and analogs thereof. In some embodiments, the therapeutic agent is a derivative of SN-38, camptothecin, topotecan, exatecan, calicheamicin, exatecan, nemorubicin, PNU-159682, anthracycline, maytansinoid, taxane, trichothecene, CC1065, elinafide, vindesine, vinblastine, PI-103, AZD8330, dolastatin, auristatin E, auristatin F, duocarmycin compounds, ispinesib, pyrrolobenzodiazepine, ARRY-520, and stereoisomers, isosteres, and analogs thereof.

[0143] In some embodiments, the therapeutic agent used in the present disclosure is a combination of two or more drugs, for example, a PI3 kinase inhibitor and a MEK inhibitor, a broad-spectrum cytotoxic compound and a platinum compound, a PARP inhibitor and a platinum compound, or a broad-spectrum cytotoxic compound and a PARP inhibitor.

[0144] In yet another embodiment, the therapeutic agent used in the present disclosure is auristatin F-hydroxypropylamido-L-alanine.

[0145] Those skilled in the art will readily understand that each of the therapeutic agents described herein can be modified in such a way that the resulting compound still retains the specificity and / or activity of the original compound.Those skilled in the art will also understand that many of these compounds can be used instead of the therapeutic agents described herein.Therefore, the therapeutic agents of the present disclosure include analogs and derivatives of the compounds described herein.

[0146] In some embodiments, the therapeutic agent has anti-proliferative activity against the target cell or pathway.

[0147] In certain embodiments, the antiproliferative activity is selected from cytostatic and / or cytotoxic activity.

[0148] In certain embodiments, the therapeutic agent is selected from anti-cancer agents, cytotoxic drugs, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antivirals, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, antiglaucoma compounds, antiparasitic and / or antiprotozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensive agents, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotics, antiemetics, and imaging agents.

[0149] In certain embodiments, the therapeutic agent comprises an amino acid-based molecule.

[0150] In certain embodiments, the amino acid-based molecule includes a peptide, polypeptide, enzyme, antibody, immunoglobulin, or functional fragment thereof.

[0151] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0152] In certain embodiments, the chemically reactive group is -COOH, primary amine, secondary amine -NHR, -OH, -SH, -C(O)H, C(O)R 14 , -C(O)NHR 15 , -C(S)OH, -S(O)2OR 15 , -P(O)2OR 15 , -CN, -NC, or -ONO, where R 14 is selected from an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Linker-polymer-drug compounds

[0153] In one embodiment, a polymer scaffold of formula (I) useful for conjugating a targeting moiety: [ka] is provided, where: the polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with said targeting moiety. p is a linking moiety comprising M a L to B a It is a stretcher that connects to the part, B a is the functional group W attached to the -NH- moiety M is a branched portion including each G 1 is independent, L P is a functional group that connects the Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are functional groups that can be independently converted to a charged state, n is an integer from 0 to 1000, m is an integer from 0 to 1000, p is an integer from 0 to 1000, and q is an integer from 0 to 1000.

[0154] In some embodiments, n is an integer from 0 to 1000, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, or 0 to 2.

[0155] In some embodiments, m is an integer between 0 and 1000, between 0 and 500, between 0 and 400, between 0 and 300, between 0 and 200, between 0 and 100, between 0 and 90, between 0 and 80, between 0 and 70, between 0 and 60, between 0 and 50, between 0 and 40, between 0 and 30, between 0 and 20, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2.

[0156] In some embodiments, p is an integer between 0 and 1000, between 0 and 500, between 0 and 400, between 0 and 300, between 0 and 200, between 0 and 100, between 0 and 90, between 0 and 80, between 0 and 70, between 0 and 60, between 0 and 50, between 0 and 40, between 0 and 30, between 0 and 20, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2.

[0157] In some embodiments, q is an integer from 0 to 1000, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 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.

[0158] In some embodiments, W p can react with a functional group on the targeting moiety via a click reaction.

[0159] In certain embodiments, W p teeth, [ka] is selected from the group consisting of:

[0160] In some embodiments, W p can react with an amino acid on the targeting moiety.

[0161] In certain embodiments, W p can react with amino acids on the targeting moiety, where the amino acids are natural amino acids, unnatural amino acids, or combinations thereof. In certain embodiments, natural amino acids can include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0162] In some embodiments, W p can react with one or more cysteines on the targeting moiety.

[0163] In certain embodiments, W p can react with one or more cysteines on the targeting moiety, and each W p teeth, [ka] wherein R 1 is a sulfur protecting group, and each R 2 are independently leaving groups.

[0164] In certain embodiments, each R 2 are independently halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0165] In some embodiments, W p can react with one or more lysines on the targeting moiety.

[0166] In certain embodiments, Wp can react with one or more lysines on the targeting moiety, and each W p is independent, [ka] is selected from the group consisting of:

[0167] In some embodiments, W p can react with one or more unnatural amino acids on the targeting moiety.

[0168] In certain embodiments, W p can react with one or more unnatural amino acids on the targeting moiety, and each W p is independent, [ka] is selected from.

[0169] In some embodiments, L is [ka] is.

[0170] In some embodiments, M a is R 3 , [ka] wherein * is the site of covalent attachment to L and ** is B a a moiety covalently bonded to the moiety, R 3 is C 1~10 Alkyl, C 1~10 Heteroalkyl, C 3~8 Cycloalkyl, -O-(C 1~8 alkyl), aryl, -C 1~10 Alkyl-aryl-, -aryl-C 1~10 Alkyl-, -C 1~10 Alkyl-(C 3~8cycloalkyl)-, -(C 3~8 Cycloalkyl-C 1~10 alkyl), 4- to 14-membered heterocycloalkyl, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-, -C 1~10 Alkyl-C(=O)-, -C 1~10 Heteroalkyl-C(=O)-, -C 3~8 Cycloalkyl-C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -aryl-C(=O)-, -C 1~10 Alkyl-aryl-C(=O)-, -aryl-C 1~10 Alkyl-C(=O)-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-C(=O)-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-C(=O)-, -4 to 14-membered heterocycloalkyl-C=(O)-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-C(=O)-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-C(=O)-, -C 1~10 Alkyl-NH-, -C 1~10 Heteroalkyl-NH-, -C 3~8 Cycloalkyl-NH-, -O-(C 1~8 Alkyl)-NH-, -aryl-NH-, -C 1~10 Alkyl-aryl-NH-, -aryl-C 1~10 Alkyl-NH-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-NH-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-NH-, -4 to 14-membered heterocycloalkyl-NH-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-NH-, -C 1~10 Alkyl-S-, -C 1~10 Heteroalkyl-S-, -C 3~8Cycloalkyl-S-, -OC 1~8 Alkyl-S-, -aryl-S-, -C 1~10 Alkyl-aryl-S-, -aryl-C 1~10 Alkyl-S-, -C 1~10 Alkyl-(C 3~8 Cycloalkyl)-S-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-S-, -4 to 14-membered heterocycloalkyl-S-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-S- or -(4- to 14-membered heterocycloalkyl)-C 1~10 alkyl-S-, Each R 4 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 5 -C(O)-NR 5a or -NR 5a -C(O)-, R 5a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-, R 6a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, Each R 6b and R 6c are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10Aryl, 5-12 membered heterocycloalkyl, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl or the side chain of a natural or unnatural amino acid; each n 1 are independently integers from 0 to 6, n 2 is an integer between 0 and 8, each n 3 are independently integers from 1 to 6, n 4 is an integer from 1 to 4, and n 5 is an integer between 1 and 4.

[0171] In certain embodiments, M a teeth, [ka] is selected from the group consisting of:

[0172] In some embodiments, G 1 teeth, [ka] wherein * is selected from the group consisting of L P Each R 7 are independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 7a is selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0173] In some embodiments, each L p independently contain unstable structures.

[0174] In certain embodiments, the labile structure is selected from a redox labile structure, a hydrolytically labile structure, or an enzyme labile structure.

[0175] In certain embodiments, the labile structure is a redox-labile structure.

[0176] In certain embodiments, the unstable structure is [ka] wherein each R 18 are independently selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl.

[0177] In certain embodiments, the labile structure is a hydrolytically labile structure.

[0178] In certain embodiments, the unstable structure is [ka] wherein * is a hydrolytically unstable structure selected from the group consisting of G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and R 8 is selected from hydrogen, alkyl, or aryl; R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0179] In certain embodiments, G 1 teeth, [ka] where * is L P is a covalently bonded moiety to R 7 is alkyl (C 1~6 alkyl, etc.).

[0180] In certain embodiments, -G 1 -L P -D is [ka] is.

[0181] In some embodiments, the labile structure is an enzyme-labile structure.

[0182] In certain embodiments, the labile structure is an enzyme-labile structure that is labile to an enzyme selected from cathepsin B, a phosphatase, a sulfatase, or a glucuronidase.

[0183] In certain embodiments, the enzyme-labile structure is sensitive to cathepsin B and is -Z-, or [ka] wherein * is selected from G 1 ** is the covalently bound site to D; Z is a substrate for cathepsin B containing 2-4 amino acids; and R 7a is alkyl.

[0184] In certain embodiments, G 1 teeth, [ka] where * is L P is a covalently bonded moiety to R 7 is alkyl (C 1~6 alkyl, etc.).

[0185] In certain embodiments, -G 1 -L P -D is [ka] is.

[0186] In certain embodiments, the enzyme-labile structure is sensitive to glucuronidase; [ka] where * is G 1 is the site covalently bonded to , and ** is the site covalently bonded to D.

[0187] In certain embodiments, G 1 teeth, [ka] where * is L P is a covalently bonded moiety to R 7 is alkyl (C 1~6 alkyl, etc.).

[0188] In certain embodiments, -G 1 -L P -D is [ka] is selected from.

[0189] In certain embodiments, the enzyme-labile structure is sensitive to phosphatases; [ka] wherein * is selected from G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 10 and R 11 is independently hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0190] In certain embodiments, G 1 teeth, [ka] is.

[0191] In certain embodiments, -G 1 -L P -D is [ka] [ka] is selected from the group consisting of:

[0192] In certain embodiments, the enzyme-labile structure is susceptible to sulfatase; [ka] where * is G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 12 and R 13 is independently hydrogen, —NH—, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0193] In some embodiments, G 1 teeth, [ka] where * is L P is a covalently bonded moiety to R 7 is alkyl (C 1~6 alkyl, etc.).

[0194] In certain embodiments, -G 1 -L P -D is [ka] is.

[0195] In some embodiments, n is an integer from 1-100, m is an integer from 1-100, and p is an integer from 1-50.

[0196] In some embodiments, the therapeutic agent has anti-proliferative activity against the target cell or pathway.

[0197] In certain embodiments, the antiproliferative activity is selected from cytostatic and / or cytotoxic activity.

[0198] In certain embodiments, the therapeutic agent is selected from anti-cancer agents, cytotoxic drugs, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antivirals, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, antiglaucoma compounds, antiparasitic and / or antiprotozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensive agents, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotics, antiemetics, and imaging agents.

[0199] In certain embodiments, the therapeutic agent comprises an amino acid-based molecule.

[0200] In certain embodiments, the amino acid-based molecule includes a peptide, polypeptide, enzyme, antibody, immunoglobulin, or functional fragment thereof.

[0201] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0202] In certain embodiments, the chemically reactive group is -COOH, primary amine, secondary amine -NHR, -OH, -SH, -C(O)H, C(O)R 14 , -C(O)NHR 15 , -C(S)OH, -S(O)2OR 15 , -P(O)2OR 15 , -CN, -NC, or -ONO, where R 14 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0203] In certain embodiments, G 2 teeth, [ka] wherein each R 16 is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In certain embodiments, each R 16 is independently hydrogen or alkyl. In certain embodiments, each R 16 are independently hydrogen or C 1~6 It is alkyl.

[0204] In some embodiments, B a teeth, [ka] and W L Stretcher M a is a functional group that connects to Each Z is independently a branch point, Each R 19 are independent and become Z and W M is a linker connecting Each W M are independently functional groups bonded to -NH-, r is an integer from 1 to 3, t is an integer from 1 to 3, In the formula, * represents M a and ** denotes a site covalently bound to the -NH- moiety.

[0205] In some embodiments, the —NH— moiety is [ka] -NH-(CH2) n6 —COOH, wherein G 1 , L p , D, G 2 , n, m, p, and q are as defined above, and n 6 is an integer from 1 to 6.

[0206] In certain embodiments, W L teeth, [ka] is selected from the group consisting of:

[0207] In certain embodiments, Z is —CH (3-r) -, -SiH (3-r) -, or -NH (2-r) -It is.

[0208] In certain embodiments, R 19 is selected from the group consisting of aliphatic, heteroaliphatic, cycloalkyl, and heterocycloalkyl. 19 is alkyl or heteroalkyl.

[0209] In some embodiments, B a teeth, [ka] is.

[0210] In certain embodiments, B a teeth, [ka] is selected from the group consisting of:

[0211] In some embodiments, B a teeth, [ka] is.

[0212] In certain embodiments, B a teeth, [ka] is selected from the group consisting of:

[0213] In certain embodiments, B a teeth, [ka] is.

[0214] In some embodiments, B a teeth, [ka] where two W M is -NH-(CH2) n6 -COOH and one W M teeth, [ka] Connect to.

[0215] In some embodiments, B a teeth, [ka] where the three W M teeth, [ka] Connect to.

[0216] In some embodiments, B a teeth, [ka] where two W M teeth, [ka] Connect to one W M teeth, [ka] Connect to.

[0217] In certain embodiments, W Lteeth, [ka] and -R 19 -W M -teeth, [ka] where ** is the site of covalent attachment to the -NH- moiety.

[0218] In certain embodiments, B a teeth, [ka] is.

[0219] In certain embodiments, the polymer scaffolds provided herein comprise: [ka] [ka] wherein: [ka] [ka] Linker-polymer compounds

[0220] The present disclosure also relates to linker-polymer compounds that can be further attached to drug moieties to form linker-polymer-drug compounds provided herein.

[0221] Thus, in a further aspect, the present disclosure provides a polymer scaffold of formula (II): [ka] where: the polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with said targeting moiety. p is a linking moiety comprising M a L to B a It is a stretcher that connects to the part, B a is the functional group W attached to the -NH- moiety M is a branched portion including each G 2 are functional groups that can be independently converted to a charged state, each G 3 independently comprise a functional group capable of reacting with a reactive group on the drug release mechanism to connect the drug release mechanism to the linear polyglycerol; n is an integer from 0 to 1000, m is an integer from 0 to 1000, p is an integer from 0 to 1000, and q is an integer from 0 to 1000.

[0222] In some embodiments, n, m, p, and q are all integers from 1-5.

[0223] In some embodiments, W p can react with a functional group on the targeting moiety via a click reaction.

[0224] In certain embodiments, W p teeth, [ka] is selected from the group consisting of:

[0225] In some embodiments, W p can react with an amino acid on the targeting moiety.

[0226] In certain embodiments, W pcan react with amino acids on the targeting moiety, where the amino acids are natural amino acids, unnatural amino acids, or combinations thereof. In certain embodiments, natural amino acids can include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0227] In some embodiments, W p can react with one or more cysteines on the targeting moiety.

[0228] In certain embodiments, W p can react with one or more cysteines on the targeting moiety, and each W p teeth, [ka] wherein R 1 is a sulfur protecting group, and each R 2 are independently leaving groups.

[0229] In certain embodiments, each R 2 are independently halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0230] In some embodiments, W p can react with one or more lysines on the targeting moiety.

[0231] In certain embodiments, W p can react with one or more lysines on the targeting moiety, and each W p is independent, [ka] is selected from the group consisting of:

[0232] In some embodiments, W pcan react with one or more unnatural amino acids on the targeting moiety.

[0233] In certain embodiments, W p can react with one or more unnatural amino acids on the targeting moiety, and each W p is independent, [ka] is selected from.

[0234] In some embodiments, L is [ka] is.

[0235] In some embodiments, M a is R 3 , [ka] wherein * is the site of covalent attachment to L and ** is B a a moiety covalently bonded to the moiety, R 3 is C 1~10 Alkyl, C 1~10 Heteroalkyl, C 3~8 Cycloalkyl, -O-(C 1~8 alkyl), aryl, -C 1~10 Alkyl-aryl-, -aryl-C 1~10 Alkyl-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-, -(C 3~8 Cycloalkyl-C 1~10 alkyl), 4- to 14-membered heterocycloalkyl, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-, -C 1~10 Alkyl-C(=O)-, -C 1~10 Heteroalkyl-C(=O)-, -C 3~8Cycloalkyl-C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -aryl-C(=O)-, -C 1~10 Alkyl-aryl-C(=O)-, -aryl-C 1~10 Alkyl-C(=O)-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-C(=O)-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-C(=O)-, -4 to 14-membered heterocycloalkyl-C=(O)-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-C(=O)-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-C(=O)-, -C 1~10 Alkyl-NH-, -C 1~10 Heteroalkyl-NH-, -C 3~8 Cycloalkyl-NH-, -O-(C 1~8 Alkyl)-NH-, -aryl-NH-, -C 1~10 Alkyl-aryl-NH-, -aryl-C 1~10 Alkyl-NH-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-NH-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-NH-, -4 to 14-membered heterocycloalkyl-NH-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-NH-, -C 1~10 Alkyl-S-, -C 1~10 Heteroalkyl-S-, -C 3~8 Cycloalkyl-S-, -OC 1~8 Alkyl-S-, -aryl-S-, -C 1~10 Alkyl-aryl-S-, -aryl-C 1~10 Alkyl-S-, -C 1~10 Alkyl-(C 3~8 Cycloalkyl)-S-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-S-, -4 to 14-membered heterocycloalkyl-S-, -C1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-S- or -(4- to 14-membered heterocycloalkyl)-C 1~10 alkyl-S-, Each R 4 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 5 -C(O)-NR 5a or -NR 5a -C(O)-, R 5a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-, R 6a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, Each R 6b and R 6c are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycloalkyl, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl or the side chain of a natural or unnatural amino acid; each n 1 are independently integers from 0 to 6, n 2 is an integer between 0 and 8, each n3 are independently integers from 1 to 6, n 4 is an integer from 1 to 4, and n 5 is an integer between 1 and 4.

[0236] In certain embodiments, M a teeth, [ka] wherein * is the site of covalent attachment to L and ** is B a The moiety is covalently bonded to the

[0237] In some embodiments, B a teeth, [ka] and W L Stretcher M a is a functional group that connects to Each Z is independently a branch point, Each R 19 are independent and become Z and W M is a linker connecting Each W M are independently functional groups bonded to -NH-, r is an integer from 1 to 3, t is an integer from 1 to 3, In the formula, * represents M a and ** denotes a site covalently bound to the -NH- moiety.

[0238] In some embodiments, the —NH— moiety is [ka] -NH-(CH2) n6 -COOH, wherein G 2 , n, m, p, and q are as defined above, and n 6 is an integer from 1 to 6.

[0239] In certain embodiments, W L teeth, [ka] is selected from the group consisting of:

[0240] In certain embodiments, Z is —CH (3-r) -, -SiH (3-r) -, or -NH (2-r) -It is.

[0241] In certain embodiments, R 19 is selected from the group consisting of aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl.

[0242] In some embodiments, B a teeth, [ka] is.

[0243] In certain embodiments, B a teeth, [ka] is selected from the group consisting of:

[0244] In some embodiments, B a teeth, [ka] is.

[0245] In certain embodiments, B a teeth, [ka] is selected from the group consisting of:

[0246] In certain embodiments, B a teeth, [ka] is.

[0247] In some embodiments, two W M is -NH-(CH2) n6 -COOH and one W M teeth, [ka] Connect to.

[0248] In some embodiments, three W M teeth, [ka] Connect to.

[0249] In certain embodiments, W L teeth, [ka] and -R 19 -W M -teeth, [ka] where ** is W L or a site covalently bonded to the -NH- moiety.

[0250] In certain embodiments, B a teeth, [ka] [ka] is.

[0251] In some embodiments, G 2 and G 3 is independent, [ka] wherein each R 17 is independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0252] In some embodiments, G 2 and G 3 is independent, [ka] wherein R 17 Ga-(CH2) 1-3 -It is.

[0253] In some embodiments, the polymer scaffolds provided herein have the structure of formula (IIa), (IIb), (IIc), (IIA), or (IIB): [ka] [ka] It has.

[0254] In some embodiments, n is an integer from 0 to 1000, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, or 0 to 2.

[0255] In some embodiments, m is an integer between 0 and 1000, between 0 and 500, between 0 and 400, between 0 and 300, between 0 and 200, between 0 and 100, between 0 and 90, between 0 and 80, between 0 and 70, between 0 and 60, between 0 and 50, between 0 and 40, between 0 and 30, between 0 and 20, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2.

[0256] In some embodiments, p is an integer between 0 and 1000, between 0 and 500, between 0 and 400, between 0 and 300, between 0 and 200, between 0 and 100, between 0 and 90, between 0 and 80, between 0 and 70, between 0 and 60, between 0 and 50, between 0 and 40, between 0 and 30, between 0 and 20, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2.

[0257] In some embodiments, q is an integer from 0 to 1000, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 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.

[0258] In some embodiments, the polymer scaffolds provided herein have the structure of formula (IId), (IIe), (IIf), (IIg), or (IIh): [ka] [ka] It has. Targeting section

[0259] The targeting moiety directs the linker-polymer-drug conjugate to a specific tissue, cell, or subcellular location. The targeting moiety can direct the modified polymer in culture, in a whole organism, or both. In each case, the targeting moiety can bind to a ligand present on the cell surface of the target cell with effective specificity, affinity, and avidity. In some embodiments, the targeting moiety targets the modified polymer to a tissue other than the liver. In other embodiments, the targeting moiety targets the modified polymer to a specific tissue, such as the liver, kidney, lung, or pancreas. The targeting moiety can target the modified polymer to a receptor expressed on cells such as cancer cells, matrix tissue, or a protein associated with cancer, such as a tumor antigen. Alternatively, cells containing tumor vasculature can be targeted. The targeting moiety can direct the modified polymer to a specific type of cell, such as hepatocytes in the liver, as opposed to Kupffer cells. In other cases, the targeting moiety can direct the modified polymer to reticuloendothelial or lymphoid cells, or to professional phagocytic cells, such as macrophages or eosinophils.

[0260] In still other embodiments, the targeting moiety can target the modified polymer to an intracellular location, such as the nucleus, cytoplasm, or endosome, etc. In certain embodiments, the targeting moiety can enhance binding to a cellular receptor, or cytoplasmic transport and entry into the nucleus, or release from an endosome or other intracellular vesicle.

[0261] In some embodiments, targeting moieties include antibodies, proteins, and peptides or peptidomimetics.

[0262] In some embodiments, the targeting moiety comprises a natural amino acid that can react with a functional group of the linking moiety of the linker-polymer-drug conjugate to form a covalent bond. In certain embodiments, the natural amino acids include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0263] In certain embodiments, the targeting moiety comprises a cysteine ​​and the targeting moiety is covalently conjugated to the linker-polymer-drug conjugate via a sulfhydryl group and a functional group on the linking portion of the linker-polymer-drug conjugate.

[0264] In certain embodiments, the targeting moiety comprises a lysine, and the targeting moiety is covalently conjugated to the linker-polymer-drug conjugate via an amino group and a functional group on the linking portion of the linker-polymer-drug conjugate.

[0265] In some embodiments, the targeting moiety may comprise an unnatural amino acid that can react with a functional group of the linking moiety of the linker-polymer-drug conjugate to form a covalent bond. In certain embodiments, the targeting moiety is covalently conjugated to the linker-polymer-drug conjugate via the amino group and the functional group of the linking moiety of the linker-polymer-drug conjugate.

[0266] In some embodiments, the targeting moiety may comprise a functional group that can react with a functional group of the linking moiety of the linker-polymer-drug conjugate via a click reaction to form a covalent bond.

[0267] In some embodiments, the targeting moiety is selected from, but not limited to, 5T4, AOC3, ALK, AXL, 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, 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, T RPV1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP, adrenergic receptor-beta 2, claudin 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, integrin (α4, α v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), 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), mucin, MUC1, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostate cancer cells, Pseudomonas aeruginosa aeruginosa), hydrophobia, RANKL, respiratory syncytial virus, Rh factor, SLAMF7, sphingosine-1-phosphate, TAG-72, T cell receptor, tenascin-C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, and the like.

[0268] In certain embodiments, antibodies or antibody or camelid antibody heavy chain fragments derived from Fab, Fab2, scFv, specific for cell surface markers include CA-125, C242, CD3, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD138, CD141, CD326, CEA, CTLA-4, EGFR (HER1), ErbB2, ErbB3, FA P, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, TAG-72, tenascin-C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate cancer cells, adrenergic receptor-beta 2, claudin 3, mucin, MUC1, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, and integrin (α v β3, α v β5, α vβ6, α1β4, α4β1, α5β1, α6β4 integrins), tenascin-C, TRAIL-R2, and vimentin.

[0269] Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO), adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), acelizumab, atlizumab (tocilizumab, Actemra, RoActemra), atrolimumab, bapineuzumab, basiliximab (Simulect), bavituximab, bectumomab (LYMPHOS CAN), 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), sitatuzumab, cixutumumab, clenoximab, clivatuzumab, conatumumab, CR6261, dace Tuzumab, daclizumab (ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab, dorlixizumab, ecloneximab, eculizumab (SOLIRIS), edovacomab, edrecolomab (PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab, ersilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab (REXOMUN), etaracizumab (ABEGRIN), exibirumab, fanolesomab (NEUTRO) SPEC), faralimomab, farletuzumab, felvizumab, fezakinumab, fizitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab, gantenerumab, gavilimomab, gemtuzumab, girentuximab, glembatumumab, golimumab (SIMPONI), golimumab, ibalizumab, ibritumomab, igovomab (INDIMACIS-125), imuciromab (MYOSCINT), infliximab (REMICADE), intetumumab, inolimomab, inotuzumab, ipilimumab,Iratumumab, keliximab, labetuzumab (CEA-CIDE), lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, lintuzumab, lucatumumab, rumiliximab, mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab, milatuzumab, minletumomab, mitumomab, morolimumab, motavizumab (NUMAX), muromonab-CD3 (ORTHOCLONE) OKT3), nacolomab, naptumomab, natalizumab (TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM), nofetumomab, ocrelizumab, ozlimomab, ofatumumab (ARZERRA), olaratumab, omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX), otelixizumab, pasivaximab, palivizumab (SYNAGIS), panitumumab Tutumumab (VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab (OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO140, rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), lobatumumab, lontalizumab, robelizumab (L EUKARREST), ruplizumab (ANTOVA), sacituzumab, satumomab pendetide, cevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab, solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab (LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab (AUREXIS) , terimomab, tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA), toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN), tremelimumab, tucotuzumab, tuvilumab, urtoxazumab, ustekinumab (STELERA), bapaliximab, vedolizumab, veltuzumab,These include beparimomab, visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), diralimumab, and zolimomab.

[0270] In some embodiments, the antibody is directed to cell surface markers for 5T4, CA-125, CEA, CD3, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD51, CTLA-4, EpCAM, HER2, EGFR (HER1), FAP, folate receptor, HGF, integrin αvβ3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, MUC1, phosphatidylserine, prostate cancer cells, PDGFRα, TAG-72, tenascin-C, TRAIL-R2, VEGF-A, and VEGFR2. In this embodiment, the antibody is selected from the group consisting of abagovomab, adecatumumab, aracizumab, altumomab, anatumomab, arcitumomab, bavituximab, bevacizumab (AVASTIN), bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, sitatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, fizitumumab, gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab, These include izumab, lexatumumab, lintuzumab, lucatumumab, matuzumab, mitumomab, naptumomab estafenatox, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab (RITUXAN), rilotumumab, lobatumumab, satumomab, sibrotuzumab, taplitumomab, tenatumomab, tenatumomab, ticilimumab (tremelimumab), tigatuzumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab, tucotuzumab-sermoreukin, volociximab, and zalutumumab.

[0271] In particular embodiments, the antibody directed against the cell surface marker for HER2 is pertuzumab or trastuzumab, for EGFR (HER1) the antibody is cetuximab or panitumumab, for CD20 the antibody is rituximab, for VEGF-A the antibody is bevacizumab, for CD-22 the antibody is epratuzumab or veltuzumab, and for CEA the antibody is labetuzumab.

[0272] Exemplary peptides or peptidomimetics include integrin targeting peptides (RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptor targeting peptides, prostate specific membrane bound antigen (PSMA) targeting peptides, lipoprotein receptor LRP1 targeting, ApoE protein derived peptides, ApoA protein peptides, somatostatin receptor targeting peptides, chlorotoxin derived peptides, and bombesin.

[0273] In certain embodiments, the peptides or peptidomimetics are LHRH receptor targeting peptides and ErbB2 (HER2) receptor targeting peptides.

[0274] Exemplary proteins include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudins, apolipoprotein E, affibody molecules such as ABY-025, ankyrin repeat proteins, ankyrin-like repeat proteins, and synthetic peptides.

[0275] In some embodiments, the targeting moiety-linker-polymer-drug conjugate comprises a broad-spectrum cytotoxin in combination with a cell surface marker for HER2, e.g., pertuzumab or trastuzumab, for EGFR, e.g., cetuximab and panitumumab, for CEA, e.g., labetuzumab, for CD20, e.g., rituximab, for VEGF-A, e.g., bevacizumab, or for CD-22, e.g., epratuzumab or veltuzumab.

[0276] In other embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a combination of two or more targeting moieties, for example, a combination of bispecific antibodies directed against EGF receptor (EGFR) on tumor cells and CD3 and CD28 on T cells, a combination of an antibody or an antibody or camelid antibody heavy chain fragment derived from Fab, Fab2, scFv and a peptide or peptidomimetic, a combination of an antibody or an antibody or camelid antibody heavy chain fragment derived from Fab, Fab2, scFv and a protein, a combination of two bispecific antibodies, for example, a CD3xCD19 plus CD28xCD22 bispecific antibody, etc.

[0277] In other embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody to antigen, such as, for example, sacituzumab, trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, veltuzumab, labetuzumab, B7-H4, B7-H3, CA125, CD33, CXCR2, EGFR, FGFR1, FGFR2, FGFR3, FGFR4, HER2, NaPi2b, c-Met, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, c-Kit, MUC1, and 5T4.

[0278] In certain embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody to 5T4, for example, a humanized anti-5T4 scFvFc antibody.

[0279] Examples of suitable 5T4 targeting ligands or immunoglobulins include those commercially available or described in patent or non-patent literature, such as U.S. Patent No. 8,044,178, U.S. Patent No. 8,309,094, U.S. Patent No. 7,514,546, EP 1036091 (commercially available as TroVax™, Oxford Biomedica), EP 2368914 A1, WO 2013041687 A1 (Amgen), US 2010 / 0173382, and those described in P. Sapra et al., Mol. Cancer Ther. 2013, 12:38-47. Anti-5T4 antibodies are disclosed in U.S. Provisional Patent Application No. 61 / 877,439, filed September 13, 2013, and U.S. Provisional Patent Application No. 61 / 835,858, filed June 17, 2013. The entire contents of each patent and scientific publication are incorporated herein by reference.

[0280] As used herein, the term "5T4 antigen-binding moiety" refers to a polypeptide sequence capable of selectively binding to the 5T4 antigen. In exemplary conjugates, the 5T4 antigen-binding moiety generally comprises a single-chain scFv-Fc form engineered from an anti-5T4 antibody. The single-chain variable region (scFv-Fc) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, connected to a linker peptide and further connected to the Fc region, including the hinge region and CH2 and CH3 regions of the antibody (any combination of such antibody moieties with each other or other peptide sequences is sometimes referred to herein as an "immunofusion" molecule). Within such scFvFc molecules, the scFv section can be C-terminally linked to the N-terminus of the Fc section via a linker peptide.

[0281] In some embodiments, the Fv portion of a 5T4 antigen-binding portion can be engineered by well-known molecular biology techniques to contain one or more amino acid substitutions in the VH region. The Fc portion of a 5T4 antigen-binding portion preferably comprises polypeptide sequences engineered from the human hinge, CH2, and CH3 regions of an anti-5T4 antibody. Conjugates

[0282] In one embodiment, a polymer scaffold of formula (III): [ka] is provided, where: the polymer scaffold comprises linear polyglycerol; PBRM is the targeting part, Each L a independently, the targeting moiety is M a is a bivalent moiety that connects to Each M a is independent, L a is a stretcher connecting the -NH- moiety to B a is the functional group W attached to the -NH- moiety M is a branched portion including each G 1 is independent, L P is a functional group that connects the Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are functional groups that can be independently converted to a charged state, n is an integer from 0 to 1000, m is an integer from 0 to 1000, p is an integer from 0 to 1000; q is an integer from 0 to 1000, and s is an integer from 1 to 8.

[0283] In some embodiments, n is an integer from 0 to 1000, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, or 0 to 2.

[0284] In some embodiments, m is an integer between 0 and 1000, between 0 and 500, between 0 and 400, between 0 and 300, between 0 and 200, between 0 and 100, between 0 and 90, between 0 and 80, between 0 and 70, between 0 and 60, between 0 and 50, between 0 and 40, between 0 and 30, between 0 and 20, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2.

[0285] In some embodiments, p is an integer between 0 and 1000, between 0 and 500, between 0 and 400, between 0 and 300, between 0 and 200, between 0 and 100, between 0 and 90, between 0 and 80, between 0 and 70, between 0 and 60, between 0 and 50, between 0 and 40, between 0 and 30, between 0 and 20, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, or between 1 and 2.

[0286] In some embodiments, q is an integer from 0 to 1000, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 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.

[0287] In some embodiments, s is an integer from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0288] In some embodiments, W p can react with a functional group on the targeting moiety via a click reaction.

[0289] In certain embodiments, W p teeth, [ka] is selected from the group consisting of:

[0290] In some embodiments, W p can react with an amino acid on the targeting moiety.

[0291] In certain embodiments, W pcan react with amino acids on the targeting moiety, where the amino acids are natural amino acids, unnatural amino acids, or combinations thereof. In certain embodiments, natural amino acids can include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0292] In some embodiments, W p can react with one or more cysteines on the targeting moiety.

[0293] In certain embodiments, W p can react with one or more cysteines on the targeting moiety, and each W p teeth, [ka] wherein R 1 is a sulfur protecting group, and each R 2 are independently leaving groups.

[0294] In certain embodiments, each R 2 are independently halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0295] In some embodiments, W p can react with one or more lysines on the targeting moiety.

[0296] In certain embodiments, W p can react with one or more lysines on the targeting moiety, and each W p is independent, [ka] is selected from the group consisting of:

[0297] In some embodiments, W pcan react with one or more unnatural amino acids on the targeting moiety.

[0298] In certain embodiments, W p can react with one or more unnatural amino acids on the targeting moiety, and each W p is independent, [ka] is selected from.

[0299] In some embodiments, L a teeth, [ka] is.

[0300] In some embodiments, M a is R 3 , [ka] wherein * is the site of covalent attachment to L and ** is B a a moiety covalently bonded to the moiety, R 3 is C 1~10 Alkyl, C 1~10 Heteroalkyl, C 3~8 Cycloalkyl, -O-(C 1~8 alkyl), aryl, -C 1~10 Alkyl-aryl-, -aryl-C 1~10 Alkyl-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-, -(C 3~8 Cycloalkyl-C 1~10 alkyl), 4- to 14-membered heterocycloalkyl, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-, -C 1~10 Alkyl-C(=O)-, -C 1~10Heteroalkyl-C(=O)-, -C 3~8 Cycloalkyl-C(=O)-, -O-(C 1~8 Alkyl)-C(=O)-, -aryl-C(=O)-, -C 1~10 Alkyl-aryl-C(=O)-, -aryl-C 1~10 Alkyl-C(=O)-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-C(=O)-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-C(=O)-, -4 to 14-membered heterocycloalkyl-C=(O)-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-C(=O)-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-C(=O)-, -C 1~10 Alkyl-NH-, -C 1~10 Heteroalkyl-NH-, -C 3~8 Cycloalkyl-NH-, -O-(C 1~8 Alkyl)-NH-, -aryl-NH-, -C 1~10 Alkyl-aryl-NH-, -aryl-C 1~10 Alkyl-NH-, -C 1~10 Alkyl-(C 3~8 cycloalkyl)-NH-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-NH-, -4 to 14-membered heterocycloalkyl-NH-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C 1~10 Alkyl-NH-, -C 1~10 Alkyl-S-, -C 1~10 Heteroalkyl-S-, -C 3~8 Cycloalkyl-S-, -OC 1~8 Alkyl-S-, -aryl-S-, -C 1~10 Alkyl-aryl-S-, -aryl-C 1~10 Alkyl-S-, -C 1~10 Alkyl-(C 3~8 Cycloalkyl)-S-, -(C 3~8 Cycloalkyl)-C 1~10Alkyl-S-, -4 to 14-membered heterocycloalkyl-S-, -C 1~10 Alkyl-(4- to 14-membered heterocycloalkyl)-S- or -(4- to 14-membered heterocycloalkyl)-C 1~10 alkyl-S-, Each R 4 are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 5 -C(O)-NR 5a or -NR 5a -C(O)-, R 5a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-, R 6a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, Each R 6b and R 6c are independently hydrogen, C 1~6 Alkyl, C 6~10 Aryl, Hydroxylated C 6~10 Aryl, Polyhydroxylated C 6~10 Aryl, 5-12 membered heterocycloalkyl, C 3~8 Cycloalkyl, Hydroxylated C 3~8 Cycloalkyl, Polyhydroxylated C 3~8 cycloalkyl or the side chain of a natural or unnatural amino acid; each n 1 are independently integers from 0 to 6, n2 is an integer between 0 and 8, each n 3 are independently integers from 1 to 6, n 4 is an integer from 1 to 4, and n 5 is an integer between 1 and 4.

[0301] In certain embodiments, M a teeth, [ka] wherein * is the site of covalent attachment to L and ** is B a The moiety is covalently bonded to the

[0302] In some embodiments, G 1 teeth, [ka] wherein * is selected from the group consisting of L P Each R 7 are independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 7a is selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety. p independently contain unstable structures.

[0303] In certain embodiments, the labile structure is selected from a redox labile structure, a hydrolytically labile structure, or an enzyme labile structure.

[0304] In certain embodiments, the unstable structure is [ka] wherein each R 18 are independently selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl.

[0305] In certain embodiments, the hydrolytically unstable structure is [ka] wherein * is selected from the group consisting of G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and R 8 is selected from hydrogen, alkyl, or aryl; R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0306] In some embodiments, G 1 teeth, [ka] where * is L P is a covalently bonded moiety to R 7 is alkyl (C 1~6 alkyl, etc.).

[0307] In some embodiments, -G 1 -L P -D is [ka] is.

[0308] In some embodiments, the labile structure is an enzyme-labile structure that is labile to an enzyme selected from cathepsin B, a phosphatase, a sulfatase, or a glucuronidase.

[0309] In certain embodiments, the enzyme-labile structure is sensitive to cathepsin B and is -Z-, [ka] wherein * is selected from G 1** is the covalently bound site to D; Z is a substrate for cathepsin B containing 2-4 amino acids; and R 7a is alkyl.

[0310] In certain embodiments, G 1 teeth, [ka] where * is L P is a covalently bonded moiety to R 7 is alkyl (C 1~6 alkyl, etc.).

[0311] In certain embodiments, -G 1 -L P -D is [ka] is.

[0312] In certain embodiments, the enzyme-labile structure is sensitive to glucuronidase; [ka] where * is G 1 is the site covalently bonded to , and ** is the site covalently bonded to D.

[0313] In certain embodiments, G 1 teeth, [ka] where * is L P is a covalently bonded moiety to R 7 is alkyl (C 1~6 alkyl, etc.).

[0314] In certain embodiments, -G 1 -L P -D is [ka] is selected from.

[0315] In certain embodiments, the enzyme-labile structure is sensitive to phosphatases; [ka] wherein * is selected from G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 10 and R 11 is independently hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0316] In certain embodiments, G 1 teeth, [ka] is.

[0317] In certain embodiments, -G 1 -L P -D is [ka] is selected from the group consisting of:

[0318] In certain embodiments, the enzyme-labile structure is sensitive to sulfatases. Receptivity, [ka] where * is G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 12 and R 13 is independently hydrogen, —NH—, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0319] In some embodiments, G 1 teeth, [ka] where R 7 is selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0320] In some embodiments, n is an integer from 1-100, m is an integer from 1-100, and p is an integer from 1-50.

[0321] In some embodiments, B a teeth, [ka] and W L Stretcher M a is a functional group that connects to Each Z is independently a branch point, Each R 19 are independent and become Z and W M is a linker connecting Each W M are independently functional groups bonded to -NH-, r is an integer from 1 to 3, t is an integer from 1 to 3, In the formula, * represents M a and ** denotes a site covalently bound to the -NH- moiety.

[0322] In some embodiments, the —NH— moiety is [ka] -NH-(CH2) n6 -COOH, wherein G 1 , L p , D, G 2 , n, m, p, and q are as defined above, and n 6 is an integer from 1 to 6.

[0323] In certain embodiments, W L teeth, [ka] is selected from the group consisting of:

[0324] In certain embodiments, Z is —CH (3-r) -, -SiH (3-r) -, or -NH (2-r) -It is.

[0325] In certain embodiments, R 19 is selected from the group consisting of aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl.

[0326] In some embodiments, B a is Equation 105

[0327] The file is TIFF2026505029000113.tif2366. In certain embodiments, B a teeth, [ka] is selected from the group consisting of:

[0328] In some embodiments, B a teeth, [ka] is.

[0329] In certain embodiments, B a teeth, [ka] is selected from the group consisting of:

[0330] In certain embodiments, B a teeth, [ka] is.

[0331] In some embodiments, two W M is -NH-(CH2)n6 -COOH and one W M teeth, [ka] Connect to.

[0332] In some embodiments, three W M teeth, [ka] Connect to.

[0333] In some embodiments, two W M teeth, [ka] Connect to one W M teeth, [ka] Connect to.

[0334] In certain embodiments, W L teeth, [ka] and -R 19 -W M -teeth, [ka] where ** is W L or a site covalently bonded to the -NH- moiety.

[0335] In certain embodiments, B a teeth, [ka] [ka] is.

[0336] In some embodiments, the therapeutic agent has anti-proliferative activity against the target cell or pathway.

[0337] In certain embodiments, the antiproliferative activity is selected from cytostatic and / or cytotoxic activity.

[0338] In certain embodiments, the therapeutic agent is selected from anti-cancer agents, cytotoxic drugs, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antivirals, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, antiglaucoma compounds, antiparasitic and / or antiprotozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensive agents, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic agents, antiemetics, and imaging agents.

[0339] In certain embodiments, the therapeutic agent comprises an amino acid-based molecule.

[0340] In certain embodiments, the amino acid-based molecule includes a peptide, polypeptide, enzyme, antibody, immunoglobulin, or functional fragment thereof.

[0341] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0342] In certain embodiments, the chemically reactive group is -COOH, primary amine, secondary amine -NHR, -OH, -SH, -C(O)H, C(O)R 14 , -C(O)NHR 15 , -C(S)OH, -S(O)2OR 15 , -P(O)2OR 15 , -CN, -NC, or -ONO, where R 14is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0343] In certain embodiments, G 2 teeth, [ka] wherein each R 16 is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0344] In some embodiments, the polymer scaffold comprises: [ka] [ka] is selected from: [ka] [ka]

[0345] In some embodiments, the targeting moiety is an antibody and / or a fragment thereof.

[0346] In certain embodiments, the targeting moieties are antibodies IgG1, IgG2, IgG3 and IgG4.

[0347] In certain embodiments, the targeting moiety is selected from the group consisting of a Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer, multispecific antibody, camelized single domain antibody, nanobody, domain antibody or bivalent domain antibody.

[0348] In certain embodiments, the targeting moiety is sacituzumab or trastuzumab. Synthesis method

[0349] Any available technique can be used to make the conjugates provided herein or compositions comprising them, as well as intermediates and components (e.g., carriers and modifiers) useful for making them. For example, semi-synthetic and total synthetic methods can be used.

[0350] The synthetic processes of the present disclosure can tolerate a wide variety of functional groups, and therefore can employ a variety of substituted starting materials. While the processes generally provide the desired final compound at or near the end of the overall process, in certain cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt, ester, or prodrug.

[0351] In some embodiments, the linker-polymer compounds provided herein can be conjugated to both a targeting moiety and a therapeutic agent (D). The linker-polymer compounds provided herein include a linking moiety suitable for connecting the targeting moiety and a linking moiety suitable for connecting the drug (D).

[0352] In some embodiments, the conjugates provided herein are formed in several steps, including: (1) modifying the polymeric carrier so that it contains functional groups capable of reacting with functional groups of a targeting moiety or a derivative thereof and functional groups capable of reacting with functional groups of a drug or a derivative thereof; (2) reacting the modified polymer with a drug or a derivative thereof to link the drug to the modified polymer; and (3) reacting the modified polymer-drug conjugate with a targeting moiety or a derivative thereof to form a conjugate provided herein.

[0353] In another embodiment, the conjugate is formed in several steps: (1) modifying the polymeric carrier such that it contains a functional group capable of reacting with a functional group on the targeting moiety or a derivative thereof and a functional group capable of reacting with a functional group on the first drug or a derivative thereof; (2) reacting the modified polymer with a first drug or a derivative thereof to link the first drug to the modified polymer; (3) modifying the polymer-drug conjugate such that the resulting polymer-drug conjugate contains a different functional group capable of reacting with a functional group on the second drug or a derivative thereof; (4) reacting the modified polymer-drug conjugate with a second drug or a derivative thereof to link the second drug to the modified polymer-drug conjugate; and (5) reacting the modified polymer-drug conjugate of step (4) with the targeting moiety or a derivative thereof to form a conjugate provided herein.

[0354] The synthetic processes of the present invention can tolerate a wide variety of functional groups, and therefore can employ a variety of substituted starting materials. While the processes generally provide the desired final compound at or near the end of the overall process, in certain cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt, ester, or prodrug.

[0355] The drug compounds used for the conjugates provided herein can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or easily prepared intermediates, utilizing standard synthetic methods and procedures known to those skilled in the art or that will become apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for organic molecule preparation and functional group transformations and manipulations can be obtained from relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic textbooks such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition, John Wiley & Sons: New York, 2001; and Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons: New York, 1999, are useful and recognized reference textbooks for organic synthesis known to those skilled in the art, and are incorporated herein by reference.

[0356] The conjugates of the present disclosure can be conveniently prepared by a variety of methods familiar to those skilled in the art. The conjugates of the present disclosure having each formula described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature procedures. The procedures show the preparation of representative conjugates of the present disclosure.

[0357] Once produced, conjugates designed, selected, and / or optimized by the methods described above can be characterized using a variety of assays known to those of skill in the art to determine whether the conjugate has biological activity. For example, conjugates can be characterized by conventional assays, including but not limited to, the assays described below, to determine whether the conjugate has the predicted activity, binding activity, and / or binding specificity.

[0358] Furthermore, high-throughput screening can be used to speed up the analysis using such assay.As a result, it is possible to use the method known in the art to rapidly screen the conjugate molecules described herein for activity.The general methodology for carrying out high-throughput screening is described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker, and U.S. Patent No. 5,763,263.High-throughput assay can use one or more different assay methods, including but not limited to those described below. Pharmaceutical Composition

[0359] For purposes of administration, in some embodiments, the conjugates provided herein are administered as the raw chemical or formulated as a pharmaceutical composition.

[0360] Thus, in one aspect, the present disclosure provides pharmaceutical compositions comprising one or more conjugates disclosed herein and acceptable carriers, such as stabilizers, buffers, etc. The conjugates can be administered or introduced into a subject by standard methods, with or without stabilizers, buffers, etc., to form pharmaceutical compositions. Administration can be topical (including to the eye and mucous membranes, including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including nebulizers, intratracheal, intranasal, epithelial and transdermal, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial, e.g., intrathecal or intraventricular administration, or oral or parenteral. The conjugates can be formulated and used as sterile solutions and / or suspensions for injectable administration; lyophilized powders for reconstitution prior to injection / infusion; topical compositions; tablets, capsules, or elixirs for oral administration; or suppositories for rectal administration, as well as other compositions known in the art.

[0361] A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., to a cell or systemically to a subject, including, for example, a human. The suitable form depends, in part, on the use or route of entry, e.g., oral, inhalation, transdermal, or injection / infusion. Such a form should not prevent the composition or formulation from reaching the target cells (i.e., cells to which drug delivery is desired). For example, a pharmacological composition injected into the bloodstream should be soluble. Other factors are known in the art and include, for example, toxicity and consideration of forms that prevent the composition or formulation from exerting its effect.

[0362] As used herein, the term "systemic administration" refers to the distribution of modified polymers throughout the body following systemic absorption or accumulation in the bloodstream in vivo.Administration routes that lead to systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular.Each of these administration routes exposes the modified polymer to accessible affected tissues.The rate at which active agents enter the bloodstream has been shown to be a function of molecular weight or size.By using the conjugates provided herein, drug delivery can be localized in specific cells, such as cancer cells, through the specificity of targeting moieties.

[0363] As used herein, the term "pharmaceutically acceptable formulation" refers to a composition or formulation that allows for the effective distribution of a conjugate in a physical location that is most suitable for the conjugate's desired activity. In some embodiments, effective delivery occurs before clearance by the reticuloendothelial system or the generation of off-target binding, which may result in reduced efficacy or toxicity. Non-limiting examples of drugs suitable for formulation with conjugates include P-glycoprotein inhibitors (e.g., Pluronic P85), which can enhance the entry of active agents into the CNS; biodegradable polymers, such as poly(DL-lactide-co-glycolide) microparticles for sustained release delivery after intracerebral implantation; and loaded nanoparticles, such as those made from polybutylcyanoacrylate, which can deliver active agents across the blood-brain barrier and alter neuronal uptake mechanisms.

[0364] Also included herein are pharmaceutical compositions prepared for storage or administration, comprising a pharmaceutically effective amount of the desired conjugate in a pharmaceutically acceptable carrier or diluent. Acceptable carriers, diluents, and / or excipients for therapeutic use are known in the pharmaceutical sciences. For example, buffers, preservatives, bulking agents, dispersing agents, stabilizers, and dyes may be provided. Additionally, antioxidants and suspending agents may be used. Suitable carriers, diluents, and / or excipients include, but are not limited to, (1) Dulbecco's phosphate-buffered saline, pH about 6.5, containing about 1 mg / ml to 25 mg / ml of human serum albumin; (2) 0.9% saline (0.9% w / v NaCl); and (3) 5% (w / v) dextrose.

[0365] As used herein, the term "pharmaceutically effective amount" refers to an amount of a pharmaceutical agent for treating, ameliorating, or preventing a specified disease or condition, or for exhibiting 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 on the subject's weight, size, and health; the nature and extent of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration. The pharmaceutically effective amount for a given situation can be determined by routine experimentation, which is within the skill and judgment of the clinician.

[0366] For any conjugate, the pharmaceutically effective amount can be estimated first either in cell culture assays, e.g., tumor cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine doses and routes useful for administration in humans. Therapeutic / prophylactic efficacy and toxicity can be determined in cell cultures or experimental animals using standard pharmaceutical procedures, e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 The therapeutic index can be determined by the dose that is lethal to 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage can vary within this range depending on the dosage form utilized, sensitivity of the patient, and the route of administration.

[0367] For example, linker-polymer-drug compounds or targeting moiety-linker-polymer-drug conjugates can be evaluated for their ability to inhibit tumor growth in several cell lines using Cell titer Glo. Dose-response curves can be generated using SoftMax Pro software, and IC 50Values ​​can be determined from a four-parameter curve fit. Cell lines utilized can include a cell line that is the target of the targeting moiety and a control cell line that is not the target of the targeting moiety contained in the conjugate being tested.

[0368] In some embodiments, the conjugates are formulated for parenteral administration by injection, including using conventional catheterization or infusion. Injectable formulations can be presented in unit dosage form, e.g., in ampoules or multi-dose containers, with added preservatives. The conjugates can be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the conjugates can be suspended or dissolved in the vehicle. Advantageously, adjuvants, such as local anesthetics, preservatives, and buffering agents, can be dissolved in the vehicle. As used herein, the term "parenteral" includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques. Additionally, pharmaceutical formulations comprising the conjugates and a pharmaceutically acceptable carrier are provided. One or more conjugates can be presented together with one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and, if desired, other active ingredients.

[0369] Sterile injectable preparations can be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly used as solvents or suspending media. For this purpose, sterile, fixed oils, including synthetic monoglycerides or diglycerides, can be used. In addition, fatty acids such as oleic acid are used to prepare injectable solutions.

[0370] The conjugates and compositions described herein can be administered in a suitable form, preferably parenterally, more preferably intravenously.For parenteral administration, the conjugates or compositions can be aqueous or non-aqueous sterile solutions, suspensions or emulsions.Propylene glycol, vegetable oils and injectable organic esters, such as ethyl oleate, can be used as solvents or vehicles.The compositions can also contain adjuvants, emulsifiers or dispersants.

[0371] Dosage levels on the order of about 0.001 mg to about 140 mg per kilogram of body weight per day are useful in treating the above-indicated conditions (about 0.05 mg to about 7 g per subject per day). In some embodiments, the dose administered to a patient is about 0.001 mg / kg to about 100 mg / kg of the subject's body weight. In some embodiments, the dose administered to a patient is about 0.01 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dose administered to a patient is about 0.1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dose administered to a patient is about 0.1 mg / kg to about 20 mg / kg of the subject's body weight. In some embodiments, the dose administered is 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 dose administered is about 1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the administered dose is about 1 mg / kg to about 10 mg / kg of the subject's body weight. The amount of conjugate that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. Unit dosage forms generally contain about 0.001 mg to about 100 mg, about 0.01 mg to about 75 mg, about 0.01 mg to about 50 mg, or about 0.01 mg to about 25 mg of conjugate.

[0372] For intravenous administration, dosage levels may range as described above, or may comprise about 0.01 to about 200 mg of conjugate per kg of animal body weight. In some embodiments, the composition may comprise about 1 to about 100 mg of conjugate per kg of animal body weight. In some embodiments, the amount administered ranges from about 0.1 to about 25 mg of compound per kg of body weight.

[0373] In some embodiments, the conjugate can be administered as follows: The conjugate can be given daily for about 5 days, either iv, as a bolus each day for about 5 days, or as a continuous infusion for about 5 days.

[0374] Alternatively, the conjugate can be administered once a week for six weeks or more. Alternatively, the conjugate can be administered every two or three weeks. A bolus dose can be administered in about 50 to about 400 ml of saline, to which about 5 to about 10 ml of human serum albumin can be added. A continuous infusion can be administered in about 250 to about 500 ml of saline, to which about 25 to about 50 ml of human serum albumin can be added every 24 hours.

[0375] In some embodiments, the patient can receive a second course of treatment about 1 to about 4 weeks after treatment. Specific clinical protocols regarding route, excipients, diluents, doses, and times of administration can be determined by one skilled in the art as required by the clinical situation.

[0376] In other embodiments, the therapeutically effective amount can be provided on another regular schedule, i.e., daily, weekly, monthly, or yearly, or on an irregular schedule with varying administration days, weeks, months, etc. Alternatively, the administered therapeutically effective amount can vary. In some embodiments, the therapeutically effective amount of the first administration is greater than the therapeutically effective amount of one or more subsequent administrations. In some embodiments, the therapeutically effective amount of the first administration is less than the therapeutically effective amount of one or more subsequent administrations. Equivalent doses can 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 2 weeks, about every 3 weeks, about every month, and about every 2 months. The number and frequency of administrations corresponding to a complete course of treatment are determined according to the recommendations of relevant regulatory authorities and the judgment of a medical professional. The therapeutically effective amount described herein refers to the total amount administered during a given period of time, i.e., if more than one different conjugate described herein is administered, the therapeutically effective amount corresponds to the total amount administered. It will be understood that the specific dose level for a particular subject will depend on a variety of factors, including the activity of the specific conjugate, 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 under treatment.

[0377] For administration to non-human animals, the conjugate can also be added to animal feed or drinking water. It may be convenient to formulate animal feed and drinking water so that the animal ingests a therapeutically effective amount of the conjugate along with its diet. It may also be convenient to present the conjugate as a mixture for addition to feed or drinking water.

[0378] The conjugates can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat a condition can increase beneficial effects while reducing the presence of side effects. In some embodiments, the conjugates are used in combination with chemotherapeutic agents, such as those disclosed in U.S. Patent No. 7,303,749. In other embodiments, the chemotherapeutic agents include, but are not limited to, letrozole, oxaliplatin, docetaxel, 5-FU, lapatinib, capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine. The present disclosure also provides pharmaceutical kits comprising one or more containers filled with a composition of the present disclosure comprising one or more conjugates and / or one or more chemotherapeutic agents. Such kits may also include, for example, other compounds and / or compositions, devices for administering the compounds and / or compositions, and instructions in a format prescribed by a government agency overseeing the manufacture, use, or sale of pharmaceutical or biological products. The compositions described herein can be packaged as a single dose or for continuous or periodic discontinuous administration.For continuous administration, the package or kit can include a conjugate in each dosage unit (e.g., a solution or other unit described above or used for drug delivery), and optionally instructions for administering the dose daily, weekly, or monthly for a predetermined period or as prescribed.If it is desired to change the composition, the concentration of the components of the composition, or the relative ratio of the conjugate or drug within the composition over time, the package or kit can contain dosage units in an order that provides the desired variety.

[0379] Numerous packages or kits for dispensing pharmaceuticals for periodic oral use are known in the art. In some embodiments, the package has indicators for each period. In some embodiments, the package is a labeled blister package, a dial dispenser package, or a bottle. The packaging means of the kit can be adapted for administration, for example, as a syringe, pipette, eye dropper, or other such device, from which the formulation is applied to the affected area of ​​the body, injected into the subject, or further applied and mixed with other components of the kit. Treatment of Disease

[0380] The targeting moiety-linker-polymer-drug conjugates provided herein can be used in methods of treating animals (e.g., mammals, such as humans, including men, women, infants, children and adults).

[0381] In some embodiments, the conjugates provided herein can be used in methods of treating animals, comprising administering a conjugate of the present disclosure to the animal. The conjugates of the present invention can be used as drug carriers and drug carrier components, in controlled drug release systems, in preparations for minimally invasive surgical procedures, and the like. The pharmaceutical formulations can be injectable, implantable, and the like.

[0382] 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 effective amount of at least one conjugate provided herein, wherein the conjugate biodegrades to release one or more therapeutic agents.

[0383] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo, and / or ex vivo to treat a subject and / or to modulate the growth of selected cell populations, including, for example, cancer.In some embodiments, specific cancer types that can be treated with the conjugates provided herein include, but are not limited to: (1) fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, 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, esophageal cancer, stomach cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma bile duct carcinoma, (1) solid tumors, including, but not limited to, acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemia (ALE), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemia (ALE), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemia (ALE), acute myeloid leukemia (AML), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemia (ALE), acute myeloid leukemia (AML), acute myeloid leukemia (AML), acute myeloid leukemia (APL), acute myeloid leukemia (APL), acute myeloid leukemia (APL), acute myeloid leukemia (APL), acute erythroleukemia (ALE), acute erythroleukemia (ALE), acute myeloid leukemia (APL ... (3) blood-borne cancers including acute myeloid leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute anaplastic leukemia, chronic myeloid leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple myeloma, acute and chronic leukemias, e.g., lymphoblastic myelogenous and lymphocytic myelocytic leukemia, and (4) lymphomas, e.g., Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and polycythemia vera.

[0384] In some embodiments, the conjugates provided herein can be administered to treat a subject in vitro, in vivo, and / or ex vivo and / or to modulate the proliferation of select cell populations in patients with anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, mouth cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, pharyngeal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, or stomach cancer.

[0385] In certain embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, non-small cell lung cancer (NSCLS), and ovarian cancer.

[0386] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo, and / or ex vivo to treat, prevent, reduce the risk of, and / or delay the onset of a particular condition, such as cancer. For example, the conjugates provided herein are useful for treating, preventing, delaying, or otherwise ameliorating the symptoms of cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, pharyngeal cancer, mouth cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, pharyngeal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, kidney cancer, and gastric cancer.

[0387] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo, and / or ex vivo to treat autoimmune diseases such as systemic lupus, rheumatoid arthritis, psoriasis, and multiple sclerosis; implant rejection such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection, and bone marrow transplant rejection; implant versus host disease; viral infections such as CMV infection, HIV infection, and AIDS; and parasitic infections such as giardiasis, amebiasis, schistosomiasis, and the like.

[0388] In some embodiments, the conjugates provided herein can also be used to manufacture a medicament useful, for example, for treating or lessening the severity of a disorder characterized by abnormal proliferation of cells (e.g., cancer).

[0389] In some embodiments, the therapeutic agent is delivered locally to a specific target cell, tissue, or organ.

[0390] In certain embodiments, provided herein conjugate can further comprise or be associated with diagnostic label.In certain embodiments, diagnostic label is selected from the group consisting of radiopharmaceuticals or radioisotopes for gamma scintigraphy and PET, contrast agents for magnetic resonance imaging (MRI), contrast agents for computed tomography, contrast agents for X-ray imaging, agents for ultrasound diagnosis, neutron activators, moieties that can reflect, scatter or act on X-rays, ultrasound, radio waves and microwaves, and fluorophores.In certain exemplary embodiments, conjugate is further monitored in vivo.

[0391] Examples of diagnostic labels include, but are not limited to, diagnostic radiopharmaceuticals or radioisotopes for gamma scintigraphy and PET, contrast agents for magnetic resonance imaging (MRI) (e.g., paramagnetic atoms and superparamagnetic nanocrystals), contrast agents for computed tomography, contrast agents for X-ray imaging, agents for ultrasound diagnostics, neutron activators, and moieties capable of reflecting, scattering, or interacting with X-rays, ultrasound, radio waves, and microwaves, fluorophores in various optical procedures, and others. Diagnostic radiopharmaceuticals include gamma-ray-emitting radionuclides, such as indium-111, technetium-99m, and iodine-131, and others. Contrast agents for MRI (magnetic resonance imaging) include magnetic compounds, such as paramagnetic ions, iron, manganese, gadolinium, lanthanides, organic paramagnetic moieties, and superparamagnetic, ferromagnetic, and antiferromagnetic compounds, such as iron oxide colloids, ferrite colloids, and others. Contrast agents for computed tomography and other X-ray-based imaging include X-ray absorbing compounds such as iodine, barium, and others. Contrast agents for ultrasound-based methods include compounds that can absorb, reflect, and scatter ultrasound, such as emulsions, crystals, bubbles, and others. Further examples include substances useful for neutron activation, such as boron and gadolinium. Additionally, labels can be utilized that can reflect, refract, scatter, or otherwise affect rays useful in X-ray, ultrasound, radio waves, microwaves, and other diagnostic procedures. Fluorescent labels can be used for photographic imaging. In certain embodiments, the modifier includes a paramagnetic ion or group.

[0392] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising preparing an aqueous formulation of at least one conjugate provided herein and parenterally injecting the formulation in a subject.

[0393] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising preparing an implant comprising at least one conjugate provided herein and implanting the implant into the subject. In certain embodiments, the implant is a biodegradable gel matrix.

[0394] In another aspect, the disclosure provides a method of treating an animal in need thereof, comprising administering a conjugate according to the methods described above.

[0395] In another aspect, the disclosure provides a method of raising an immune response in an animal, the method comprising administering a conjugate as in the methods described above.

[0396] In another aspect, the disclosure provides a method of diagnosing a disease in an animal, the method comprising administering a conjugate as in the method described above, wherein the conjugate comprises a detectable molecule, and detecting the detectable molecule.

[0397] In some embodiments, detecting the detectable molecule is performed non-invasively, hi some embodiments, detecting the detectable molecule is performed using suitable imaging equipment.

[0398] In some embodiments, a method of treating an animal includes administering a conjugate provided herein as a packing for a surgical wound from which a tumor or growth has been removed, the packing of the conjugate replacing the tumor site during recovery and degrading and dissipating as the wound heals.

[0399] In certain embodiments, the conjugates provided herein are associated with a diagnostic label for in vivo monitoring.

[0400] The conjugates provided herein can be used in therapeutic, prophylactic, and analytical (diagnostic) treatments of animals. The conjugates are generally intended for parenteral administration, although in some cases they can be administered by other routes.

[0401] In some embodiments, soluble or colloidal conjugates are administered intravenously. In some embodiments, soluble or colloidal conjugates are administered via local (e.g., subcutaneous, intramuscular) injection. In some embodiments, solid conjugates (e.g., particles, implants, drug delivery systems) are administered via implantation or injection.

[0402] In some embodiments, a conjugate containing a detectable label is administered to examine the pattern and kinetics of label distribution within an animal.

[0403] In certain embodiments, any one or more of the conjugates provided herein can be used in practicing any of the methods described above.

[0404] Throughout the description, when a composition is described as having, including, or containing a specific compound, it is also contemplated that the composition consists essentially of, or consists of, the recited components. Similarly, when a method or process is described as having, including, or containing specific process steps, the process also consists essentially of, or consists of, the recited process steps. Furthermore, it should be understood that the order of steps or the order in which certain actions are performed is not important so long as the invention remains operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0405] All publications and patent documents cited herein are incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference herein. The citation of publications and patent documents is not intended as an admission that any is pertinent prior art, and does not constitute any admission as to the contents or date of the same. Example

[0406] For illustrative purposes, 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 understood that these examples do not limit the disclosure, but are merely meant to suggest a method of practicing the disclosure. Those skilled in the art will recognize that the described chemical reactions can be easily adapted to prepare numerous other compounds or conjugates of the disclosure, and alternative methods of preparing the compounds or conjugates of the disclosure are considered to be within the scope of the disclosure. Those skilled in the art will also understand that the individual steps or separate batches of compounds described herein can be combined. Alternatively, it will be understood that other reactions disclosed herein or known in the art have applicability for preparing other compounds of the disclosure. Therefore, the following description is not intended to limit the scope of the disclosure, which is defined by the claims appended hereto. Example 1 Synthesis of Amine Intermediate Compound 1 [ka]

[0407] To a 250 mL flask under N2, 4-methoxybenzyl alcohol (5.0 g, 36.2 mmol), t-BuOK (7.3 mL, 7.2 mmol), and DME (25 mL) were added. The mixture was stirred at room temperature for 30 minutes, and then 2-((1-ethoxyethoxy)methyl)oxirane (23.8 g, 169 mmol) was added. The mixture was stirred at 80 °C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to give compound 1-1 (6.0 g, 8.3 mmol, 23%) as a colorless oil.

[0408] To a solution of 2,3-dihydrofuran (90.0 g, 1.3 mol) in DCM (900 mL) was added MeOH (82.3 mL, 2.6 mol) and BF3.Et2O (2.5 mL, 19.5 mmol) at room temperature. The reaction mixture was stirred at room temperature for 10 minutes, and then 1,3-dimercaptopropane (138.5 g, 1.3 mol) was added. The reaction mixture was stirred at room temperature for 2 hours, then diluted with DCM (1.5 L) and washed with saturated sodium bicarbonate solution (500 mL) and water (500 mL × 3). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel to give compound 1-2 (210.0 g, 1.2 mol, 92%) as a white solid.

[0409] To a solution of compound 1-2 (210.0 g, 1.6 mol) in anhydrous DCM (2.0 L) was added EtN (241.3 g, 2.4 mmol) and MsCl (236.7 g, 2.1 mol) dropwise at 0 °C. The mixture was stirred at room temperature for 2 h. Water (2 L) was added to quench the reaction, and the mixture was extracted with DCM (1 L × 3). The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give compound 1-3 (326.0 g, 1.6 mol, 98%) as a pale yellow oil.

[0410] To a solution of compound 1-2 (518.0 g, 2.9 mol) and compound 1-3 (833.0 g, 4.0 mol) in anhydrous DMF (4 L) was added NaH (335 g, 8.4 mol) portionwise under N at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The reaction was quenched by adding water and extracted with EtOAc (1 L × 3). The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give compound 1-4 (713.0 g, 2.4 mol, 84%) as a pale yellow oil.

[0411] To a solution of compound 1-4 (100.0 g, 342.0 mol) in anhydrous THF (800 mL) was added n-BuLi (274 mL, 2.5 M in hexane, 684.0 mmol) dropwise at −60° C. under N. After the mixture was stirred at −60° C. for 40 min, methyl disulfide (64.4 g, 684.0 mmol) was added. The reaction mixture was stirred at −10° C. for 16 h. The reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc (1 L × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give compound 1-5 (91.0 g, 269.0 mol, 78%) as a pale yellow oil.

[0412] To a solution of compound 1-5 (91.0 g, 269.0 mol) in HO (500 mL) was added dropwise AcOH (500 mL) at 0 °C. The mixture was stirred at 0 °C for 4 h. The reaction was quenched with saturated aqueous NaHCO and extracted with DCM (1 L × 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give compound 1-6 (61.0 g, 204.4 mol, 76%) as a pale yellow oil.

[0413] To a solution of compound 1-6 (27.8 g, 93.2 mmol) in toluene (270 mL) was added TrtCl (26.0 g, 93.2 mmol) and EtN (18.9 g, 186.4 mmol). The reaction mixture was stirred at 110 °C for 3 h. The mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography on silica gel to give compound 1-7 (43.8 g, 81.1 mmol, 87%) as a colorless oil.

[0414] To a solution of compound 1-7 (76.0 g, 140.5 mmol) in anhydrous DMF (800 mL) was added NaH (6.7 g, 168.6 mmol) at 0 °C under N. The mixture was stirred at 0 °C for 1 h, and then allyl bromide (20.4 g, 168.6 mmol) was added. The reaction mixture was stirred at room temperature for 3 h. The reaction was quenched with water and extracted with EtOAc (1 L × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on silica gel to give the desired product, compound 1-8 (75.0 g, 129.1 mmol, 92%), as a colorless oil.

[0415] To a solution of compound 1-8 (75.0 g, 129.1 mmol) in anhydrous THF (85 mL) and anhydrous MeOH (750 mL) was added TsOH (4.4 g, 25.8 mmol) at room temperature under N. The mixture was stirred at room temperature for 2 h. The reaction was quenched with saturated aqueous NaHCO and extracted with DCM (1 L × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on silica gel to give the desired product, compound 1-9 (35.4 g, 104.6 mmol, 81%), as a colorless oil.

[0416] To a solution of compound 1-9 (35.4 g, 104.6 mmol) in anhydrous DCM (350 mL) was added TsCl (21.2 g, 209.2 mmol), EtN (21.2 g, 209.2 mmol), and DMAP (1.27 g, 10.5 mmol) at room temperature under N. The mixture was stirred at room temperature for 4 h. The reaction was quenched with water and extracted with DCM (500 mL × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a residue, which was purified by flash column chromatography on silica gel to give the desired product, compound 1-10 (47.7 g, 96.8 mmol, 90%), as a pale yellow oil.

[0417] To a solution of compound 1-1 (4.4 g, 8.4 mmol) in DMF (30 mL) was added NaH (1.0 g, 24.5 mmol) at room temperature under N. After the mixture was stirred for 1 h, TBAI (0.5 g, 1.2 mmol) and compound 1-10 (6.0 g, 12.3 mmol) were added to the solution. The reaction mixture was heated to 90 °C and stirred for 16 h. The reaction was quenched with water at 0 °C and extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated in vacuo to give the crude product, which was purified by silica gel chromatography to give compound 1-11 (7.5 g, 7.2 mmol, 85%) as a yellow oil.

[0418] Compound 1-11 (7.5 g, 7.2 mmol), Pd(PPh3)4 (4.2 g, 3.6 mmol), and K2CO3 (3.0 g, 21.6 mmol) were dissolved in MeOH / THF (55 mL, 6:1 v / v). The reaction was stirred under N2 at 60 °C for 16 h. The mixture was concentrated in vacuo to give the crude product, which was purified by silica gel chromatography to give compound 1-12 (5.4 g, 5.3 mmol, 74%) as a colorless oil.

[0419] To a solution of compound 1-1 (20.0 g, 27.7 mmol) in anhydrous DMF (150 mL) was added NaH (2.2 g, 55.3 mmol) at room temperature under N. After the mixture was stirred at room temperature for 1 hour, iodomethane (3.5 mL, 55.3 mmol) was added. The reaction mixture was stirred at room temperature under N for 2 hours. The mixture was concentrated to give a residue, which was purified by reverse-phase chromatography to give the desired product, compound 1-13 (20.2 g, 27.4 mmol, 98%), as a white solid.

[0420] To a solution of compound 1-13 (20.2 g, 27.4 mmol) in MeOH (200 mL) was added 10% Pd / C (10.0 g) and 10% Pd(OH) / C (200 mg) at room temperature. The reaction mixture was stirred under H at 40 °C for 16 h. The mixture was filtered and concentrated to give the desired product, compound 1-14 (16.3 g, 26.4 mmol, 96%), as a colorless oil.

[0421] To a solution of compound 1-14 (16.3 g, 26.4 mmol) in anhydrous DCM (6 mL) was added EtN (15.0 mL, 105.7 mmol), DMAP (323.0 mg, 2.6 mmol), and TsCl (10.1 g, 52.9 mmol) at room temperature under N. The mixture was stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure to give a residue, which was purified by silica gel chromatography to give the desired product, compound 1-15 (17.8 g, 23.1 mmol, 87% yield), as a pale yellow solid.

[0422] To a solution of compound 1-12 (5.4 g, 5.4 mmol) in NMP (50 mL) was added NaH (0.9 g, 21.4 mmol) at room temperature under N. After the mixture was stirred for 1 h, TBAI (400 mg, 1.07 mmol) and compound 1-15 (8.2 g, 10.7 mmol) were added to the solution. The reaction mixture was heated to 90 °C and stirred for 16 h. The reaction was quenched with water at 0 °C and extracted with EtOAc. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated in vacuo to give the crude product, which was purified by silica gel chromatography to give compound 1-16 (7.1 g, 4.4 mmol, 83%) as a yellow oil.

[0423] To a solution of compound 1-16 (7.1 g, 4.4 mmol) and NaHCO (3.4 g, 39.9 mmol) in MeOH / DCM / HO (300 mL, 40:20:1 v / v / v) was added I (3.4 g, 13.3 mmol) slowly at 0 °C. The reaction was stirred at 0 °C for 10 min. NaHCO (50 mL) was added to the mixture and quenched with NaSO. Water was added to the reaction, which was extracted with DCM. The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated in vacuo to give the crude product, which was purified by silica gel column chromatography to give compound 1-17 (5.3 g, 3.5 mmol, 80%) as a yellow oil.

[0424] To a solution of compound 1-17 (5.3 g, 3.5 mmol) in THF (200 mL) was added 10% Pd / C (3.7 g) and 10% Pd(OH) / C (3.7 g) under H. The reaction was stirred at 50 °C for 20 h. The mixture was filtered and concentrated in vacuo to give the crude product, which was purified by silica gel chromatography to give compound 1-18 (3.4 g, 2.4 mmol, 70%) as a yellow oil.

[0425] To a solution of compound 1-18 (3.4 g, 2.4 mmol) in THF (55 mL) was added PPh3 (1.9 g, 7.3 mmol), DEAD (1.3 g, 7.3 mmol), and DPPA (2.4 g, 8.5 mmol) at 0 °C. The reaction was stirred at 0 °C for 2 h. After completion, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give compound 1-19 (3.3 g, 2.3 mmol, 96%) as a yellow oil.

[0426] To a solution of compound 1-19 (3.3 g, 2.3 mmol) in THF / HO (30 mL, 2:1 v / v) was added PPh (1.3 g, 4.8 mmol). The reaction was stirred at room temperature for 15 h. Upon completion, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give compound 1-20 (2.3 g, 1.7 mmol, 71%) as a colorless oil.

[0427] To a solution of Fmoc-alanine (500 mg, 1.7 mmol) in DMF (20 mL) under N2, DIPEA (0.6 g, 5.0 mmol), compound 1-20 (2.3 g, 1.7 mmol), and HATU (1.0 g, 2.5 mmol) were added. The reaction was stirred at room temperature for 16 hours. The reaction was purified by flash chromatography on silica gel to give compound 1-21 (2.7 g, 1.6 mmol, 97%) as a colorless oil.

[0428] To a solution of compound 1-21 (2.7 g, 1.6 mmol) in DCM (25 mL) was added piperidine (5 mL) slowly at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. Water (50 mL) was poured into the reaction mixture, and the reaction mixture was extracted with DCM (50 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product, which was purified by silica gel chromatography to give compound 1 (2.1 g, 1.4 mmol, 87%) as a colorless oil. Example 2 Synthesis of intermediate compound 2 [ka]

[0429] To a solution of 2-1 (2.9 g, 7.4 mmol), 2-2 (2.5 g, 6.7 mmol), and NMM (1.3 mL, 11.3 mmol) in DCM (60 mL) was added IBCF (1.2 mL, 9.4 mmol) slowly at 0 °C. The reaction mixture was stirred at room temperature under N for 16 h. The mixture was concentrated to give a residue. The residue was diluted with EtOAc (150 mL) and water (150 mL), and the aqueous layer was extracted with EtOAc (100 mL × 3). The combined organic layers were washed with 0.5 M citric acid, saturated NaHCO (aq), and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography to give compound 2-3 (3.3 g, 4.3 mmol, 64%) as a pale yellow oil.

[0430] To a mixture of compound 2-3 (7.4 g, 9.7 mmol) in MeOH (50 mL) / HO (50 mL) was added NaOH (1.2 g, 29.0 mmol) at room temperature. The reaction mixture was stirred for 16 h at room temperature. The pH was adjusted to 3 with 0.5 N HCl. The aqueous layer was extracted with DCM (200 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography to give compound 2-4 (6.5 g, 9.0 mmol, 93%) as a colorless solid.

[0431] To a solution of compound 2-4 (2.1 g, 2.9 mmol) in DCM (18 mL) / DMF (2 mL) was added amine (2.9 g, 7.4 mmol), DIPEA (2.9 mL, 17.5 mmol), EDCI (1.9 g, 9.6 mmol), and HOAT (1.3 g, 9.6 mmol) at room temperature under N2. The mixture was stirred for 5 minutes, and then methyl 3-amino-2-hydroxypropanoate hydrochloride (1.4 g, 10.2 mmol) was added. The reaction mixture was stirred at room temperature under N2 for 16 hours. The mixture was concentrated under reduced pressure, and the residue was purified by C18 chromatography to give compound 2-5 (2.6 g, 2.6 mmol, 90%) as a colorless oil.

[0432] To a solution of compound 2-5 (2.6 g, 2.6 mmol) in MeOH (35 mL) was added 10% Pd / C (1.3 g) and (Boc)O (1.7 g, 7.9 mmol) at room temperature. The reaction mixture was stirred under H at 35 °C for 16 h. The mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel chromatography to give compound 2-6 (2.1 g, 2.2 mmol, 85%) as a colorless oil.

[0433] To a mixture of compound 2-6 (2.1 g, 2.2 mmol) in MeOH (15 mL) and HO (15 mL) was added NaOH (400 mg, 10 mmol) at room temperature. The reaction mixture was stirred for 16 h at room temperature. Citric acid was added to adjust the pH to approximately 3. The aqueous layer was extracted with DCM (200 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the desired product, compound 2-7 (1.7 g, 1.9 mmol, 83%), as a colorless solid.

[0434] To a solution of compound 2-7 (150.0 mg, 0.2 mmol) in DCM, DIPEA (180.0 μL, 1.0 mmol), EDC (140.0 μL, 1.0 mmol), HOBt (100.0 mg, 1.0 mmol), and compound 1 (1.2 g, 1.0 mmol) were added sequentially. The reaction mixture was stirred under N2 at room temperature for 16 hours. Water was poured into the reaction mixture, and the reaction mixture was extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product, which was treated with lithium hydroxide in THF / water. Saturated NH4Cl solution was poured into the reaction mixture, and the reaction mixture was extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give compound 2 (1.08 g, 75%) as a colorless oil. Example 3 Synthesis of intermediate compound 3 [ka]

[0435] To a solution of compound 2-5 (150.0 mg, 0.2 mmol) in DCM, DIPEA (36.0 μL, 0.2 mmol), EDC (28.0 μL, 0.2 mmol), HOBt (20.0 mg, 0.2 mmol), and compound 1 (300 mg, 0.2 mmol) were added sequentially. The reaction mixture was stirred under N2 at room temperature for 16 hours. Water was added to the reaction, and the reaction was extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product, which was treated with lithium hydroxide in THF / water. Saturated NH4Cl solution was added to the reaction, and the reaction was extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give compound 3 (148.0 g, 50%) as a colorless oil. Example 4 Synthesis of Compound 4 [ka]

[0436] To a solution of compound 4-2 (2.1 g, 12.0 mmol) and NaHCO (1.7 g, 20.6 mmol) in a mixture of HO (40 mL) and THF (20 mL) was added compound 4-1 (5.0 g, 11.5 mmol) in DME (40 mL). The reaction mixture was stirred at 15 °C for 16 h. The reaction mixture was adjusted to pH 10 with saturated aqueous KCO and then washed with EtOAc (100 mL × 2). The aqueous layer was acidified to pH 4 with 0.1 N HCl, and the precipitate was filtered. The cake was dried to give compound 4-3 (3.9 g, 8.1 mmol, 70%) as a white solid.

[0437] To a solution of (4-aminophenyl)methanol (1.0 g, 8.4 mmol) in DCM (40 mL) and methanol (20 mL) was added EEDQ (5.2 g, 20.9 mmol) at room temperature. After stirring for 5 min, compound 4-3 (2.0 g, 4.2 mmol) was added, and the resulting solution was stirred for 36 h under dark conditions. The solvent was removed in vacuo, and the residue was triturated with MTBE (200 mL) and ethyl acetate (200 mL) to give compound 4-4 (1.9 g, 3.1 mmol, 73%) as a pale yellow solid.

[0438] To a solution of compound 4-4 (2.5 g, 4.2 mmol) and bis(4-nitrophenyl)carbonate (2.5 g, 8.3 mmol) in DMF (50 mL) was added DIEA (1.1 g, 8.3 mmol) at 20 °C under N. The reaction was stirred at room temperature for 16 h. The reaction was concentrated in vacuo to give a residue, which was triturated with diethyl ether (10 mL). The resulting precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum to give compound 4-5 (3.2 g, 4.1 mmol, 100%) as a yellow solid.

[0439] To a solution of compound 4-5 (500 mg, 0.6 mmol) in DMF (10 mL) was added MMAE (700 mg, 0.8 mmol) at 20 °C. Then, HOBT (18.0 mg, 0.1 mmol) and DIEA (200 mg, 1.9 mmol) were added at 20 °C. The resulting solution was stirred for 48 h. Water (50 mL) was added to the reaction solution, and the mixture was extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound 4-6 (512 mg, 0.3 mmol, 51%) as a white solid.

[0440] To a solution of compound 4-6 (500 mg, 0.4 mmol) in DMF (5 mL) was added piperidine (60.7 mg, 0.7 mmol) at 20 °C. The reaction mixture was then stirred for 18 h. The reaction mixture was purified by preparative HPLC to give compound 4-7 (197 mg, 0.2 mmol, 47%) as a white solid.

[0441] To a solution of compound 2 (100.0 mg, 2 μmol) in DCM, DIPEA (20.0 μl, 10 μmol), EDC (20.0 μl, 10 μmol), HOBt (15.8 mg, 10 μmol), and compound 4-7 (120.0 mg, 10 μmol) were added sequentially. The reaction mixture was stirred under N2 at room temperature for 16 hours. The crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN to remove the Boc and EE protecting groups, and then with LiOH to hydrolyze the methyl ester group. After the reaction, the mixture was purified by preparative HPLC to give compound 4-8 (153 mg, 75%).

[0442] To a solution of compound 4-8 (50.0 mg, 7.5 μmol) in DMF was added malimido-PEG4-NHS (4.0 mg, 7.5 μmol). The crude product was purified by reverse phase chromatography to give compound 4 (26 mg, 50%). Example 5 Synthesis of Compound 5 [ka]

[0443] To a solution of compound 4-8 (50.0 mg, 7.5 μmol) in DMF was added DBCO-PEG4-NHS (5.0 mg, 7.5 μmol). The crude product was purified by reverse-phase chromatography to give compound 5 (24 mg, 50%). Example 6 Synthesis of Compound 6 [ka]

[0444] To a solution of compound 2 (100.0 mg, 2.0 μmol) in DCM, DIPEA (8.0 μL, 4 μmol), EDC (8.0 μL, 4 μmol), HOBt (6.0 mg, 4 μmol), and compound 4-7 (48.0 mg, 4 μmol) were added. The reaction mixture was stirred at room temperature under N for 16 hours. The crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN to remove the Boc and EE protecting groups, and then with LiOH. After the reaction, the mixture was purified by preparative HPLC to give compound 6-1 (65.0 mg, 40%).

[0445] To a solution of compound 6-1 (50.0 mg, 8.5 μmol) in DMF was added malimido-PEG-NHS (5.0 mg, 8.5 μmol). The crude product was purified by reverse phase chromatography to give compound 6 (28 mg, 50%). Example 7 Synthesis of Compound 7 [ka]

[0446] To a solution of compound 3 (100.0 mg, 2 μmol) in DCM, DIPEA (4.0 μL, 2 μmol), EDC (4.0 μL, 2 μmol), HOBt (3.2 mg, 2 μmol), and compound 4-7 (24.0 mg, 2 μmol) were added sequentially. The reaction mixture was stirred at room temperature under N for 16 hours. The crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN to remove the Boc and EE protecting groups, and then with LiOH. After the reaction, the mixture was purified by preparative HPLC to give compound 7-1 (102.0 mg, 80%).

[0447] To a solution of compound 7-1 (50.0 mg, 1.8 μmol) in DMF was added malimido-PEG4-NHS (8.0 mg, 1.8 μmol). The crude product was purified by reverse phase chromatography to give compound 7 (41.0 mg, 80%). Example 8 Synthesis of Compound 8 [ka]

[0448] To a solution of compound 8-1 (8.0 g, 20.1 mmol) in MeCN (160 mL) was added aldehyde (3.4 g, 20.1 mmol) and AgO (5.1 g, 22.1 mmol) under N. After stirring at room temperature in the dark for 15 h, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 8-2 (4.8 g, 9.9 mmol) as a white solid.

[0449] To a solution of compound 8-2 (4.8 g, 9.9 mmol) and silica gel (2.1 g) in CHCl (50 mL) and i-PrOH (10 mL) was added NaBH (564 mg, 14.91 mmol) under N at 0 °C. After stirring at 25 °C for 1.5 h, the mixture was filtered and the solvent was evaporated to give compound 8-3 (5.0 g) as a colorless oil, which was used directly in the next step without further purification.

[0450] To a solution of compound 8-3 (5.0 g, 10.3 mmol) in DMF (50 mL) under N2, imidazole (5.2 g, 9.3 mmol) and DMAP (0.3 g, 2.3 mmol) were added, followed by a solution of TBSCl (2.3 g, 15.5 mmol) in DMF (30 mL). After stirring at room temperature for 16 h, the reaction mixture was diluted with EtOAc (100 mL) and washed with NH4Cl (3 × 50 mL) and brine (3 × 50 mL). The combined organic layers were dried over Na2SO4, filtered, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography to give compound 8-4 (6.2 g, 10.3 mmol) as a white solid.

[0451] To a solution of compound 8-4 (4.2 g, 7.0 mmol) in EtOH (50 mL) was added 10% Pd / C (420 mg) at room temperature. The mixture was stirred under H (1 atm) at 25 °C for 16 h. After completion, the mixture was filtered through Celite and washed with EtOH (50 mL). The filtrate was concentrated in vacuo to give compound 8-5 (3.8 g, 6.7 mmol) as a white solid.

[0452] To a solution of compound 8-5 (3.8 g, 6.7 mmol) and carboxylic acid (1.4 g, 7.3 mmol) in DCM (50 mL) was added EEDQ (6.6 g, 26.7 mmol). After stirring at room temperature for 16 h under N2 atmosphere, the reaction mixture was diluted with water (100 mL) and extracted with DCM (3 × 100 mL). The organic phase was washed with brine (100 mL), dried over Na2SO4, and concentrated in vacuo to give the crude product, which was purified by flash chromatography to give compound 8-6 (2.4 g, 3.2 mmol, 49%) as a white solid.

[0453] To a solution of compound 8-6 (2.4 g, 3.3 mmol) in THF (20 mL) was added TEA 3HF (2 mL). The reaction mixture was stirred at room temperature for 1.5 hours. After completion, the solvent was removed and the residue was purified by silica gel chromatography to give compound 8-7 (1.8 g, 2.8 mmol, 86%) as a white solid.

[0454] To a solution of compound 8-7 (1.1 g, 1.8 mmol) and TEA (400 mg, 3.5 mmol) in DCM (50 mL) was added acid chloride (500 mg, 2.6 mmol) under N. After stirring at 25 °C for 16 h, the solvent was removed and the residue was purified by silica gel chromatography to give compound 8-8 (802 mg, 1.0 mmol, 57%) as a white solid.

[0455] To a solution of compound 8-8 (802 mg, 1.0 mmol) in MeOH (20 mL) was added NaOMe (200 mg, 3.0 mmol) under N. After stirring at 25 °C for 16 h, the solvent was removed and the residue was purified by flash chromatography on silica gel to give compound 8-9 (795 mg, 0.6 mmol, 60%) as a white solid.

[0456] To a solution of compound 8-9 (200 mg, 0.30 mmol) in DMF (5 mL) was added MMAE (200 mg, 0.30 mmol), HOBT (6.7 mg, 0.03 mmol), and TEA (50 mg, 0.50 mmol) under N. After stirring at 25 °C for 16 h, the mixture was diluted with water and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over NaSO, filtered, and concentrated in vacuo to give the crude product, which was purified by preparative HPLC to give compound 8-10 (196 mg, 0.15 mmol, 59%).

[0457] To a solution of compound 8-10 (196 mg, 0.15 mmol) in DCM (20 mL) was added TFA (1 mL). After stirring at 25 °C for 16 h, the solvent was removed to give compound 8-11 (193 mg, 0.16 mmol) as a white solid.

[0458] To a solution of compound 3 (100.0 mg, 2 μmol) in DCM, DIPEA (20.0 μL, 10 μmol), EDC (20.0 μL, 10 μmol), HOBt (15.8 mg, 10 μmol), and compound 8-11 (130.0 mg, 10 μmol) were added sequentially. The reaction mixture was stirred at room temperature under N for 16 hours. The crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN to remove the protecting groups and then with LiOH to hydrolyze the methyl ester group. After the reaction, the residue was purified by preparative HPLC to give compound 8-12 (140.0 mg, 70%).

[0459] Compound 8-12 (55.0 mg, 7.5 × 10) in DMF -3To a solution of 4.0 mg of methylparaben-PEG-NHS (7.5 μmol) was added and stirred for 2 hours. The crude mixture was purified by reverse phase chromatography to give compound 8 (26.0 mg, 50%). Example 9 Synthesis of Compound 9 [ka]

[0460] Compound 8-12 (55.0 mg, 7.5 × 10) in DMF -3 ) solution, DBCO-PEG4-NHS (5.0 mg, 7.5 × 10 -3 After stirring for 1 hour, the crude product was purified by reverse phase chromatography to give compound 9 (28.0 mg, 50%). Example 10 Synthesis of Compound 10 [ka]

[0461] Compound 2-7 (100.0 mg, 2.0 × 10) in DCM -3 A solution of 1.0 mmol of HCl was added to a solution of 1.0 mmol of HCl in 10 mL of HCl solution containing DIPEA (4.0 μL, 2 μmol), EDC (4.0 μL, 2 μmol), and HOBt (3.2 mg, 2.0 × 10 -3 To the reaction mixture were added 10-1 (28.0 mg, 2 μmol), followed by compound 8-11 (28.0 mg, 2 μmol). The reaction mixture was stirred under N2 at room temperature for 16 hours. The solvent was removed, and the crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN to remove the protecting groups, and then with LiOH to hydrolyze the methyl ester group. After the reaction, the crude product was purified by preparative HPLC to give compound 10-1 (90.0 mg, 70%).

[0462] To a solution of compound 10-1 (55.0 mg, 18 μmol) in DMF was added malimido-PEG4-NHS (8.0 mg, 1.8 μmol). After stirring for 1 h, the crude mixture was purified by reverse-phase chromatography to give compound 10 (35.0 mg, 65%). Example 11 Synthesis of Compound 11 [ka]

[0463] To a solution of compound 11-1 (1.0 g, 0.8 mmol) in DCM (2 mL) was added TFA (0.5 mL). After stirring at 25° C. for 16 h, the solvent was removed to give compound 11-2 (900 mg, 0.8 mmol) as a white solid.

[0464] Compound 3 (100.0 mg, 2.0 × 10) in DCM -3 To a solution of 11-2 (24.0 mg, 2 μmol), DIPEA (4.0 μL, 2 μmol), EDC (4.0 μL, 2 μmol), HOBt (3.2 mg, 2 μmol), and compound 11-2 (24.0 mg, 2 μmol) were added sequentially. The reaction mixture was stirred under N2 at room temperature for 16 h. The crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN. After the reaction, the residue was purified by preparative HPLC to give compound 11-3 (88 mg, 85%).

[0465] To a solution of compound 11-3 (60.0 mg, 7.5 μmol) in DMF was added malimido-PEG4-NHS (4.0 mg, 7.5 μmol). After stirring for 1 h, the crude product was purified by reverse-phase chromatography to give compound 11 (32.0 mg, 55%). Example 12 Synthesis of Compound 12 [ka]

[0466] To a solution of compound 11-3 (60.0 mg, 7.5 μmol) in DMF was added DBCO-PEG4-NHS (5.0 mg, 7.5 μmol). After 1 h, the crude product was purified by reverse-phase chromatography to give compound 12 (32.0 mg, 50%). Example 13 Synthesis of Compound 13 [ka]

[0467] To a solution of compound 13-1 (1.0 g, 1.6 mmol) and bis(4-nitrophenyl)carbonate (700 mg, 2.4 mmol) in DMF (5 mL) was added DIEA (300 mg, 2.4 mmol) at 20 °C under N. The reaction was stirred at room temperature for 16 h. The reaction was concentrated in vacuo, and diethyl ether (10 mL) was added to the residue. The resulting precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum to give compound 13-2 (1.3 g, 1.6 mmol, 100%) as a yellow solid.

[0468] To a solution of compound 13-2 (1.3 g, 1.6 mmol) in NMP (5 mL) was added 13-3 (1.2 g, 2.4 mmol) at 20 °C. Then, TMP (700 mg, 2.4 mmol) and HOAt (0.2 g, 1.6 mmol) were added at 20 °C. The resulting solution was stirred for 48 h. Water (50 mL) was added to the reaction solution, and it was extracted with EtOAc (50 mL × 3). The combined organic layer was washed with brine (50 mL × 3), dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC to give compound 13-4 (1.2 g, 1.1 mmol, 70%) as a white solid.

[0469] To a solution of compound 13-4 (1.2 g, 1.1 mmol) in DCM (20 mL) was added pepperonidine (2 mL). After stirring at 25 °C for 18 h, the solvent was removed and the residue was purified by C18 chromatography to give compound 13-5 (1.1 g, 95%) as a white solid.

[0470] To a solution of compound 2 (100.0 mg, 2 μmol) in DCM, DIPEA (20.0 μL, 10 μmol), EDC (20.0 μL, 10 μmol), HOBt (15.8 mg, 10 μmol), and compound 13-5 (130.0 mg, 10 μmol) were added sequentially. The reaction mixture was stirred at room temperature under N for 16 h. The crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN to remove the protecting groups and then with LiOH to hydrolyze the methyl ester group. After the reaction, the residue was purified by preparative HPLC to give compound 13-6 (158 mg, 72%).

[0471] To a solution of compound 13-6 (65.0 mg, 7.5 μmol) in DMF was added DBCO-PEG4-NHS (5.0 mg, 7.5 μmol). After 1 h, the crude product was purified by reverse-phase chromatography to give compound 13 (40.6 mg, 55%). Example 14 Synthesis of Compound 14 [ka]

[0472] To a solution of compound 14-1 (80.0 mg, 0.3 mmol) in DMF (3 mL) was added DIEA (108.0 mg, 0.8 mmol), Fmoc-ethylenediamine (158.0 mg, 0.6 mmol), and HATU (160.0 mg, 0.4 mmol). The reaction mixture was stirred at room temperature for 16 hours. The mixture was purified by flash chromatography to give compound 14-2 (123.0 mg, 0.2 mmol, 79%) as a white solid.

[0473] To a solution of compound 14-2 (123.0 mg, 0.2 mmol) in DMF (2 mL), diethylamine (0.2 mL) was added slowly. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated in vacuo to give compound 14-3 (70.0 mg) as a colorless oil.

[0474] To a solution of (S,E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((S)-3-methyl-2-(methylamino)-3-phenylbutanamido)butanamido)hex-2-enoic acid (50.0 mg, 0.1 mmol) in DMF (2 mL) was added DIEA (34 mg, 0.26 mmol), compound 14-3 (70.0 mg, 0.2 mmol), and HATU (52.0 mg, 0.1 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction was purified by flash chromatography to give compound 14-4 (90.0 mg) as a colorless oil.

[0475] To a solution of compound 14-4 (90.0 mg, 0.11 mmol) in DCM (2 mL) was added TFA (0.5 mL) slowly. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. The reaction mixture was concentrated in vacuo to give compound 14-5 (78.0 mg, 0.1 mmol, 99%) as a colorless oil.

[0476] To a solution of compound 3 (100.0 mg, 2.0 μmol) in DCM, DIPEA (4.0 μL, 2.0 μmol), EDC (4.0 μL, 2.0 μmol), HOBt (3.2 mg, 2.0 μmol), and compound 14-5 (9.0 mg, 2.0 μmol) were added sequentially. The reaction mixture was stirred at room temperature under N for 16 h. The crude product was purified by reverse-phase chromatography. The purified product was first treated with 0.5 N HCl / MeCN to remove the protecting groups and then with LiOH to hydrolyze the methyl ester group. After the reaction, the crude product was purified by preparative HPLC to give compound 7-6 (80.0 mg, 75%).

[0477] To a solution of compound 7-6 (60.0 mg, 7.5 μmol) in DMF, DBCO-PEG-NHS (5.0 mg, 7.5 × 10 -3 ) was added. The crude product was purified by reverse phase chromatography to give compound 14 (37.0 mg, 75%). Example 15 Synthesis of ADC PLAD-042

[0478] To a solution of mAb02 (5 mg, 2.5 mg / mL) in conjugation buffer (PBS buffer), 2 equivalents of TCEP were added. The mixture was shaken at room temperature for 1 hour. To the reduced antibody solution, 6 equivalents of compound 4 were added. The reaction mixture was mixed well and shaken for 1 hour. Next, 50 equivalents of L-cysteine ​​solution were added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a gel filtration column (3 mg, 2 mg / mL). The DAR was determined by UV spectroscopy. Example 16 Synthesis of ADC PLAD-114

[0479] To a solution of mAbB854 (5 mg, 2.5 mg / ml) in conjugation buffer (PBS buffer), 3 equivalents of TCEP were added. The mixture was shaken at room temperature for 1 hour. 10 equivalents of compound 6 were added to the reduced antibody solution. The reaction mixture was mixed well and shaken for 1 hour. 50 equivalents of L-cysteine ​​solution were then added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a cation exchange column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 17 Synthesis of ADC PLAD-085

[0480] To a solution of mAbB854 (5 mg, 2.5 mg / ml) in conjugation buffer (PBS buffer), 3 equivalents of TCEP were added. The mixture was shaken at room temperature for 1 hour. To the reduced antibody solution, 10 equivalents of compound 8 were added. The reaction mixture was mixed well and shaken for 1 hour. Next, 50 equivalents of L-cysteine ​​solution were added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a cation exchange column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 18 Synthesis of ADC PLAD-141

[0481] To a solution of mAbA149-32 (5 mg, 2.5 mg / ml) in conjugation buffer (PBS buffer), 3 equivalents of TCEP were added. The mixture was shaken at room temperature for 1 hour. 10 equivalents of compound 10 were added to the reduced antibody solution. The reaction mixture was mixed well and shaken for 1 hour. 50 equivalents of L-cysteine ​​solution were then added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a cation exchange column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 19 Synthesis of ADC PLAD-134

[0482] To a solution of mAbA149-32 (5 mg, 2.5 mg / ml) in conjugation buffer (PBS buffer), 3 equivalents of TCEP were added. The mixture was shaken at room temperature for 1 hour. 10 equivalents of compound 11 were added to the reduced antibody solution. The reaction mixture was mixed well and shaken for 1 hour. 50 equivalents of L-cysteine ​​solution were then added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a cation exchange column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 20 Synthesis of ADC PLAD-135

[0483] To a solution of mAb A149-32 (5 mg, 2.5 mg / ml) in conjugation buffer (PB buffer), 0.1 wt% endoglycosidase and 10 equivalents of a disaccharide oxazoline (oxa-bis-N3) bearing an azide group were added. The mixture was shaken at room temperature for 1 hour. To the azide-modified antibody solution, 20 equivalents of compound 12 were added. The reaction mixture was mixed well and shaken for 6 hours. The ADC was then purified via a cation exchange column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 21 Synthesis of ADC PLAD-072

[0484] To a solution of mAb B854 (5 mg, 2.5 mg / ml) in conjugation buffer (PB buffer), 0.1 wt% endoglycosidase and 10 equivalents of a disaccharide oxazoline (oxa-bis-N3) bearing an azide group were added. The mixture was shaken at room temperature for 1 hour. To the azide-modified antibody solution, 20 equivalents of compound 13 were added. The reaction mixture was mixed well and shaken for 6 hours. The ADC was then purified via a gel filtration column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 22 Synthesis of ADC PLAD-136

[0485] To a solution of mAbB854 (5 mg, 2.5 mg / ml) in conjugation buffer (PB buffer), 0.1 wt% endoglycosidase and 10 equivalents of a disaccharide oxazoline (oxa-bis-N3) bearing an azide group were added. The mixture was shaken at room temperature for 1 hour. To the azide-modified antibody solution, 20 equivalents of compound 14 were added. The reaction mixture was mixed well and shaken for 6 hours. The ADC was then purified via a gel filtration column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 23 Synthesis of ADC PLAD-043

[0486] mAb02 (5 mg, 2.5 mg / ml) in conjugation buffer (PBS buffer) and 3 equivalents of TCEP were added to the antibody solution. The mixture was shaken at room temperature for 1 hour. 10 equivalents of compound 4 were added to the reduced antibody solution. The reaction mixture was mixed well and shaken for 1 hour. 50 equivalents of L-cysteine ​​solution were then added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a gel filtration column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 24 Synthesis of ADC PLAD-047

[0487] mAbS001 (5 mg, 2.5 mg / ml) in conjugation buffer (PB buffer), 0.1 wt% endoglycosidase, and 10 equivalents of a disaccharide oxazoline (oxa-bis-N3) bearing an azide group were added to the antibody solution. The mixture was shaken at room temperature for 1 hour. 12 equivalents of compound 9 were added to the azide-modified antibody solution. The reaction mixture was mixed well and shaken for 6 hours. The ADC was then purified via a gel filtration column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 25 Synthesis of ADC PLAD-053

[0488] mAbS002 (5 mg, 2.5 mg / ml) in conjugation buffer (PB buffer), 0.1 wt% endoglycosidase, and 10 equivalents of a disaccharide oxazoline (oxa-bis-N3) bearing an azide group were added to the antibody solution. The mixture was shaken at room temperature for 1 hour. 12 equivalents of compound 9 were added to the azide-modified antibody solution. The reaction mixture was mixed well and shaken for 6 hours. The ADC was then purified via a gel filtration column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 26 Synthesis of ADC PLAD-119

[0489] mAbA149-32 (5 mg, 2.5 mg / ml) in conjugation buffer (PBS buffer) and 3 equivalents of TCEP were added to the antibody solution. The mixture was shaken at room temperature for 1 hour. 10 equivalents of compound 6 were added to the reduced antibody solution. The reaction mixture was mixed well and shaken for 1 hour. Next, 50 equivalents of L-cysteine ​​solution were added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a cation exchange column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 27 Synthesis of ADC PLAD-120

[0490] mAb3-7 (5 mg, 2.5 mg / ml) in conjugation buffer (PBS buffer) and 2 equivalents of TCEP were added to the antibody solution. The mixture was shaken at room temperature for 1 hour. 6 equivalents of compound 6 were added to the reduced antibody solution. The reaction mixture was mixed well and shaken for 1 hour. Next, 50 equivalents of L-cysteine ​​solution were added to the reaction mixture to quench the reaction. The reaction mixture was shaken at room temperature for an additional 30 minutes. The ADC was then purified via a cation exchange column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 28 Synthesis of ADC PLAD-142

[0491] mAb A149-32 (5 mg, 2.5 mg / ml) in conjugation buffer (PB buffer), 0.1 wt% endoglycosidase, and 10 equivalents of a disaccharide oxazoline (oxa-bis-N3) bearing an azide group were added to the antibody solution. The mixture was shaken at room temperature for 1 hour. 15 equivalents of compound 7 were added to the azide-modified antibody solution. The reaction mixture was mixed well and shaken for 6 hours. The ADC was then purified via a gel filtration column (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy. Example 29 Synthesis of Amine Intermediate Compound 15 [ka]

[0492] A solution of N3NBu4 (1.98 g, 6.98 mmol), EGE (61 g, 418.6 mmol), and t-BGA (39.3 g, 209.3 mmol) in diglyme (200 mL) was added under N2. The mixture was cooled to -40 °C, and then Al(iBu)3 (30 ml, 29.7 mmol, 1 M in hexane) was added slowly. The reaction was allowed to warm to room temperature and stirred under N2 for 16 h. The reaction was quenched with EtOH, diluted with water, and extracted with EA. The combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 15-1 (103 g) as a colorless oil with a Mw of 27,000 as determined by GPC using PS as the standard.

[0493] To a solution of compound 15-1 (50 g, 1.85 mmol) in DCM (500 mL), HCl (200 mL, 1 M in 1,4-dioxane) was slowly added, and the reaction mixture was stirred at room temperature for 16 h. After completion, the solvent was removed under reduced pressure, and the residue was dissolved in 10% NaOH solution. After adjusting the pH to 7 with 4 N HCl, the total volume of the solution was adjusted to 500 mL with water to give compound 15-2 as an aqueous solution.

[0494] To the solution of 15-2, Na2S.9H2O (30 g, 125 mmol) was added and the reaction mixture was heated at 100 °C for 18 h. Upon completion, the reaction was cooled to room temperature and the pH was adjusted using 4 N HCl. The mixture was dialyzed against a 3 KD MWCO to give compound 15-3 as an aqueous solution (300 mL).

[0495] To the solution of 15-3 was added NaOH (5 g, 125 mmol) and (Boc)O (30 g, 125 mmol), and the reaction mixture was stirred at room temperature for 18 h. Upon completion, the mixture was dialyzed against a 3 KD MWCO to give compound 15 as an aqueous solution (800 mL, 25 mg / mL).

[0496] 1H NMR (400MHz, D2O) δ3.95(s, 100H), 3.87-3.57(br, 800H), 1.43(s, 9H). Example 30 Synthesis of Compound 16 [ka]

[0497] To a solution of 16-1 in THF (200 mL) was added pyridine (12 mL, 148 mmol) and BocO (1.44, 6.59 mmol). The reaction mixture was stirred at 15 °C for 18 h. The reaction mixture was washed with 0.5 N HCl (3 × 100 mL) and saturated NaHCO (100 mL). The organic phase was dried over MgSO, filtered, and concentrated in vacuo to give the desired product 16-2 (2.45 g) as a white solid.

[0498] To a solution of 16-2 (2.5 g, 5.38 mmol) in DCM (50 mL) was added (tert-butoxycarbonyl)-L-alanine (2.5 g, 13.2 mmol), EDCI (1.99 g, 10.4 mmol), and DMAP (197.3 mg, 1.61 mmol). The reaction mixture was stirred at 15 °C for 16 h. The reaction mixture was washed with 0.5% NaHCO (2 × 400 mL), water (400 mL), and 0.1 N HCl (2 × 400 mL). The organic phase was dried over anhydrous MgSO, filtered, and evaporated in vacuo. The residue was purified by silica gel column chromatography to give the desired product 16-3 (3.0 g, 87.7% yield) as a white solid.

[0499] To a solution of 16-3 (1.5 g, 2.36 mmol) in DCM (30 mL) was added TFA (10 mL). After stirring at 15 °C for 16 h, the solvent was removed in vacuo and the residue was purified by preparative HPLC (TFA) to give the desired product 16-4 (627 mg) as a yellow solid.

[0500] To the aqueous solution of compound 15, a DMF solution of NHS (0.12 mg, 1.05 μmol), EDC (20 mg, 105.4 μmol), and 16-4 (23.4 mg, 42.2 μmol) was slowly added at 0° C., and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed against a 5 KD MWCO to give compound 25-5 as an aqueous solution (25 mL, 5 mg / mL).

[0501] To a solution of compound 16-5 in water (25 mL, 5 mg / mL) was added 4N HCl (1.6 mL) slowly at 0° C., and the reaction mixture was stirred at room temperature for 16 h. After completion, the pH of the mixture was adjusted to 7 with saturated NaHCO (aq) to give compound 16, which was used directly in the next step. Example 31 In vitro cytotoxicity assay of ADCs PLAD-042 and PLAD-043

[0502] The cytotoxicity of ADCs PLAD-042 and PLAD-043 was investigated using KG1α cells (acute myeloid leukemia, ATCC No. CCL-246.1). Briefly, KG1α cells were plated at 5,000 cells / well in 90 μL of basal culture medium into a clear, flat-bottom, white 96-well assay plate (catalog no. #BS-MP-96W) (except for the edge wells, which contained only medium or PBS) and grown in a humidified incubator at 37°C under a 5% CO2 atmosphere. After overnight incubation, each ADC was added to each well in a 10 μl volume of 10× test concentrations ranging from 100 nM to 0.015 nM. After an additional 72 hours of incubation, the plate was removed from the incubator and allowed to equilibrate to room temperature. Approximately 30 minutes later, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (Catalog No. G7573, Promega) was added to each well. Plates were shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking. Fluorescence was then measured on an Envision plate reader (Instrument No. 2104, PerkinElmer) using an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 1, ADCs PLAD-042 and PLAD-043 exhibited desirable cell inhibitory and cytotoxic activities. Example 32 In vitro cytotoxicity assay of ADCs PLAD-047 and PLAD-053

[0503] The cytotoxicity of ADCs PLAD-047 and PLAD-053 was investigated using NCI-H358 cells (lung cancer, Procell #CL-0400), MDA-MB-231 cells (breast cancer, ATCC #HTB-26), and MCF-7 cells (breast cancer, ATCC #HTB-22). Briefly, cancer cells were plated at 5,000 cells / well in 90 μL of basal medium into a clear, flat-bottom, white 96-well assay plate (catalog #BS-MP-96W) (excluding edge wells containing medium or PBS only) and grown at 37°C in a humidified 5% CO2 atmosphere. After overnight incubation, each ADC was added to each well in a 10x test concentration of 100 nM to 0.015 nM in a 10 μl volume. After a further 72 hours of incubation, the plate was removed from the incubator and then equilibrated to room temperature. Approximately 30 minutes later, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (Catalog No. G7573, Promega) was added to each well. The plate was shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking. Fluorescence was then measured using an Envision plate reader (Instrument No. 2104, PerkinElmer) with an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 2, ADCs PLAD-047 and PLAD-053 exhibited desirable cell-inhibitory activity against the NCI-H358 cell line. Meanwhile, ADC PLAD-047 failed to effectively inhibit cell proliferation in both MDA-MB-231 and MCF-7 cell lines. ADC PLAD-053 had limited cytotoxic activity against the MCF-7 cell line at the highest concentration and no effect on the MDA-MB-231 cell line, suggesting that ADCs PLAD-047 and PLAD-053 exhibited selectivity for inhibiting cell proliferation in specific cell lines. Example 33 In vitro cytotoxicity assay of ADC PLAD-072

[0504] The cytotoxicity of ADC PLAD-072 was investigated using IGROV1 (ovarian cancer, CTCC No. 009-0048), OVCAR3 (ovarian-derived, ATCC No. HTB-161), KB (cervical cancer, CTCC No. 001-0248), JEG3 (placenta, CTCC No. 4000250), and SKOV3 (ovarian cancer, ATCC No. HTB-77). Briefly, cancer cells were plated at 5,000 cells / well in 90 μL of basal culture medium into a clear, flat-bottom, white 96-well assay plate (catalog no. #BS-MP-96W) (excluding edge wells containing medium or PBS only) and grown at 37°C in a humidified incubator with a 5% CO atmosphere. After overnight incubation, each ADC was added to each well in a 10x test concentration ranging from 100 nM to 0.015 nM in a volume of 10 μl. After an additional 72 hours of incubation, the plates were removed from the incubator and allowed to equilibrate to room temperature. Approximately 30 minutes later, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (Catalog No. G7573, Promega) was added to each well. The plates were shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking. Fluorescence was then measured using an Envision plate reader (Instrument No. 2104, PerkinElmer) with an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 3, ADC PLAD-072 can effectively inhibit tumor cell proliferation, particularly in KB, JEG3, and IGROV1 cells. Example 34 In vitro cell binding assay of ADC PLAD-072

[0505] SKOV3 cells were used for in vitro cell binding assessment of mAb B854, ADC PLAD-072, and hIgG1. In the binding assay, the native human IgG1 antibody B854 (Sanyou Bio) was used as a free antibody control. Briefly, SKOV3 cells were maintained in McCoy's 5a medium (Cat. No. 30-2007, ATCC) supplemented with 10% FBS (Cat. No. FSP500, Excell Bio) and 1% penicillin-streptomycin solution (Biosharp Life Sciences, BL505A). Cells were cultured at 37°C in a 5% CO2 atmosphere in air and harvested from flasks for assays when they reached 70%-80% confluence. 2 x 10 cells were cultured per well in a 96-well cell culture plate. 6 100 μL of cells at 100 μM / mL were incubated with B854, PLAD-072, and hIgG1 in a 5-fold serial dilution starting from 100 nM to 0.00128 nM on ice for 1 hour. After washing twice with 200 μL of FACS buffer per well, the cells were incubated with 100 μL of 1x secondary antibody PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) on ice for 30–60 minutes. The cells were washed twice with 200 μL of FACS buffer and resuspended in 200 μL of FACS buffer for further detection by CytoFlex Cytometry (Beckman Coulter). Data were analyzed using the built-in analysis software, CytoFlex. The results are shown in Figure 4, demonstrating that the ADC PLAD-072 effectively bound to SKOV3 cells, almost as effectively as the native antibody B854. This indicates that conjugation has little effect on binding to the target compared to the native antibody. Example 35 Internalization Assay of ADC PLAD-072

[0506] SKOV3 was also used in the following internalization assay: Cells were harvested with 0.25% Tricine / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 2 × 10 using 1 × PBS containing 1% FBS buffer. 6The concentration was adjusted to cells / mL. Then, 100 μL of cells per well were plated into a 96-well cell culture plate and incubated with B854 and ADC PLAD-072 at a final concentration of 100 nM for seven time points (0, 0.5, 1, 2, 3, and 4 hours). The mixture was incubated at 4°C in an air-to-5% CO2 atmosphere. At specific time points, the cells were immediately washed twice and suspended in 100 μL of cold wash buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for an additional hour at 4°C and 5% CO2. After two washes with wash buffer, the cells were resuspended in 100 μL of FACS buffer, and the fluorescent signal of the cells was detected using CytoFlex Cytometry (Beckman Coulter). The internalization rate at several time points was calculated as the percentage loss of the MFI value at a particular time point at time 0. The internalization rate versus incubation time (hours) curve was plotted using Graphpad Prism Software. The results are shown in Figure 5, which demonstrates that ADC PLAD-072 can effectively mediate internalization into SKOV3 cells, with an internalization rate of over 60% within 4 hours. The internalization rate was comparable to that of naked antibodies. Example 36 In vitro cytotoxicity assay of ADC PLAD-085

[0507] The cytotoxicity of ADC PLAD-085 was investigated using IGROV1 (ovarian cancer, CTCC No. 009-0048), OVCAR3 (ovarian-derived, ATCC No. HTB-161), JEG3 (placenta, CTCC No. 4000250), and SKOV3 (ovarian cancer, ATCC No. HTB-77). Briefly, cancer cells were plated at 5,000 cells / well in 90 μL of basal culture medium into a clear, flat-bottom, white 96-well assay plate (catalog no. #BS-MP-96W) (except for the edge wells, which contained only medium or PBS) and grown at 37°C in a humidified incubator with a 5% CO atmosphere. After overnight incubation, each ADC was added to each well in a 10x test concentration of 100 nM to 0.015 nM in a 10 μL volume. After an additional 72 hours of incubation, the plates were removed from the incubator and allowed to equilibrate to room temperature. Approximately 30 minutes later, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (Catalog No. G7573, Promega) was added to each well. The plates were shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking. Fluorescence was then measured using an Envision plate reader (Instrument No. 2104, PerkinElmer) with an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 6, ADC PLAD-085 can effectively inhibit the proliferation of different types of cancer cells. Example 37 In vitro cytotoxicity assay of ADC PLAD-114

[0508] The cytotoxicity of ADC PLAD-114 was investigated using IGROV1 (ovarian cancer, CTCC No. 009-0048), OVCAR3 (ovarian-derived, ATCC No. HTB-161), JEG3 (placenta, CTCC No. 4000250), and SKOV3 (ovarian cancer, ATCC No. HTB-77). Briefly, cancer cells were plated at 5,000 cells / well in 90 μL of basal culture medium into a clear, flat-bottom, white 96-well assay plate (catalog no. BS-MP-96W) (except for the edge wells, which contained only medium or PBS) and grown at 37°C in a humidified incubator with a 5% CO atmosphere. After overnight incubation, each ADC was added to each well in a 10× test concentration ranging from 100 nM to 0.015 nM in a volume of 10 μl. After an additional 72 hours of incubation, the plates were removed from the incubator and allowed to equilibrate to room temperature. Approximately 30 minutes later, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (Catalog No. G7573, Promega) was added to each well. The plates were shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking. Fluorescence was then measured using an Envision plate reader (Instrument No. 2104, PerkinElmer) with an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 7, ADC PLAD-114 can effectively inhibit the proliferation of different types of tumor cells. Example 38 In vitro cell binding assay of ADC PLAD-114

[0509] SKOV3 cells were used for in vitro cell binding evaluation of mAb B854, PLAD-0114, and hIgG1. In the binding assay, the native human IgG1 antibody B854 (Sanyou Bio) was used as a free antibody control. Briefly, SKOV3 cells were maintained in McCoy's 5a medium (Cat. No. 30-2007, ATCC) supplemented with 10% FBS (Cat. No. FSP500, Excell Bio) and 1% penicillin-streptomycin solution (Cat. No. BL505A, Biosharp Life Sciences). Cells were cultured at 37°C in a 5% CO2 atmosphere in air and harvested from flasks for assays when they reached 70%-80% confluence. 2 × 10 cells were cultured per well in a 96-well cell culture plate. 6 100 μL of cells / mL were incubated with B854, ADC PLAD-114, and hIgG1 in a 5-fold serial dilution starting from 100 nM to 0.00128 nM for 1 hour on ice. After washing twice with 200 μL of FACS buffer per well, the cells were incubated with 100 μL of 1x secondary antibody PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for 30–60 minutes on ice. The cells were washed twice with 200 μL of FACS buffer and resuspended in 200 μL of FACS buffer for further detection by CytoFlex Cytometry (Beckman Coulter). Data were analyzed using the built-in analysis software, CytoFlex. The results are shown in Figure 8, indicating that ADC PLAD-114 bound to SKOV3 cells more effectively than B854. This indicates that conjugation does not affect target binding compared to native antibodies. Example 39 Internalization Assay of ADC PLAD-114

[0510] SKOV3 was also used in the following internalization assay: Cells were harvested with 0.25% Tricine / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 2 × 10 using 1 × PBS containing 1% FBS buffer. 6The concentration was adjusted to cells / mL. Then, 100 μL of cells per well were plated into a 96-well cell culture plate and incubated with B854 and ADC PLAD-114 at a final concentration of 100 nM for seven time points (0, 0.5, 1, 2, 3, and 4 hours). The mixture was incubated at 4°C in an air-to-5% CO2 atmosphere. At specific time points, the cells were immediately washed twice and suspended in 100 μL of cold wash buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for an additional hour at 4°C and 5% CO2. After two washes with wash buffer, the cells were resuspended in 100 μL of FACS buffer, and the fluorescent signal of the cells was detected using CytoFlex Cytometry (Beckman Coulter). The internalization rate at several time points was calculated as the percentage loss of the MFI value at a particular time point at time 0. The internalization rate versus incubation time (hours) curve was plotted using Graphpad Prism Software. The results are shown in Figure 9, which shows that ADC PLAD-114 could effectively mediate internalization into SKOV3 cells, with an internalization rate of approximately 80% within 4 hours. Conjugation could not alter target-mediated antibody internalization. Example 40 In vitro cytotoxicity assay of ADC PLAD-119

[0511] The cytotoxicity of the ADC PLAD-119 was investigated using OVCAR3 (ovarian-derived, ATCC No. HTB-161). Briefly, cancer cells were plated at 5,000 cells / well in 90 μL of basal culture medium into a clear, flat-bottom, white 96-well assay plate (catalog no. #BS-MP-96W) (except for the edge wells, which contained only medium or PBS) and grown at 37°C in a humidified incubator with a 5% CO2 atmosphere. After overnight incubation, the ADC was added to each well in a 10x test concentration of 100 nM to 0.015 nM in a 10 μL volume. After an additional 72 hours of incubation, the plate was removed from the incubator and allowed to equilibrate to room temperature. After approximately 30 minutes, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (catalog no. G7573, Promega) was added to each well. The plate was shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking, after which fluorescence was measured on an Envision plate reader (instrument number 2104, PerkinElmer) using an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 10, ADC PLAD-119 can effectively inhibit tumor cell proliferation. Example 41 In vitro cell binding assay of ADC PLAD-119

[0512] OVCAR3 cells were used for in vitro cell binding evaluation of A149-32, ADC PLAD-119, and hIgG1. In the binding assay, the native human IgG1 antibody A149-32 (Sanyou Bio) was used as a free antibody control. Briefly, OVCAR3 cells were maintained in RPMI 1640 medium (catalog no. abs9468, Absin) supplemented with 20% FBS (catalog no. FSP500, Excell Bio) and 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences). Cells were cultured at 37°C in a 5% CO2 atmosphere in air and harvested from flasks for assays when they reached 70%–80% confluence. 2 × 10 cells were cultured per well in a 96-well cell culture plate. 6 100 μL of cells at 100 μM / mL were incubated with A149-32, ADC PLAD-119, and hIgG1 in a 5-fold serial dilution starting from 100 nM to 0.00128 nM on ice for 1 hour. After washing twice with 200 μL of FACS buffer per well, the cells were incubated with 100 μL of 1x secondary antibody PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) on ice for 30–60 minutes. The cells were washed twice with 200 μL of FACS buffer and resuspended in 200 μL of FACS buffer for further detection by CytoFlex Cytometry (Beckman Coulter). Data were analyzed using the built-in analysis software, CytoFlex. The results are shown in Figure 11, demonstrating that the ADC PLAD-119 effectively bound to OVCAR3 cells, comparable to the native antibody A149-32. Example 42 Internalization Assay of ADC PLAD-119

[0513] OVCAR3 was also used in the following internalization assay: Cells were harvested with 0.25% Tricine / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 2 × 10 using 1 × PBS containing 1% FBS buffer. 6The concentration of cells was adjusted to 100 cells / mL. Then, 100 μL of cells per well were plated into a 96-well cell culture plate and incubated with B854 and ADC PLAD-119 at a final concentration of 100 nM for seven time points (0, 0.5, 1, 2, 3, and 4 hours). The mixture was incubated at 4°C in an air-to-air 5% CO2 atmosphere. At specific time points, the cells were immediately washed twice and suspended in 100 μL of cold wash buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for an additional hour at 4°C and 5% CO2. After two washes with wash buffer, the cells were resuspended in 100 μL of FACS buffer, and the fluorescent signal of the cells was detected using CytoFlex Cytometry (Beckman Coulter). The internalization rate at several time points was calculated as the percentage loss of the MFI value at a particular time point at time 0. The internalization rate versus incubation time (hours) curve was plotted using Graphpad Prism Software. The results are shown in Figure 12, which shows that ADC PLAD-119 could effectively mediate internalization into OVCAR3 cells, with an internalization rate of over 80% within 4 hours. The internalization rate was enhanced compared to the naked antibody. Example 43 In vitro cytotoxicity of ADCs PLAD-134 and PLAD-135

[0514] The cytotoxicity of the ADCs PLAD-134 and PLAD-135 was investigated using OVCAR3 (ovarian-derived, ATCC No. HTB-161) and HEK293-overexpressing cells (in-house). Briefly, cancer cells were plated at 5,000 cells / well in 90 μL of basal culture medium into a clear, flat-bottom, white 96-well assay plate (catalog no. BS-MP-96W) (excluding edge wells containing medium or PBS only) and grown at 37°C in a humidified 5% CO2 atmosphere. After overnight incubation, each ADC was added to each well in a 10x test concentration of 100 nM to 0.015 nM in a 10 μl volume. After an additional 72 hours of incubation, the plate was removed from the incubator and allowed to equilibrate to room temperature. Approximately 30 minutes later, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (Catalog No. G7573, Promega) was added to each well. The plate was shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking. Fluorescence was then measured using an Envision plate reader (Instrument No. 2104, PerkinElmer) with an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 13, the ADCs PLAD-134 and PLAD-135 could effectively inhibit the proliferation of OVCAR3 and HEK293-overexpressing cells. Meanwhile, the HEK293-overexpressing cell line was more sensitive to these ADCs than the OVCAR3 cell line. Example 44 In vitro cell binding assay of ADCs PLAD-134 and PLAD-135

[0515] OVCAR3 cells were used for in vitro cell binding evaluation of A149-32, ADC PLAD-134, ADC PLAD-135, and hIgG1. In the binding assay, the native human IgG1 antibody A149-32 (Sanyou Bio) was used as a free antibody control. Briefly, OVCAR3 cells were maintained in RPMI 1640 medium (catalog no. abs9468, Absin) supplemented with 20% FBS (catalog no. FSP500, Excell Bio) and 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences). Cells were cultured at 37°C in a 5% CO2 atmosphere in air and harvested from flasks for assays when they reached 70%-80% confluence. 2 x 10 cells were cultured per well in a 96-well cell culture plate. 6 100 μL of cells / mL were incubated with A149-32, PLAD-134, PLAD-135, and hIgG1 in a 5-fold serial dilution starting from 400 nM to 0.00512 nM on ice for 1 hour. After washing twice with 200 μL of FACS buffer per well, the cells were incubated with 100 μL of 1x secondary antibody PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) on ice for 30–60 minutes. The cells were washed twice with 200 μL of FACS buffer and resuspended in 200 μL of FACS buffer for further detection by CytoFlex Cytometry (Beckman Coulter). Data were analyzed using the built-in analysis software, CytoFlex. The results are shown in Figure 14, demonstrating that the ADC PLAD-134 was able to bind to OVCAR3 cells as effectively as its native antibody, A149-32. This indicates that the conjugation method has little effect on binding to the target compared to the native antibody. Example 45 Internalization Assay of ADCs PLAD-134 and PLAD-135

[0516] OVCAR3 was also used in the following internalization assay: Cells were harvested with 0.25% Tricine / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 2 × 10 using 1 × PBS containing 1% FBS buffer. 6 The concentration was adjusted to cells / mL. Then, 100 μL of cells per well were plated into a 96-well cell culture plate and incubated with A149-32, ADC PLAD-134, ADC PLAD-135, and hIgG1 at a final concentration of 400 nM for seven time points (0, 0.5, 1, 2, 3, and 4 hours). The mixture was incubated at 4°C in an air-to-air 5% CO2 atmosphere. At specific time points, the cells were immediately washed twice and suspended in 100 μL of cold wash buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for an additional hour at 4°C and 5% CO2. After two washes with wash buffer, the cells were resuspended in 100 μL of FACS buffer, and the fluorescent signal of the cells was detected using CytoFlex Cytometry (Beckman Coulter). The internalization rate at several time points was calculated as the percentage loss of the MFI value at a particular time point at time 0. The internalization rate versus incubation time (hours) curve was plotted using Graphpad Prism Software. The results are shown in Figure 15, which shows that the ADCs could effectively mediate internalization into OVCAR3 cells, with the internalization rate exceeding 60% within 4 hours. Among these ADCs, the internalization rates of ADCs PLAD-134 and PLAD-135 were enhanced compared to the naked antibody. Example 46 In vitro cytotoxicity of ADCs PLAD-141 and PLAD-142

[0517] The cytotoxicity of the ADCs PLAD-141 and PLAD-142 was investigated using OVCAR3 (ovarian-derived, ATCC No. HTB-161), OV90 (ovarian-derived, ATCC No. CRL-11732), and HEK293-overexpressing cells (in-house). Briefly, cancer cells were plated at 5,000 cells / well in 90 μL of basal culture medium into a clear, flat-bottom, white 96-well assay plate (catalog no. BS-MP-96W) (excluding edge wells containing medium or PBS only) and grown in a humidified incubator at 37°C in a 5% CO2 atmosphere. After overnight incubation, each ADC was added to each well in a 10x test concentration of 100 nM to 0.015 nM in a 10 μl volume. After an additional 72 h of incubation, the plate was removed from the incubator and allowed to equilibrate to room temperature. Approximately 30 minutes later, 50 μL of Cell counting-lite 2.0 Luminescent Cell Viability Assay (Catalog No. G7573, Promega) was added to each well. The plate was shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking. Fluorescence was then measured using an Envision plate reader (Instrument No. 2104, PerkinElmer) with an integration time of 250 ms per well. Fluorescence versus concentration (nM) curves were fitted using GraphPad Prism Software. As shown in Figure 16, the ADCs PLAD-141 and PLAD-142 can effectively inhibit tumor cell proliferation. The HEK293 overexpressing cell line was more sensitive to the ADCs than the OVCAR3 and CV90 cell lines. Furthermore, the ADC PLAD-141 showed superior inhibitory effects against the three cell lines compared with the ADC PLAD-142. Example 47 In vitro cell binding assay of ADCs PLAD-141 and PLAD-142

[0518] OVCAR3 cells were used for in vitro cell binding evaluation of A149-32, ADC PLAD-141, ADC PLAD-142, and hIgG1. In the binding assay, the native human IgG1 antibody A149-32 (Sanyou Bio) was used as a free antibody control. Briefly, OVCAR3 cells were maintained in RPMI 1640 medium (catalog no. abs9468, Absin) supplemented with 20% FBS (catalog no. FSP500, Excell Bio) and 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences). Cells were cultured at 37°C in a 5% CO2 atmosphere in air and harvested from flasks for assays when they reached 70%-80% confluence. 2 x 10 cells were cultured per well in a 96-well cell culture plate. 6 100 μL of cells at 100 μL / mL were incubated with A149-32, PLAD-141, PLAD-142, and hIgG1 in a 5-fold serial dilution starting at 400 nM and ending at 0.00512 nM on ice for 1 hour. After washing twice with 200 μL of FACS buffer per well, cells were incubated with 100 μL of 1x secondary antibody PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for 30–60 minutes on ice. Cells were washed twice with 200 μL of FACS buffer and resuspended in 200 μL of FACS buffer for further detection by CytoFlex Cytometry (Beckman Coulter). Data were analyzed using the built-in analysis software, CytoFlex. The results are shown in Figure 17, and show that ADCs PLAD-141 and PLAD-142 were able to effectively bind to OVCAR3 cells with weaker binding capacity than A149-32. Example 48 Internalization Assay of ADCs PLAD-141 and PLAD-142

[0519] OVCAR3 was also used in the following internalization assay: Cells were harvested with 0.25% Tricine / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 2 × 10 using 1 × PBS containing 1% FBS buffer. 6The concentration was adjusted to cells / mL. Then, 100 μL of cells per well were plated into a 96-well cell culture plate and incubated with A149-32, PLAD-141, and PLAD-142 at a final concentration of 400 nM for seven time points (0, 1, 2, 3, 4, and 5 hours). The mixture was incubated at 4°C in an air-to-air 5% CO2 atmosphere. At specific time points, the cells were immediately washed twice and suspended in 100 μL of cold wash buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for an additional hour at 4°C and 5% CO2. After two washes with wash buffer, the cells were resuspended in 100 μL of FACS buffer, and the fluorescent signal of the cells was detected using CytoFlex Cytometry (Beckman Coulter). The internalization rate at several time points was calculated as the percentage loss of the MFI value at a particular time point at time 0. The internalization rate versus incubation time (hours) curve was plotted using Graphpad Prism Software. The results are shown in Figure 18, which shows that ADCs PLAD-141 and PLAD-142 could effectively mediate internalization into OVCAR3 cells, with internalization rates exceeding 90% within 5 hours. The internalization rates of these ADCs were superior to those of naked antibodies.

[0520] The foregoing description is considered as illustrative only of the principles of the disclosure. Moreover, 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 above. Accordingly, all suitable modifications and equivalents may be considered to be within the scope of the invention as defined by the following claims.

[0521] The words "comprise", "comprising", "include", "including" and "includes", when used in this specification and in the claims that follow, are intended to specify the presence of stated features, integers, components or steps, but they do not exclude the presence or addition of one or more other features, integers, components, steps or groups thereof.

Claims

1. Polymer scaffolds of formula (I) useful for conjugating targeting moieties: 【Chemistry 1】 And, the polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with said targeting moiety. ｐ is a linking moiety comprising M ａ L to B ａ It is a stretcher that connects to the part, B ａ is a functional group W connected to the —NH— moiety Ｍ is a branched portion including Each G １ is independent, L Ｐ is a functional group that connects the Each L Ｐ are independently therapeutic moieties D and G １ This is the drug release mechanism between each D is independently a therapeutic moiety; Each G ２ are functional groups that can be independently converted to a charged state, n is an integer from 0 to 1000, m is an integer from 0 to 1000; p is an integer from 0 to 1000, and A polymer scaffold wherein q is an integer from 0 to 1000.

2. W ｐ The polymer scaffold of claim 1 , wherein said polymer scaffold is capable of reacting with a functional group on said targeting moiety via a click reaction.

3. W ｐ but, 【Chemistry 2】 3. The polymer scaffold of claim 2, selected from the group consisting of:

4. W ｐ The polymer scaffold of claim 1 , wherein

5. The polymer scaffold of claim 4 , wherein the amino acids are natural amino acids, unnatural amino acids, or combinations thereof.

6. The polymer scaffold of claim 5 , wherein the natural amino acids include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

7. W ｐ The polymer scaffold of claim 4 , wherein:

8. W ｐ but, 【Transformation 3】 wherein R １ is a sulfur protecting group, and each R ２ The polymer scaffold of claim 7 , wherein:

9. Each R ２ are independently halo or R ２ａ C(O)O—, where R ２ａ 9. The polymer scaffold of claim 8, wherein is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

10. W ｐ is capable of reacting with one or more lysines on the targeting moiety, and each W ｐ became independent, 【Chemistry 4】 5. The polymer scaffold of claim 4, selected from the group consisting of:

11. W ｐ The polymer scaffold of claim 4 , wherein

12. W ｐ but, 【Transformation 5】 The polymer scaffold of claim 11 , wherein the polymer scaffold is selected from:

13. L 【Transformation 6】 10. The polymer scaffold according to any one of the preceding claims, wherein

14. M ａ but, 【Transformation 7】 wherein * is a site covalently bonded to L and ** is B ａ a moiety covalently bonded to the moiety, R ３ is C １～１０ Alkyl, C １～１０ Heteroalkyl, C ３～８ cycloalkyl, —O—(C １～８ alkyl), aryl, -C １～１０ Alkyl-aryl-, -aryl-C １～１０ Alkyl-, -C １～１０ Alkyl-(C ３～８ cycloalkyl)-, -(C ３～８ Cycloalkyl-C １～１０ alkyl)-, 4- to 14-membered heterocycloalkyl, —C １～１０ Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C １～１０ Alkyl-, -C １～１０ Alkyl-C(=O)-, -C １～１０ Heteroalkyl-C(=O)-, -C ３～８ Cycloalkyl-C(=O)-, —O—(C １～８ alkyl)-C(=O)-, -aryl-C(=O)-, -C １～１０ Alkyl-aryl-C(═O)-, -aryl-C １～１０ Alkyl-C(=O)-, -C １～１０ Alkyl-(C ３～８ cycloalkyl)-C(=O)-, -(C ３～８ Cycloalkyl)-C １～１０ Alkyl-C(═O)-, -4 to 14-membered heterocycloalkyl-C═(O)-, -C １～１０ Alkyl-(4- to 14-membered heterocycloalkyl)-C(═O)-, -(4- to 14-membered heterocycloalkyl)-C １～１０ Alkyl-C(=O)-, -C １～１０ Alkyl-NH-, -C １～１０ Heteroalkyl-NH-, -C ３～８ Cycloalkyl-NH-, —O—(C １～８ alkyl)-NH-, -aryl-NH-, -C １～１０ Alkyl-aryl-NH-, -aryl-C １～１０ Alkyl-NH-, -C １～１０ Alkyl-(C ３～８ cycloalkyl)-NH-, -(C ３～８ Cycloalkyl)-C １～１０ Alkyl-NH-, -4 to 14-membered heterocycloalkyl-NH-, -C １～１０ Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C １～１０ Alkyl-NH-, -C １～１０ Alkyl-S-, -C １～１０ Heteroalkyl-S-, -C ３～８ Cycloalkyl-S-, —O—C １～８ Alkyl-S-, -aryl-S-, -C １～１０ Alkyl-aryl-S-, -aryl-C １～１０ Alkyl-S-, -C １～１０ Alkyl-(C ３～８ cycloalkyl)-S-, -(C ３～８ Cycloalkyl)-C １～１０ Alkyl-S-, -4 to 14-membered heterocycloalkyl-S-, -C １～１０ alkyl-(4- to 14-membered heterocycloalkyl)-S-, or -(4- to 14-membered heterocycloalkyl)-C １～１０ alkyl-S-, Each R ４ are independently hydrogen, C １～６ Alkyl, C ６～１０ Aryl, C ３～８ Cycloalkyl, —COOH or —COO—C １～６ is alkyl, R ５ is -C(O)-NR ５ａ or -NR ５ａ -C(O)-, R ５ａ is hydrogen, C １～６ Alkyl, C ６～１０ Aryl, C ３～８ Cycloalkyl, —COOH or —COO—C １～６ is alkyl, R ６ is a bond or -NR ６ａ - (CR ６ｂ R ６ｃ )—C(O)—, R ６ａ is hydrogen, C １～６ Alkyl, C ６～１０ Aryl, C ３～８ Cycloalkyl, —COOH or —COO—C １～６ is alkyl, Each R ６ｂ and R ６ｃ are independently hydrogen, C １～６ Alkyl, C ６～１０ Aryl, Hydroxylated C ６～１０ Aryl, Polyhydroxylated C ６～１０ aryl, 5- to 12-membered heterocycloalkyl, C ３～８ Cycloalkyl, hydroxylated C ３～８ Cycloalkyl, polyhydroxylated C ３～８ cycloalkyl or the side chain of a natural or unnatural amino acid; each n １ are independently integers from 0 to 6, n ２ is an integer from 0 to 8, each n ３ are independently integers from 1 to 6, n ４ is an integer from 1 to 4, and n ５ 2. The polymer scaffold of claim 1, wherein is an integer from 1 to 4.

15. M ａ but, 【Transformation 8】 15. The polymer scaffold of claim 14, selected from the group consisting of:

16. G １ but, 【Chemistry 9】 wherein * is selected from the group consisting of L Ｐ and each R ７ are independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R ７ａ is selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

17. Each L ｐ The polymer scaffold of claim 1 , wherein each of the polymer scaffolds independently comprises an unstable structure.

18. 18. The polymer scaffold of claim 17, wherein the labile structure is selected from a redox labile structure, a hydrolytically labile structure, or an enzyme labile structure.

19. The unstable structure is 【Chemistry 10】 wherein each R １８ is independently selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl.

20. The unstable structure is 【Chemistry 11】 wherein * is a hydrolytically unstable structure selected from the group consisting of G １ is a moiety covalently bonded to D, ** is a moiety covalently bonded to D, and R ８ is selected from hydrogen, alkyl, or aryl; R ９ is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

21. G １ but, 【Chemistry 12】 where * is L Ｐ is a moiety covalently bonded to R ７ The polymer scaffold of claim 20 , wherein is alkyl.

22. -G １ -L Ｐ -D is, 【Chemistry 13】 22. The polymer scaffold of claim 21 , wherein:

23. 19. The polymer scaffold of claim 18, wherein the labile structure is an enzyme-labile structure that is sensitive to an enzyme selected from cathepsin B, a phosphatase, a sulfatase, or a glucuronidase.

24. the enzyme-labile structure is sensitive to cathepsin B, -Z-, 【Chemistry 14】 wherein * is selected from G １ ** is a site covalently bound to D; Z is a substrate for cathepsin B containing 2-4 amino acids; R ７ａ 24. The polymer scaffold of claim 23, wherein is alkyl.

25. G １ but, 【Chemistry 15】 where * is L Ｐ is a moiety covalently bonded to R ７ 25. The polymer scaffold of claim 24, wherein is alkyl.

26. -G １ -L Ｐ -D is, 【Chemistry 16】 26. The polymer scaffold of claim 25, wherein:

27. the enzyme-labile structure is sensitive to glucuronidase; 【Chemistry 17】 where * is G １ 24. The polymer scaffold of claim 23, wherein: is a moiety covalently bonded to: and ** is a moiety covalently bonded to D.

28. G １ but, [Chemistry 18] where * is L Ｐ is a moiety covalently bonded to R ７ 28. The polymer scaffold of claim 27, wherein is alkyl.

29. -G １ -L Ｐ -D is, 【Chemistry 19】 28. The polymer scaffold of claim 27, selected from:

30. the enzyme-labile structure is sensitive to phosphatases; 【Chemistry 20】 wherein * is selected from G １ , ** is a site covalently bonded to D, and each R １０ and R １１ is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

31. G １ but, 【Chemistry 21】 31. The polymer scaffold of claim 30, wherein:

32. -G １ -L Ｐ -D is, 【Chemistry 22】 32. The polymer scaffold of claim 31 , selected from the group consisting of:

33. the enzyme-labile structure is susceptible to sulfatase; 【Chemistry 23】 where * is G １ , ** is a site covalently bonded to D, and each R １２ and R １３ are independently hydrogen, -NH-, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

34. G １ but, 【Chemistry 24】 where * is L Ｐ is a moiety covalently bonded to R ７ 34. The polymer scaffold of claim 33, wherein is alkyl.

35. -G １ -L Ｐ -D is, 【Chemistry 25】 29. The polymer scaffold of claim 28, wherein:

36. n is an integer from 1 to 100, m is an integer from 1 to 100, and 2. The polymer scaffold of claim 1, wherein p is an integer from 1 to 50.

37. The polymer scaffold of claim 1 , wherein the therapeutic agent has antiproliferative activity against a target cell or pathway.

38. 38. The polymer scaffold of claim 37, wherein said antiproliferative activity is selected from cytostatic and / or cytotoxic activity.

39. 2. The polymer scaffold of claim 1, wherein the therapeutic agent is selected from anticancer substances, cytotoxic drugs, radionuclides, vitamins, anti-AIDS substances, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antivirals, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian substances, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, antiglaucoma compounds, antiparasitic and / or antiprotozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic substances, antiemetics, contrast agents.

40. The polymer scaffold of claim 1 , wherein the therapeutic agent comprises an amino acid-based molecule.

41. 41. The polymer scaffold of claim 40, wherein the amino acid-based molecule comprises a peptide, polypeptide, enzyme, antibody, immunoglobulin, or functional fragment thereof.

42. The polymer scaffold of claim 1 , wherein the therapeutic agent has a chemically reactive group.

43. The chemically reactive group is -COOH, primary amine, secondary amine -NHR, -OH, -SH, -C(O)H, C(O)R １４ , —C(O)NHR １５ , -C(S)OH, -S(O) ２ OR １５ , -P(O) ２ OR １５ , —CN, —NC, or —ONO, wherein R １４ is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R １５ 43. The polymer scaffold of claim 42, wherein is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

44. G ２ but, 【Chemistry 26】 wherein each R １６ is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

45. B ａ but, 【Chemistry 27】 and W Ｌ Stretcher M ａ is a functional group that connects to each Z is independently a branch point; Each R １９ became independent and became Z and W Ｍ is a linker connecting Each W Ｍ are independently functional groups bonded to —NH—, r is an integer from 1 to 3; t is an integer from 1 to 3, In the formula, * represents M ａ and ** is a site covalently bonded to the —NH— moiety.

46. The -NH- moiety is 【Chemistry 28】 -NH-(CH ２ ) ｎ６ —COOH, wherein G １ , L ｐ , D.G. ２ , n, m, p and q are as defined in claim 1, and n ６ 46. ​​The polymer scaffold of claim 45, wherein is an integer from 1 to 6.

47. W Ｌ but, 【Chemistry 29】 46. ​​The polymer scaffold of claim 45, selected from the group consisting of:

48. Z is -CH （３－ｒ） -, -SiH （３－ｒ） -or-NH （２－ｒ） 46. ​​The polymer scaffold of claim 45, wherein:

49. R １９ 46. ​​The polymer scaffold of claim 45, wherein is selected from the group consisting of aliphatic, heteroaliphatic, cycloalkyl, and heterocycloalkyl.

50. B ａ but, 【Transformation 30】 46. ​​The polymer scaffold of claim 45, wherein:

51. B ａ but, 【Chemistry 31】 50. The polymer scaffold of claim 49, selected from the group consisting of:

52. B ａ but, 【Chemistry 32】 46. ​​The polymer scaffold of claim 45, wherein:

53. B ａ but, 【Transformation 33】 53. The polymer scaffold of claim 52, selected from the group consisting of:

54. B ａ but, 【Transformation 34】 54. The polymer scaffold of any one of claims 1 to 53, wherein

55. Two Ws Ｍ -NH-(CH ２ ) ｎ６ -COOH and one W Ｍ but, 【Chemistry 35】 55. The polymer scaffold of claim 54, wherein the polymer scaffold is connected to

56. W Ｍ but, 【Transformation 36】 55. The polymer scaffold of claim 54, wherein the polymer scaffold is connected to

57. Two Ws Ｍ but, 【Chemistry 37】 Connect to one W Ｍ but, 【Transformation 38】 55. The polymer scaffold of claim 54, wherein the polymer scaffold is connected to

58. W Ｌ but 【Chemistry 39】 and -R １９ -W Ｍ -but, 【Chemistry 40】 where ** is W Ｌ or a moiety covalently attached to the -NH- moiety.

59. B ａ but, 【Chemistry 41-1】 【Chemistry 41-2】 45. The polymer scaffold of claim 44, wherein: 【Request Item 60】 【Chemistry 42-1】 【Chemistry 42-2】 A polymer scaffold selected from the group consisting of: 【Chemistry 43-1】 【Chemistry 43-2】 The polymer scaffold of claim 1 .

61. A polymer scaffold of formula (II): 【Chemistry 44】 And, the polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with said targeting moiety. ｐ is a linking moiety comprising M ａ L to B ａ It is a stretcher that connects to the part, B ａ is a functional group W connected to the -NH- moiety Ｍ is a branched portion including Each G ２ are functional groups that can be independently converted to a charged state, Each G ３ independently comprise a functional group capable of reacting with a reactive group on the drug release mechanism to connect the drug release mechanism to the linear polyglycerol; n is an integer from 0 to 1000, m is an integer from 0 to 1000; p is an integer from 0 to 1000, and A polymer scaffold wherein q is an integer from 0 to 1000.

62. n is an integer from 1 to 5, m is an integer from 1 to 5; p is an integer from 1 to 5, and 62. The polymer scaffold of claim 61, wherein q is an integer from 1 to 5.

63. G ２ and G ３ became independent, 【Chemistry 45】 wherein each R １７ are independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

64. Structure of formula (IIa), (IIb), (IIc), (IIA) or (IIB): 【Chemistry 46-1】 【Chemistry 46-2】 62. The polymer scaffold of claim 61 , having

65. Structure of formula (IId), (IIe), (IIf), (IIg) or (IIh): 【Chemistry 47-1】 【Chemistry 47-2】 62. The polymer scaffold of claim 61 , having

66. A polymer scaffold of formula (III): 【Chemistry 48】 And, the polymer scaffold comprises linear polyglycerol; PBRM is the targeting part, Each L ａ independently connects the targeting moiety to M ａ is a bivalent moiety that connects to Each M ａ is independent, L ａ B ａ It is a stretcher that connects to the part, B ａ is a functional group W connected to the -NH- moiety Ｍ is a branched portion including Each G １ is independent, L Ｐ is a functional group that connects the Each L Ｐ are independently a fragment of therapeutic agent D and a fragment of G １ This is the drug release mechanism between each D is independently a fragment of a therapeutic agent; Each G ２ are functional groups that can be independently converted to a charged state, n is an integer from 0 to 1000, m is an integer from 0 to 1000; p is an integer from 0 to 1000; q is an integer from 0 to 1000, and A polymer scaffold wherein s is an integer from 1 to 8.

67. 67. The polymer scaffold of claim 66, wherein the targeting moiety is an antibody and / or a fragment thereof.

68. 68. The polymer scaffold of claim 67, wherein the targeting moieties are antibodies IgG1, IgG2, IgG3 and IgG4.

69. 67. The polymer scaffold of claim 66, wherein the targeting moiety is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody (dsdiabody), single chain antibody molecule (scFv), scFv dimer, multispecific antibody, camelized single domain antibody, nanobody, domain antibody or bivalent domain antibody.

70. 70. A pharmaceutical composition comprising one or more polymer scaffolds according to any one of claims 1 to 60 and 66 to 69 and an acceptable carrier.

71. 70. A method of treating a disorder in a subject in need thereof, comprising administering to a subject a polymer scaffold or a polymeric scaffold according to any one of claims 1 to 60 and 66 to 69.

63. A method comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 63.

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