Tetrazine conjugates for targeted delivery of payloads in vivo

Abstract

The present disclosure relates generally to targeting moieties for bioorthogonal delivery of a payload to a targeted location in a subject. The compositions and methods have applications in the treatment of cancer, tumor growth, and immunotherapy.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 273,792, filed October 29, 2021, U.S. Provisional Application No. 63 / 273,785, filed October 29, 2021, U.S. Provisional Application No. 63 / 315,482, filed March 1, 2022, and U.S. Provisional Application No. 63 / 391,136, filed July 21, 2022, each of which is incorporated herein by reference in its entirety.

[0002] [Reference to electronic sequence listing] The contents of the electronic sequence listing (2022-10-31_Sequence_Listing_63XT-342805-WO.xml, size: 18193 bytes, and creation date: October 31, 2022) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates generally to tetrazine conjugates, including antibody-tetrazine conjugates, for bioorthogonal delivery of payloads to targeted locations in a subject, with applications, for example, in the treatment of cancer, tumor growth, and immunotherapy. [Background technology]

[0004] Bioorthogonal conjugation or click reactions are selective and orthogonal (non-interacting) functionalities found in biological systems that have been used in a variety of applications in chemistry, chemical biology, molecular diagnostics, and medicine, where they can be used to facilitate the selective manipulation of molecules, cells, particles, and surfaces, as well as the tagging and tracking of biomolecules in vitro and in vivo. These reactions include the Staudinger ligation reaction, the azide-cyclooctyne cycloaddition reaction, and the inverse electron-demand Diels-Alder reaction. Summary of the Invention

[0005] Provided herein are targeting moieties comprising an antibody or antibody fragment moiety covalently linked to one or more tetrazine moieties. The targeting moieties described herein are designed to localize to a target site within a subject upon administration to the subject. The targeting moieties can be administered locally or systemically. Once administered, a prodrug comprising a payload or therapeutic agent and one or more complementary bioorthogonal moieties (i.e., a trans-cyclooctene moiety) can be administered, which enables targeted delivery of the payload or therapeutic agent upon contact with the targeting moiety in vivo. In some embodiments, the targeting moiety is a therapeutic targeting moiety. In some embodiments, the targeting moieties described herein comprise a diagnostic agent, and thus the targeting moieties described herein can be used in the diagnosis of a condition or disease, with or without the administration of a payload or therapeutic agent.

[0006] In some embodiments, a method of treating cancer is provided, comprising administering to a subject in need of treatment a support composition described herein at a target location, and administering to the subject a conjugate or a pharmaceutically acceptable salt thereof or composition described herein.

[0007] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

[0008] In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the cancer is a hematopoietic malignancy. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows the SDS-Page of methyltetrazine-trastuzumab targeting moiety. [Figure 2] FIG. 1 shows LCMS of methyltetrazine-trastuzumab targeting moiety. [Figure 3] FIG. 1 shows SDS-PAGE of methyltetrazine-Fab targeting moieties. [Figure 4] FIG. 1 shows LCMS of methyltetrazine-Fab targeting moiety. [Figure 5] FIG. 1 shows the efficacy of methyltetrazine-Fab targeting moiety with doxorubicin-TCO prodrug (TCO-modified doxorubicin drug) in a mouse HCC1954 xenograft model. [Figure 6] FIG. 1 shows LC-MS analysis of conjugated trastuzumab-Me-Tet-PEG9 ADC. [Figure 7] FIG. 1 shows FACS analysis of the binding effect of trastuzumab Fab, Fab-Me-Tet-PEG9 ADC, and IgG (as a negative control) to the NCI-N87 cell line. [Figure 8] FIG. 1 shows tumor volume in the NCI-N87 model. [Figure 9] FIG. 1 shows tumor growth inhibition curves in the NCI-N87 model. [Figures 10A-10B] FIG. 1 shows the xenotransplantation results and % of body weight loss (BWL) for Ab-Tz conjugates prepared in Example 6. [Figure 11]FIG. 1 shows a schematic of the tetrazine-antigen binding protein targeting moiety prepared in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0010] The following description sets forth exemplary embodiments of the present technology, however, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but rather is provided as a description of exemplary embodiments.

[0011] 1.Definition It is understood that certain features of the present disclosure, which are for clarity described in the context of separate embodiments, may also be provided in combination in a single embodiment. In contrast, various features of the present disclosure, which are for brevity described in the context of a single embodiment, may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed, to the extent that such combinations encompass subject matter that is, for example, a stable compound (i.e., a compound that can be made, isolated, characterized, and tested for biological activity). Furthermore, all subcombinations of various embodiments and elements thereof (e.g., elements of chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present disclosure and are disclosed herein as if each and every subcombination were individually and explicitly disclosed herein.

[0012] A.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0013] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0014] The modifier "about" used in connection with a quantity is inclusive of the specified value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses a range of "2 to 4." The term "about" can refer to plus or minus 10% of the specified number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" (e.g., rounding) may be apparent from the context; for example, "about 1" can also mean 0.5 to 1.4.

[0015] The conjunction term "or" includes any and all combinations of one or more listed elements associated by the conjunction term. For example, the phrase "a device comprising A or B" may refer to a device comprising A but not B, a device comprising B but not A, or a device comprising both A and B. The phrase "at least one of A, B, ... and N" or "at least one of A, B, ... N, or combinations thereof" is defined in its broadest sense to mean any combination including one or more elements selected from the group comprising A, B, ... and N, i.e., one or more of the elements A, B, ..., or N, including any one element alone or in combination with one or more other elements, which may include additional unlisted elements in combination.

[0016] The definitions of specific functional groups and chemical terms are explained in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS version) on the inside cover of the Handbook of Chemistry and Physics (75th Edition), and specific functional groups are generally defined as described therein. Furthermore, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and 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 are incorporated herein by reference).

[0017] The term "alkyl" as used herein means a straight or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. The term "lower alkyl" or "C1-C6-alkyl" means a straight or branched hydrocarbon chain containing 1 to 6 carbon atoms. The term "C1-C3-alkyl" means a straight or branched hydrocarbon chain containing 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0018] The term "alkoxy," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0019] As used herein, the term "alkenyl" refers to a hydrocarbon chain containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond. An alkenyl group may be substituted or unsubstituted. For example, an alkenyl group may be substituted with an aryl group such as phenyl.

[0020] As used herein, the term "alkynyl" refers to a linear or branched monovalent hydrocarbyl group having 2 to 30 carbon atoms, e.g., 2 to 20 or 2 to 10 carbon atoms, and at least one site of triple bond unsaturation. The term "alkyne" also includes non-aromatic cycloalkyl groups of 5 to 20 carbon atoms, e.g., 5 to 10 carbon atoms, having mono- or polycyclic rings and at least one triple bond. Examples of such alkynyl groups include, but are not limited to, acetylenyl (-C≡CH) and propargyl (-CHC≡CH), and cycloalkynyl moieties, such as, but not limited to, substituted or unsubstituted cyclooctyne moieties.

[0021] The term "alkoxyalkyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0022] The term "alkylene," as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 30 carbon atoms, for example, 2 to 10 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH-, -CH(CH)-, -C(CH)-, -CHCH-, -CH(CH)CH-, -C(CH)CH-, -CHCHCH-, -CH(CH)CHCH-, -C(CH)CHCH-, -CHC(CH)CH-, -CHCHCHCHCH-, and -CHCHCHCHCHCH-.

[0023] The term "amino acid" refers to both natural and unnatural amino acids, protected natural and unnatural amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids include the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Unnatural amino acids refer to amino acid analogs that have the same basic chemical structure as a naturally occurring amino acid, i.e., by way of example only, an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may retain the same basic chemical structure as a natural amino acid but have modified R groups (e.g., norleucine, by way of example only) or a modified peptide backbone. Non-limiting examples of unnatural amino acids or amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, homophenylalanine, ornithine, formylglycine, phenylglycine, para-azidophenylglycine, para-azidophenylalanine, para-acetophenylalanine, 4-(3-methyl-(1,2,4,5-tetrazine))-phenylglyine, and 4-(3-methyl-(1,2,4,5-tetrazine))-phenylalanine.

[0024] The term "aryl" as used herein refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or multiple rings (e.g., bicyclic or tricyclic), including fused systems. Representative examples of aryl include, but are not limited to, phenyl, naphthyl, and anthracenyl. Monocyclic aryl, bicyclic aryl, and tricyclic aryl are connected to the parent molecular moiety through any carbon atom contained within the ring and can be unsubstituted or substituted. An aromatic bicyclic or tricyclic ring system does not contain a non-aromatic ring. Therefore, if a bicyclic or tricyclic ring system contains a non-aromatic ring, the ring system is cycloalkyl or heterocyclyl, depending on whether a heteroatom is present in the non-aromatic ring, regardless of the point of attachment to the rest of the molecule.

[0025] In some embodiments, the term "aryl" as used herein refers to a phenyl group, or a bicyclic aryl fused ring system or a tricyclic aryl fused ring system. A bicyclic fused ring system is exemplified by a phenyl group attached to the parent molecular moiety and fused to the phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to the parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryls include, but are not limited to, naphthyl. Representative examples of tricyclic aryls include, but are not limited to, anthracenyl. Monocyclic aryls, bicyclic aryls, and tricyclic aryls are connected to the parent molecular moiety through any carbon atom contained within the ring and can be unsubstituted or substituted.

[0026] As used herein, the term "azido" refers to the functional group -N3.

[0027] The term "cycloalkyl," as used herein, refers to a non-aromatic carbocyclic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10 carbon atoms and no heteroatoms. Cycloalkyl ring systems may contain one or more double bonds as long as the ring is not aromatic; therefore, the term cycloalkyl includes cycloalkenyl ring systems. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl. "Cycloalkyl" also includes carbocyclic ring systems in which a cycloalkyl group is fused to an aryl or heteroaryl, as defined herein, regardless of the point of attachment to the remainder of the molecule.

[0028] In some embodiments, the term "cycloalkyl" as used herein refers to a carbocyclic ring system containing 3 to 10 carbon atoms, no heteroatoms, and no double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. "Cycloalkyl" also includes a carbocyclic ring system in which a cycloalkyl group is appended to a parent molecular moiety and is fused to an aryl group, as defined herein, a heteroaryl group, as defined herein, or a heterocycle, as defined herein.

[0029] As used herein, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0030] The term "cyclooctene" as used herein refers to a substituted or unsubstituted non-aromatic cyclic alkyl group of 8 carbon atoms having a single ring with a double bond. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).

[0031] The term "fluoroalkyl" as used herein means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven, or eight hydrogen atoms have been replaced by fluorine. Representative examples of fluoroalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl, such as 3,3,3-trifluoropropyl.

[0032] The term "alkoxyfluoroalkyl," as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.

[0033] The term "fluoroalkoxy" as used herein means at least one fluoroalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of fluoroalkyloxy include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0034] The term "halogen" or "halo" as used herein means Cl, Br, I, or F.

[0035] The term "haloalkyl" as used herein means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by halogen.

[0036] The term "haloalkoxy," as used herein, means at least one haloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0037] The term "heteroalkyl," as used herein, refers to an alkyl group, as defined herein, in which one or more carbon atoms are replaced by a heteroatom selected from S, Si, O, P, and N. The heteroatoms may be oxidized. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, and alkyl sulfides.

[0038] The term "heteroaryl" as used herein refers to an aromatic group having a single ring, multiple rings, or multiple condensed rings, with one or more ring heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the term "heteroaryl" as used herein refers to an aromatic monocyclic ring, an aromatic bicyclic ring system, or an aromatic tricyclic ring system. The aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl. Representative examples of bicyclic heteroaryls include, but are not limited to, chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic heteroaryls, bicyclic heteroaryls, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring and can be unsubstituted or substituted. In some embodiments, an aromatic bicyclic or tricyclic ring system does not contain a non-aromatic ring. Thus, if a bicyclic or tricyclic ring system contains a non-aromatic ring, the ring system is a cycloalkyl or heterocyclyl, depending on whether a heteroatom is present in the non-aromatic ring, regardless of the point of attachment to the rest of the molecule.

[0039] In some embodiments, the 5-membered aromatic monocyclic ring has two double bonds, and the 6-membered aromatic monocyclic ring has three double bonds. In some embodiments, exemplary bicyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring attached to a parent molecular moiety and fused to a monocyclic cycloalkyl group as defined herein, a monocyclic aryl group as defined herein, a monocyclic heteroaryl group as defined herein, or a monocyclic heterocycle as defined herein. In some embodiments, tricyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring attached to a parent molecular moiety and fused to two of a monocyclic cycloalkyl group as defined herein, a monocyclic aryl group as defined herein, a monocyclic heteroaryl group as defined herein, or a monocyclic heterocycle as defined herein.

[0040] As used herein, the terms "heterocyclyl," "heterocycle," or "heterocyclic" refer to a non-aromatic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10 carbon atoms, at least one (e.g., 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) heteroatom, and optionally one or more oxo and / or double bonds. The terms "heterocyclyl," "heterocycle," or "heterocyclic" include monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridged ring systems, provided that at least one non-aromatic ring system containing at least one heteroatom is present. In some embodiments, the monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, the 3- or 4-membered ring contains zero double bonds or one double bond and one heteroatom selected from the group consisting of O, N, and S. In some embodiments, the 5-membered ring contains zero double bonds or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the 6-membered ring contains zero double bonds or one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the 7- and 8-membered rings contain zero double bonds or one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S.Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, o- ... Examples include oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl. A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl.A tricyclic heterocycle is a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane). Monocyclic heterocycles, bicyclic heterocycles, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring, and can be unsubstituted or substituted.

[0041] As used herein, the term "hydroxyl" refers to an --OH group.

[0042] The term "hydroxyalkyl," as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven, or eight hydrogen atoms have been replaced by a hydroxyl group.

[0043] In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix "C x ~C y -" or "C x~y " where x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms in the substituent. Thus, for example, "C1-C3-alkyl" and "C 1~3 "Alkyl" refers to an alkyl substituent containing 1 to 3 carbon atoms. x ~Cy -" and "C x~y " notation are used interchangeably and have the same meaning.

[0044] The term "substituted" refers to a group that may be further substituted with one or more non-hydrogen substituents, including, but not limited to, halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0045] The term "tetrazine" refers to a substituted or unsubstituted aromatic cyclic group of two carbon atoms and four nitrogen atoms, having a single ring with three double bonds. Examples of tetrazine groups include 1,2,3,4-tetrazine and 1,2,4,5-tetrazine. As used herein, 1,2,4,5-tetrazine is referred to as a "Tz" group.

[0046] The term "selectively deliver" refers to the delivery of an agent (e.g., payload) to an organ or tissue (or portion thereof) requiring treatment or diagnosis without significant binding to other non-target organs or tissues (or portions thereof). In some embodiments, the targeting moieties or therapeutic targeting moieties described herein do not themselves have a therapeutic effect, but rather are designed to enable selective or targeted delivery of a therapeutic agent. However, it is also possible for a targeting moiety to have a therapeutic effect, and such constructs are not excluded by the present disclosure.

[0047] The term "payload" refers to an agent that is delivered to a target site in a subject. Payloads include therapeutic agents.

[0048] The term "therapeutic agent" refers to an agent capable of treating and / or ameliorating a condition or disease, or one or more symptoms thereof, in a subject. Therapeutic agents of the present disclosure also include prodrug forms of the therapeutic agent.

[0049] The term "diagnostic agent" refers to an agent that aids in the diagnosis of a condition or disease. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, radionuclides, etc. Paramagnetic agents are imaging agents that exhibit magnetism under an externally applied field. Examples of paramagnetic agents include, but are not limited to, iron particles, including iron nanoparticles and iron microparticles. Optical probes are fluorescent compounds that can be detected by excitation with radiation at one wavelength and detection with radiation at a second, different wavelength. Optical probes of the present disclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate), and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include, but are not limited to: 3 H, 11 C. 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 89 Zr, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In,123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu, and 241 Am is one example.

[0050] The term "targeting agent" refers to a chemical or biological agent that specifically binds to a target (e.g., a targeted organ or tissue), thereby forming a stable association between the targeting agent and the particular target. "Stably associated" or "stable association" means that a moiety binds to or associates with another moiety or structure under standard physiological conditions. Bonds can include covalent or non-covalent interactions such as, but not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. A targeting agent can be one member of a specific binding pair such as, but not limited to, one member of a receptor / ligand pair, a ligand-binding portion of a receptor, one member of an antibody / antigen pair, an antigen-binding fragment of an antibody, a hapten, one member of a lectin / carbohydrate pair, one member of an enzyme / substrate pair, biotin / avidin, biotin / streptavidin, digoxin / antidigoxin, one member of a DNA or RNA aptamer binding pair, one member of a peptide aptamer binding pair, etc. Targeting agents include ligands that specifically bind (or substantially specifically bind) to a particular clinically relevant target receptor or cell surface target. The ligand can be an antibody, peptide, nucleic acid, phage, bacterium, virus, or other molecule that has specific affinity for the target receptor or cell surface target. Examples of receptors and cell surface targets include, but are not limited to, PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, LT4, a human monoclonal antibody against the inhibitory immune checkpoint receptor immunoglobulin-like transcript 4 (ILT4), leukocyte immunoglobulin-like receptor superfamily B member 2 (LILRB2), lymphocyte immunoglobulin-like receptor 2 (LIR2), monocyte / macrophage immunoglobulin-like receptor 10 (MIR-10), CD85d, or other cell receptors or cell surface targets. Additional examples are included in the various embodiments disclosed herein.

[0051] The term "target organ or tissue" refers to an organ or tissue that is targeted for delivery of a payload. Exemplary targeted organs and tissues include organs and tissues that can be targeted by chemical or biological targeting agents, as well as organs and tissues that cannot be targeted by chemical or biological targeting agents.

[0052] The term "implant" refers to surgical implantation into the body of a subject.

[0053] The terms "contacting" or "contacting" refer to the process of bringing at least two different species into contact so that they may interact with each other, for example, in a non-covalent or covalent interaction or binding reaction. However, it should be understood that the resulting complex or reaction product may be produced directly from the interaction or reaction between the added reagents, or may be produced from an intermediate that may be produced in the contacting mixture that is derived from one or more of the added reagents or moieties.

[0054] The term "binding agent" refers to an agent having a functional group capable of forming a covalent bond with a complementary functional group of another binding agent in a biological environment. Bonding between binding agents in a biological environment is sometimes referred to as a bioconjugation reaction. Binding agents include bioorthogonal binding agents, which are binding agents having a bioorthogonal functional group. The bioorthogonal functional group of a bioorthogonal binding agent selectively reacts with the complementary bioorthogonal functional group of another bioorthogonal binding counterpart. Selective reaction between bioorthogonal binding counterparts can minimize side reactions with other binding agents, biological compounds, or other non-complementary bioorthogonal binding agents or non-complementary bioorthogonal functional groups. Bioorthogonal moieties or functional groups of bioorthogonal binding agents include, but are not limited to, azides and alkynes that form triazoles via click chemistry, trans-cyclooctene (TCO), and tetrazines (Tz) (e.g., 1,2,4,5-tetrazine), etc. Binding agents useful in the present disclosure may have high reactivity with the corresponding binding agent, resulting in a rapid reaction.

[0055] The term "functionalized" refers to a moiety having a functional group attached thereto, e.g., a moiety having a binding agent functional group (e.g., a bioorthogonal functional group) attached thereto.

[0056] The term "administering" refers to any suitable route of administration to a subject, including, but not limited to, oral administration, administration as a suppository, topical contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal or subcutaneous administration, intrathecal administration, or implantation of a sustained release device, e.g., a mini-osmotic pump, into a subject.

[0057] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0058] The term "leaving group" refers to an atom (or group of atoms) with electron-withdrawing ability that can be displaced as a stable chemical species and with it accept a bonding electron. Examples of suitable leaving groups include halides (e.g., Br, Cl, I), sulfonate esters (e.g., triflate, mesylate, tosylate, and brosylate), and nitrophenols.

[0059] The terms "pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more symptoms thereof and / or prevent or reduce the risk of onset or recurrence of a disease or disorder or symptom(s) thereof. With respect to tumorigenic proliferative disorders, a pharmaceutically effective amount or therapeutically effective amount includes, inter alia, an amount sufficient to shrink a tumor or reduce the rate of tumor growth.

[0060] As used herein, the terms "subject," "patient," or "organism" include humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which the agent(s) of the present disclosure may be administered include mammals, particularly primates, and especially humans. For veterinary applications, suitable subjects may include, for example, livestock such as cattle, sheep, goats, cows, pigs, etc.; poultry such as chickens, ducks, geese, turkeys, etc.; and domesticated animals, particularly pets, such as dogs and cats. For diagnostic or research applications, suitable subjects may include mammals, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, such as inbred pigs.

[0061] The term "treat" or "treatment" as used herein means treating or curing a disease or condition or symptom(s) thereof in a patient, such as a mammal (particularly a human), including (a) ameliorating the disease or condition or symptom(s), e.g., eliminating or causing the disease or condition or symptom(s) thereof in the patient; (b) inhibiting the disease or condition or symptom(s), e.g., by slowing or arresting the onset of the disease or condition or symptom(s) thereof in the patient; or (c) alleviating the symptom of the disease or condition or symptom(s) thereof in the patient.

[0062] The term "physiological conditions" is intended to encompass conditions compatible with living cells, eg, primarily aqueous conditions of temperature, pH, salinity, etc., compatible with living cells.

[0063] For the compounds described herein, the groups and substituents thereof may be chosen in accordance with the allowed valences of the atoms and substituents, such that the selection and substitution results in stable compounds, e.g., compounds that do not spontaneously undergo transformations such as by rearrangement, cyclization, elimination, and the like.

[0064] When a range of values ​​is given, it is understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0065] When describing ranges of values ​​herein, each intervening value therebetween is expressly contemplated to the same degree of precision, for example, in the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0066] Compounds may exist as stereoisomers where asymmetric or chiral centers exist. Stereoisomers are "R" or "S" depending on the configuration of substituents around the chiral carbon atom. As used herein, the terms "R" and "S" refer to the configurations defined in the IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem., 1976, 45: 13-30. Various stereoisomers and mixtures thereof are contemplated and specifically included within the scope of this disclosure. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of a compound can be prepared by synthesis from commercially available starting materials containing asymmetric or chiral centers, or by preparation of racemic mixtures followed by resolution methods well known to those skilled in the art. These resolution methods are described in (1) Furniss, Hannaford, Smith, and Tatchell, "Vogel's Textbook of Practical Organic Chemistry," 5 thedition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, by the addition of a mixture of enantiomers to a chiral auxiliary, separation of the resulting diastereomeric mixture by recrystallization or chromatography, and optionally liberation of the optically pure product from the auxiliary; or (2) direct separation of a mixture of optical enantiomers on a chiral chromatographic column; or (3) fractional recrystallization methods.

[0067] It is understood that compounds may have geometric isomers as well as tautomeric forms and these also form an aspect of the present invention.

[0068] The present disclosure also includes isotopically labeled compounds, which are identical to the compounds recited herein except that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in the compounds of the present disclosure include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, for example, but not limited to, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 Cl. Deuterium (i.e., 2 Substitution with heavier isotopes, such as H, may be preferable in some circumstances as it may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased half-life in vivo or reduced dosage requirements. The compounds may also include positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that may be included include: 11 C. 13 N, 15 O, and18 F. Isotopically labeled compounds disclosed herein can generally be prepared by conventional techniques known to those skilled in the art, or by methods analogous to those described in the accompanying examples, using an appropriate isotopically labeled reagent in place of a non-isotopically labeled reagent.

[0069] B. Targeting moiety Provided herein are targeting moieties comprising a biocompatible support, an antibody, or antibody fragment moiety, or in certain embodiments, an antibody or antibody fragment moiety, covalently bound to one or more tetrazine moieties. The targeting moieties described herein are designed to localize to a target site within a subject upon administration. The targeting moiety can be administered locally or systemically. In some embodiments, the targeting moiety is a therapeutic targeting moiety. Once administered, a prodrug containing a complementary bioorthogonal moiety (i.e., a trans-cyclooctene moiety) can be administered, which enables targeted drug delivery of a payload or therapeutic agent upon contact with the targeting moiety in vivo. In some embodiments, the targeting moieties described herein comprise a diagnostic agent, and thus the targeting moieties described herein can be used in the diagnosis of a condition or disease, with or without the administration of a payload or therapeutic agent.

[0070] Formula I, Formula II, or Formula V: [ka] (In the formula, Ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; The dotted line is R 3 and R 4 represents an additional bond that forms a tetrazine when both are absent, or R 3 and R 4 When both are present, they represent an additional bond to form a dihydrotetrazine, except that when ring A is aryl, R 3 and R 4 Both exist, X is a biocompatible support, an antibody, or an antibody fragment moiety, with the proviso that for Formula I and Formula II, X is not a biocompatible support; p is 1 to 150; L, independently at each occurrence, is a linker; R 1 is independently at each occurrence hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R' and NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally selected from the group consisting of 1 to 3 Z 1 is replaced by R 2is independently at each occurrence halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; and 1 is replaced by R 3 and R 4 are both absent or R 3 and R 4 are each independently hydrogen or a group that is removable after a triggering event; R 20 is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- is selected from the group consisting of R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R22 is, independently in each occurrence, a linker of 1 to 100 linked atoms, optionally containing one or more ethyleneoxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups; R 30 is independently at each occurrence halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; R a , R 31a , and R 31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl, Each Z 1 are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- selected from R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; and t at each occurrence is independently 0, 1, 2, 3, or 4).

[0071] Compounds of Formula I, Formula II, Formula IIA, Formula IIB, Formula IIC, and Formula III: In one embodiment, Formula I: [ka] (In the formula, X is an antibody or antibody fragment moiety; p is 1 to 16; L, independently at each occurrence, is a linker; R 20 is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- is selected from the group consisting of R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R 22 is, in each occurrence, independently a linker of 1 to 100 linked atoms optionally containing one or more ethyleneoxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups; and R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; wherein R''' at each occurrence is independently selected from aryl and alkyl).

[0072] In one embodiment, a compound of formula II: [ka] (In the formula, X is an antibody or antibody fragment moiety; p is 1 to 16; L, independently at each occurrence, is a linker; R 20 is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- is selected from the group consisting of R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R 30 is independently at each occurrence halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; R a , R 31a , and R 31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl, R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; and and t at each occurrence is independently 0, 1, 2, 3, or 4).

[0073] In one embodiment, R 22 is, in each occurrence, independently a linker of 1 to 100 linked atoms, which may include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functionalities.

[0074] In one embodiment, a compound of formula IIA: [ka] (Wherein L, p, X, and R 20 are each independently as defined herein).

[0075] In one embodiment, a compound of formula IIB: [ka] wherein L, p, and X are each independently as defined herein.

[0076] In one embodiment, a compound of formula IIC: [ka] wherein L, p, and X are each independently as defined herein.

[0077] In one embodiment, a compound of formula III: [ka] (In the formula, X is an antibody or antibody fragment moiety; p is 1 to 16; L, independently at each occurrence, is a linker; R 20 is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- is selected from the group consisting of R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R 30 is independently at each occurrence halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; and and t is independently 0, 1, 2, 3, or 4).

[0078] In some embodiments, the compound of formula IID: [ka] (Wherein, X and R20 and R are each independently as defined herein. 20 is methyl. In some embodiments, X is an antigen binding protein. In some embodiments, X is an antigen binding protein that targets HER2.

[0079] Also, Formula IIE: [ka] wherein p and X are each independently as defined herein.

[0080] Also, the formula IIF: [ka] In some embodiments, a targeting moiety of the formula: is provided: wherein p and X are each independently as defined herein.

[0081] Also, Formula IIG: [ka] In some embodiments, a targeting moiety of the formula: is provided: wherein p and X are each independently as defined herein.

[0082] In some embodiments of Formula IIA, [ka] At least one of [ka] is.

[0083] In some embodiments of Formula IIA, [ka] At least one of [ka] (In the formula, R 20 is as defined herein).

[0084] In some embodiments of Formula IIA, [ka] At least one of [ka] is.

[0085] In some embodiments of Formula IIA, [ka] At least one of [ka] (In the formula, R 20 is as defined herein).

[0086] In some embodiments of Formula IIA, [ka] At least one of [ka] is.

[0087] In some embodiments, p is 1 to 12. In some embodiments, X is an antibody. In some embodiments, p is 1 to 6 or 5 to 6.2. In some embodiments, p is 1 to 16, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments, X is an antibody fragment portion (e.g., Fab).

[0088] Compounds of Formula V, Formula VI, and Formula VII: In one embodiment, Formula V: [ka] (In the formula, Ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; The dotted line is R 3 and R 4 represents an additional bond that forms a tetrazine when both are absent, or R 3 and R 4 When both are present, they represent an additional bond to form a dihydrotetrazine, except that when ring A is aryl, R 3 and R 4 Both exist, X is a biocompatible support, an antibody, or an antibody fragment moiety; p is 1 to 150; L, independently at each occurrence, is a linker; R 1is independently at each occurrence hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R' and NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally selected from the group consisting of 1 to 3 Z 1 is replaced by R 2 is independently at each occurrence halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; and 1 is replaced by R 3 and R 4are both absent or R 3 and R 4 are each independently hydrogen or a group that is removable after a triggering event; Each Z 1 are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- selected from R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; and t at each occurrence is independently 0, 1, 2, 3, or 4).

[0089] Formula V: [ka] (In the formula, Ring A is cycloalkyl, heterocyclyl, or heteroaryl; The dotted line is R 3 and R 4 represents an additional bond that forms a tetrazine when both are absent, or R 3 and R 4represent an additional bond that, when both are present, forms a dihydrotetrazine; X is a biocompatible support, an antibody, or an antibody fragment moiety; p is 1 to 150; L, independently at each occurrence, is a linker; R 1 is independently at each occurrence hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R' and NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally selected from the group consisting of 1 to 3 Z 1 is replaced by R 2is independently at each occurrence halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; and 1 is replaced by R 3 and R 4 are both absent, or R 3 is a group that is removable after a triggering event, R 4 is hydrogen or R 3 and Each Z 1are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- selected from R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; and t at each occurrence is independently 0, 1, 2, 3, or 4).

[0090] In some embodiments, the compound of formula VI: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , each of rings A, L, p, t, and X is independently as defined herein).

[0091] In some embodiments, R 4 is hydrogen.

[0092] In the targeting moieties described herein, R 3is a group that can be removed after a trigger event. In some embodiments, the trigger event occurs in vivo. 3 is removed, the dihydrotetrazine moiety is oxidized to give the compound of formula VII: [ka] (In the formula, R 1 , R 2 , each of rings A, L, p, t, and X is independently as defined herein.

[0093] The triggering event is initiated after administration of the targeting moiety to a subject and can be initiated by any means, such as internal means (e.g., via enzymatic cleavage of a functional group, optionally followed by degradation) or external means (e.g., a photocleavable linker). 3 comprises a targeting moiety such as an antibody or antibody fragment described herein.

[0094] In some embodiments, R 3 contains an amino acid sequence specific for cleavage by a protease or esterase.

[0095] In some embodiments, R 3 contains an amino acid sequence specific for cleavage by the proteases shown in Table 1A.

[0096] TIFF2024542021000027.tif202170TIFF2024542021000028.tif204170TIFF2024542021000029.tif73170

[0097] In some embodiments, R 3 comprises an amino acid sequence specific for cleavage by a cathepsin, a matrix metalloproteinase (MMP), or PSMA. For example, in some embodiments, R 3comprises Val-Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly for cleavage by cathepsins. 3 includes Ac-γE-PLG-S(OBn)YL or Ac-PLG-HofOrnL, where Hof is homophenylalanine and Orn is ornithine for cleavage by MMPs. 3 comprises the amino acid sequence shown in Table 1B.

[0098] TIFF2024542021000030.tif204170TIFF2024542021000031.tif207170TIFF20245420210 00032.tif211170TIFF2024542021000033.tif208170TIFF2024542021000034.tif180170

[0099] Additional cleavable groups are described in Choi, et al., Theranostics. 2012; 2(2): 156-178, Table 2 therein, which is incorporated herein by reference.

[0100] In some embodiments, R 3 is photolabile. In some embodiments, the photolabile group becomes unstable or decomposes when exposed to light of a wavelength that matches the absorbance profile of the photolabile group.

[0101] In some embodiments, R 3 teeth, [ka] and L 5 is a direct bond or a linker, and X 1 is an optionally substituted peptide unit comprising -NO2, an optionally substituted sugar moiety, or one or more natural or unnatural amino acids.

[0102] In some embodiments, the moiety: [ka] At least one of [ka] TIFF2024542021000038.tif129170 (in the formula, R 1 , R 2 , R 3 , and R 4 are independently as defined herein, and optionally, the ring A moiety is selected from one or more R 2 and the compound is represented by a formula selected from the group consisting of:

[0103] In some embodiments, the moiety: [ka] At least one of [ka] (In the formula, X 2 is alkyl (e.g., methyl) optionally substituted with PEG, amino acid, ester, amide, amine, —C(O)OH, —SO2, —SO3, —PO3, —PO4, or other solubility enhancing substituents, and L, ring A, R 1 , R 2 , t, p, and X are independently as defined herein.

[0104] In some embodiments, Ring A is cycloalkyl. In some embodiments, Ring A is heterocyclyl. In some embodiments, Ring A is heteroaryl. In some embodiments, Ring A is aryl.

[0105] In some embodiments, Ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl.

[0106] In some embodiments, ring A is phenyl.

[0107] In some embodiments, the moiety: [ka] At least one of [ka] (In the formula, R 1 and R 2 each of which is independently as defined herein.

[0108] In some embodiments, R 1 is independently at each occurrence hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl, wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally followed by one to three Z 1 is replaced by .

[0109] In some embodiments, R 1 is, in each occurrence, independently hydrogen or 1 to 3 Z 1 is alkyl optionally substituted with

[0110] In some embodiments, Z 1 is independently selected at each occurrence from halo, hydroxy, alkoxy, and OC(=O)OR'.

[0111] In some embodiments, R 2 is, independently at each occurrence, halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl. 2 is, independently at each occurrence, halo, alkyl, or haloalkyl. 2 is independently at each occurrence halo or alkyl.

[0112] In some embodiments, t is 0 at each occurrence.

[0113] Also, the formula VA: [ka] wherein p and X are each independently as defined herein. Also provided are targeting moieties of formula VB: [ka] In some embodiments, a targeting moiety of the formula: is provided: wherein p and X are each independently as defined herein.

[0114] In some embodiments, X is a biocompatible support.

[0115] In some embodiments, Ring A is other than pyridyl. In some embodiments, Ring A is other than aryl. In some embodiments, Ring A is other than phenyl.

[0116] In some embodiments, X is a biocompatible support comprising a particle, a polymer, a viscous or non-viscous liquid material, a gel, a hydrogel, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, or a protein.

[0117] In some embodiments, X is a biocompatible support comprising a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, chondroitin sulfate, heparin, a suitable sugar-based biomaterial, polyphosphazene, polyanhydride, polyacetal, poly(orthoester), polyphosphate ester, polycaprolactone, polyurethane, polylactide, polycarbonate, polyamide, polyether, blends / composite / copolymers thereof, collagen, gelatin, elastin, elastin-like polypeptide, albumin, fibrin, poly(γ-glutamic acid), poly(L-lysine), poly(L-glutamic acid), or poly(aspartic acid). In some embodiments, X is a biocompatible support comprising hyaluronic acid having a molecular weight of about 5 kD to 25 kD, or 26 kD to 75 kD, or 76 kD to 200 kD, or greater than 201 kD.

[0118] In some embodiments, X is an antibody or antibody fragment moiety.

[0119] Antibody and antibody fragment moieties In certain embodiments, the targeting agent, i.e., X, is selected from the group consisting of CD25 (NCBI Gene ID 3559), CEA (NCBI Gene ID 634), CEACAM5 (NCBI Gene ID 1048), ASPH (NCBI Gene ID 444), EGFR (NCBI Gene ID 1956), EPCAM (NCBI Gene ID 4072), VEGFR (NCBI Gene ID 3791), PDGFR (NCBI Gene ID 5159), TROP2 (NCBI Gene ID 4070), Nectin4 (NCBI Gene ID 81607), ), PSMA (NCBI Gene ID 2346), BCMA (NCBI Gene ID 608), CD22 (NCBI Gene ID 933), CD20 (NCBI Gene ID 920), CD19 (NCBI Gene ID 930), CD79b (NCBI Gene ID 974), CD38 (NCBI Gene ID 952), CD45 (NCBI Gene ID 5788), endoglin (NCBI Gene ID 2022), FGFR2 (NCBI Gene ID 14183), C4.4A (NCBI Gene ID 27076), and claudin-18.2 (NCBI gene ID 51208), MMP9 (NCBI gene ID 4318), folate receptor (NCBI gene ID 2348), DLL3 (NCBI gene ID 10683), CD138 (NCBI gene ID 6382), CD56 (NCBI gene ID 4684), CD37 (NCBI gene ID 951), CD74 (NCBI gene ID 972), mesothelin (NCBI gene ID 10232), IL-6R (NCBI gene ID 3570), SLAMF7 (NCBI gene ID 10232), and IL-6R (NCBI gene ID 3570). NCBI Gene ID 57823), BAFF (NCBI Gene ID 10673), MUC1 (NCBI Gene ID 4582), GPC3 (NCBI Gene ID 2719), HER2 (NCBI Gene ID 2064), HER3 (NCBI Gene ID 2065), CD30 (NCBI Gene ID 943), CD33 (NCBI Gene ID 945), CD123 (NCBI Gene ID 3563), GPNMB (NCBI Gene ID 10457), cMET (NCBI Gene ID 10458), and HER2 (NCBI Gene ID 2065). D4233), CD142 (NCBI Gene ID 2152), NaPi2B (NCBI Gene ID 10568), GCC (NCBI Gene ID 2984), STEAP1 (NCBI Gene ID 26872), MUC16 (NCBI Gene ID 94025), CD70 (NCBI Gene ID 970), CD44 (NCBI Gene ID 960), (NCBI Gene ID), antibody fragments (NCBI Gene ID), vWF (NCBI Gene ID 7450), TNF (NC and antibodies or antibody fragment portions that target one or more of: IL-6R (NCBI Gene ID 7124), IL-6R (NCBI Gene ID 3570), BCMA (NCBI Gene ID 608), ADAMTS5 (NCBI Gene ID 11096), CX3CR1 (NCBI Gene ID 1524), CXCR4 (NCBI Gene ID 7852), TfR1 (NCBI Gene ID 7037), VEGFR (NCBI Gene ID 3791), or PSMA (NCBI Gene ID 2346).

[0120] In some embodiments, X is selected from the group consisting of CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, CD25, CLDN4 (NCBI gene ID 1364), TNC (NCBI gene ID 3371), FN1 (NCBI gene ID 2335), ITGAV (NCBI gene ID 3685), TACSTD2 (NCBI gene ID 4070), CD174 (NCBI gene ID 2525), GPN An antibody or antibody fragment portion that targets MB (NCBI Gene ID 10457), GPC1 (NCBI Gene ID 2817), ITGB6 (NCBI Gene ID 3694), SEZ6 (NCBI Gene ID 124925), SLITRK6 (NCBI Gene ID 84189), NaPi-2b (NCBI Gene ID 20531), ZIP6 (NCBI Gene ID 25800), ROR1 (NCBI Gene ID 4919), or ROR2 (NCBI Gene ID 4920).

[0121] In certain embodiments, X is selected from the group consisting of CD25 (NCBI Gene ID 3559), CEA (NCBI Gene ID 634), CEACAM5 (NCBI Gene ID 1048), ASPH (NCBI Gene ID 444), EGFR (NCBI Gene ID 1956), EPCAM (NCBI Gene ID 4072), VEGFR (NCBI Gene ID 3791), PDGFR (NCBI Gene ID 5159), TROP2 (NCBI Gene ID 4070), Nectin4 (NCBI Gene ID 81607), PSMA (NCBI Gene ID 2346), BCMA (NCBI Gene ID 608), CD22 (NCBI Gene ID 933), CD20 (NCBI Gene ID 920), CD19 (NCBI Gene ID 930), CD79b (NCBI Gene ID 974), CD38 (NCBI Gene ID 952), CD45 (NCBI Gene ID 5788), endoglin (NCBI Gene ID 2022), FGFR2 (NCBI Gene ID 14183), C4.4A (NCBI Gene ID 27076), and claudin-18.2 (NCBI gene ID 51208), MMP9 (NCBI gene ID 4318), folate receptor (NCBI gene ID 2348), DLL3 (NCBI gene ID 10683), CD138 (NCBI gene ID 6382), CD56 (NCBI gene ID 4684), CD37 (NCBI gene ID 951), CD74 (NCBI gene ID 972), mesothelin (NCBI gene ID 10232), IL-6R (NCBI gene ID 3570), SLAMF7 (NCBI gene ID 57 823), BAFF (NCBI gene ID 10673), MUC1 (NCBI gene ID 4582), GPC3 (NCBI gene ID 2719), HER2 (NCBI gene ID 2064), HER3 (NCBI gene ID 2065), CD30 (NCBI gene ID 943), CD33 (NCBI gene ID 945), CD123 (NCBI gene ID 3563), GPNMB (NCBI gene ID 10457), cMET (NCBI gene ID 4233), CD142 (NCBI gene ID 1 D2152), NaPi2B (NCBI Gene ID 10568), GCC (NCBI Gene ID 2984), STEAP1 (NCBI Gene ID 26872), MUC16 (NCBI Gene ID 94025), CD70 (NCBI Gene ID 970), CD44 (NCBI Gene ID 960), (NCBI Gene ID), antibody fragment (NCBI Gene ID), vWF (NCBI Gene ID 7450), TNF (NCBI Gene ID 7124), IL-6R (NCBI Gene ID 3570 ), BCMA (NCBI Gene ID 608), ADAMTS5 (NCBI Gene ID 11096), CX3CR1 (NCBI Gene ID 1524), CXCR4 (NCBI Gene ID 7852), TfR1 (NCBI Gene ID 7037), VEGFR (NCBI Gene ID 3791), PSMA (NCBI Gene ID 2346), ANTXR1 (NCBI Gene ID 84168), or FAP (NCBI Gene ID 2191).

[0122] In some embodiments, X is an antibody or antibody fragment moiety that targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

[0123] In some embodiments, X is an antibody or antibody fragment moiety that targets HER2, TROP2, nectin-4, claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5.

[0124] In some embodiments, X is an antibody or antibody fragment moiety that targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, or CD25.

[0125] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD25, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment moiety derived therefrom.

[0126] In certain embodiments, X is an antibody or antibody fragment moiety that targets CEA, such as labetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment moiety derived therefrom.

[0127] In certain embodiments, X is an antibody or antibody fragment moiety that targets CEACAM5, such as Tusamitiamab or CC4, or an antibody fragment moiety derived therefrom.

[0128] In certain embodiments, X is an antibody or antibody fragment moiety that targets ASPH, such as PAN-622, or an antibody fragment moiety derived therefrom.

[0129] In certain embodiments, X is an antibody or antibody fragment moiety that targets EGFR, such as cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, altumumab, tomzotuximab, or laprituximab, or an antibody fragment moiety derived therefrom.

[0130] In certain embodiments, X is an antibody or antibody fragment moiety that targets EPCAM, such as oportuzumab, sitatuzumab, tucotuzumab, catumaxomab, edrecolomab, or adecatumumab, or an antibody fragment moiety derived therefrom.

[0131] In certain embodiments, X is an antibody or antibody fragment moiety that targets a VEGFR, such as ramucizumab, ramucirumab, or blinacimab, or an antibody fragment moiety derived therefrom.

[0132] In certain embodiments, X is an antibody or antibody fragment moiety that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment moiety derived therefrom.

[0133] In certain embodiments, X is an antibody or antibody fragment moiety that targets TROP2, such as sacituzumab or Pr1E11, or an antibody fragment moiety derived therefrom.

[0134] In certain embodiments, X is an antibody or antibody fragment moiety that targets Nectin-4, such as enfortumab, or an antibody fragment moiety derived therefrom.

[0135] In certain embodiments, X is an antibody or antibody fragment moiety that targets PSMA, such as J591 or MLN591, or an antibody fragment moiety derived therefrom.

[0136] In certain embodiments, X is an antibody or antibody fragment moiety that targets BCMA, such as belantamab, or an antibody fragment moiety derived therefrom.

[0137] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD22, such as moxetumomab, inotuzumab, epratuzumab, or pinatuzumab, or an antibody fragment moiety derived therefrom.

[0138] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD20, such as ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, or ibritumomab, or an antibody fragment moiety derived therefrom.

[0139] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD19, such as loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, or MDX-1342, or an antibody fragment moiety derived therefrom.

[0140] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD79b, such as polatuzumab, or an antibody fragment moiety derived therefrom.

[0141] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD38, such as isatuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment moiety derived therefrom.

[0142] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD45, such as I-131-BC8 or Iomab-B, or an antibody fragment moiety derived therefrom.

[0143] In certain embodiments, X is an antibody or antibody fragment moiety that targets endoglin, such as carotuximab, or an antibody fragment moiety derived therefrom.

[0144] In certain embodiments, X is an antibody or antibody fragment moiety that targets FGFR2, such as bemarituzumab or apultuzumab, or an antibody fragment moiety derived therefrom.

[0145] In certain embodiments, X is an antibody or antibody fragment moiety that targets C4.4A, such as rupartumab, or an antibody fragment moiety derived therefrom.

[0146] In certain embodiments, X is an antibody or antibody fragment moiety that targets claudin-18.2, such as zolbetuximab or claudiximab, or an antibody fragment moiety derived therefrom.

[0147] In certain embodiments, X is an antibody or antibody fragment moiety that targets MMP9, such as andecaliximab, or an antibody fragment moiety derived therefrom.

[0148] In certain embodiments, X is an antibody or antibody fragment moiety that targets the folate receptor, such as mirvetuximab, farletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment moiety derived therefrom.

[0149] In certain embodiments, X is an antibody or antibody fragment moiety that targets DLL3, such as rovalpituzumab, or an antibody fragment moiety derived therefrom.

[0150] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD138, such as indatuximab, or an antibody fragment moiety derived therefrom.

[0151] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment moiety derived therefrom.

[0152] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD37, such as BI 836826, otlertuzumab, or naratuximab, or an antibody fragment moiety derived therefrom.

[0153] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD74, such as milatuzumab, or an antibody fragment moiety derived therefrom.

[0154] In certain embodiments, X is an antibody or antibody fragment moiety that targets mesothelin, such as anetumab, amatuximab, or MMOT-0530A, or an antibody fragment moiety derived therefrom.

[0155] In certain embodiments, X is an antibody or antibody fragment moiety that targets IL-6R, such as tocilizumab or sarilumab, or an antibody fragment moiety derived therefrom.

[0156] In certain embodiments, X is an antibody or antibody fragment moiety that targets SLAMF7, such as elotuzumab, or an antibody fragment moiety derived therefrom.

[0157] In certain embodiments, X is an antibody or antibody fragment moiety that targets BAFF, such as belimumab, or an antibody fragment moiety derived therefrom.

[0158] In certain embodiments, X is an antibody or antibody fragment portion that targets MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, or cantuzumab, or an antibody fragment portion derived therefrom.

[0159] In certain embodiments, X is an antibody or antibody fragment moiety that targets GPC3, such as codrituzumab, ECT204, or MDX-1414, or an antibody fragment moiety derived therefrom.

[0160] In certain embodiments, X is an antibody or antibody fragment moiety that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment moiety derived therefrom.

[0161] In certain embodiments, X is an antibody or antibody fragment moiety that targets HER3, such as patritumab, seribantumab, lumletuzumab, elgemtumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment moiety derived therefrom.

[0162] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD30, such as brentuximab, or an antibody fragment moiety derived therefrom.

[0163] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD33, such as gemtuzumab, BI 835858, vadastuximab, or lintuzumab, or an antibody fragment moiety derived therefrom.

[0164] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment moiety derived therefrom.

[0165] In certain embodiments, X is an antibody or antibody fragment moiety that targets GPNMB, such as glembatumumab, or an antibody fragment moiety derived therefrom.

[0166] In certain embodiments, X is an antibody or antibody fragment moiety that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment moiety derived therefrom.

[0167] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD142, such as tisotumab, or an antibody fragment moiety derived therefrom.

[0168] In certain embodiments, X is an antibody or antibody fragment moiety that targets NaPi2B, such as rifastuzumab, or an antibody fragment moiety derived therefrom.

[0169] In certain embodiments, X is an antibody or antibody fragment moiety that targets GCC, such as indusatumab, or an antibody fragment moiety derived therefrom.

[0170] In certain embodiments, X is an antibody or antibody fragment moiety that targets STEAP1, such as bundurumab, or an antibody fragment moiety derived therefrom.

[0171] In certain embodiments, X is an antibody or antibody fragment moiety that targets MUC16, such as sofituzumab, or an antibody fragment moiety derived therefrom.

[0172] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD70, such as borsetuzumab, or an antibody fragment moiety derived therefrom.

[0173] In certain embodiments, X is an antibody or antibody fragment moiety that targets CD44, such as bivatuzumab, or an antibody fragment moiety derived therefrom.

[0174] In certain embodiments, X is an antibody or antibody fragment moiety that targets vWF, such as caplacizumab, or an antibody fragment moiety derived therefrom.

[0175] In certain embodiments, X is an antibody or antibody fragment moiety that targets TNF, such as ozoralizumab, V565, or PF-05230905, or an antibody fragment moiety derived therefrom.

[0176] In certain embodiments, X is an antibody or antibody fragment moiety that targets IL-6R, such as bovalilizumab, or an antibody fragment moiety derived therefrom.

[0177] In certain embodiments, X is an antibody or antibody fragment moiety that targets BCMA, such as LCAR-B38M, or an antibody fragment moiety derived therefrom.

[0178] In certain embodiments, X is an antibody or antibody fragment moiety that targets ADAMTS5, such as M6495, or an antibody fragment moiety derived therefrom.

[0179] In certain embodiments, X is an antibody or antibody fragment moiety that targets CX3CR1, such as BI 655088, or an antibody fragment moiety derived therefrom.

[0180] In certain embodiments, X is an antibody or antibody fragment moiety that targets CXCR4, such as AD-214 or ALX-0651, or an antibody fragment moiety derived therefrom.

[0181] In certain embodiments, X is an antibody or antibody fragment moiety that targets TfR1, such as TXB4, or an antibody fragment moiety derived therefrom.

[0182] In certain embodiments, X is an antibody or antibody fragment moiety that targets a VEGFR, such as CDP791, or an antibody fragment moiety derived therefrom.

[0183] In certain embodiments, X is an antibody or antibody fragment moiety that targets PSMA, such as GY1, or an antibody fragment moiety derived therefrom.

[0184] In certain embodiments, X is an antibody or antibody fragment moiety that targets FN1, such as L19 or NJB2, or an antibody fragment moiety derived therefrom.

[0185] In certain embodiments, X is an antibody or antibody fragment moiety that targets a FAP, such as F19, OMTX005, or sibrotuzumab, or an antibody fragment moiety derived therefrom.

[0186] In certain embodiments, X is an antibody or antibody fragment moiety that targets TNC, such as F16 or R6N, or an antibody fragment moiety derived therefrom.

[0187] In some embodiments, X is an antibody.

[0188] In some embodiments, the antibody is selected from the group consisting of daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitiamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, altumumab, tomzotuximab, raprituximab, oportuzumab, sitatuzumab, tucotuzumab, catumaxomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, blinacimab, olaratumab, ramucirumab, sacituzumab, Pr1E11, enfortumab, J591, MLN591, belantam , moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MOR20 2, TAK-079, I-131-BC8, iomab-B, carotuximab, bemarituzumab, apruzumab, rupartumab, zolbetuximab, claudiximab, andecaliximab, mirvetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, rovalpituzumab, indatuximab, lorvotuzumab, promiximab, BI 836826, otlertuzumab, naratuximab, milatuzumab, anetuzumab, amatuximab, MMOT-0530A, sarilumab, elotuzumab, belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, cantuzumab, codrituzumab, ECT204, MDX-1414, pertuzumab, trastuzumab, margetuximab, patritumab, seribantumab, lumletuzumab, elgemtumab, AV-203, CDX-3379, GSK284933, brentuximab, gemtuzumab, BI 835858, vadastuximab, lintuzumab, KHK2823, taclotuzumab, G4723A, glembatumumab, telisotuzumab, onartuzumab, SAIT301, tisotuzumab, rifastuzumab, indusatumab, bundletuzumab, sofituzumab, borsetuzumab, bivatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, sibrotuzumab, F16, or R6N.

[0189] In some embodiments, X is an antibody selected from atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, and trastuzumab.

[0190] In some embodiments, X is an antibody fragment moiety.

[0191] In some embodiments, X, or the antibody fragment portion, is selected from the group consisting of a single-chain variable fragment (scFv), a bivalent (or divalent) single-chain variable fragment (di-scFv, bi-scFv), an antigen-binding fragment (Fab), a single domain antibody (sdAb), an antigen-binding protein, a DotBody, an affibody, a DARPin, a DART, a TandAb, a diabody, a ribobody, a centrin, a knottin, an affilin, an affimer, an alphabody, an anticalin, an atrimer, an avimer, a fynomer, a Kunitz domain, an obody, a pronectin, a repebody, and a bicyclic peptide, or a humabody.

[0192] In some embodiments, X is an antibody fragment moiety selected from the group consisting of a single-chain variable fragment (scFv), a bivalent (or bivalent single-chain variable fragment (di-scFv, bi-scFv), an antigen-binding fragment (Fab), a single domain antibody (sdAb), and a single domain antibody (sdAb).

[0193] In some embodiments, the antibody fragment portion is an antigen-binding protein DotBody, affibody, DARPin, DART, TandAb, diabody, ribobody, centirin, knottin, affilin, affimer, alphabody, anticalin, atrimer, avimer, fynomer, Kunitz domain, obody, pronectin, repebody, bicyclic peptide, or humabody.

[0194] In some embodiments, X or the antibody fragment portion is an antigen-binding fragment (Fab). Fab is the region of an antibody that binds to an antigen and is composed of one constant domain and one variable domain from each of the heavy and light chains. In some embodiments, Fab comprises four domains: VH, CH1, VL, and CL1. In some embodiments, Fab comprises 400 to 500 amino acids, or 440 to 480 amino acids. In some embodiments, Fab has a molecular weight of about 50 kDa, or 40 kDa to 55 kDa, or 45 kDa to 50 kDa, or 45 kDa to 55 kDa.

[0195] In some embodiments, the Fab of trastuzumab, enfortumab, brentuximab, sacituzumab, L19 that binds to FN-1 (gene ID 2335), F16 that binds to TNC (gene ID 3371), or NJB2 (ECM targeting sequence).

[0196] In some embodiments, the antibody fragment portion comprises one or more PEG units, which can extend circulatory life.

[0197] In some embodiments, the antibody fragment portion is an antigen-binding protein. Antigen-binding proteins are proteins that are engineered to be antibody mimetics and exhibit high affinity and specificity for a given target. In some embodiments, the antigen-binding proteins are single-chain antigen-binding proteins, which are novel recombinant polypeptides composed of an antibody variable light amino acid sequence (VL) tethered to a variable heavy sequence (VH) by an engineered peptide that links the carboxyl terminus of the VL to the amino terminus of the VH.

[0198] In some embodiments, the antigen binding protein is about 5 kDa to 10 kDa, or about 7 kDa. In some embodiments, the antigen binding protein is about 50 to 80, or 60 to 70, or 66 amino acids in length. In some embodiments, the antigen binding protein contains cysteines only at the N-terminus or C-terminus. In some embodiments, the antigen binding protein contains cysteines only at the N-terminus. In some embodiments, the antigen binding protein contains cysteines only at the C-terminus.

[0199] In some embodiments, the antibody fragment portion is an antigen binding protein that targets TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, or CD25. In some embodiments, the antibody fragment portion is an antigen binding protein that targets HER2. Antigen binding proteins can be prepared and tested according to standard methods or purchased from commercial sources (e.g., Affilogic).

[0200] In some embodiments, the antibody fragment portion is selected from the group consisting of daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitiamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, fuligin ... Tuximab, gc1118, imgatuzumab, panitumumab, altumumab, tomzotuximab, raprituximab, oportuzumab, sitatuzumab, tucotuzumab, catumaxomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, blinacimab, olaratumab, ramucirumab, sacituzumab, Pr1E11, enfortumab, J591, MLN591, bevacizumab Lantamab, moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MOR 202, TAK-079, I-131-BC8, iomab-B, carotuximab, bemarituzumab, apruzumab, rupartumab, zolbetuximab, claudiximab, andecaliximab, mirvetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, rovalpituzumab, indatuximab, lorvotuzumab, promiximab, BI 836826, otlertuzumab, naratuximab, milatuzumab, anetuzumab, amatuximab, MMOT-0530A, sarilumab, elotuzumab, belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, cantuzumab, codrituzumab, ECT204, MDX-1414, pertuzumab, trastuzumab, margetuximab, patritumab, seribantumab, lumletuzumab, elgemtumab, AV-203, CDX-3379, GSK284933, brentuximab, gemtuzumab, BI Derived from 835858, vadastuximab, lintuzumab, KHK2823, taclotuzumab, G4723A, glembatumumab, telisotuzumab, onartuzumab, sait301, tisotuzumab, rifastuzumab, indusatumab, bundletuzumab, sofituzumab, borsetuzumab, bivatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, sibrotuzumab, F16, or R6N.

[0201] In some embodiments, X is an antibody fragment moiety derived from atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, or trastuzumab.

[0202] In certain embodiments, X is an antibody or antibody fragment moiety that targets vWF, such as caplacizumab.

[0203] In certain embodiments, X is an antibody or antibody fragment moiety that targets TNF, such as ozoralizumab, V565, or PF-05230905.

[0204] In certain embodiments, X is an antibody or antibody fragment moiety that targets IL-6R, such as bovalilizumab.

[0205] In certain embodiments, X is an antibody or antibody fragment moiety that targets BCMA, such as LCAR-B38M.

[0206] In certain embodiments, X is an antibody or antibody fragment moiety that targets ADAMTS5, such as M6495.

[0207] In certain embodiments, X is an antibody or antibody fragment moiety that targets CX3CR1, such as BI 655088.

[0208] In certain embodiments, X is an antibody or antibody fragment moiety that targets CXCR4, such as AD-214 or ALX-0651.

[0209] In certain embodiments, X is an antibody or antibody fragment moiety that targets TfR1, such as TXB4.

[0210] In certain embodiments, X is an antibody or antibody fragment moiety that targets VEGFR, such as CDP791.

[0211] In certain embodiments, X is an antibody or antibody fragment moiety that targets PSMA, such as GY1.

[0212] In some embodiments, the antibody fragment portion is caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, M6495, BI 655088, AD-214, ALX-0651, TXB4, CDP791, or GY1.

[0213] In some embodiments, X further comprises an imaging contrast agent. In some embodiments, the imaging contrast agent is a protein.

[0214] Linker part In some embodiments, L is attached to X via a cystine or lysine residue on X.

[0215] In some embodiments, L is a non-cleavable linker.

[0216] In some embodiments, L is a cleavable linker.

[0217] In some embodiments, L comprises one or more amino acids.

[0218] In some embodiments, L comprises a polypeptide.

[0219] In some embodiments, L comprises one or more of a hydrazone, a hydrazide, a disulfide, N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), 4-(4'-acetylphenoxy)butanoic acid (AcBut), one or more linear or branched natural or unnatural amino acids, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety.

[0220] In some embodiments, L contains 1 to 100 linked atoms, or 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0221] In some embodiments, L comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety is independently selected from oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4 It may be optionally substituted with 1 to 5 substituents independently selected from haloalkyl.

[0222] In some embodiments, L is an alkylene linker that optionally includes one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.

[0223] In some embodiments, L has the formula: -Y 10 -(CHR 130 ) n’ -Y 20 -(CHR 140 ) n’’ -Y 30 -(CHR 150 ) m’’ -Y 40 - (In the formula, Y 10 , Y 20 , Y 30 , and Y 40 each independently represents a bond, -NR 110 -, -O-, -S(O) 0~2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120-, -C(O)-, -OC(O)-, -OC(O)O-, -(CH2CH2O) 1~5 -, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene, wherein each alkylene, alkenylene, alkynylene, arylene, or heteroarylene independently is oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4 optionally substituted with 1 to 5 substituents independently selected from haloalkyl; Each R 110 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R 120 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R 130 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; Each R 140 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; Each R 150 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; and n′, n″, and m″ are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8).

[0224] In some embodiments, L has the formula: -Y 10-(CH2) n’ -Y 20 -(CH2) m’’ -Y 30 - (In the formula, Y 10 , Y 20 , and Y 30 each independently represents a bond, -NR 110 -, -O-, -S(O) 0~2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene independently is oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4 optionally substituted with 1 to 5 substituents independently selected from haloalkyl; Each R 110 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R 120 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8).

[0225] In certain embodiments, each R 110 are independently hydrogen, C1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and each R 120 are independently hydrogen, C 1~4 Alkyl, C 1~4 It is haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. In certain embodiments, the linker is not a bond.

[0226] The linker L can include one or more polyethylene glycol (e.g., PEG having an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diylbis(methylcarbamate), arylene (i.e., phenylene), ethyleneoxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.

[0227] In some embodiments, the linker is [ka] Contains one or more of the following:

[0228] In some embodiments, the linker is [ka] Contains one or more of the following:

[0229] In some embodiments, the linker is [ka] Contains one or more of the following:

[0230] In some embodiments, the linker comprises one or more of: [ka] In some embodiments, the linker comprises one or more of: [ka] Includes.

[0231] In some embodiments, the linker comprises one or more of: [ka] is or contains them.

[0232] In some embodiments, the linker comprises one or more of: [ka] is or contains them.

[0233] In some embodiments, the linker comprises one or more natural or unnatural amino acids, sometimes referred to as a peptide linker. The linker may be a peptide linker composed of a carboxylic acid acyl unit and one or more amino acids that make up a protein or peptide sequence. The linker may also include a self-immolative spacer that separates the drug from the protein peptide sequence.

[0234] In some embodiments, the linker is "AYZX 2 -W" (wherein "A" is a carboxylic acid acyl unit, "Y" and "Z" are each one or more natural or unnatural amino acids, which together form a peptide sequence, and "X" is a 2 " and "W" is any additional linker having 1-50 linked atoms, or 5-10 linked atoms, or 1-10 linked atoms separating the peptide from the payload, D, or bioorthogonal moiety. In certain embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0235] In some embodiments, Y can be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In some embodiments, Y can be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0236] In some embodiments, Z can be at least one amino acid selected from the group consisting of alanine, lysine, acetyl- or formyl-protected lysine, arginine, tosyl- or nitro-protected arginine, histidine, ornithine, acetyl- or formyl-protected ornithine, and citrulline. In some embodiments, Z can be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0237] Exemplary YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0238] In some embodiments, A is —OC(O)—.

[0239] In some embodiments, X 2 is -OC(O)-.

[0240] In some embodiments, W is -OC(O)-. 2 is absent and W is -OC(O)-.

[0241] In some embodiments, the moiety -X 2 -W is [ka] In some embodiments, the moiety -X 2 teeth, [ka] is.

[0242] In some embodiments, -XW is [ka] is.

[0243] In some embodiments, -XW is [ka] is.

[0244] In some embodiments, the peptide linker is specifically tailored to be selectively cleaved (eg, enzymatically cleaved) by, for example, one or more tumor-associated proteases to release the drug.

[0245] In some embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., a dipeptide, tripeptide, or tetrapeptide), although it will be understood that peptide linkers of up to 5, up to 6, up to 7, or up to 8 amino acid residues may also be suitably used.

[0246] In some embodiments, the peptide linker is selected from the group consisting of Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-N 9 -Tosyl-Arg or Phe-N 9-nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0247] In some embodiments, the linker L is [ka] It is or contains one or more of: TIFF2024542021000057.tif186170TIFF2024542021000058.tif254170.

[0248] In some embodiments, the linker L is [ka] Contains one or more of the following:

[0249] In some embodiments, the linker L is [ka] Contains one or more of the following:

[0250] The above-mentioned linkers may be attached to the amino acid side chain present on X, such as lysine or cysteine ​​(e.g., [ka] ).

[0251] In some embodiments, the linker L is —C(O)L 4 -or-C(O)C 1~6 Alkylene C(O)L 4 - and L 4 is a bond, -N(R 12 )-C 2~3 Alkylene-N(R13 )C(O)-, -CH(NHC(O)R 14 )C 1~4 Alkylene-SSC 1~4 Alkylene -OC(O)-, -NHNHC(O)CH(NHC(O)R 15 )CH2C(O)-, -C 1~6 Alkylene-CH(G x )OC(O)-, [ka] and R 12 , R 13 , R 14 , R 15 , and R 19 are each independently hydrogen or C 1~4 is alkyl, R 16 is hydrogen, C 1~4 Alkyl, -C 1~4 Alkylene -OH, -C 1~4 Alkylene-OC 1~4 Alkyl, -C 1~4 Alkylene -COH or -C 1~4 alkylene-CONH2, and G x is a halogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 and phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0252] In some embodiments, the linker L comprises a carbonyl moiety that conjugates the tetrazine moiety to the linker or X. For example, the linker may comprise a polypeptide moiety (PPM) having a lysine residue and a lysine side chain, and the PPM may also have an additional lysine or other amino acid side chain conjugated to the carbonyl moiety. In some embodiments, the linker L comprises [ka] may include:

[0253] In some embodiments, the linker L is [ka] is or includes one or more of:

[0254] In some embodiments, the linker L is [ka] is or includes one or more of:

[0255] In some embodiments, the linker L is [ka] is or includes one or more of:

[0256] In some embodiments, the linker L is [ka] is.

[0257] In some embodiments, the linker L is [ka] is.

[0258] In some embodiments, the linker L is [ka] is or includes one or more of:

[0259] In some embodiments, the linker L is [ka] is or includes one or more of:

[0260] In some embodiments, the linker L is [ka] is or includes one or more of:

[0261] In some embodiments, the linker L is [ka] is or includes one or more of:

[0262] In some embodiments, the linker L is [ka] is or includes one or more of:

[0263] In some embodiments, the linker L is [ka] is or includes one or more of:

[0264] In some embodiments, the linker L is [ka] is or includes one or more of:

[0265] In some embodiments, the linker L is [ka] is or includes one or more of:

[0266] In some embodiments, the linker L is [ka] is or includes one or more of:

[0267] In some embodiments, the linker L is [ka] is or includes one or more of:

[0268] In some embodiments, the linker L is [ka] is or includes one or more of:

[0269] In one embodiment, Formula I: [ka] (In the formula, p is 1 to 16; R 20 is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- is selected from the group consisting of R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R 22is, independently in each occurrence, a linker of 1 to 100 linked atoms, optionally containing one or more ethyleneoxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups; R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; X is an antibody or antibody fragment moiety that targets HER2, TROP2, nectin-4, claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5; and L, in each occurrence, independently: [ka] is provided a targeting moiety of

[0270] In one embodiment, a compound of formula II: [ka] (In the formula, X is an antibody or antibody fragment moiety; p is 1 to 16; L, independently at each occurrence, is a linker; R 20is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- is selected from the group consisting of R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R 30 is independently at each occurrence halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; R a , R 31a , and R 31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl, R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; t is independently 0, 1, 2, 3, or 4; X is an antibody or antibody fragment moiety that targets HER2, TROP2, nectin-4, claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5; and L, in each occurrence, independently: [ka] is provided a targeting moiety of

[0271] In one embodiment, Formula V: [ka] (In the formula, Ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; The dotted line is R 3 and R 4 represents an additional bond that forms a tetrazine when both are absent, or R 3 and R 4 When both are present, they represent an additional bond to form a dihydrotetrazine, except that when ring A is aryl, R 3 and R 4 Both exist, X is a biocompatible support, an antibody, or an antibody fragment moiety; p is 1 to 15; L, independently at each occurrence, is a linker; R 1is independently at each occurrence hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R' and NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally selected from the group consisting of 1 to 3 Z 1 is replaced by R 2 is independently at each occurrence halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; and 1 is replaced by R 3 and R 4are both absent or R 3 and R 4 are each independently hydrogen or a group that is removable after a triggering event; Each Z 1 are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S- selected from R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; t is independently 0, 1, 2, 3, or 4 at each occurrence; X is an antibody or antibody fragment moiety that targets HER2, TROP2, nectin-4, claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5; and L, in each occurrence, independently: [ka] is provided a targeting moiety of

[0272] In some embodiments, Ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl, each of which is optionally substituted.

[0273] In some embodiments, X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin-4, FN1, FAP, TNC, or ECM.

[0274] In some embodiments, X is zolbetuximab, claudiximab, andecaliximab, anetumab, amatuximab, MMOT-0530A, L19, NJB2, F19, OMTX005, sibrotuzumab, F16, or R6N, trastuzumab, enfortumab, or sacituzumab, or an antibody fragment moiety derived therefrom.

[0275] In some embodiments, X is L19, NJB2, F19, OMTX005, sibrotuzumab, F16, or R6N, trastuzumab, enfortumab, or sacituzumab, or an antibody fragment moiety derived therefrom.

[0276] In some embodiments, p is 1 to 100. In some embodiments, p is 1 to 75. In some embodiments, p is 1 to 50. In some embodiments, p is 1 to 30. In some embodiments, p is 1 to 20. In some embodiments, p is 1 to 10. In some embodiments, p is 5 to 10. In some embodiments, p is 1 to 15. In some embodiments, p is 8 to 12.

[0277] In some embodiments, p is 1 to 12. In some embodiments, X is an antibody. In some embodiments, p is 1 to 6 or 5 to 6. In some embodiments, X is an antibody fragment portion (e.g., Fab).

[0278] In some embodiments, p is 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 2-10, or 2-9, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 2-3, or 3-10, or 3-9, or 3-8, or 3-7, or 3-6, or 3-5, or 3-4, or 4-10, or 4-9, or 4-8, or 4-7, or 4-6, or 4-5, or 5-10, or 5-9, or 5-8, or 5-7, or 5-6, 6-10, or 6-9, or 6-8, or 6-7, 7-10, or 7-9, or 7-8, 8-10, or 8-9, or 9-10. In some embodiments, X is an antibody or antibody fragment moiety.

[0279] In some embodiments, p is 1 to 16, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments, X is an antibody fragment moiety (e.g., Fab).

[0280] In certain embodiments, p is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, 7 to 10, or 7 to 9, or 7 to 8, 8 to 10, or 8 to 9, or 9 to 10, and X is an antibody or antibody fragment portion of 15 kDa to 75 kDa, or 25 kDa to 75 kDa, or 45 kDa to 55 kDa, or less than 25 kDa, or less than 35 kDa, or less than 45 kDa, or about 50 kDa, or less than 55 kDa, or less than 65 kDa, or less than 75 kDa, or more than 75 kDa.

[0281] In certain embodiments, p depends on the size and / or number of available binding sites on X that form a covalent bond with L. In certain embodiments, when X is an antibody or antibody fragment moiety of greater than 75 kDa, p is 2 to 6. In certain embodiments, when X is an antibody or antibody fragment moiety of between 25 kDa and 75 kDa, p is 1 to 4. In certain embodiments, when X is an antibody or antibody fragment moiety of between 45 kDa and 55 kDa, p is 1 to 4. In certain embodiments, when X is an antibody or antibody fragment moiety of less than 25 kDa, p is 1 to 3, or 2 to 3, or 1 to 2, or about 1, about 2, or about 3.

[0282] C. Support composition The support composition includes a support. In certain embodiments, the support composition is a therapeutic support composition. The support may be a biocompatible support composition, i.e., compatible with the subject's body. In some cases, the support is non-toxic to the subject and does not substantially react with tissues or biological compounds in the subject. For example, the support may be, inter alia, a hydrogel. The support can be implanted into the subject's body and can support not only a binding agent (e.g., a tetrazine-containing group) but also a payload after the binding agent is conjugated. Exemplary supports include, but are not limited to, polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, crosslinked polymer matrices, metals, ceramics, plastics, bone graft materials, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and the like. Supports also include particles such as nanoparticles, microparticles, and the like.

[0283] The hydrogel can be a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, chondroitin sulfate, heparin, etc. Other suitable sugar-based biomaterials include those described in Polymer Advanced Technology, 2014, 25, 448-460. Polymers that can be used as supports include, but are not limited to, polyphosphazenes, polyanhydrides, polyacetals, poly(orthoesters), polyphosphates, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, as well as blends / composites / copolymers thereof. Exemplary polyethers include, but are not limited to, poly(ethylene glycol) (PEG), polypropylene glycol (PPG), triblock pluronics ([PEG]), and the like. n -[PPG] m -[PEG] n), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA). Supports can also include proteins and other poly(amino acids), such as collagen, gelatin, elastin and elastin-like polypeptides, albumin, fibrin, poly(γ-glutamic acid), poly(L-lysine), poly(L-glutamic acid), poly(aspartic acid), etc.

[0284] In some embodiments, the support is a hydrogel. In some embodiments, the support is alginate. In some embodiments, the support is chitin. In some embodiments, the support is hyaluronic acid (e.g., non-hydrogel hyaluronic acid having substantially no crosslinks). In some embodiments, the support is chitosin.

[0285] In certain embodiments, the support is a particle. The particles of the present disclosure may have a diameter of 2 cm or less, for example, 1.5 cm or less, or 1 cm or less, or 0.5 cm or less. For example, the particles may be nanoparticles or microparticles. Nanoparticles include particles having an average size on the nanometer scale (e.g., 1000 nm or less). Microparticles are particles having an average size on the micrometer scale (e.g., 1000 μm or less). "Average" refers to the arithmetic mean. In some embodiments, the nanoparticles have a diameter in the range of 1 nm to 1 μm, e.g., 10 nm to 1 μm, or 25 nm to 1 μm, or 50 nm to 1 μm, or 75 nm to 1 μm, or 100 nm to 1 μm, or 150 nm to 1 μm, or 200 nm to 1 μm, or 250 nm to 1 μm, or 300 nm to 1 μm, or 350 nm to 1 μm, or 400 nm to 1 μm, or 450 nm to 1 μm, or 500 nm to 1 μm. In other embodiments, the microparticles have diameters ranging from 1 μm to 1 mm, e.g., 10 μm to 1 mm, or 25 μm to 1 mm, or 50 μm to 1 mm, or 75 μm to 1 mm, or 100 μm to 1 mm, or 150 μm to 1 mm, or 200 μm to 1 mm, or 250 μm to 1 mm, or 300 μm to 1 mm, or 350 μm to 1 mm, or 400 μm to 1 mm, or 450 μm to 1 mm, or 500 μm to 1 mm. In further embodiments, small particles having diameters on the order of 10 nm to 100 nm may aggregate to form larger complexes, such as clusters or aggregates on the order of 1 μm to 10 μm. The particles of the present disclosure may be substantially spherical, thus having a substantially circular cross-section. Other particle shapes may be used, including, but not limited to, ellipsoids, cubes, cylinders, cones, needles, or other irregular shapes.

[0286] A "particle" can take the form of any manufactured material, molecule, cryptophane, virus, phage, etc. Particles may be composed of materials such as, but not limited to, metal, ceramic, plastic, glass, composite, polymer, hydrogel, etc. For example, particles may be composed of an inert material such as alginate or iron oxide. In some instances, particles may be magnetic and may be formed from paramagnetic, superparamagnetic, or ferromagnetic materials, or other materials that respond to magnetic fields. Furthermore, particles may be of any shape, e.g., spheres, rods, asymmetric shapes, etc. Particles, or groups of particles in a composite, may be functionalized with receptors that have specific affinity to bind to or interact with clinically relevant substrates. The receptors may be inherent to the particles themselves. For example, the particles themselves may be viruses or phage that have inherent affinity for a particular substrate. Additionally or alternatively, particles may be functionalized by covalently or otherwise attaching or associating receptors that specifically bind to or otherwise recognize particular clinically relevant substrates. The functionalized receptor can be an antibody, peptide, nucleic acid, phage, bacterium, virus, or any other molecule with a defined affinity for the target substrate. Examples of materials that can be used for the "particles" and / or "carriers" include polylactic acid, polyglycolic acid, PLGA polymers, alginate and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pluronic polyols, poloxamers, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of any of the above. These examples do not limit their concentration, their cross-linking with different agents, their method of administration, their tailored degradation profile, and other characteristics known to those skilled in the art.

[0287] The particle, or a group of particles in the complex, may be functionalized with a targeting agent (e.g., a ligand or antibody) that specifically binds (or substantially specifically binds) to a target (e.g., a target receptor or cell surface target, such as a clinically relevant receptor or cell surface target (e.g., an antigen)). The targeting agent may be directly attached to the particle itself. The targeting agent may be an antibody, peptide, nucleic acid, phage, bacterium, virus, or any other molecule that has specific affinity for the target receptor or cell surface target. In some cases, the receptor or cell surface target is PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, or other cell receptor or cell surface target. Other compounds or molecules, such as fluorophores or autofluorescent or luminescent markers, that may aid in the detection of the particle (e.g., in vivo detection) may also be attached to the particle. The ligand and / or detectable label may be attached directly to the particle or via a bioorthogonal functional group as described herein.

[0288] In certain embodiments, the support is a bone graft material, such as a bone graft substitute material. Bone graft substitute materials are materials structurally similar to bone. In some cases, bone graft substitute materials are bioabsorbable, meaning that they can dissolve or be absorbed by the body over time. In some cases, bone graft substitute materials can be osteoconductive, promoting vascularization and new bone formation within the bone graft substitute material. In some cases, bone graft substitute materials are osteoinductive, promoting new bone formation through the active recruitment of mesenchymal stem cells from surrounding tissues. For example, growth factors such as bone morphogenetic proteins can be included in the bone graft substitute material. Bone graft substitute materials include, but are not limited to, hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone chips, and the like, and combinations thereof.

[0289] In certain embodiments, the support composition has the formula: [ka] wherein the dashed line represents the bond to L, or a salt thereof.

[0290] In some embodiments, the support composition has the formula: [ka] wherein the dashed line represents the bond to L, or a salt thereof.

[0291] Hyaluronic acid derivatives include hyaluronic acid that has a plurality of glucuronic acid units and tetrazine-containing groups that are linked or directly linked to the glucuronic acid units of hyaluronic acid.Hyaluronic acid may have a plurality of N-acetylglucosamine units.In certain embodiments, the N-acetylglucosamine units of hyaluronic acid are not linked or conjugated to tetrazine-containing groups.

[0292] The tetrazine-containing group can be linked through the carboxylic acid of the glucuronic acid unit or can be directly attached. The tetrazine-containing group can be incorporated into hyaluronic acid at about 0.1% to about 80%, as measured by the % of carboxylic acid linked or conjugated to the tetrazine-containing group, e.g., about 1% to about 75%, about 5% to about 75%, about 10% to about 50%, or about 40% to about 75%, as measured by the % of carboxylic acid linked or conjugated to L of the tetrazine-containing group.

[0293] Additional support compositions are exemplified in WO 2017 / 044983, WO 2015 / 139025, and WO 2014 / 205126, the entire contents of each of which are incorporated herein by reference in their entirety.

[0294] D. trans-Cyclooctene Functionalized Prodrugs Trans-cyclooctene functionalized prodrugs, including prodrugs of anticancer drugs, are known in the art, as described in WO 2018 / 187740, WO 2014 / 205126, WO 2015 / 139025, and WO 2017 / 044983, which are incorporated herein by reference. Further embodiments using trans-cyclooctene functionalized prodrugs follow below.

[0295] In some embodiments, the trans-cyclooctene functionalized prodrug is a conjugate comprised of a payload linked to one or more trans-cyclooctene moieties.

[0296] In some embodiments, the conjugate (or trans-cyclooctene-functionalized prodrug) comprises an immunomodulatory payload, such as, for example, an immunomodulatory payload selected from the group consisting of a cytokine, a chemokine, a chemokine antagonist, a therapeutic monoclonal antibody, and an immune checkpoint payload, or a pharmaceutically acceptable salt thereof.

[0297] In some embodiments, the immunomodulatory payload is an inhibitor of a cytokine payload, or a pharmaceutically acceptable salt thereof.

[0298] In some embodiments, the inhibitor of the cytokine payload is an inhibitor of TNF-α, infliximab, certolizumab, TGF-β, galunisertib, fresolimumab, M7824, CSF-1, pexidartinib, or cabilalizumab.

[0299] In some embodiments, the conjugate comprises a monoclonal antibody, or a pharmaceutically acceptable salt thereof.

[0300] In some embodiments, the conjugate comprises a therapeutic protein payload, or a pharmaceutically acceptable salt thereof.

[0301] In some embodiments, the therapeutic protein payload is an antibody-based drug, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an artificial protein scaffold, an enzyme, a growth factor, a hormone, an interferon, an interleukin, or a thrombolytic drug.

[0302] In some embodiments, the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen.

[0303] In some embodiments, the therapeutic protein payload is selected from the group consisting of erythropoietin (EPO, e.g., natural EPO or synthetic EPO (see, e.g., U.S. Patent Application Publication No. 2003 / 0191291), including, but not limited to, PROCRIT™, EPREX™, or EPOGEN™ (epoetin-α), ARANESP™ (darbepoetin-α), NEORECORMON™, EPOGIN™ (epoetin-β), etc.), growth hormone (e.g., somatotropin, e.g., GENOTROPIN™), , NUTROPIN™, NORDITROPIN™, SAIZEN™, SEROSTIM™, HUMATROPE™, etc.), therapeutic monoclonal antibodies (e.g., atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, trastuzumab, etc.), human growth hormone (hGH), bovine growth hormone (bGH), follicle-stimulating hormone (FSH), interferons (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω, IFN-τ, consensus interferon, etc.), insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.), insulin-like growth factors (e.g., IGF-I, IGF-II), blood factors (e.g., factor X, tissue plasminogen activator (TPA), etc., including, but not limited to, ACTIVA SE™ (alteplase) tissue plasminogen activator, NOVOSEVEN™ (recombinant human Factor VIIa), Factor VIIa, Factor VIII (e.g., KOGENATE™), Factor IX, beta globin, hemoglobin, etc.), colony stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g., NEUPOGEN™ (filgrastim)), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), Neulasta (pegfilgrastim), granulocyte-monocyte colony stimulating factor,megakaryocyte colony-stimulating factor, etc.), transforming growth factors (e.g., TGF-β, TGF-α), interleukins (e.g., IL-1, IL-2 (e.g., Proleukin™), IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, etc.), growth factors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF, e.g., REGRANEX™ (beclamethasone)), fibroblast growth factor (FG), FGF, e.g., aFGF, bFGF, e.g., FIBLAST™ (trafermin), glial cell line-derived growth factor (GDNF), nerve growth factor (NGF), stem cell factor (e.g., STEMGEN™ (ansestim)), keratinocyte growth factor, hepatocyte growth factor, etc.), soluble receptors (e.g., TNF-α binding soluble receptors, e.g., ENBREL™ (etanercept), soluble VEGF receptors, soluble interleukin receptors, soluble gamma / delta T cell receptors, etc.), enzymes (e.g., α-glucosidase, CERAZYME™ (imiglucarase, β-glucocerebrosidase, CEREDASE™ (alglucerase)), enzyme activators (e.g., tissue plasminogen activator), chemokines (e.g., IP-10, Mig, Groα / IL-8, regulated and normal T cell expressed and secreted secreted) (RANTES), MIP-1α, MIP-1ρ, MCP-1, PF-4, etc.), angiogenic agents (e.g., vascular endothelial growth factor (VEGF), anti-angiogenic agents (e.g., soluble VEGF receptors), protein vaccines, neuroactive peptides such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, galanin, growth hormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptides, etc., other proteins such as thrombolytic agents, atrial natriuretic peptide,Payloads such as bone morphogenetic proteins, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human alpha-1 antitrypsin, leukemia inhibitory factor, transforming growth factor, tissue factor, insulin-like growth factor, luteinizing hormone, follicle-stimulating hormone, macrophage-activating factor, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, tissue inhibitor of metalloproteinases, vasoactive intestinal peptide, angiogenin, angiotropin, fibrin, hirudin, leukemia inhibitory factor, or IL-1 receptor antagonists (e.g., Kineret™ (anakinra)).

[0304] In some embodiments, the conjugate has formula X: [ka] (In the formula, m is an integer from 1 to 150; G, at each occurrence, is independently an optionally substituted trans-cyclooctene moiety; D is the payload, L 1 is, in each occurrence, independently a linker), or a pharmaceutically acceptable salt thereof.

[0305] In some embodiments of the conjugates described herein, each trans-cyclooctene moiety is independently [ka] (In the formula, R 1A are, in each case independently, C 1~4 Alkyl, C 1~4 Haloalkyl, and C 1~4 alkoxy; q is 0, 1, or 2; q1 is 0 or 1, R 1B are, in each case independently, G 1 , -OH, -NR 1c-C 1~4 Alkylene-G 1 , -NR 1c -C 1~4 Alkylene-N(R 1d )2, -NR 1c -C 1~6 Alkylene-N(C 1~4 Alkyl)3 + , -N(R 1c )CHR 1e CO2H, -N(R 1c )-C 1~6 Alkylene -COH, -N(R 1f )-C 2~4 Alkylene-(N(C 1~4 Alkylene-COH)-C 2~4 alkylene) n -N(C 1~4 Alkylene -CO2H)2, -N(R 1c )CHR 1e C(O)OC 1~6 Alkyl, -N(R 1c )-C 1~6 Alkylene-C(O)OC 1~6 Alkyl, -N(R 1f )-C 2~4 Alkylene-(N(C 1~4 Alkylene-C(O)OC 1~6 Alkyl)-C 2~4 alkylene) n -N(C 1~4 Alkylene-C(O)OC 1~6 alkyl)2, -N(R 1c )-C 1~6 Alkylene -SO3H, -N(R 1c )-(CH2CH2O) 1~3 -CH2CH2N((CH2CH2O) 1~3 -C 1~6 Alkylene -COH) and -N(R 1c )-CH(CHO-(CHCHO) 0~2 -C 1~6 alkylene-COH), R 1c and R 1d is, in each occurrence, independently hydrogen or C 1~4 is alkyl, R1e In each case, independently, -C 1~4 Alkylene -COH, -C 1~4 Alkylene -CONH2 or -C 1~4 alkylene-OH, R 1f is, in each case independently, hydrogen, C 1~6 Alkyl or C 1~4 alkylene-COH, n is independently at each occurrence 0, 1, 2, or 3; L 2 is, in each occurrence, independently -C(O)- and C 1~3 alkylene; and G 1 is independently at each occurrence an optionally substituted heterocyclyl.

[0306] In some embodiments, the conjugate has formula X: [ka] (In the formula, G is a trans-cyclooctene moiety, and G is, independently at each occurrence: [ka] and L 1 is, independently in each occurrence, a linker, m is an integer from 1 to 150; D is the payload, R 1A are, in each case independently, C 1~4 Alkyl, C 1~4 Haloalkyl, and C 1~4 alkoxy; q is 0, 1, or 2; q1 is 0 or 1, R 1B are, in each case independently, G 1 , OH, -NR 1c -C1~4 Alkylene-G 1 , -NR 1c -C 1~4 Alkylene-N(R 1d )2, -NR 1c -C 1~6 Alkylene-N(C 1~4 Alkyl)3 + , -N(R 1c )CHR 1e CO2H, -N(R 1c )-C 1~6 Alkylene -COH, -N(R 1f )-C 2~4 Alkylene-(N(C 1~4 Alkylene-COH)-C 2~4 alkylene) n -N(C 1~4 Alkylene -CO2H)2, -N(R 1c )CHR 1e C(O)OC 1~6 Alkyl, -N(R 1c )-C 1~6 Alkylene-C(O)OC 1~6 Alkyl, -N(R 1f )-C 2~4 Alkylene-(N(C 1~4 Alkylene-C(O)OC 1~6 Alkyl)-C 2~4 alkylene) n -N(C 1~4 Alkylene-C(O)OC 1~6 alkyl)2, -N(R 1c )-C 1~6 Alkylene -SO3H, -N(R 1c )-(CH2CH2O) 1~3 -CH2CH2N((CH2CH2O) 1~3 -C 1~6 Alkylene -COH) and -N(R 1c )-CH(CHO-(CHCHO) 0~2 -C 1~6 alkylene-COH), R 1c and R 1d is, in each occurrence, independently hydrogen or C 1~4 is alkyl, R1e In each case, independently, -C 1~4 Alkylene -COH, -C 1~4 Alkylene -CONH2 or -C 1~4 alkylene-OH, R 1f is, in each case independently, hydrogen, C 1~6 Alkyl or C 1~4 alkylene-COH, n is independently at each occurrence 0, 1, 2, or 3; L 2 is, in each occurrence, independently -C(O)- and C 1~3 alkylene; and G 1 is independently at each occurrence an optionally substituted heterocyclyl), or a pharmaceutically acceptable salt thereof.

[0307] In some embodiments, q1 is 1.

[0308] In some embodiments, the payload is an immunomodulatory payload.

[0309] In some embodiments, the payload is a therapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, and immune checkpoint inhibitor payload, or a pharmaceutically acceptable salt thereof.

[0310] In some embodiments, the payload is a therapeutic agent for treating cancer (e.g., doxorubicin, daunorubicin, PNU-159682, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, baccatin III, gemcitabine, podophyllotoxin, carmustine, ixabepilone, patupilone (epothelone class), platinum agents, exatecan, auristatins (dolastatin 10, MMAE, MMAD, MMAF), duocarmycin, pyrrolobenzodiazapene dimers, mitomycin C, bleomycin, calicheamicin, staurosporine, hemispheric acid, erythromycin, ribozyme, ribozyme-containing steroids ... therapies, immunosuppressants (e.g., cyclosporine A, rapamycin, etc.), antifungals (e.g., amphotericin, etc.), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, etc.), matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidylcholine (c-AMP).

[0311] In some embodiments, the payload is a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepilone, patupilone (epothelon class), platinum agents, exatecan, auristatins (dolastatin 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterin, etc.), immunosuppressants (e.g., cyclosporin A, rapamycin, etc.), antifungal agents (e.g., amyloid β, erythromycin ... amphotericin, etc.), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, etc.), lurbinectedin, gardiquimod, matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidylcholine (c-AMP).

[0312] Reference to a payload means that one or more atoms, including hydrogen or non-hydrogen atoms, of the original, unmodified payload have been replaced by a covalent bond to one or more linkers. The payload is derived from a known core payload and modified to be covalently attached to at least one optionally substituted trans-cyclooctene via a linker. After modification to arrive at the compounds described herein, the payload maintains biological activity equivalent to that observed in the original, unmodified payload. In certain embodiments, the payload exhibits binding activity or inhibition that is at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the binding activity or inhibition observed in the original, unmodified payload.

[0313] In certain embodiments, a hydrogen atom bonded to a heteroatom (e.g., N, O, or S) of the original, unmodified payload is replaced by a covalent bond to a linker. In certain embodiments, a halogen atom on the payload is replaced and attached to the remainder of the compound. In certain embodiments, a hydrogen atom on the payload is replaced and attached to the remainder of the compound. In certain embodiments, the hydrogen atom is on a heteroatom. In certain embodiments, the hydrogen atom is on a nitrogen. In certain embodiments, the hydrogen atom is on an oxygen. In certain embodiments, the hydrogen atom is on a carbon.

[0314] In some embodiments, G is, independently at each occurrence: [ka] is.

[0315] In some embodiments, G is, independently at each occurrence: [ka] is.

[0316] In some embodiments, the payload is a monoclonal antibody payload. A monoclonal antibody used herein as a payload can be a whole monoclonal antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In some embodiments, the antibody is an immune cell engager and will itself induce or elicit an immune response. In some embodiments, the monoclonal antibody or fragment thereof is selected from the group consisting of CD3 (NCBI Gene ID 916), CD28 (NCBI Gene ID 940), CD137 (4-1BB) (NCBI Gene ID 3604), CD16 (NCBI Gene ID 2214), NKG2D (NCBI Gene ID 22914), CD64 (NCBI Gene ID 2209), GITR / TNFRSF18 (NCBI Gene ID 8487), CD25 (NCBI Gene ID 3559), CD40 (NCBI Gene ID 958), CD4 (NCBI Gene ID 920), CXCR4 (NCBI Gene ID 7852), G-CSFR (NCBI Gene ID 1441), GM-CSFR (NCBI Gene ID 1438), CD122 (N and targeting one or more of: PD1 (NCBI Gene ID 3560), PD2 (NCBI Gene ID 5133), CTLA4 (NCBI Gene ID 1493), LAG3 (NCBI Gene ID 3902), TIGIT (NCBI Gene ID 201633), NCR1 (NCBI Gene ID 9437), TIM3 (NCBI Gene ID 84868), VISTA (NCBI Gene ID 64115), CD134 (NCBI Gene ID 7293), CD27 (NCBI Gene ID 939), CD40L (NCBI Gene ID 959), ICOS (NCBI Gene ID 29851), BAFFR (NCBI Gene ID 115650), LFA-1 (NCBI Gene ID 3689), or BTLA (NCBI Gene ID 151888).

[0317] In certain embodiments, the payload is an antibody or antibody fragment that targets CD3, such as OKT3, SP34, UCHT1, teplizumab, otelixizumab, visilizumab, or foralumab, or an antibody fragment derived therefrom.

[0318] In certain embodiments, the payload is an antibody or antibody fragment that targets CD28, such as celalizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.

[0319] In certain embodiments, the payload is an antibody or antibody fragment that targets CD137 (4-1BB), such as utomilumab, urelumab, LVGN6051, or AGEN2373, or an antibody fragment derived therefrom.

[0320] In certain embodiments, the payload is an antibody or antibody fragment that targets CD16, such as AFM13, or an antibody fragment derived therefrom.

[0321] In certain embodiments, the payload is an antibody or antibody fragment that targets NKG2D, such as NNC0152-0002 or JNJ-64304500, or an antibody fragment derived therefrom.

[0322] In certain embodiments, the payload is an antibody or antibody fragment that targets CD64, such as H22, or an antibody fragment derived therefrom.

[0323] In certain embodiments, the payload is an antibody or antibody fragment that targets GITR / TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.

[0324] In certain embodiments, the payload is an antibody or antibody fragment that targets CD25, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0325] In certain embodiments, the payload is an antibody or antibody fragment that targets CD40, such as iscalimab, ABBV-323, bleselumab (ASKP-1240), BI-655064, FFP-104, BMS986090, dacetuzumab, or lucatumumab, or an antibody fragment derived therefrom.

[0326] In certain embodiments, the payload is an antibody or antibody fragment that targets CD4, such as MAX.16H5, IT1208, zanolimumab (HuMax-CD4), UB-421, or MTRX1011A, or an antibody fragment derived therefrom.

[0327] In certain embodiments, the payload is an antibody or antibody fragment that targets CXCR4, such as F50067, or an antibody fragment derived therefrom.

[0328] In certain embodiments, the payload is an antibody or antibody fragment that targets G-CSFR, such as CSL324, or an antibody fragment derived therefrom.

[0329] In certain embodiments, the payload is an antibody or antibody fragment that targets GM-CSFR, such as mavrilimumab, or an antibody fragment derived therefrom.

[0330] In certain embodiments, the payload is an antibody or antibody fragment that targets CD122, such as Hu-Mik(β)1, or an antibody fragment derived therefrom.

[0331] In certain embodiments, the payload is an antibody or antibody fragment that targets PD-1, such as CC-90006, cemiplimab, camrelizumab, or TSR-042, or an antibody fragment derived therefrom.

[0332] In certain embodiments, the payload is an antibody or antibody fragment that targets CTLA4, such as tremelimumab or ipilimumab, or an antibody fragment derived therefrom.

[0333] In certain embodiments, the payload is an antibody or antibody fragment that targets LAG3, such as leratolimab (BMS-986016), GSK2831781, cemiplimab (REGN3767), favezelimab, yelamilimab, or mavezelimab, or an antibody fragment derived therefrom.

[0334] In certain embodiments, the payload is an antibody or antibody fragment that targets TIGIT, such as BMS-986207, tiragolumab, vibostolimab, etigilimab, domvanalimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.

[0335] In certain embodiments, the payload is an antibody or antibody fragment that targets NCR1, such as hNKp46.02, or an antibody fragment derived therefrom.

[0336] In certain embodiments, the payload is an antibody or antibody fragment that targets TIM3, such as covolimab, Sym023, LY3321367, BMS-986258, SHR-1702, sabatolimab, or INCAGN02390, or an antibody fragment derived therefrom.

[0337] In certain embodiments, the payload is an antibody or antibody fragment that targets VISTA, such as SG7, K01401-020, CI-8993, or JNJ-61610588, or an antibody fragment derived therefrom.

[0338] In certain embodiments, the payload is an antibody or antibody fragment that targets CD134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.

[0339] In certain embodiments, the payload is an antibody or antibody fragment that targets CD27, such as varlilumab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.

[0340] In certain embodiments, the payload is an antibody or antibody fragment that targets CD40L, such as dapirolizumab, or an antibody fragment derived therefrom.

[0341] In certain embodiments, the payload is an antibody or antibody fragment that targets ICOS, such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.

[0342] In certain embodiments, the payload is an antibody or antibody fragment that targets BAFFR, such as ianalumab, or an antibody fragment derived therefrom.

[0343] In certain embodiments, the payload is an antibody or antibody fragment that targets LFA-1, such as efalizumab, or an antibody fragment thereof.

[0344] In certain embodiments, the payload is an antibody or antibody fragment that targets a BTLA, such as icatolimab, or an antibody fragment derived therefrom.

[0345] In some embodiments, the payload is an anti-CD3 (αCD3) monoclonal antibody, or a derivative or analog thereof. In some embodiments, the anti-CD3 (αCD3) monoclonal antibody is SP34, UCHT1, or OKT3, or a derivative or analog thereof.

[0346] In some embodiments, at least one payload is selected from inhibitors of poly(ADP-ribose) polymerase (PARP), duocarmycin, pyrrolobenzodiazepines (PBDs), hemiasterin, HTI-286, anti-CD3 (αCD3) monoclonal antibodies, lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, exatecan, and MMAE, or derivatives or analogs thereof.

[0347] In some embodiments, D is a payload selected from an inhibitor of poly(ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterin, HTI-286, and an anti-CD3 (αCD3) monoclonal antibody, or a derivative or analog thereof.

[0348] In some embodiments, at least one payload is selected from lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, exatecan, seco-duocarmycin SA, and MMAE, or a derivative or analog thereof.

[0349] In some embodiments, the payload is an inhibitor of poly(ADP-ribose) polymerase (PARP), or a derivative or analog thereof. In some embodiments, the inhibitor of poly(ADP-ribose) polymerase (PARP inhibitor) is niraparib, talazoparib, olaparib, pamiparib, rucaparib, veliparib, iniparib, 3-aminobenzamide, CEP-9722, E7016, or a derivative or analog thereof.

[0350] In some embodiments, the payload is [ka] is.

[0351] In some embodiments, the payload is a duocarmycin, or a derivative or analog thereof, hi some embodiments, the duocarmycin is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, carzelesin, bizeresin, or a derivative or analog thereof.

[0352] In some embodiments, the payload is [ka] is.

[0353] In some embodiments, the payload is a pyrrolobenzodiazepine (PBD) or a derivative or analog thereof. In some embodiments, the pyrrolobenzodiazepine (PBD) is [1,2]diazepino[3,4-e]indole or a derivative or analog thereof.

[0354] In some embodiments, the payload is [ka] is.

[0355] In some embodiments, the payload is an inhibitor of tubulin polymerization, hi some embodiments, the payload is hemiasterlin, HTI-286, or a derivative or analog thereof.

[0356] In some embodiments, the payload is [ka] is derived from

[0357] In some embodiments, the payload is [ka] is.

[0358] In some embodiments, the payload comprises a topoisomerase inhibitor. In some embodiments, the payload comprises camptothecin, or a derivative or analog thereof. In some embodiments, the payload comprises topotecan, irinotecan, ciratecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, or rubitecan.

[0359] In some embodiments, the payload is [ka] Includes.

[0360] In some embodiments, the payload is [ka] Includes.

[0361] In some embodiments, the payload is [ka] Includes.

[0362] In some embodiments, the payload is [ka] Includes.

[0363] In some embodiments, the payload is [ka] Includes.

[0364] In some embodiments, the payload is [ka] Includes.

[0365] In some embodiments, the payload is [ka] Includes.

[0366] In some embodiments, the payload comprises a polypeptide. In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues. In some embodiments, the linker L 1 is covalently attached to a lysine, serine, threonine, or tyrosine residue present on the payload. In some embodiments, the polypeptide comprises one or more lysine residues. In some embodiments, the linker L 1 is covalently attached to a lysine residue present on the payload.

[0367] In some embodiments, the payload comprises an N-terminal amino acid, wherein the linker L 1 is covalently attached to the N-terminal amino acid.

[0368] In some embodiments, m is 1-20.

[0369] In some embodiments, the payload is an immunomodulatory payload.

[0370] In some embodiments, the immunomodulatory agent payload is an antibody payload.

[0371] In some embodiments, the immunomodulatory agent payload is an immune checkpoint inhibitor payload, ie, pidilizumab, sintilimab, AMP-224, atezolizumab, durvalumab, BMS-936559, tremelimumab, indoximod, epacadostat, a TIGIT inhibitor (e.g., LAG-3, such as an anti-LAG-3 antibody, TIM-3, such as an anti-TIM-3 antibody), a B7 molecule, or a BTLA pathway antagonist payload.

[0372] In some embodiments, the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload. In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload. In some embodiments, the PD-1 inhibitor payload is a payload of nivolumab, pembrolizumab, pidilizumab, sintilimab, or AMP-224.

[0373] In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload, ie, the PD-L1 inhibitor payload is atezolizumab, avelumab, durvalumab, or BMS-936559.

[0374] In some embodiments, the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload, hi some embodiments, the CTLA4 inhibitor payload is an ipilimumab or tremelimumab payload.

[0375] In some embodiments, the immune checkpoint inhibitor payload is an indoleamine 2,3-dioxygenase (IDO) inhibitor payload. In some embodiments, the IDO inhibitor payload is an indoximod or epacadostat payload.

[0376] In some embodiments, the immunomodulatory payload is a cytokine payload.

[0377] In some embodiments, the cytokine payload is an interferon, interleukin, tumor necrosis factor, erythropoietin, MIP3a, ICAM, macrophage colony-stimulating factor, erythropoietin (EPO), granulocyte colony-stimulating factor (GCSF), or granulocyte-macrophage colony-stimulating factor payload.

[0378] In some embodiments, the interleukin payload is selected from IL-1 through IL-40. In some embodiments, the interleukin payload is IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0379] In some embodiments, the immunomodulatory payload is a type 1 cytokine (IL-2, IL-12, TNF-B, IFN-g).

[0380] In some embodiments, the cytokine payload is selected from the group consisting of IFN-α, IFN-β, IFN-γ, PEGylated IFN-α, and apolipoprotein AI fusion proteins with IFN-α, interleukins, IL-2, IL-2 covalently linked to an immunoglobulin (e.g., sergutuzumab amnaleukin, RO6874281), IL-2 covalently linked to a PEG molecule (e.g., NKTR-214), IL-10, PEGylated IL-10 (e.g., pegilodecakin), IL-7, IL-12, IL-15, recombinant non-glycosylated IL-15, and IL-15 with the binding domain of IL-15Rα. fusion proteins (e.g., RLI), a triple fusion protein comprising human IL-15, the binding domain of IL-15Rα, and apolipoprotein AI, ALT-803 (IL-15 fused to the Fc domain of IgG1), IL-18, IL-21, tumor necrosis factors (TNF-α, TNF-β), erythropoietin (EPO), MIP3α, ICAM, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF, and talimogene laherparepvec.

[0381] In some embodiments, the immunomodulatory agent payload is a chemokine payload.

[0382] In some embodiments, the chemokine payload is a payload of CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CXCL14.

[0383] In some embodiments, the immunomodulatory agent payload is a chemokine antagonist payload. In some embodiments, the chemokine antagonist payload is a plerixafor payload.

[0384] In some embodiments, the immunomodulatory agent is a monoclonal antibody specific for a cytokine or cytokine receptor.

[0385] In some embodiments, the immunomodulatory payload comprises a polypeptide.

[0386] In some embodiments, the polypeptide comprises one or more lysine residues.

[0387] In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.

[0388] In some embodiments, the trans-cyclooctene is linked to one of the one or more lysine residues.

[0389] In some embodiments, the trans-cyclooctene is independently linked to one or more lysine, serine, threonine, or tyrosine residues.

[0390] In some embodiments, the polypeptide comprises an N-terminal amino acid, wherein the bioorthogonal moiety present is linked to the N-terminal amino acid.

[0391] In some embodiments, m is 1 to 20. In some embodiments, m is 1 to 10. In some embodiments, m is 1 to 5. In some embodiments, m is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, m is 1.

[0392] In some embodiments, the compound of formula XI: [ka] (In the formula, R 1a is, in each case independently, hydrogen, C 1~4 Alkyl, and C 1~4 haloalkyl; R 1b is, in each case independently, hydrogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C(O)OH, C(O)OC 1~4 Alkyl, C(O)N(R 1c )CHR 1e CO2H, C(O)N(R 1c )CHR 1e C(O)OC 1~4 Alkyl, C(O)N(R 1c )-C 1~6 Alkylene-COH, and C(O)N(R 1c )-C 1~6 Alkylene-C(O)OC 1~4 is selected from the group consisting of alkyl, R 1c is, in each occurrence, independently hydrogen or C 1~4 is alkyl, R 1e In each case, independently, -C 1~4 Alkylene -COH, -C 1~4 Alkylene -CONH2 or -C1~4 alkylene-OH, D is, in each case independently, the payload, L 1 is, independently in each occurrence, a linker, p' is independently 0, 1, or 2 at each occurrence; and p'' is independently 1, 2, or 3 at each occurrence, or a pharmaceutically acceptable salt thereof.

[0393] In some embodiments, D, at each occurrence, is independently selected from the group consisting of an anti-cancer drug payload, a toll-like receptor (TLR) agonist payload, and a stimulator of interferon genes (STING) agonist payload.

[0394] In some embodiments, R 1a is hydrogen.

[0395] In some embodiments, R 1a is C 1~4 It is alkyl.

[0396] In some embodiments, R 1a is CH3.

[0397] In some embodiments, R 1b are C(O)OH and C(O)OC 1~4 Alkyl, C(O)N(R 1c )CHR 1e CO2H, C(O)N(R 1c )CHR 1e C(O)OC 1~4 Alkyl, C(O)N(R 1c )-C 1~6 Alkylene-COH, and C(O)N(R 1c )-C 1~6 Alkylene-C(O)OC 1~4 alkyl.

[0398] In some embodiments, R1b is C(O)OH, C(O)N(R 1c )CHR 1e CO2H, and C(O)N(R 1c ) CH2CO2H.

[0399] In some embodiments, R 1b is -NR 1c -CH2CH2-N(CH3)3 + , -N(R 1c )-CH2CH2-SO3H, -N(R 1c )-(CH2CH2O)3-CH2CH2N((CH2CH2O)3-CH2CH2-CO2H)2, and -N(R 1c )—CH(CH2O—CH2CH2—CO2H)2.

[0400] In some embodiments, the trans-cyclooctene moiety (G) is [ka] is.

[0401] In some embodiments, the trans-cyclooctene moiety is [ka] is.

[0402] In some embodiments, the trans-cyclooctene moiety is [ka] In some embodiments, the trans-cyclooctene moiety is [ka] is.

[0403] In some embodiments, the trans-cyclooctene moiety is [ka] In some embodiments, the trans-cyclooctene moiety is [ka] is.

[0404] In some embodiments, the trans-cyclooctene moiety is [ka] is.

[0405] In some embodiments, the trans-cyclooctene moiety is [ka] is.

[0406] In some embodiments, the trans-cyclooctene moiety is [ka] is.

[0407] In some embodiments, the trans-cyclooctene moiety is [ka] is.

[0408] In some embodiments, the trans-cyclooctene moiety is [ka] and R 2 is —OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R 2 may optionally be further substituted with a polyether.

[0409] In some embodiments, the trans-cyclooctene moiety is [ka] Includes.

[0410] In some embodiments, the trans-cyclooctene moiety is [ka] Includes.

[0411] In some embodiments, the trans-cyclooctene moiety is [ka] Includes.

[0412] In some embodiments, the trans-cyclooctene moiety is [ka] Includes.

[0413] In some embodiments, R 1e is -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH.

[0414] In some embodiments, R 1e Ha-C 1~4 It is alkylene-CO2H.

[0415] In some embodiments, R 1e is -CH2CO2H.

[0416] In some embodiments, R 1b is -C(O)N(R 1c )-C 1~6 It is alkylene-CO2H.

[0417] In some embodiments, R 1b is -C(O)N(R 1c )CH2CO2H.

[0418] In some embodiments, R 1c is hydrogen.

[0419] In some embodiments, R 1b is hydrogen.

[0420] In some embodiments, R 1b is C(O)OH.

[0421] In some embodiments, the linker L 1 may have 1 to 100 linking atoms and may include ethyleneoxy, amine, ester, amide, carbamate, carbonate, and ketone functional groups. For example, the linker may have 1 to 50 linking atoms, or 5 to 50 linking atoms, or 10 to 50 linking atoms, or 1 to 40 linking atoms, or 1 to 30 linking atoms, or 1 to 20 linking atoms, or 1 to 10 linking atoms, or 1 to 5 linking atoms, or 5 to 30 linking atoms, or 10 to 30 linking atoms, or 5 to 40 linking atoms, or 5 to 50 linking atoms, or 10 to 50 linking atoms.

[0422] In some embodiments, the linker L 1 may contain one or more (e.g., 1 to 10 or 1 to 5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1 to 10 or 1 to 5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, where each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety independently may be selected from the group consisting of oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4It may be optionally substituted with 1 to 5 substituents independently selected from haloalkyl.

[0423] In some embodiments, the linker L 1 is the expression: -Y 10 -(CH2) n’ -Y 20 -(CH2) m’’ -Y 30 - (In the formula, Y 10 , Y 20 , and Y 30 each independently represents a bond, -NR 110 -, -O-, -S(O) 0~2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene independently is oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4 optionally substituted with 1 to 5 substituents independently selected from haloalkyl; Each R 110 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R 120 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and n' and m'' may each independently be 0, 1, 2, 3, 4, 5, 6, 7, or 8).

[0424] In certain embodiments, the linker is a bond.

[0425] In certain embodiments, the linker is not a bond. 110 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and each R 120 are independently hydrogen, C 1~4 Alkyl, C 1~4 It is haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.

[0426] Exemplary linkers include, but are not limited to, those shown below: [ka] Examples include:

[0427] Exemplary linkers include, but are not limited to, those shown below: [ka] Examples include:

[0428] In some embodiments, the linker L 1 may include one or more polyethylene glycol (e.g., PEG having an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diylbis(methylcarbamate), arylene (i.e., phenylene), ethyleneoxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. In some embodiments, the linker L 1 teeth, [ka] may include:

[0429] In some embodiments, the linker L 1 may contain one or more natural or unnatural amino acids, sometimes referred to as a peptide linker. When the drug (D) contains an amino moiety, the linker may be attached to the amino moiety using a peptide linker composed of a carboxylic acid acyl unit and one or more amino acids that make up the protein or peptide sequence. In some embodiments, the linker L 1 may also include a self-immolative spacer separating the drug and the protein peptide sequence.

[0430] In some embodiments, the linker L 1 may be a peptide linker represented by "AYZXW" (where "A" is a carboxylic acid acyl unit, "Y" and "Z" are each one or more natural or unnatural amino acids that together form a peptide sequence, and "X" and "W" are 1-50 linked atoms, or 5-10 linked atoms, or optional additional linkers having 1-10 linked atoms that separate the peptide from the drug, D, or bioorthogonal moiety). In certain embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0431] In some embodiments, Y can be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In some embodiments, Y can be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0432] In some embodiments, Z can be at least one amino acid selected from the group consisting of alanine, lysine, acetyl- or formyl-protected lysine, arginine, tosyl- or nitro-protected arginine, histidine, ornithine, acetyl- or formyl-protected ornithine, and citrulline. In some embodiments, Z can be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0433] In some embodiments, exemplary YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0434] In certain embodiments, A is —OC(O)—.

[0435] In certain embodiments, X is —OC(O)—.

[0436] In certain embodiments, W is -OC(O)-. In certain embodiments, X is absent and W is -OC(O)-.

[0437] In certain embodiments, -XW is [ka] is.

[0438] In certain embodiments, -XW is [ka] is.

[0439] In certain embodiments, the peptide linker is specifically tailored to be selectively cleaved (eg, enzymatically cleaved) by, for example, one or more tumor-associated proteases to release the drug.

[0440] In certain embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., a dipeptide, tripeptide, or tetrapeptide), although it will be understood that peptide linkers of up to 5, up to 6, up to 7, or up to 8 amino acid residues may also be suitably used.

[0441] In certain embodiments, the peptide linker is selected from the group consisting of Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly [SEQ ID NO:], Ala-Leu-Ala-Leu [SEQ ID NO:], Phe-N 9 -Tosyl-Arg or Phe-N 9 -nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0442] In certain embodiments, the linker L 1 teeth, [ka] is.

[0443] The linker described above may be attached to the right side of the amino acid side chain of D, such as lysine or cysteine ​​(e.g., [ka] ).

[0444] In some embodiments, the payload is covalently attached to the linker via an amide bond. For example, the payload may be an amine-containing payload that is attached to a carbonyl group of the linker, or in other cases, the payload may be a carboxyl-containing payload that is attached to an amine group of the linker. In some cases, the payload and the linker together form a carbamate group. For example, the payload may be an amine-containing payload that is attached to an acyloxy group of the linker. In some cases, the payload and the linker together form a carbonate group. For example, the payload may be a hydroxy-containing payload that is attached to an acyloxy group of the linker.

[0445] In some embodiments, L 1 teeth, [ka] L 3a is a bond or C 1~6 is alkylene, L 4a is the bond, -NHN:, -N(R 10 )-C 2~6 Alkylene-N(R 11 )-, -N(R 12 )-C 2~3 Alkylene-N(R 13 )C(O)-, -N(R 10 )-C 1~6 Alkylene -C(O)NHN: -NHNHC(O)C 1~6 Alkylene -C(O)NHN: -CH(NHC(O)R 14 )C 1~4 Alkylene-SSC 1~4 Alkylene -OC(O)-, -NHNHC(O)CH(NHC(O)R 15 )CH2C(O)-, -C 1~6 Alkylene-CH(G x )OC(O)-, [ka] and R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 19 are each independently hydrogen or C 1~4 is alkyl, R 16 is hydrogen, C 1~4 Alkyl, -C 1~4 Alkylene -OH, -C 1~4 Alkylene-OC 1~4 Alkyl, -C 1~4 Alkylene -COH or -C 1~4 alkylene-CONH2, R 17 is independently in each occurrence hydrogen or —CH2OC(O)—, and G x is a halogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 and phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0446] In certain embodiments, the linker L 1 is -OC(O)-.

[0447] In some embodiments, L 1 teeth, [ka] and L 3a is a bond, L 4a teeth, [ka] and R 12 and R 13 are each independently hydrogen or C 1~4 It is alkyl.

[0448] In some embodiments, p'' is 1. In some embodiments, p' is 1.

[0449] In some embodiments, [ka] teeth, [ka] TIFF2024542021000135.tif155170, R 18 is independently in each occurrence hydrogen or —CHOC(O)NHD′, R D is a hydrogen atom on the nitrogen atom of the payload or C 1~4 alkyl, and D and D' are independently payload moieties.

[0450] In some embodiments, D or D' is a cyclic dinucleotide payload moiety, an imidazo[4,5-c]quinolin-4-amine payload moiety, a TLR agonist payload moiety, a STING agonist payload moiety, or an anticancer drug payload moiety.

[0451] In some embodiments, [ka] teeth, [ka] and R 12 and R 13 are each independently hydrogen or C 1~4 alkyl, and D and D' are independently payload moieties (eg, anti-cancer drug payload moieties).

[0452] In some embodiments, p' is 0.

[0453] In some embodiments, p'' is 2 or 3.

[0454] In some embodiments, p is 2, and [ka] teeth, [ka] is.

[0455] Those skilled in the art will recognize that the payload (D or D') attached to a linker does not refer to the payload molecule itself, but rather to the portion of the payload molecule attached to the linker. Release of the payload (D or D') from the prodrug results in release of the payload itself.

[0456] The payload (D or D') can be an anti-cancer drug payload of any of the anti-cancer drugs described herein.

[0457] In some embodiments, the payload comprises a TLR7 / 8 agonist and X is a biocompatible support. In some embodiments, the payload comprises gardiquimod and X is a biocompatible support.

[0458] In some embodiments, the payload comprises a TLR7 / 8 agonist and X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin 4, or the extracellular matrix (ECM). In some embodiments, the payload comprises gardiquimod and X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin 4, or the extracellular matrix (ECM).

[0459] In some embodiments, the payload comprises camptothecin or a derivative thereof and X is a biocompatible support. In some embodiments, the payload comprises exatecan and X is a biocompatible support.

[0460] In some embodiments, the payload comprises camptothecin or a derivative thereof, and X is an antibody or antibody fragment moiety that targets HER2, TROP2, nectin 4, or the extracellular matrix (ECM). In some embodiments, the payload comprises exatecan, and X is an antibody or antibody fragment moiety that targets HER2, TROP2, nectin 4, or the extracellular matrix (ECM).

[0461] In some embodiments, the payload comprises MMAE and X is a biocompatible support.

[0462] In some embodiments, the payload comprises MMAE or a derivative thereof, and X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin 4, or the extracellular matrix (ECM).

[0463] In some embodiments, the payload comprises paclitaxel or a derivative thereof and X is a biocompatible support.

[0464] In some embodiments, the payload comprises paclitaxel or a derivative thereof, and X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin 4, or the extracellular matrix (ECM).

[0465] In some embodiments, the payload comprises docetaxel or a derivative thereof and X is a biocompatible support.

[0466] In some embodiments, the payload comprises docetaxel or a derivative thereof, and X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin 4, or the extracellular matrix (ECM).

[0467] In some embodiments, the payload has the structure: [ka] TIFF2024542021000141.tif192170TIFF2024542021000142.tif196170TIFF2024542021000143.tif205170TIFF2024542021000144.tif170170TIFF2024542021000145.tif199170TIFF2024542021000146.tif114170

[0468] In certain embodiments, the trans-cyclooctene functionalized prodrug is [ka] is selected from.

[0469] E. Treatment method Aspects of the present disclosure include methods of delivering a payload to a target location in a subject. In certain embodiments, the methods include selectively delivering a payload to a target location in a subject. Selective delivery of a payload includes delivering the payload to a target location (e.g., an organ or tissue, or portion thereof) without targeting other locations in the subject that do not require administration of the payload (e.g., other organs or tissues, or portions thereof). Selective delivery of a payload can be achieved through the use of targeting moieties and functionalized payloads described herein.

[0470] In some cases, the targeting moiety of the present disclosure can be localized to a desired target location in a subject. For example, the method of the present disclosure can include administering a targeting moiety described herein to a subject. The targeting moiety can be administered to the subject at the desired target location in the subject. In some cases, the targeting moiety can be locally injected into the subject at the desired target location in the subject. In some embodiments, the targeting moiety is administered systemically. In these embodiments, the targeting moiety can be localized to the desired target location in the subject through specific binding of the targeting agent to its target (e.g., antibody-antigen interaction, etc.), or can be localized on the surface (e.g., cell surface) of the desired target through specific binding of the targeting agent to its target (e.g., antibody-antigen interaction, etc.).

[0471] As described herein, selective binding between bioorthogonal binding counterparts (e.g., between the tetrazine of the targeting moiety and its complementary trans-cyclooctene of the prodrug) can occur. For localized administration of the targeting moiety to a desired location in a subject, as described above, selective binding between the trans-cyclooctene of the prodrug and its complementary binding agent results in the payload being localized to the desired target location.

[0472] Provided herein are methods of treating cancer, comprising administering to a subject in need of treatment a therapeutically effective amount of a targeting moiety described herein or a pharmaceutically acceptable salt thereof and a trans-cyclooctene prodrug.

[0473] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

[0474] In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer.

[0475] In some embodiments, the cancer is a solid tumor.

[0476] In some embodiments, the cancer is a soft tissue sarcoma.

[0477] In some embodiments, the soft tissue sarcoma is fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0478] In some embodiments, the method also includes enhancing or eliciting an immune response, hi some embodiments, the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0479] In some embodiments, the method further comprises administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, wherein the anti-cancer agent, immunomodulatory agent, and a trans-cyclooctene prodrug thereof are known in the art.

[0480] Indications for this approach include both hematopoietic and solid cancers. In certain embodiments, this approach can be used to treat and / or diagnose soft tissue sarcomas, including rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma, and all the various subtypes of soft tissue sarcoma, as well as osteosarcoma. The composition can be used to treat and / or diagnose pigmented villonodular synovitis.

[0481] In certain embodiments, this approach can be used to treat and / or diagnose hematopoietic malignancies, such as myelodysplastic syndromes, acute myeloid leukemia, myelodisplastic syndromes, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin's lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt's lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte-predominant Hodgkin's lymphoma, etc.

[0482] The compositions of the present disclosure are used in the treatment and / or diagnosis of a condition or disease in a subject amenable to treatment or diagnosis by administration of a payload (e.g., a parent drug (i.e., the drug prior to conjugation to the composition)). "Treatment" means achieving at least amelioration of symptoms associated with the condition from which the subject suffers, where amelioration is used in a broad sense to refer to at least a reduction in a parameter associated with the condition being treated, e.g., the magnitude of the symptoms. Thus, treatment also includes a situation in which a pathological condition, or at least symptoms associated therewith, are completely arrested, e.g., prevented from developing, or arrested, e.g., terminated, such that the subject is no longer afflicted by the condition, or at least symptoms characterizing the condition. Treatment can include arresting clinical symptoms, i.e., halting their progression or further progression, e.g., mitigating or completely arresting active disease. Treatment can also include buffering clinical symptoms, i.e., causing their regression. For example, in the context of cancer, the term "treating" includes any or all of the following: reducing the growth of solid tumors; inhibiting the replication of cancer cells; reducing the total tumor burden; increasing survival; and ameliorating one or more symptoms associated with cancer.

[0483] The subject to be treated may be a subject in need of therapy, where the subject to be treated is a subject suitable for treatment with the parent drug. Thus, a variety of subjects may be suitable for treatment with the compositions disclosed herein. Generally, such subjects are "mammals," with human beings being of interest. Other subjects may include household pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., animal models of disease), as well as non-human primates (e.g., chimpanzees and monkeys, etc.).

[0484] In certain embodiments, additional therapeutic agents and methods are used to treat, but are not limited to, melanoma (e.g., unresectable metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer, such as metastatic epithelial ovarian cancer), endometrial cancer, breast cancer (e.g., triple-negative breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer, among others. Solid tumors, including cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal carcinoma, and cutaneous T-cell lymphoma, can be treated, prevented, and / or diagnosed. The disclosed approach is well suited as an adjuvant / neoadjuvant system. For example, the particles disclosed herein can be placed during a biopsy, and once the results from the study are returned, the physician can deliver the appropriate cocktail to the desired site in the body. This minimizes tumor size, especially in the setting of tumors that are surgically resectable. At the end of the surgery, the surgeon can then administer additional targeting moieties to the subject to target the surgical cavity and treat the patient with additional therapeutic doses (e.g., chemotherapy according to the disclosed approaches), minimizing the risk that any cancer cells may be missed at the surgical margins.

[0485] In certain embodiments, the targeting moieties disclosed herein can be administered, and the physician can deliver the appropriate cocktail to the desired location in the body. This minimizes the size of the tumor, especially in the situation of tumors that can be surgically removed. Then, at the end of the surgery, the surgeon can administer additional targeting moieties to the subject to target the surgical cavity and treat the patient with additional therapeutic doses (e.g., chemotherapy according to the disclosed approach), minimizing the risk that some cancer cells may be missed at the surgical margin.

[0486] In certain embodiments, the disclosed methods provide the ability to deploy the particles disclosed herein at the time of biopsy, and once the results are returned, a physician can deliver an immunomodulatory agent to the biopsy site.

[0487] In certain embodiments, the disclosed methods provide physicians the ability to deliver immunomodulatory agents, such as TLR agonists, STING agonists, chemokines (agents that attract cancerous cells and / or immune cells), and adjuvants, to enhance the immune system, similar to the combination of chemotherapy and immunotherapy, but with fewer side effects. This combination approach can benefit patients. While chemotherapy agents treat solid tumors or specific sites, an enhanced immunotherapy response can be useful at distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can be used or utilized with anthracyclines, taxanes, gemcitabine, and other agents that enhance the efficacy of one or more immunomodulatory agents, such as ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer).

[0488] cancer The disclosed methods can be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that can involve maintaining growth signaling, evading growth inhibition, resisting cell death, achieving immortality through replication, inducing angiogenesis, and activating invasion and metastasis. The disclosed methods can enhance or induce an immune response against cancer in a subject. The immune response can cause an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0489] Cancers that may be treated by the disclosed methods include, but are not limited to, astrocytoma, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, brain stem cancer, brain stem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse intrinsic pontine glioma, ductal carcinoma, endometrial cancer, ependymoma, Ewing's sarcoma, esophageal cancer, eye cancer, fibrosarcoma, gallbladder cancer, stomach cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, macroglobulinemia, melanoma, melanoma, leukemia ... The cancers include, but are not limited to, rhabdomyosarcoma, oral cancer, multiple myeloma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thyroid cancer, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, non-small cell lung cancer (NSCLC), diffuse large B-cell lymphoma (DLBCL), or oral tongue squamous cell carcinoma (OTSCC).

[0490] In some embodiments, the cancer that may be treated by the disclosed methods is melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a soft tissue cancer. In some embodiments, the cancer is a fibrosarcoma. In some embodiments, the cancer is diffuse intrinsic pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0491] In some embodiments, the cancer that can be treated by the disclosed methods is a hematopoietic malignancy, such as myelodysplastic syndrome, acute myeloid leukemia, myelodysplastic syndrome, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte-predominant Hodgkin lymphoma, and the like.

[0492] Without being bound by any particular theory, localized release of certain anticancer drugs using the compounds and methods of the present disclosure may cause or contribute to immunogenic cell death (ICD). For example, certain anticancer drugs (e.g., anthracyclines, cyclophosphamide, and oxaliplatin) have been reported to induce ICD. Kroemer et al. Annu. Rev. Immunol. 2013 (31), 51-72. Immunogenic apoptosis of cancer cells can induce effective antitumor immune responses through the activation of dendritic cells (DCs) and the resulting activation of specific T cell responses. ICD is characterized by the secretion of damage-associated molecular patterns (DAMPs). Three important DAMPs are exposed on the cell surface during ICD. One DAMP molecule, calreticulin (CRT), is normally located in the lumen of the endoplasmic reticulum (ER) and moves to the surface of dying cells after induction of immunogenic apoptosis, where it functions as an "eat me" signal for professional phagocytes. Other important surface-exposed DAMPs are heat shock proteins (HSPs), namely HSP70 and HSP90, which also translocate to the plasma membrane under stress conditions. At the cell surface, they have immunostimulatory effects based on their interaction with many antigen-presenting cell (APC) surface receptors, such as CD91 and CD40, and also promote cross-presentation of tumor cell-derived antigens on MHC class I molecules, which then translocate to CD8. + It triggers T cell responses. Other important DAMPs characteristic of ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is considered a late apoptosis marker, and its release into the extracellular space is thought to be necessary for optimal release and presentation of tumor antigens to dendritic cells. HMGB1 binds to several pattern recognition receptors (PRRs), such as Toll-like receptor (TLR) 2 and TLR4, expressed on APCs. A more recently discovered DAMP released during immunogenic cell death is ATP, which, when secreted, functions as a "find me" signal for monocytes, inducing their attraction to the site of apoptosis. Kroemer et al. Curr. Op. Immunol. 2008 (20), 504-511.

[0493] Thus, local delivery of ICD-inducing factors using the compounds and methods of the present disclosure can be advantageously combined with one or more immunomodulatory agents.

[0494] In certain embodiments, targeting moieties can be used to treat, prevent, and / or diagnose solid tumors, including, but not limited to, melanoma (e.g., unresectable metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer such as metastatic epithelial ovarian cancer), breast cancer (e.g., triple-negative breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, among others.

[0495] The disclosed approach is well suited as an adjuvant / neoadjuvant system. For example, a targeting moiety disclosed herein can be placed into a biopsy, and once the results from the study are back, a physician can administer the appropriate cocktail to deliver the treatment to the desired location in the body (a compound disclosed herein and any additional therapeutic agent(s)). The biopsy results can indicate the amount and type of treatment delivered to the tumor site. For example, chemokines (agents that attract cancerous cells and / or immune cells) and adjuvants can be delivered and combined with immunotherapeutic agents to strengthen the immune system, similar to chemotherapy but with fewer side effects.

[0496] The disclosed methods can include single or multiple systemic doses of a targeting moiety that concentrates at one or more locations. The disclosed methods can be used to deliver functionalized payloads to these locations through systemic or local administration. In some embodiments, the targeting moiety is delivered systemically. In some embodiments, both the targeting moiety and the payload (i.e., the TCO-labeled payload) are delivered systemically. In some embodiments, the targeting moiety is delivered locally.

[0497] The disclosed compounds and compositions can be administered before surgical resection. The disclosed methods can minimize tumor size before surgical resection, thereby minimizing tumor size, particularly in the setting of surgically resectable tumors. The disclosed conjugates, compounds, and compositions can be administered during surgical resection. The disclosed conjugates, compounds, and compositions can be administered after surgical resection. At the end of surgical resection, targeting moieties can be placed around the resection margins, and the subject can then be treated with additional therapeutic doses to minimize the risk that any cancer cells may be missed at the resection margins.

[0498] The disclosed methods can include multiple systemic doses of a functionalized payload that concentrates at one location. The disclosed methods can be used to deliver a second payload. If the tumor is resistant to the first payload, the disclosed methods can be used to administer a second functionalized payload. The second payload can be a TCO-labeled payload of gemcitabine or docetaxel. A TCO-labeled payload of gemcitabine, paclitaxel, or docetaxel can be administered in combination with doxorubicin. The second functionalized payload can be activated by the targeting moiety used in the first prodrug.

[0499] The functionalized payloads disclosed herein can function as adjuvants. This combination approach can be beneficial to patients. Chemotherapeutic agents may treat solid tumors or specific locations and enhance or induce an immune response, while functionalized payloads and / or separate agents may enhance the immunotherapeutic response at distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can be used or utilized with anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, camptothecin analogs, and other agents that enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer).

[0500] The disclosed methods can be used to treat diffuse intrinsic pontine glioma. Diffuse intrinsic pontine glioma (DIPG) is a pediatric brainstem tumor that can be aggressive and difficult to treat. There is no known curative treatment for DIPG, and survival rates have remained dismal over the past 40 years. Patients with DIPG have a median overall survival of only 11 months, with a 2-year survival rate of less than 10%. DIPG accounts for 75% to 80% of pediatric brainstem tumors, affecting an estimated 200 to 300 children in the United States each year. Research has been hampered by the rarity of this devastating disease and the lack of experimental model systems to date, resulting in a plateau in survival rates over the past 40 years. Diagnosis of DIPG begins with clinical symptoms and can be confirmed by MRI. The disease may begin with several months of systemic symptoms, including behavioral changes and academic difficulties, diplopia, abnormal or limited eye movements, asymmetric smiling, loss of balance, and weakness. Alternatively, severe neurologic deterioration may occur earlier, with symptoms presenting for less than a month before diagnosis. Clinical examination may reveal a triad of multiple cranial nerve deficits, tract signs such as hyperreflexia and clonus, and ataxia. Enlargement of the pontine portion of the brainstem may lead to obstructive hydrocephalus and elevated intracranial pressure.

[0501] Nuclei important for vital functions such as breathing and heartbeat are located within the pons, and untreated, DIPGs can impair breathing and heartbeat.

[0502] The disclosed methods can include multiple systemic doses of a functionalized payload that concentrates at one location. The disclosed methods can be used to deliver a second payload. If a tumor is resistant to the first payload, the disclosed methods can be used to administer a second functionalized payload. The second payload can be a TCO-labeled payload of gemcitabine or docetaxel. A TCO-labeled payload of gemcitabine or docetaxel can be administered in combination with doxorubicin. The second functionalized payload can be activated by the targeting moiety used in the first prodrug.

[0503] Administration method Therapeutic methods can include any number of administration modes of the disclosed conjugates, compounds, or compositions, including tablets, pills, dragees, hard and soft gel capsules, granules, pellets, skin patches, skin creams, skin gels, aqueous, lipid, oily, or other liquids, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixirs, solid emulsions, solid solutions, or dispersible powders. In pharmaceutical compositions, the conjugates, compounds, or compositions disclosed herein can be dispersed in microparticles, e.g., nanoparticulate compositions.

[0504] For parenteral administration, the conjugates, compounds, or compositions disclosed herein may be dissolved or suspended in a physiologically acceptable diluent such as water, a buffer, an oil, or the like, with or without a solubilizing agent, surfactant, dispersing agent, or emulsifying agent. Suitable oils can include, for example, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil, and sesame oil. For parenteral administration, the conjugates, compounds, or compositions disclosed herein can be administered in the form of an aqueous, lipid, oily, or other type of solution or suspension, or can also be administered in the form of a liposome or nanosuspension.

[0505] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0506] The amount of the composition to be administered to a subject can be initially determined based on the dosage and / or dosing regimen guidelines of the parent drug. Generally, the composition can provide targeted delivery and / or extended serum half-life of the conjugated drug, thereby resulting in at least one of a reduced dose or reduced administration in the dosing regimen. Thus, the composition can provide a reduced dose and / or reduced administration in the dosing regimen compared to the parent drug before conjugation in the composition of the present disclosure.

[0507] Pharmaceutical preparations may be provided in unit dosage form.In this form, the pharmaceutical preparations may be subdivided into unit doses containing appropriate amounts of the compositions of the present disclosure.The unit dosage form may be a packaged preparation, and the package contains individual amounts of preparations, such as tablets, capsules, and powders, which are divided into pouches, vials, or ampoules.

[0508] In some embodiments, a kit is provided that includes a targeting moiety described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, and instructions for its use.

[0509] In some embodiments, the kit further comprises a prodrug.

[0510] The compositions of the present disclosure can be present in any suitable amount, which may depend on a variety of factors, including but not limited to, the weight and age of the subject, the disease state, etc. Suitable dosage ranges for the compositions of the present disclosure include 0.1 mg to 10,000 mg, or 1 mg to 1,000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For example, suitable dosages for compositions of the present disclosure include 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg.

[0511] In some embodiments, multiple doses of the composition are administered. The number of times the composition is administered can vary depending on any of a variety of factors, such as the severity of the symptoms, the condition of the subject, etc. For example, in some embodiments, the composition is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0512] The compositions of the present disclosure can be administered at any suitable frequency, interval, and period.For example, the compositions of the present disclosure can be administered once an hour, or 2 or 3 times or more per hour, once a day, or 2 or 3 times or more per day, or once every 2, 3, 4, 5, 6, or 7 days, to provide the subject with the desired dosage level.When the compositions of the present disclosure are administered more than once a day, representative intervals include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes, as well as 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, and 24 hours. The compositions of the present disclosure can be administered once, twice, or three or more times over the course of 1 hour, 1 hour to 6 hours, 1 hour to 12 hours, 1 hour to 24 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day, 1 day to 7 days, 1 week, 1 week to 4 weeks, 1 month, 1 month to 12 months, 1 year or more, or even indefinitely.

[0513] The compositions of the present disclosure can be co-administered with another active agent. Co-administration includes administering the compositions of the present disclosure and the active agent within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours of each other. Co-administration also includes administering the compositions of the present disclosure and the active agent simultaneously or nearly simultaneously (e.g., within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or about 30 minutes of each other), or sequentially in any order. Furthermore, the compositions of the present disclosure and the active agent can each be administered once a day, or two, three, or more times per day to provide the desired daily dosage level.

[0514] Co-administration can be achieved by co-implantation or co-injection.

[0515] In some embodiments, co-administration can be achieved by co-formulation, e.g., preparing a single pharmaceutical formulation that includes both the composition of the present disclosure and the active agent. In other embodiments, the composition of the present disclosure and the active agent can be formulated separately and co-administered to a subject.

[0516] The composition of the present disclosure and the active agent can be present in the formulation in any suitable weight ratio, such as 1:100-100:1 (wt / wt), or 1:50-50:1, or 1:25-25:1, or 1:10-10:1, or 1:5-5:1 (wt / wt). The composition of the present disclosure and the other active agent can be present in any suitable weight ratio, such as 1:100 (wt / wt), 1:75, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 (wt / wt). Other dosage amounts and dosage ratios of the composition of the present disclosure and the active agent are suitable for the formulations and methods described herein.

[0517] Combination therapy In one aspect, there is provided a method of treating cancer or enhancing or eliciting an immune response, comprising administering to a subject in need of treatment or enhancing or eliciting a therapeutically effective amount of a targeting moiety of the present disclosure or a pharmaceutically acceptable salt or composition thereof, a prodrug such as those described herein, and optionally, a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0518] The present disclosure also provides pharmaceutical combinations comprising a targeting moiety or a pharmaceutically acceptable salt thereof or composition described herein, a prodrug described herein, and optionally an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, for use in the treatment or prevention of cancer or for use in enhancing or eliciting an immune response.

[0519] The present disclosure also provides for the use of a pharmaceutical combination comprising a targeting moiety described herein or a pharmaceutically acceptable salt or composition thereof, a prodrug such as those described herein, and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, for the treatment or prevention of cancer or for use in enhancing or eliciting an immune response.

[0520] In the methods and uses described herein, the components of the pharmaceutical combination may be administered / used simultaneously, separately, or sequentially in any order, and these components may be administered separately or as a fixed combination. For example, delaying or treating disease progression according to the present disclosure may involve administering a first active ingredient in free form or in pharmaceutically acceptable salt form and a second active ingredient in free form or in pharmaceutically acceptable salt form simultaneously or sequentially in any order in therapeutically effective or effective amounts taken together, for example, in daily dosage amounts corresponding to the amounts described herein. The individual active ingredients of the pharmaceutical combination may be administered separately at different times during the course of treatment or simultaneously in divided or single dosage forms. Therefore, the present disclosure should be understood to encompass all such regimes of simultaneous or alternating treatment, and the term "administering" should be interpreted accordingly. Thus, the pharmaceutical combination as used herein defines either a fixed combination in one unit dosage form, or separate dosage forms in the case of combined administration, where the combined administration may be independently simultaneous or at different times. As a further example, the targeting moiety (or therapeutic targeting moiety) and prodrug can be administered / used simultaneously (e.g., by co-injection or co-implantation), separately, or sequentially, followed by administration of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0521] Methods of and uses in treating cancer include administering / localizing a targeting moiety to a tumor. In the methods and uses disclosed herein, administration of a prodrug or a pharmaceutically acceptable salt or composition thereof, a targeting moiety, and optionally an additional therapeutic agent may inhibit tumor growth.

[0522] The additional therapeutic agent(s) can be administered simultaneously or sequentially with the disclosed conjugates and compositions. Sequential administration includes administration before or after the disclosed conjugates and compositions. The additional therapeutic agent can be administered before the disclosed conjugates and compositions. The additional therapeutic agent can be administered after the disclosed conjugates and compositions. The additional therapeutic agent can be administered simultaneously with the disclosed conjugates and compositions. In some embodiments, the additional therapeutic agent can be administered in the same composition as the disclosed conjugates. In other embodiments, there can be a time interval between the administration of the additional therapeutic agent and the administration of the disclosed conjugates or compositions. In some embodiments, administering the disclosed conjugates or compositions with an additional therapeutic agent can allow for lower doses and / or less frequent administration of the other therapeutic agent. When used in combination with one or more other active ingredients, the disclosed conjugates or compositions and the other active ingredients can be used in lower doses than when each is used alone. Thus, pharmaceutical compositions of the present disclosure include those containing one or more other active ingredients in addition to the disclosed conjugates.

[0523] anticancer drugs Exemplary anticancer agents include, but are not limited to, abiraterone acetate, avitrexate (methotrexate), Abraxane (an albumin-stabilized nanoparticle formulation of paclitaxel), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), adrsil (fluorouracil), afatinib dimaleate, and afinitor (everolimus). ), Aldara (imiquimod), aldesleukin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), ambochlorin (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), arsenic trioxide, Arzera (ofatumumab), Erwinia chrysanthemi asparaginase, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexar (tositumomab and I 131Iodine tositumomab), bicalutamide, bleomycin, bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib S-malate, CAF, Camptosar (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomib, Casodex (bicalutamide), CeeNU (lomustine), Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV) V bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, Cometriq (cabozantinib S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, liposomal cytarabine, Cytosar-U (cytarabine), Cytoxan (cytarabine) Clofosfamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DepoCyt (liposomal cytarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), efdex X (fluorouracil), ERYTECH (rasburicase), Ellence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (Erwinia chrysanthemi asparaginase), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Ebacet (doxorubicin hydrochloride liposome), everolimus,Evista (raloxifene hydrochloride), exemestane, Fairston (toremifene), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), Forfiri, forfiri-bevacizumab, forfiri-cetuximab, Forfirinox, Forfox (leucovorin, fluorouracil, oxaliplatin), Folotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, Gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Jilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), recombinant HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, Hycamtin (topotecan hydrochloride), Hy Per-CVAD, ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosfamide), ifosfamide, ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Inlyta (axitinib), Intron A (recombinant interferon alpha-2b), iodine, 131Tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepivance (palifermin), Kyprolis (carfilzomib), lapatinib ditosylate, lenalidomide, letrozole, leucovorin calcium, Leukeran (chlorambucil), leucovorin acetate Prolide, Levran (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), liposomal cytarabine, lomustine, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lupron Depot (3 months) (leuprolide acetate), Lupron Depot (4 months) (leuprolide acetate), Marchibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, Megase (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), metazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), mitomycin C, Mitozytrex (mitomycin C), MOPP, Mozobil (plelixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (brinjal) Sulfane), Myrosal (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (albumin-stabilized nanoparticle formulation of paclitaxel), Navelbine (vinorelbine tartrate), nelarabine, Neosal (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, ofatumumab, omacetaxine mepeccate, Oncaspar (pegaspargase),Ontak (denileukin diftitox), OEPA, OPPA, oxaliplatin, paclitaxel, albumin-stabilized nanoparticle formulation of paclitaxel, palifermin, palonosetron hydrochloride, pamidronate disodium, panitumumab, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegaspargase, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pemafibrate Trexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), Provenzi (sipuleucel-T), Purinetol (mercaptopurine), radium dichloride, 223, raloxifene hydrochloride, rasburicase, R-CHOP, R-CVP, recombinant HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, recombinant interferon alfa-2b, regorafenib, Revlimid (lenalidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, sclerosol intrapleural aerosol (talc), sipuleucel-T, sorafenib tosylate, Sprycel (dasatinib), Stanford V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate , Sutent (sunitinib malate), Silatron (peginterferon alfa-2b), Sinovir (thalidomide), Synribo (omacetaxine mepeccate), Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and I 131Iodine tositumomab, Totect (dexrazoxane hydrochloride), trametinib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), vandetanib, VAMP, Vectibix (panitumumab), VelP, Velban (vinblastine sulfate), Velcade (bortezomib), Versal (vinblastine sulfate), vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate) ), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine liposome sulfate, vinorelbine tartrate, vismodegib, Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xerox, Xgeva (denosumab), Xofigo (radium dichloride) 223 ), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), and Zytiga (abiraterone acetate).

[0524] The anticancer drug can be a PBD dimer, calicheamicin, speromycin, tubulysin B, rhizoxin, dolastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothilone B, taxol, cryptophycin, SN38, velcade, bruceanthin, DAVLBH, DM1, filanthoside, alimta, T2 toxin, MMC, vantaranib, vinorelbine, brefeldin, sunitinib, daunomycin, semaxanib, tarceva, Iressa, irinotecan, LY-541503, geldanomycin, gemcitabine, methotrexate, Gleevec, topotecan, bleomycin, doxorubicin, cisplatin, nitrogen mustard, etoposide, or 5-FU.

[0525] In certain embodiments, the anticancer agent is an anthracycline. In certain embodiments, the anticancer agent is a taxane. In certain embodiments, the anticancer agent is gemcitabine. In certain embodiments, the anticancer agent is doxorubicin. In certain embodiments, the anticancer agent is docetaxel. In certain embodiments, the anticancer agent is SN38. In certain embodiments, the anticancer agent is monomethyl auristatin E.

[0526] Compound synthesis Targeting moieties can be prepared using the methods disclosed herein and their routine modifications, which will be apparent in light of the disclosure herein and methods well known in the art. In addition to the teachings herein, conventional well-known synthetic methods can be used. The synthesis of typical targeting moieties described herein can be achieved as described in the following examples. Where available, reagents and starting materials can be purchased commercially, for example, from Sigma Aldrich or other chemical suppliers.

[0527] It will also be understood that where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are given, other process conditions can be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0528] Furthermore, conventional protecting groups may be required to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups, as well as suitable conditions for protecting and deprotecting specific functional groups, are well known in the art. For example, Wuts, PGM, Greene, TW, & Greene, TW (2006). Greene's protective groups in organic synthesis. Hoboken, NJ, Wiley-Interscience, and the references cited therein, describe numerous protecting groups.

[0529] As shown in Scheme I, a compound of formula V (wherein the dashed line, R 1 , R 2 , R 3 , R 4 , each of rings A, t, L, p, and X is independently defined herein, and R 50can be prepared by coupling a compound of formula I-1 with a suitable functionalized biocompatible support, antibody, or antibody fragment moiety X (X is a leaving group, e.g., a synthetic handle for attachment to L, such as halo, or a portion of L or a further portion of L that can be linked to X, e.g., hydroxy, amino, methylamino, etc.). Suitable methods can be found in the literature (see, for example, WO 2020 / 077140, WO 2018 / 187740, WO 2017 / 044983, WO 2015 / 139025, and WO 2014 / 205126). Compounds of formula I-2 can be prepared by reacting compound I-1 with a precursor of L under suitable coupling reaction conditions. In some embodiments, X is an antibody or antibody fragment. Suitable coupling methods include, but are not limited to, the use of a succinimide functional group capable of forming an amide bond with a primary amine on the antibody or antibody fragment, or L can be functionalized with a group such as pyrrole-2,5-dione that can form a covalent bond with a cysteine ​​residue on the antibody or antibody fragment.

[0530] [ka]

[0531] Compounds of formula I-1 can be prepared according to Scheme II, where the dotted line, R 1 , R 2 , R 3 , R 4 , each of rings A, t, and L is independently defined herein, and R 50 is a leaving group, e.g., a synthetic handle for attachment to L, such as halo or thioether, or a portion of L or a further portion of L that can be linked to X, e.g., hydroxy, amino, methylamino, etc., and each LG is independently a leaving group, e.g., halo.

[0532] [ka]

[0533] As shown in Scheme II, compound II-1 and compound II-2 are coupled in the presence of N2H4 to give compound II-3. Compound II-3 can be further modified with compound II-4 and / or compound II-6 under standard coupling conditions to give compound II-5 and / or compound II-7. Alternatively, compound II-3 can be obtained by coupling compound II-8 and compound II-9 in the presence of N2H4. Compound II-10 can be obtained by contacting compound II-3 with a suitable oxidizing agent (e.g., NaNO2). Alternatively, compound II-3 can be obtained by contacting compound II-10 with thiourea dioxide.

[0534] Upon completion of each reaction, each intermediate or final compound can be recovered and optionally purified by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration, and the like.

[0535] It should be understood that any of the compounds or intermediates shown in Scheme I or Scheme II can be prepared using conventional methods or purchased from commercial sources. Additionally, any of the intermediates or any product obtained by the process outlined in Scheme I or Scheme II can be derivatized at any step to provide various compounds of Formula V.

[0536] Exemplary payloads that can be prepared can be prepared according to methods cited in the literature (see, e.g., WO 2022 / 032191, WO 2021 / 007160, WO 2020 / 077140, WO 2018 / 187740, WO 2017 / 044983, WO 2015 / 139025, and WO 2014 / 205126, which methods are incorporated herein by reference in their entireties). Exemplary procedures for MMAE payloads are shown in Examples A, B, and C. These procedures can be adapted to prepare other payloads, such as those disclosed herein. [Example]

[0537] The following examples are included to demonstrate specific embodiments of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that work well in the practice of the present disclosure and can therefore be considered to constitute specific modes for its practice. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0538] LCMS analytical method: Test articles were subjected to PNGaseF (IgG) and DTT or DTT alone (Fab) in a RapiGest according to the manufacturer's protocol. Samples were diluted to 100 μg / mL with water and centrifuged at an RCF of 16.1 kJ for 10 minutes at 4°C. Samples were then analyzed by LCMS (LC-Q-TOF) and mass spectra were reconstructed from the charge ladder.

[0539] Example 1: Synthesis of tetrazine-trastuzumab targeting moiety Trastuzumab (22.1 mg / mL, 1.1 mL) in 0.01 M PBS was mixed with 20 equivalents of methyltetrazine-PEG4-NHS (Clickchemtools no. 1069-10).

[0540] [ka]

[0541] The reaction was thoroughly mixed and aged at room temperature for 1 hour, at which point the reaction was quenched by adding 1 volume of 0.1 M Tris buffer. The resulting solution was buffer-exchanged into 0.01 M PBS to remove excess reagents and buffer salts. The resulting targeting moiety solution (6.3 mg / mL, 1.6 mL) was analyzed by SDS-Page (Figure 1) and LCMS (Figure 2) to confirm the formation of the targeting moiety and was thus used in subsequent studies. Based on the analysis, it appears that up to 12 methyltetrazine-PEG4 units have been covalently attached to the antibody.

[0542] Example 2: Tetrazine-Fab targeting moiety Fab was prepared from trastuzumab using a commercially available kit (Pierce™ Fab Preparation Kit #44985) according to the manufacturer's protocol and purified on Protein G resin (BioVision #6511-25). The purified Fab in 0.01 M PBS was mixed with 20 equivalents of methyltetrazine-PEG4-NHS (Clickchemtools #1069-10). The reaction was mixed thoroughly and aged at room temperature for 1 hour, at which point the reaction was quenched by adding 1 volume of 0.1 M Tris buffer. The resulting solution was buffer-exchanged into 0.01 M PBS to remove excess reagents and buffer salts. The resulting targeting moiety solution (1.0 mg / mL, 10.3 mL) was analyzed by SDS-Page (Figure 3) and LCMS (Figure 4) to confirm the formation of the targeting moiety and was thus used in subsequent studies. Based on the analysis, it appears that up to about six methyltetrazine-PEG4 units are covalently attached to the Fab.

[0543] Example 3: Tetrazine-Fab targeting moiety Fab was prepared from enfortumab using the following method: 0.1 mg of papain was pretreated with 1 mM DTT and 2 mM EDTA at a concentration of 0.5 mg / ml by incubation at 37°C for 30 minutes. The antibody was prepared in PBS buffer (pH 7.4) (10 mg, 0.5 mg / mL). The pretreated papain and antibody were mixed at a molar ratio of 1:100 and incubated at 37°C for 2 hours. The digestion mixture was loaded onto an anti-CH1 affinity column, washed with 25 mM Tris, 150 mM NaCl (pH 8.0), and eluted with 50 mM sodium citrate, 150 mM NaCl (pH 3.0). The filtrate containing the product Fab was dialyzed against PBS. The purified Fab in PBS was concentrated to 0.2 mg / mL. Fab and Me-Tet-PEG9-NHS (prepared at 10 mM in DMSO) were mixed in a 3:1 molar ratio and incubated for 2 hours at 37° C. The resulting conjugate was analyzed by LCMS, and the DAR was calculated to be 2.66.

[0544] Example 4: Tetrazine-Fab targeting moiety Fab was prepared from brentuximab using the following method: 0.1 mg of papain was pretreated by incubation with 1 mM DTT and 2 mM EDTA at a concentration of 0.5 mg / ml for 30 minutes at 37°C. The antibody was prepared in PBS buffer (pH 7.4) (10 mg, 0.5 mg / mL). The pretreated papain and antibody were mixed at a molar ratio of 1:100 and incubated at 37°C for 2 hours. The digestion mixture was loaded onto an anti-CH1 affinity column, washed with 25 mM Tris, 150 mM NaCl (pH 8.0), and eluted with 50 mM sodium citrate, 150 mM NaCl (pH 3.0). The filtrate containing the product Fab was dialyzed against PBS. The purified Fab in PBS was concentrated to 0.2 mg / mL. Fab and Me-Tet-PEG-NHS (prepared at 10 mM in DMSO) were mixed at a 3:1 molar ratio and incubated for 2 hours at 37° C. The resulting conjugate was analyzed by LCMS, and the DAR was calculated to be 4.57.

[0545] Example 5: Tetrazine-Fab targeting moiety Fabs were prepared from sacituzumab using a commercially available kit (Pierce™ Fab Preparation Kit No. 44985) according to the manufacturer's protocol and purified using Protein G resin (BioVision No. 6511-25). A 10 mM Me-Tet-PEG9-NHS solution of the purified Fab was prepared in DMSO. The two components were reacted at a 3:1 drug-to-protein molar ratio for 2 hours at 25°C, after which it was dialyzed against PBS (pH 7.4) to remove excess Me-Tet-PEG9-NHS compound from the protein component. The resulting targeting moiety solution was analyzed by SDS-Page and LCMS to confirm targeting moiety formation. Approximately two methyltetrazines were expected to be covalently attached to each Fab, on average, as confirmed by LCMS.

[0546] Example 6: Tetrazine-antigen binding protein targeting moiety Antigen-binding proteins are engineered proteins capable of binding to antigens. These proteins are approximately 66 amino acids in length and have a molecular weight of 7 kDa. The proteins can be expressed in E. coli, can contain cysteine ​​residues at the N- or C-terminus of the sequence that can be conjugated to a cysteine-reactive group, and can be purchased from commercial sources (e.g., Nanofitins™ from Affilogic).

[0547] An antigen-binding protein targeting HER2 with a C-terminal cysteine ​​is expressed in E. coli and purified to homogeneity. The protein is treated with a reducing agent such as TCEP at ambient temperature or on ice, followed by buffer exchange into a new buffer. The protein is then treated with maleimide-PEG-tetrazine (n=3). After the reaction is complete, the protein is exchanged into a new buffer to remove excess reagent, yielding the conjugate (Ab-Tz, Figure 11). The conjugate is analyzed by SDS-PAGE, analytical HPLC, and mass spectrometry to confirm its expected properties. The conjugate can also be treated with a trans-cyclooctene-functionalized fluorophore to confirm the reactivity of the tetrazine.

[0548] A non-binding control protein with a C-terminal cysteine ​​is expressed in E. coli and purified to homogeneity. The protein is treated with a reducing agent such as TCEP at ambient temperature or on ice, followed by buffer exchange into a new buffer. The protein is then treated with maleimide-PEG-tetrazine (n=3). After the reaction is complete, the protein is exchanged into a new buffer to remove excess reagent, yielding the conjugate (Ab-Tz). The conjugate is analyzed by SDS-PAGE, analytical HPLC, and mass spectrometry to confirm the expected properties. The conjugate can also be treated with a trans-cyclooctene-functionalized fluorophore to confirm the reactivity of the tetrazine.

[0549] Example 7: HCC1954 xenograft model Animal experiments were performed according to IACUC protocols in accordance with AAALAC guidelines. Female Balb / c nude mice were implanted with log-phase grown HCC1954 cells (5 × 10) in 0.2 mL of PBS into the right flank. 6 cells + Matrigel). Tumor volume was approximately 200 mm 3Animals were randomized when the animals reached 100 mg / kg / day. Animals were dosed intravenously with saline (days 1-4), the methyltetrazine-Fab targeting moiety from Example 2 (day 0, 5 mg / kg) plus saline (days 1-4), or the methyltetrazine-Fab targeting moiety from Example 2 (day 0, 5 mg / kg) plus doxorubicin-TCO prodrug (days 1, 2, 3, and 4, 120 mg / kg).

[0550] The doxorubicin-TCO prodrug has the structure shown below and was prepared according to the method described in WO 2020 / 077140.

[0551] [ka]

[0552] Tumor volume was measured twice weekly in two dimensions using calipers and this volume was calculated using the formula: V = 0.5a × b 2 (where a and b are the long and short diameters of the tumor, respectively) 3 Figure 5 shows tumor growth over a 33 day period. The reduction in tumor growth upon administration of the prodrug demonstrates the efficacy of the system described therein.

[0553] Prodrugs suitable for use in the methods disclosed herein can be prepared and administered as described in WO 2020 / 077140, WO 2018 / 187740, WO 2017 / 044983, WO 2015 / 139025, and WO 2014 / 205126.

[0554] Example 8: General procedure for the preparation of compound A [ka]

[0555] To a solution of MMAE (1.40 g, 1.95 mmol) and DIEA (690 mg, 5.34 mmol) in DMF (4.00 mL) was added compound 2 (800 mg, 1.79 mmol) in DMF (4.00 mL) at 0° C., and the mixture was stirred at 25° C. for 16 h. HOBt (480 mg, 3.56 mmol) in DMF (0.50 mL) was then added to the above reaction mixture at 0° C., and the reaction mixture was stirred at 25° C. for 1.0 h. The reaction mixture was then cooled to 0° C., and TBAF (1 M in THF, 4.45 mL) was added. The mixture was stirred at 25° C. for 2.0 h. Another batch of TBAF (1 M in THF, 4.45 mL) was then added at 0° C., and the reaction mixture was continued to stir at 25° C. for 12.0 h. LC-MS indicated one major peak was the desired mass. The resulting reaction mixture was purified by preparative HPLC (column: Welch XB-C18 7 μm 110 Å 250 mm × 50 mm; mobile phase: [water (0.1% TFA)-ACN]; B%: 50% → 70% in 40 min, injections: 2, retention time: 37 min, flow rate: 60 mL / min) to give compound A (450 mg, 99.0% purity; 64.6 mg, 99.2%, 31.2% yield).

[0556] LCMS (m / z): 928.6 [M+H] +

[0557] 1HNMR: (400 MHz, DMSO-d6): δ8.46 - 8.28 (m, 1H), 8.03 - 7.84 (m, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.35 - 7.23 (m, 4H), 7.21 - 7.13 (m, 1H), 6.05 - 5.57 (m, 2H), 5.10 (s, 1H), 4.81 - 4.39 (m, 3H), 4.35 - 4.19 (m, 1H), 4.05 - 3.92 (m, 2H), 3.40 - 3.09 (m, 11H), 3.08 - 2.83 (m, 5H), 2.48 - 2.37 (m, 2H), 2.31 - 2.09 (m, 5H), 2.07 - 1.89 (m, 3H), 1.88 - 1.64 (m, 6H), 1.63 - 1.33 (m, 4H), 1.32 - 1.16 (m, 1H), 1.08 - 0.97 (m, 9H), 0.90 - 0.68 (m, 18H).

[0558] Example 9: General preparation procedure for compound B

change

[0559] General recipe for compound 4

change

[0560] To a solution of compound 2 (1.00 g, 5.43 mmol) in DCM (10 mL) were added DIEA (2.10 g, 16.3 mmol), EDCI (2.08 g, 10.9 mmol), and DMAP (1.33 g, 10.9 mmol), and compound 3 (1.61 g, 8.14 mmol). The mixture was stirred at 25 °C for 16 h. TLC showed that compound 2 was completely consumed and one new spot had formed. The reaction mixture was partitioned between DCM (20 mL) and HO (10 mL). The organic phase was separated and washed with saturated aqueous citric acid (3 mL) and brine (20 mL), then dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1 → 1 / 1) to give compound 4 (700 mg, 39.4% yield).

[0561] 1 HNMR (400MHz, CDCl3): δ ppm 1.12 (s, 3 H), 1.60 (dd, J =15.45, 6.19 Hz, 1 H), 1.79 - 1.87 (m, 2 H), 1.92 (br d, J = 5.88 Hz, 1 H), 1.95 (s, 1 H), 1.98 - 2.00 (m, 1 H), 2.02 (br d, J = 4.13 Hz, 1 H), 2.26 (dd, J = 11.63, 3.88 Hz, 1 H), 2.30 - 2.36 (m, 1 H), 2.77 - 2.89 (m, 1 H), 2.88 - 2.88 (m, 1 H), 3.00 (dd, J = 16.95, 4.57 Hz, 1 H), 3.70 (s, 4 H), 3.75 (s, 3 H), 4.80 (dt, J = 8.00, 4.50 Hz, 1 H), 5.66 (dd, J = 16.63, 2.38 Hz, 1 H), 6.02 - 6.12 (m, 1 H), 6.54 (br d, J = 7.88 Hz, 2 H).

[0562] General procedure for the preparation of compound 6 [ka]

[0563] To a solution of compound 4 (700 mg, 2.14 mmol) in DCM (5 mL) was added Py (846 mg, 10.7 mmol) and compound 5 (1.72 g, 8.55 mmol) in DCM (5 mL). The mixture was stirred at 25 °C for 1 h. TLC showed that compound 4 was completely consumed and one new spot had formed. The reaction mixture was partitioned between DCM (20 mL) and HO (10 mL). The organic phase was separated, washed with saturated aqueous citric acid (3 mL) and brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1 → 1 / 1) to give compound 6 (490 mg, 46.5% yield).

[0564] 1 HNMR (400MHz, CDCl3): δ ppm 1.17 (s, 3 H), 1.55 - 1.61 (m, 1 H), 1.58 (br s, 1 H), 1.76 (dd, J = 14.76, 6.25 Hz, 1 H), 1.87 - 2.03 (m, 3 H), 2.06 - 2.15 (m, 1 H), 2.19 - 2.41 (m, 3 H), 2.82 (dd, J = 17.13, 4.50 Hz, 1 H), 3.03 (dd, J = 17.07, 4.44 Hz, 1 H), 3.72 (s, 3 H), 3.77 (s, 3 H), 4.78 - 4.86 (m, 1 H), 5.67 (dd, J = 16.70, 2.44 Hz, 1 H), 6.03 - 6.14 (m, 1 H), 6.58 (br d, J = 7.88 Hz, 1 H), 7.40 - 7.45 (m, 2 H), 8.27 - 8.33 (m, 2 H).

[0565] General procedure for the preparation of compound 7 [ka]

[0566] To a solution of compound 6 (490 mg, 995 μmol) and MMAE (714 mg, 995 μmol) in DMF (4 mL) was added DIEA (64.3 mg, 497 μmol) and HOBt (202 mg, 1.49 mmol). The mixture was stirred at 25 °C for 16 h. LC-MS showed that compound 6 was completely consumed, with one major peak having the desired mass. The residue was purified by preparative HPLC (0.1% TFA) to give compound 7 (500 mg, 46.9% yield).

[0567] 11H NMR (400 MHz, CDCl3): δ ppm 0.84 (broad d, J = 6.75 Hz, 4 H), 0.89 (broad d, J = 4.50 Hz, 5 H), 0.92 (broad d, J = 6.63 Hz, 4 H), 0.98 (broad d, J = 6.25 Hz, 3 H), 1.04 (broad d, J = 6.88 Hz, 3 H), 1.16 (s, 3 H), 1.25 - 1.27 (m, 3 H), 1.59 - 1.74 (m, 3 H), 1.88 (broad d, J = 9.38 Hz, 4 H), 2.07 (broad d, J = 8.38 Hz, 5 H), 2.27 (broad s, 4 H), 2.36 - 2.43 (m, 2 H), 2.45 - 2.53 (m, 1 H), 2.89 (broad s, 6 H), 2.95 - 3.01 (m, 4 H), 3.04 (broad s, 2 H), 3.29 - 3.34 (m, 3 H), 3.36 - 3.47 (m, 5 H), 3.67 - 3.73 (m, 4 H), 3.76 (s, 3 H), 3.82 - 3.89 (m, 1 H), 4.05 - 4.19 (m, 3 H), 4.28 (broad s, 1 H), 4.63 - 4.86 (m, 3 H), 4.96 (d, J = 2.50 Hz, 1 H), 5.24 (broad s, 1 H), 5.63 (broad d, J = 18.14 Hz, 1 H), 5.82 (broad s, 1 H), 6.53 - 6.74 (m, 3 H), 7.30 - 7.41 (m, 5 H).

[0568] General Preparation Procedure of Compound B [Chemical Structure Diagram]

[0569] To a solution of compound 7 (500 mg, 467 μmol) in MeOH (5 mL) was added LiOH·HO (196 mg, 4.67 mmol) in HO (2 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed that compound 7 was completely consumed, with one major peak having the desired mass. The residue was adjusted to a pH of approximately 2 with saturated aqueous citric acid and then purified by preparative HPLC (0.1% TFA) to give compound B (265 mg, 53.4% ​​yield).

[0570] 1 HNMR (400MHz, CDCl3): δ ppm 0.80 - 1.04 (m, 25 H), 1.09 (s, 3 H), 1.24 (d, J = 6.88 Hz, 3 H), 1.69 - 1.82 (m, 2 H), 1.88 - 1.94 (m, 3 H), 2.02 - 2.11 (m, 4 H), 2.16 (br d, J = 18.64 Hz, 1 H), 2.11 - 2.24 (m, 2 H), 2.32 (br d, J = 5.25 Hz, 2 H), 2.40 - 2.45 (m, 1 H), 2.52 (br d, J = 5.50 Hz, 2 H), 2.81 (br dd, J = 14.01, 4.88 Hz, 1 H), 2.94 - 3.04 (m, 2 H), 3.08 (s, 2 H), 3.14 - 3.27 (m, 6 H), 3.33 (s, 1 H), 3.39 (s, 3 H), 3.48 - 3.57 (m, 2 H), 3.94 (br d, J = 1.25 Hz, 1 H), 4.05 - 4.18 (m, 4 H), 4.30 (br dd, J = 6.19, 4.82 Hz, 2 H), 4.54 - 4.67 (m, 4 H), 4.91 (br d, J = 2.00 Hz, 2 H), 5.30 (br s, 1 H), 5.64 - 5.73 (m, 1 H), 5.79 - 5.89 (m, 1 H), 6.61 (br d, J = 7.38 Hz, 1 H), 7.30 - 7.42 (m, 5 H), 7.55 - 7.64 (m, 1 H).

[0571] Example 10: Alternative Route to Compound B and Synthesis of Compound C [ka]

[0572] General procedure for the preparation of compound 2 [ka]

[0573] To a solution of compound 1 (20.0 g, 83.2 mmol) in MeOH (80 mL) was added KOH (8.19 g, 124 mmol) in HO (80 mL). The mixture was stirred at 25 °C for 24 h. The reaction was monitored by TLC (compound 1, PE / EtOAc = 5 / 1, R f =0.5). The reaction mixture was extracted with MTBE (3 × 400 mL). The combined organic layers were washed with water (100 mL), dried over NaSO, filtered, and concentrated in vacuo to give the undesired ester. While cooling in an ice-water bath (T below 7 °C), the aqueous layer was acidified with 1 M HCl to pH = 4. The aqueous layer was extracted with MTBE (3 × 400 mL). The combined MTBE layers were dried over NaSO, filtered, and concentrated in vacuo to give compound 2 (5.50 g, 35.9% yield). The crude product was used in the subsequent step without further purification.

[0574] 1 H NMR: (400 MHz, DMSO-d6): δ ppm 11.9 (br s, 1 H), 5.81 - 5.94 (m, 1 H), 5.58 (dd, J = 16.45, 2.31 Hz, 1 H), 4.65 (br s, 1 H), 4.24 (br s, 1 H), 2.04 - 2.24 (m, 2 H), 1.87 - 2.03 (m, 1 H), 1.61 - 1.86 (m, 4 H), 1.36 - 1.46 (m, 1 H), 0.97 (s, 3 H).

[0575] General procedure for the preparation of compound 3 [ka]

[0576] To a solution of compound 2 (8.00 g, 43.4 mmol) in MeCN (160 mL) was added DIEA (39.3 g, 304 mmol) and DSC (47.8 g, 186.4 mmol). The mixture was stirred at 40° C. for 14 hours. Completion of the reaction was confirmed by TLC (compound 2, DCM / MeOH=10 / 1, R f =0.5). After pouring the reaction mixture into water (400 mL), the temperature rose from 20 to 27 °C. After 15 minutes, the mixture was cooled to 17 °C in an ice-water bath and stirred for 15 minutes. The solid was filtered, washed with water (3 × 20 mL), and dried under vacuum at 35 °C for 4 hours to give crude compound 3 (9.60 g). Acetonitrile (20 mL) was added to the crude, and the mixture was heated at 40 °C for 1 hour using mechanical stirring. The heating was stopped, and the mixture was cooled to 8 °C in an ice-water bath for 15 minutes. The solid was filtered, washed with acetonitrile (2 × 10 mL), and dried under vacuum at 35 °C for 3 hours to give compound 3 (6.65 g, 36.3% yield).

[0577] 1 H NMR: (400 MHz, CDCl3): δ 6.03 - 6.14 (m, 1 H), 5.60 - 5.67 (m, 1 H), 5.29 (br s, 1 H), 2.80 - 2.88 (m, 8 H), 2.25 - 2.47 (m, 4 H), 1.94 - 2.18 (m, 4 H), 1.29 (s, 3 H).

[0578] General procedure for the preparation of compound 4 [ka]

[0579] To a solution of compound 3 (100 mg, 0.24 mmol) in DMF (20 mL) was added MMAE (136 mg, 0.19 mmol) and DIEA (61.2 mg, 0.47 mmol). The mixture was stirred at 25 °C for 16 h. LC-MS showed that one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% FA)-ACN) to give compound 4 (41.0 mg, 16.9% yield).

[0580] LCMS (m / z): 1025.6 (M+H) + .

[0581] General procedure for preparing compound B [ka]

[0582] To a solution of compound 4 (500 mg, 0.49 mmol) and compound 4-1 (519 mg, 3.90 mmol) in DMF (10 mL) was added DIEA (378 mg, 2.93 mmol) and DMAP (119 mg, 0.97 mmol). The mixture was stirred at 25 °C for 12 h. LC-MS showed that one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound B (161 mg, 31.6% yield).

[0583] 1H NMR: (400 MHz, MeOD): δ 7.73 - 8.00 (m, 1 H), 7.20 - 7.39 (m, 4 H), 5.78 - 5.96 (m, 1 H), 5.73 (br s, 1 H), 5.20 - 5.28 (m, 1 H), 5.13 - 5.20 (m, 1 H), 4.49 - 4.74 (m, 3 H), 4.17 - 4.28 (m, 2 H), 4.04 - 4.10 (m, 1 H), 3.85 - 3.90 (m, 1 H), 3.50 - 3.80 (m, 2 H), 3.31 - 3.50 (m, 9 H), 3.28 - 3.30 (m, 3 H), 2.77 - 3.12 (m, 6 H), 2.44 - 2.58 (m, 2 H), 1.78 - 2.36 (m, 13 H), 1.56 - 1.72 (m, 2 H), 1.22 - 1.48 (m, 3 H), 1.08 - 1.23 (m, 9 H), 0.80 - 1.07 (m, 18H).

[0584] LCMS (m / z): 1043.62 (M+H) + ; 1065.61 (M+Na) + .

[0585] General procedure for the preparation of compound C [ka]

[0586] To a solution of compound 4 (320 mg, 0.31 mmol) and compound 4-2 (187 mg, 2.50 mmol) in DMF (3.2 mL) was added DIEA (242 mg, 1.87 mmol) and DMAP (76.3 mg, 0.62 mmol). The mixture was stirred at 25 °C for 12 h. LC-MS showed that compound 4 was completely consumed and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound C (92.0 mg, 29.9% yield).

[0587] 1 H NMR (400 MHz, MeOD): δ 7.86 - 8.00 (m, 1 H), 7.15 - 7.45 (m, 5 H), 5.67 - 5.98 (m, 2 H), 5.17 (br s, 1 H), 4.50 - 4.74 (m, 2 H), 4.03 - 4.29 (m, 3 H), 3.81 - 3.89 (m, 2 H), 3.51 - 3.77 (m, 2 H), 3.46 - 3.50 (m, 1 H), 3.33 - 3.45 (m, 5 H), 3.30 (br s, 4 H), 3.20 (dt, J = 11.57, 7.47 Hz, 1 H), 3.02 - 3.15 (m, 3 H), 2.90 - 3.01 (m, 1 H), 2.41 - 2.57 (m, 2 H), 1.65 - 2.39 (m, 15 H), 1.52 - 1.64 (m, 1 H), 1.26 - 1.51 (m, 2 H), 1.08 - 1.23 (m, 9 H), 0.82 - 1.07 (m, 18 H).

[0588] LCMS (m / z): 985.6 (M+H) + .

[0589] Example 11: Synthesis of Compound D

change

[0590] General recipe for compound 6

change

[0591] To a solution of compound 5 (150 g, 689 mmol, HCl) in NaOH (1.38 L, 1 M) and NaHCO (1.38 L, 1 M) was added (2,5-dioxopyrrolidin-1-yl) 2,2,2-trichloroethyl carbonate (210 g, 723 mmol) in dioxane (1 L). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove dioxane. The residue was extracted with MTBE (5 L), and the aqueous phase was adjusted to a pH of about 4 with saturated aqueous KHSO and extracted with EtOAc (5 L). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give the residue. To a solution of the above crude material in MeOH (2 L) was added SOCl (90.2 g, 758 mmol), and the mixture was stirred at 25 °C for 2 h. LC-MS showed the reaction was complete, with one major peak having the desired mass. The reaction mixture was adjusted to a pH of approximately 9-10 with saturated aqueous NaHCO3 and then extracted with EtOAc (5 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was precipitated with PE (10 vol) to give compound 6 (190 g, 74.4% yield).

[0592] 1 H NMR: (400 MHz, CDCl3): δ 3.25 (br s, 1 H) 3.85 (s, 3 H) 4.64 - 4.83 (m, 2 H) 5.30 (dd, J = 9.51, 1.13 Hz, 1 H) 5.92 (br d, J = 9.38 Hz, 1 H) 7.30 - 7.45 (m, 5H).

[0593] LCMS (m / z): 391.9 / 393.9 (M+H) + .

[0594] General procedure for the preparation of compound 7 [ka]

[0595] To a solution of compound 6 (185 g, 499 mmol) in toluene (1.9 L) was added 4-methylbenzenesulfonic acid pyridine (3.90 g, 15.4 mmol) and 4-methoxybenzaldehyde dimethyl acetal (121 g, 666 mmol). The mixture was stirred at 110 °C for 4 hours. LC-MS showed that one major peak with the desired mass was detected. The reaction mixture was then cooled to 25 °C. The reaction mixture was concentrated under reduced pressure to remove toluene. The residue was diluted with HO (500 mL) and then extracted with EtOAc (500 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give compound 7 (285 g, crude), which was carried on directly.

[0596] General procedure for the preparation of compound 8 [ka]

[0597] To a solution of compound 7 (285 g, crude) in MeOH (2000 mL) was added KOH (42.5 g, 758 mmol) in HO (1000 mL). The mixture was stirred at 25 °C for 1 h. LC-MS showed that compound 7 was completely consumed, with one major peak having the desired mass. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was extracted with MTBE (5 L). The aqueous layer was diluted with saturated aqueous KHSO (1 L) and extracted with EtOAc (5 L). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give the crude product. The crude product was precipitated with PE (10 vol) to give compound 8 (95.0 g, 34.3% yield).

[0598] 1H NMR (400 MHz, MeOD): δ 3.82 (s, 3 H) 4.41 - 4.47 (m, 1 H) 4.50 - 4.56 (m, 1 H) 4.60 (d, J = 4.88 Hz, 1 H) 5.47 (d, J = 4.75 Hz, 1 H) 6.46 (s, 1 H) 6.86 - 6.94 (m, 2 H) 7.34 - 7.46 (m, 7 H).

[0599] LCMS (m / z): 495.9 (M+Na) + .

[0600] General procedure for the preparation of 7-Troc-baccatin III [ka]

[0601] To a solution of baccatin III (30.0 g, 51.1 mmol) in DCM (300 mL) was added DMAP (625 mg, 5.11 mmol), pyridine (14.2 g, 179 mmol), and 2,2,2-trichloroethyl carbonochloridate (15.2 g, 71.6 mmol). The mixture was stirred at 25 °C for 0.5 h. LC-MS showed that baccatin III was completely consumed, with one major peak having the desired mass being detected. The residue was diluted with water (300 mL), extracted with DCM (300 mL), washed with water (200 mL) and brine (200 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give 7-Troc-baccatin III (45.0 g, 34.3% yield).

[0602] LCMS (m / z): 761.5 / 763.5 (M+Na) + .

[0603] General procedure for the preparation of compound 9 [ka]

[0604] To a solution of 7-Troc-baccatin III (26.0 g, 34.1 mmol) and compound 8 (32.4 g, 68.2 mmol) in DCM (1000 mL) was added DMAP (4.20 g, 34.1 mmol) and DCC (21.1 g, 102 mmol). The mixture was stirred at 0 °C for 1 h. LC-MS showed that compound 8 was completely consumed, with one major peak having the desired mass being detected. The reaction mixture was filtered. The crude product was washed with saturated aqueous NH4Cl (100 mL) and water (1000 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 9 (35.0 g, crude).

[0605] LCMS (m / z): 1240.0 / 1242.0 (M+Na) + .

[0606] General procedure for the preparation of compound 10 [ka]

[0607] To a solution of compound 9 (80.0 g, 65.6 mmol) in MeOH (350 mL) was added 4-methylbenzenesulfonic acid hydrate (24.9 g, 131 mmol). The mixture was stirred at 25 °C for 16 h. LC-MS showed that approximately 50% of compound 9 remained, and one major peak with the desired mass was detected. The reaction mixture was filtered and concentrated, and the residue was purified by preparative HPLC (water (0.1% TFA)-ACN). The eluate was concentrated under reduced pressure to remove the solvent, followed by extraction with EtOAc (500 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 10 (13.0 g, 17.9% yield).

[0608] LCMS (m / z): 1120.2 (M+Na) + .

[0609] General procedure for the preparation of compound 11 [ka]

[0610] Compound 10 (13.0 g, 11.8 mmol), DMAP (722 mg, 5.90 mmol), EDCI (2.70 g, 14.2 mmol), and benzoic acid (1.70 g, 14.2 mmol) were dissolved in DCM (260 mL). The mixture was stirred at 25 °C for 1 h. LC-MS showed that compound 10 was completely consumed, with one major peak having the desired mass being detected. The reaction mixture was washed with saturated aqueous citric acid (100 mL), saturated aqueous NaHCO (100 mL), and water (200 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give compound 11 (11.0 g, 77.3% yield).

[0611] LCMS (m / z): 1204.1 (M+H) + .

[0612] General procedure for the preparation of compound 12 [ka]

[0613] To a solution of compound 11 (20.0 g, 16.6 mmol) in MeOH (200 mL) and AcOH (200 mL) was added zinc dust (21.6 g, 331 mmol). The mixture was stirred at 25 °C for 1 h. LC-MS showed that compound 11 was completely consumed, with one major peak having the desired mass being detected. The reaction mixture was filtered, diluted with HO (500 mL), and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with saturated aqueous NaHCO (200 mL) and brine (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give the crude product. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound 12 (5.0 g, 21% yield).

[0614] LCMS (m / z): 854.3 (M+H) + .

[0615] General procedure for the preparation of compound 13 [ka]

[0616] Compound 12 (5.00 g, 5.90 mmol), DIEA (1.50 g, 11.7 mmol), and compound 3 (3.90 g, 8.80 mmol) were dissolved in DMF (50 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed that approximately 50% of compound 12 remained, and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound 13 (505 mg, 7.4% yield).

[0617] LCMS (m / z): 1161.4 (M+H) + .

[0618] General procedure for the preparation of compound D [ka]

[0619] To a solution of compound 13 (150 mg, 0.13 mmol) in DMF (1.50 mL), DMAP (94.7 mg, 0.78 mmol), compound 13-1 (66.7 mg, 0.65 mmol), and DIEA (100 mg, 0.78 mmol) were added. The mixture was stirred at 25 °C for 16 h. LC-MS showed that compound 13 was completely consumed, with one major peak having the desired mass being detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound D (75.0 mg, 50.5% yield).

[0620] LCMS (m / z): 1148.5 (M) + .

[0621] Example 12: General procedure for the preparation of compound E [ka]

[0622] Compound 13 (350 mg, 0.30 μmol), DMAP (221 mg, 1.81 mmol), and compound 14 (249 mg, 0.39 mmol, HCl) were dissolved in DMF (0.3 mL). The mixture was stirred at 25° C. for 16 hours. LC-MS showed that compound 13 was completely consumed, and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound E (205 mg, 41.3% yield).

[0623] LCMS (m / z): 1646.5 (M+H) + .

[0624] Example 13: 3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-tetrazine [ka]

[0625] N-Boc-3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-tetrazine (2). To a solution of N-Boc-2-cyano-5-aminomethyl-pyrimidine (1) in dry acetonitrile was added hydrazine and nickel(II) triflate. The reaction mixture was then heated overnight, after which the starting material was consumed by TLC. Sodium nitrite (dissolved in water) was added to the reaction mixture, followed by 1 M hydrochloric acid. The reaction mixture was then stirred at ambient temperature until the reaction was complete as determined by HPLC. The reaction mixture was then partitioned between ethyl acetate and water. The organic layer was washed with water (3x) and then brine (1x) before drying over sodium sulfate. The solution was filtered, and the filtrate was concentrated under reduced pressure to give the product, which could be carried forward without further purification.

[0626] 3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-tetrazine (3). To a solution of 2 in dioxane is added hydrochloric acid (4 M in dioxane). The reaction mixture is then stirred at ambient temperature until the starting material is consumed. The product is isolated by filtration, and the precipitate is washed with diethyl ether to give the product, optionally as the HCl salt.

[0627] The compounds and methods described herein can also utilize 6-(6-methyl-1,2,4,5-tetrazin-3-yl)-3-pyridinemethanamine, which can be prepared according to the art or purchased from commercial sources (e.g., Enamine US Inc., New Jersey, USA).

[0628] [ka]

[0629] Example 14: Val-Cit-PABC-dihydrotetrazine [ka]

[0630] N-Boc-3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-dihydrotetrazine (4). In a sealed flask, thiourea dioxide is added to a solution of tetrazine 2 in DMF / HO (v / v = 10 / 1) at ambient temperature. The reaction mixture is then heated with stirring until the color of the solution changes from pink to colorless. The reaction mixture is concentrated under reduced pressure, and the resulting residue is dried under vacuum to give dihydrotetrazine 4, which can be used directly without further purification.

[0631] To the solution of dihydrotetrazine 4 is added a solution of nitrophenyl carbonate 5 in toluene. The reaction mixture is then stirred at ambient temperature. Upon completion, the reaction mixture is concentrated under reduced pressure and the resulting residue is purified by flash chromatography to give compound 6.

[0632] 1-(N-Acyl-(Val-Cit)-PABC)-3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-dihydrotetrazine (7). To a solution of 6 in dioxane is added hydrochloric acid (4 M in dioxane). The reaction mixture is then stirred at ambient temperature until the starting material is consumed. The product is isolated by filtration, and the precipitate is washed with diethyl ether to give the product, optionally as the HCl salt.

[0633] Example 15: Dihydrotetrazine (Target 5) [ka]

[0634] To a solution of N2H4·HO (9.74 g, 190 mmol, 9.44 mL, 98% purity, 7.08 equiv.) in EtOH (35.0 mL) was added compound 1 (5.00 g, 26.9 mmol, 1.00 equiv., HCl) and compound 2 (2.55 g, 26.9 mmol, 1.00 equiv., HCl) at 20 °C. The mixture was stirred at 78 °C for 3 h. LCMS analysis of the reaction mixture indicated that compound 1 was completely consumed. After adding HO (100 mL) to the reaction mixture at 20 °C, the resulting solution was concentrated under reduced pressure at 40 °C to remove EtOH. The reaction mixture was extracted with 150 mL of ethyl acetate (50.0 mL × 3), and the combined organic layers were washed with 20.0 mL of brine (20.0 mL × 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , n-heptane / ethyl acetate=100 / 1→1 / 1) and then further purified by preparative HPLC (HCl condition) to give target 5 (100 mg, 2.87 mmol) as a white solid. 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.94 - 8.13 (m, 2H), 7.53 - 7.75 (m, 3H), 2.64 (s, 3H). LCMS: M+H + = 175.1

[0635] Example 16: 3-methyl-6-(1-methyl-1H-imidazol-4-yl)-1,2,4,5-tetrazine (Target 4) [ka]

[0636] 3-Methyl-6-(1-methyl-1H-imidazol-4-yl)-1,2,4,5-tetrazine (Target 4): A solution of 1-methylimidazole-4-carbonitrile (200 mg, 1.87 mmol), MeCN (268 mg, 6.54 mmol), and zinc trifluoromethanesulfonate (68 mg, 0.19 mmol) in dioxane (1 mL) was treated with NHNH at 25 °C. 20 HO (2.34 g, 46.68 mmol) was added, and the mixture was stirred at 65 °C under N for 16 h. The mixture was then cooled to 25 °C, and a solution of NaNO (387 mg, 5.60 mmol) in HO (3 mL) was added dropwise at 25 °C. The mixture was stirred at 25 °C for 3 h. The mixture was cooled to room temperature and adjusted to pH = 3 with 1 M aqueous hydrochloric acid. The aqueous phase was extracted with DCM (3 × 5 mL). The combined organics were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC (SiO, DCM:MeOH = 10:1) to give 3-methyl-6-(1-methyl-1H-imidazol-4-yl)-1,2,4,5-tetrazine (target 4) (20.1 mg, 6.1%).

[0637] LCMS (ESI+): m / z = 177.2 [M+H] +

[0638] 1 H NMR (400MHz, CDCl3) (ET60578-12-P1M): δ = 8.00 (s, 1H), 7.68 (s, 1H), 3.85 (s, 3H), 3.06 (s, 3H).

[0639] Example 17: 3-(6-methyl-1,2,4,5-tetrazin-3-yl)isoxazole (Target 8) [ka]

[0640] Isoxazole-3-carboxamide (2): To a solution of isoxazole-3-carboxylic acid (10 g, 88.44 mmol) in DMF (646 mg, 8.84 mmol) and DCM (100 mL) was added oxalyl dichloride (13.47 g, 106.13 mmol) at 0 °C under N. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in THF (50 ml) and adjusted to pH = 9 with NH.HO at 0 °C. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give isoxazole-3-carboxamide (2) (3.3 g, 33.3%).

[0641] 1 H NMR (400MHz, DMSO): δ = 9.05 (d, J = 1.6 Hz, 1H), 8.12 (s, 1H), 7.82 (s, 1H), 6.85 (d, J = 1.6 Hz, 1H)

[0642] Isoxazole-3-carbonitrile (3): To a solution of isoxazole-3-carboxamide (500 mg, 4.46 mmol) in pyridine (18 mL) was added POCl (1.03 g, 6.69 mmol) at 20 °C under N. The mixture was stirred at 20 °C for 2 h. After stirring for 2 h, the mixture was cooled in an ice bath and water (10 mL) was added. The aqueous phase was adjusted to pH = 4 by adding 3 M aqueous HCl and extracted with MTBE (3 × 10 mL). The combined organics were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give isoxazole-3-carbonitrile (3) (280 mg, 66.7%).

[0643] 1 H NMR (400MHz, MeOD): δ 9.06 (d, J = 1.6 Hz, 1H), 7.02 (d, J = 1.2 Hz, 1H)

[0644] 3-(6-Methyl-1,2,4,5-tetrazin-3-yl)isoxazole (Target 8): To a solution of isoxazole-3-carbonitrile (220 mg, 2.34 mmol) in EtOH (2 mL) was added NH2NH2·HO (1.87 g, 37.42 mmol) at 20 °C for 0.5 h. Then, MeCN (384 mg, 9.35 mmol) and 3-sulfanylpropanoic acid (248 mg, 2.34 mmol) were added to the mixture at 20 °C under N2. The mixture was stirred at 45 °C for 12 h. Then, NaNO2 (500 mg) was added, and the reaction mixture was stirred at 20 °C for 0.5 h. 3 M hydrochloric acid was added dropwise to pH = 1, and the reaction mixture was stirred at 20 °C for 0.5 h. The aqueous phase was extracted with EtOAc (3 × 10 mL). The combined organic phase was washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (FA) under the following conditions: column: Phenomenex Luna C18 100 mm × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; B%: 5% → 45% in 8 min to give 3-(6-methyl-1,2,4,5-tetrazin-3-yl)isoxazole (12.45 mg, 3.26%). LCMS (ESI+): m / z = 164.1 [M+H] +

[0645] 1 H NMR (400MHz, MeOD): δ 9.01 (d, J = 1.6 Hz, 1H), 7.34 (d, J = 1.6 Hz, 1H), 3.11 (s, 3H).

[0646] Example 18: 3-Methyl-6-(1H-pyrazol-1-yl)-1,2,4,5-tetrazine (Target 13) [ka]

[0647] Methyl (E)-hydrazinecarbohydrazonothioate (2): To a mixture of 1,3-diaminothiourea (100 g, 942.06 mmol) in MeOH (500 mL) was added MeI (160.46 g, 1.13 mol) at 25 °C, and the mixture was stirred at 80 °C under N for 1.5 h. H NMR showed the reaction was complete. The resulting pale yellow solution was cooled to room temperature until a solid precipitated. It was then diluted with MTBE (500 mL). The mixture was cooled in ice for 2 h and filtered. The collected solid was washed with MTBE and dried under reduced pressure to give 1,3-diamino-2-methyl-isothiourea hydroiodide (compound 2) (132 g, 56.48% yield, HI).

[0648] 1 H NMR (400MHz, DMSO): δ = 10.96 - 9.08 (m, 1H), 5.80 - 4.81 (m, 2H), 2.37 (s, 3H)

[0649] 3-Methyl-6-(methylthio)-1,2,4,5-tetrazine (4): To a solution of 1,3-diamino-2-methyl-isothiourea (30 g, 249.63 mmol) in DMF (750 mL) was added 1,1,1-triethoxyethane (44.55 g, 274.60 mmol) at 25 °C, and the mixture was stirred at 25 °C for 5 minutes under N2. Then, TEA (25.26 g, 249.63 mol) was added to the mixture at 25 °C, and the mixture was stirred at 50 °C for 3 hours. TLC showed the reaction was complete. The mixture was poured into water (1000 mL) and extracted with EtAc (3 × 500 mL). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=10 / 1→5 / 1) to give 3-methyl-6-methylsulfanyl-1,2,4,5-tetrazine (compound 4) (5.8 g, 16.3%).

[0650] 1 H NMR (400MHz, CDCl3): δ = 2.99 (s, 3H), 2.74 (s, 3H).

[0651] 3-Methyl-6-(1H-pyrazol-1-yl)-1,2,4,5-tetrazine (Target 13): To a mixture of 1H-pyrazole (144 mg, 2.11 mmol) in THF (2 mL) was added NaH (84 mg, 2.11 mmol, 60% purity) at 0 °C, and the mixture was stirred at 0 °C for 15 min under N. Then, 3-methyl-6-methylsulfanyl-1,2,4,5-tetrazine (200 mg, 1.41 mmol) in THF (1 mL) was added to the mixture at 0 °C, and the mixture was stirred at 25 °C under N for 2 h. TLC showed the reaction was complete. The mixture was poured into saturated NH Cl (5 mL) and extracted with EtAc (3 × 3 mL). The combined organic layers were washed with brine (3 mL), dried over Na SO , filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC (SiO, PE: EtOAc = 3:1) to give 3-methyl-6-pyrazol-1-yl-1,2,4,5-tetrazine (target 13) (20 mg, 8.8%). LCMS (ESI+): m / z = 163.1 [M+H] +

[0652] 1 H NMR (400MHz, CDCl3): δ = 8.74 (d, J = 2.8 Hz, 1H), 8.02 (d, J = 1.2 Hz, 1H), 6.68 (dd, J = 1.6, 2.8 Hz, 1H), 3.14 (s, 3H).

[0653] Example 19: (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 15) [ka]

[0654] tert-Butyl (4-cyano-2-(trifluoromethyl)benzyl)carbamate (2): To a solution of 4-(aminomethyl)-3-(trifluoromethyl)benzonitrile (300 mg, 1.50 mmol) in DCM (10 mL) was added BocO (360 mg, 1.65 mmol) and TEA (227 mg, 2.25 mmol) at 20 °C under N. The mixture was stirred at 20 °C for 2 h. The mixture was cooled and poured into HO (10 mL), and the aqueous phase was extracted with DCM (3 × 10 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (PE: EtOAc = 1:0 to 3:1) to give tert-butyl (4-cyano-2-(trifluoromethyl) benzyl) carbamate (2) (400 mg, 88.9%).

[0655] 1 H NMR (400MHz, CDCl3): δ 7.94 (s, 1H), 7.87 - 7.81 (m, 1H), 7.79 - 7.73 (m, 1H), 5.00 (s, 1H), 4.56 (d, J = 6.0 Hz, 2H), 1.47 (s, 9H).

[0656] tert-Butyl (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (3): To a solution of tert-butyl N-[[4-cyano-2-(trifluoromethyl)phenyl]methyl]carbamate (150 mg, 0.50 mmol) in EtOH (0.5 mL) was added NH2NH2·HO (400 mg, 7.99 mmol) at 20 °C for 0.5 h. Then, MeCN (82 mg, 2.00 mmol) and 3-sulfanylpropanoic acid (53 mg, 0.50 mmol) were added to the mixture at 20 °C under N2. Then, NaNO2 (500 mg) was added, and the reaction mixture was stirred at 20 °C for 0.5 h. 3 M hydrochloric acid was added until pH = 1, and the reaction mixture was stirred at 20 °C for 0.5 h. The aqueous phase was extracted with EtOAc (3 × 10 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by preparative TLC (SiO, PE: EtOAc = 3:1) to give tert-butyl N-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methyl)carbamate (3) (50 mg, 14.1%).

[0657] 1 H NMR (400MHz, CDCl3): δ 8.91 (s, 1H), 8.78 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 5.11 - 4.96 (m, 1H), 4.63 (d, J = 6.8 Hz, 2H), 3.14 (s, 3H), 1.49 (s, 9H).

[0658] (4-(6-Methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 15): To a solution of tert-butyl N-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methyl)carbamate (50 mg, 0.14 mmol) in MeOH (2 mL) was added HCl / MeOH (2 mL, 4 M) at 20° C. under N. The mixture was stirred at 20° C. for 2 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (FA) under the following conditions (Phenomenex Luna 80 mm × 30 mm × 3 μm phase: [water (FA)-ACN]; B%: 1% → 25% in 8 min) to give (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (4.8 mg, 15.9%).

[0659] LCMS (ESI+): m / z = 270.0 [M+H] +

[0660] 1 H NMR (400MHz, MeOD): δ 8.93 (s, 1H), 8.89 (d, J = 8.4 Hz, 1H), 8.49 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 4.37 (s, 2H), 3.09 (s, 3H).

[0661] Example 20: (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 17) [ka]

[0662] 3-((1,3-Dioxoisoindolin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile (2): To a solution of 3-(hydroxymethyl)-4-(trifluoromethyl)benzonitrile (4.5 g, 22.38 mmol) in THF (50 mL) was added isoindoline-1,3-dione (3.3 g, 22.38 mmol), PPh (11.7 g, 44.76 mmol), and DIAD (6.79 g, 33.57 mmol) at 0 °C under N. The resulting mixture was then stirred at 20 °C for 16 h. The reaction mixture was quenched by the addition of water (15 mL) and extracted with EtOAc (3 × 10 mL). The combined organic phase was washed with brine (15 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a residue, which was purified by column chromatography to give 3-((1,3-dioxoisoindolin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile (2) (5.1 g, 69%).

[0663] 1 H NMR (400 MHz, CDCl3) δ = 7.91-8.03 (m, 2H), 7.84 (dd, J = 5.6, 2.8 Hz, 3H), 7.70 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 5.13 (s, 2H).

[0664] 3-(Aminomethyl)-4-(trifluoromethyl)benzonitrile (3): To a solution of 3-((1,3-dioxoisoindolin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile (1 g, 3.03 mmol) in EtOH (20 mL) was added NH2NH2·H2O (3.79 g, 60.56 mmol, 80% purity) at 20 °C, and the mixture was stirred at 20 °C for 12 h. 6 M hydrochloric acid was added until pH = 1, and the reaction mixture was stirred at 20 °C for 2 h. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was poured into water (50 mL), and the pH was adjusted to 11 with 6 M aqueous NaOH (20 mL). The mixture was then extracted with EtOAc (3×50 mL), and the organic layer was washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give 3-(aminomethyl)-4-(trifluoromethyl)benzonitrile (3) (600 mg, 99.0%).

[0665] 1 H NMR (400 MHz, CDCl3): δ = 8.09 (s, 1H), 7.72-7.80 (m, 1H), 7.62-7.71 (m, 1H), 4.12 ppm (s, 2H).

[0666] tert-Butyl (5-cyano-2-(trifluoromethyl)benzyl)carbamate (4): To a solution of 3-(aminomethyl)-4-(trifluoromethyl)benzonitrile (0.3 g, 1.50 mmol) and NaOH (180 mg, 4.50 mmol) in dioxane (6 mL) and HO (3 mL) was added BocO (654 mg, 3.00 mmol) at 20 °C, and the mixture was stirred at 20 °C for 2 h. The reaction mixture was quenched with HO (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE: EtOAc = 1:0 → 0:1) to give tert-butyl (5-cyano-2-(trifluoromethyl) benzyl) carbamate (4) (300 mg, 66.6%).

[0667] 1 H NMR (400 MHz, CDCl3): δ = 7.89 (s, 1H), 7.75 (s, 1H), 7.65-7.71 (m, 1H), 4.88-5.19 (m, 1H), 4.54 (br d, J = 6.0 Hz, 2H), 1.48 ppm (s, 9H).

[0668] tert-Butyl (5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (5): To a solution of tert-butyl (5-cyano-2-(trifluoromethyl)benzyl)carbamate (500 mg, 1.67 mmol), MeCN (273 mg, 6.66 mmol), and 3-sulfanylpropanoic acid (177 mg, 1.67 mmol) in EtOH (3 mL) was added N2H4·HO (1.33 g, 26.64 mmol) at 25 °C, and the mixture was stirred at 65 °C under N2 for 16 h. The mixture was then cooled to room temperature, and a solution of NaNO2 (345 mg, 5.00 mmol) in HO (2.5 mL) was added dropwise at 25 °C. The mixture was stirred at 25 °C for 3 h. The mixture was cooled to room temperature and adjusted to pH = 3 with 1 M aqueous hydrochloric acid. The mixture was extracted with DCM (20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EtOAc = 20: 1 → 1: 1) to give tert-butyl (5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (5) (140 mg, 22.8%).

[0669] 1 H NMR (400 MHz, CDCl3): δ = 8.78 (s, 1H), 8.60 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 5.08 (s, 1H), 4.67 (s, 1H), 3.15 (s, 3H), 1.49 (s, 9H).

[0670] (5-(6-Methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 15): To a solution of tert-butyl N-[[5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl]methyl]carbamate (5) (140 mg, 0.38 mmol) in MeOH (2 mL), HCl / MeOH (2 mL, 4 M) was added at 20 °C under N. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative TLC (SiO, DCM:MeOH = 10:1) to give [5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl]methanamine (18.0 mg, 17.7%).

[0671] LCMS (ESI+): m / z = 270.0 [M+H] +

[0672] 1 H NMR (400MHz, MeOD): δ 8.88 (s, 1H), 8.60 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 4.11 (s, 2H), 3.08 (s, 3H)

[0673] Example 21: 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (Target 6) [ka]

[0674] tert-Butyl (2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)carbamate: To a mixture of 4-iodobenzonitrile (15 g, 65.50 mmol) in EtOH (140 mL), tert-butyl N-(2-cyanoethyl)carbamate (44.59 g, 261.99 mmol), 3-sulfanylpropanoic acid (6.95 g, 65.50 mmol), and NHNH·HO (59.02 g, 1.18 mol) were added at 0 °C under N. The mixture was stirred at 45 °C for 16 h. The mixture was then cooled to 20 °C, and a solution of NaNO in HO (40 mL) was added dropwise at 20 °C. The mixture was stirred at 20 °C for 1 h. The pH was adjusted to 3 with 1 M aqueous hydrochloric acid under ice-cooling, followed by extraction with DCM (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EtOAc = 10:1 → 1:1) to give tert-butyl (2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)carbamate (14 g, 50%).

[0675] LCMS: m / z = 372.0 [M-BuOH+H] + .

[0676] 2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethan-1-amine: A mixture of tert-butyl (2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)carbamate (6.5 g, 5.85 mmol) in HCl / EtOAc (4 M, 100 mL) was stirred for 1 hour at 25° C. The reaction was concentrated under reduced pressure to give 2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethan-1-amine hydrochloride (5.8 g, 80%).

[0677] LCMS: m / z = 328.2 [M+H] + .

[0678] 1-(2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea: To a mixture of CDI (3.35 g, 20.63 mmol) in DCM (45 mL) was added TEA (4.18 g, 41.27 mmol) and 2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethan-1-amine (5.8 g, 13.76 mmol) at −40° C. under N. The mixture was stirred at −40° C. for 1 h. Then, MeNH (2 M, 17 mL) and TEA (4.17 g, 41.24 mmol) were added at 0° C. under N. The mixture was stirred at 25° C. for 12 h. The reaction was concentrated under reduced pressure to give a residue. The residue was triturated with DCM. The resulting solid was collected by filtration, washed with DCM (40 mL), and dried under reduced pressure to give 1-[2-[6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl]ethyl]-3-methylurea (3 g, 57%). LCMS: m / z = 385.0 [M+H] + .

[0679] 1 H NMR (400 MHz, DMSO): δ = 8.24 (d, J = 8.4 Hz, 2 H) 8.06 (d, J = 8.4 Hz, 2 H) 6.09 (br t, J = 5.6 Hz, 1 H) 5.77 (br d, J = 4.8 Hz, 1 H) 3.53 - 3.57 (m, 2 H) 3.36 - 3.40 (m, 2 H) 2.45 (d, J = 4.4 Hz, 3 H).

[0680] tert-Butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate: To a mixture of 1-[2-[6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl]ethyl]-3-methyl-urea (1 g, 2.60 mol) and (tert-butoxycarbonylamino)methyl-trifluoro-borate; potassium hydride (926 mg, 3.90 mmol) in 2-methyl-2-butanol (4 mL) and HO (1 mL) was added CsCO (1.70 g, 5.21 mmol) and di-tert-butyl(cyclopentyl)phosphane; dichloropalladium; iron (170 mg, 0.26 mmol) under N at 25 °C. The mixture was stirred at 80 °C for 16 h. The mixture was diluted with HO (15 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EtOAc = 5:1 → 0:1) to give tert-butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate (200 mg, 20%).

[0681] LCMS: m / z = 388.3 [M+H] + .

[0682] 1 H NMR (400 MHz, DMSO): δ = 8.44 (br d, J = 8.0 Hz, 2 H), 7.52 (br d, J = 7.6 Hz, 3 H), 6.08 (br t, J = 5.6 Hz, 1 H), 5.78 (br s, 1 H), 4.26 (br d, J = 5.6 Hz, 2 H), 3.54 - 3.58 (m, 2 H), 3.38 (br d, J = 6.4 Hz, 2 H), 2.45 (d, J = 4.4 Hz, 3 H), 1.41 (s, 9 H).

[0683] 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (Target 6): To a mixture of tert-butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate (150 mg, 0.39 mmol) was added HCl / EtOAc (4 M, 3 mL) under N at 25° C. The mixture was stirred at 25° C. for 1 h. The reaction was concentrated under reduced pressure. The crude product was purified by preparative HPLC under the following conditions (Column: Phenomenex C18 100 mm x 30 mm x 5 μm; Mobile phase: A: 10 mM NH4HCO3 in water, B: MeCN; % B in A: 20% to 50% in 10 min) to give 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (63 mg, 29%). LCMS: m / z = 288.2 [M+H] + .

[0684] 1 H NMR (400 MHz, DMSO): δ = 8.52 (d, J = 8.0 Hz, 2 H), 8.45 (br s, 2 H), 7.77 (d, J = 8.2 Hz, 2 H), 6.14 (br t, J = 5.6 Hz, 1 H), 5.76 - 5.83 (m, 1 H), 4.18 (br s, 2 H), 3.56 (q, J = 6.4 Hz, 2 H), 3.38 - 3.41 (m, 2 H), 2.44 (d, J = 4.4 Hz, 3 H).

[0685] Example 22: 4-((S)-2-((S)-2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl-6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (Target 1b) [ka]

[0686] 3-Methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (2): To a solution of benzonitrile (10 g, 96.97 mmol), ACN (31.85 g, 775.79 mmol), and 3-mercaptopropanoic acid (10.29 g, 96.97 mmol) in EtOH (100 mL) was added NHNH·HO (79.26 g, 1.55 mol) dropwise at 0 °C under N. The mixture was stirred at 40 °C for 16 h. The mixture was quenched with water (100 mL), adjusted to pH = 4 by the addition of HCl (1 M), and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The mixture was slurried with MTBE:EtOAc (10:1, 100 mL), filtered, and the solid was collected. The solid was purified by p-HPLC (FA) under the following conditions: column: Phenomenex luna C18 (250 mm × 70 mm, 15 μm); mobile phase: [water (FA)-ACN]; B%: 8% to 35% in 22 min to give 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (10 g, 29%).

[0687] LCMS, m / z = 175.1 [M+H] +

[0688] 1 H NMR (400MHz, DMSO): δ 8.50 (br s, 1H), 8.29 (br s, 1H), 7.74 (dd, J = 1.6, 7.6 Hz, 2H), 7.47-7.35 (m, 3H), 1.78 (s, 3H).

[0689] 4-Nitrophenyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (4): To a solution of 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (3 g, 17.22 mmol) in DCM (30 mL) was added DIEA (6.68 g, 51.66 mmol) followed by (4-nitrophenyl)carbonochloridate (3.64 g, 18.08 mmol) at 0 °C under N. The mixture was stirred at 25 °C for 2 h. The mixture was poured into water (45 mL) and extracted with DCM (3 × 15 mL). The combined organic layers were washed with brine (15 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE: EtOAc = 1:0 → 0:1) to give 4-nitrophenyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (1 g, 17%).

[0690] LCMS, m / z = 340.0 [M+H] +

[0691] 1 H NMR (400MHz, CDCl3): δ 8.36-8.28 (m, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.56-7.42 (m, 5H), 6.30-6.02 (m, 2H), 2.44 (s, 3H).

[0692] 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl-6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (6): To a solution of 4-nitrophenyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (705 mg, 1.66 mmol) and (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (1 g, 1.66 mmol) in DMF (10 mL) was added DIEA (644 mg, 4.99 mmol) at 25 °C under N. The mixture was heated to 80° C. and stirred for 2 h. The crude product was purified by p-HPLC (neutral) under the following conditions (column: Phenomenex C18 250 mm × 50 mm × 10 μm; mobile phase: [water(NH4HCO3)-ACN]; B%: 14% to 44% in 8 min) to give 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl-6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (240 mg, 25%).

[0693] LCMS, m / z = 580.2 [M+H] +

[0694] 1H NMR (400MHz, DMSO): δ 9.97 (s, 1H), 9.84 (s, 1H), 8.42 (d, J = 7.6 Hz, 1H), 7.88-7.79 (m, 2H), 7.60-7.47 (m, 5H), 7.23 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 5.98 (br t, J = 6.0 Hz, 1H), 5.47-5.35 (m, 2H), 5.12-5.04 (m, 1H), 4.43 (d, J = 5.6 Hz, 3H), 4.26 (dd, J = 5.6, 8.8 Hz, 1H), 3.07-2.94 (m, 2H), 2.18 (s, 3H), 2.14-2.04 (m, 1H), 1.75-1.55 (m, 2H), 1.51-1.30 (m, 2H), 0.92 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H)

[0695] 4-((S)-2-((S)-2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl-6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (Target 1b): To a solution of 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (100 mg, 0.17 mmol) and DIEA (45 mg, 0.35 mmol) in THF (0.5 mL) was added DMAP (4 mg, 0.03 mmol) followed by AcO (23 mg, 0.22 mmol) at 0 °C under N. The mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure. The crude product was purified by p-HPLC (neutral) under the following conditions (column: Waters xbridge 150 mm × 25 mm × 10 μm; mobile phase: [water(NH4HCO3)-ACN]; B%: 24% → 54% in 8 min) to give 4-((S)-2-((S)-2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (58.9 mg, 55%).

[0696] LCMS, m / z = 622.2 [M+H] +

[0697] 1H NMR (400MHz, DMSO): δ 10.09 (s, 1H), 9.85 (s, 1H), 8.44 (d, J = 7.2 Hz, 1H), 7.87-7.77 (m, 2H), 7.64-7.47 (m, 5H), 7.30 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 5.99 (br t, J = 5.6 Hz, 1H), 5.41 (s, 2H), 5.00 (s, 2H), 4.47-4.36 (m, 1H), 4.27 (dd, J = 5.6, 8.7 Hz, 1H), 3.10-2.88 (m, 2H), 2.18 (s, 3H), 2.14-2.06 (m, 1H), 2.04 (s, 3H), 1.77-1.57 (m, 2H), 1.52-1.32 (m, 2H), 0.92 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H).

[0698] Example 23: LCMS analysis of drug release by tetrazine A solution of each tetrazine in PBS (final concentration of 20 μM) is mixed with TCO-MMAE (final concentration of 10 μM). The solution is mixed thoroughly and an aliquot is removed at the appropriate time for analysis by LCMS. The results are reported in the table below.

[0699] TIFF2024542021000187.tif98170

[0700] Based at least on the above data, it is believed that by adjusting the structure of the bicyclic tetrazine or dihydrotetrazine portion of the targeting moiety, the drug release profile can be modified, enhanced, or attenuated.

[0701] Example 24: Preparation and FACS analysis of trastuzumab Fab-Me-Tet-PEG9 conjugates Trastuzumab Fab was synthesized by plasmid construction, HEK293 cell expression, and purification. Fab-tetrazine conjugates (ADCs) were prepared by reacting Me-Tet-PEG9-NHS (structure shown below, purchased from SiChem, catalog number SC-8808) with primary amines on the Fab to form stable amide bonds. The ADCs were tested by flow cytometry (FACS) to compare binding to HER2-positive cells compared to unconjugated Fabs.

[0702] [ka]

[0703] Synthesis of trastuzumab Fab Vector Construction: The coding sequences (listed below) were synthesized and subcloned into expression vectors. The constructed plasmids were transformed and propagated in E. coli. Large-scale plasmid production was performed using the NucleoBond Xtra Maxi Plus EF kit. Purified plasmids were verified by agarose gel and confirmed by sequencing.

[0704] HC sequence of trastuzumab-Fab: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 3).

[0705] Trastuzumab-Fab LC sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).

[0706] Protein expression: Constructs containing the heavy and light chains of Fab were co-transfected into HEK293 cells using PEI. Culture medium was collected 6-7 days after transfection.

[0707] Protein purification: Conditioned medium expressing the target Fab was collected by centrifugation and filtration and then loaded onto a KappaSelect affinity column (Mabselect Prism). The loading buffer was 25 mM Tris (pH 8.0) containing 150 mM NaCl. The column was washed with 25 mM Tris buffer (pH 8.0) containing 150 mM NaCl and 0.2% Triton X-100 / 114, and eluted with 100 mM sodium citrate buffer (pH 2.5) containing 150 mM NaCl. The collected solution was neutralized with 1 M arginine, 400 mM succinate buffer (pH 9.0). The affinity-purified protein was further purified by gel filtration using Superdex S-200 column chromatography. The purified Fab was analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0708] Conjugate preparation: 120 mg of Fab protein was dialyzed overnight against PBS (pH 7.4) with one buffer change at approximately 4 hours from the start. 10 mM Me-Tet-PEG9-NHS was prepared in DMSO. The two components were reacted at a 3:1 drug-to-protein molar ratio for 2 hours at 25°C, after which it was dialyzed against PBS (pH 7.4) to remove excess Me-Tet-PEG9-NHS compound from the protein component.

[0709] FACS binding assay of trastuzumab Fab, Fab-Me-Tet-PEG9-NHS conjugate to HER2-positive cell lines: Cells (NCI-N87 cell line) cultured in RPMI1640 + 10% FBS were collected by centrifugation and resuspended in FACS buffer (PBS with 2% FBS, pH 7.4) at 2 × 10 per mL. 6 The density was adjusted to 100 μL (2 × 10 5 Cells (number of cells) were seeded into a 96-well plate, centrifuged at 400g for 5 minutes, the supernatant discarded, and resuspended in a 1:10 serially diluted Fab solution from a 20µL / mL stock solution. After incubating the antibody and cells for 1 hour at 4°C, the cells were processed by centrifugation at 400g for 5 minutes, the supernatant removed, and washed three times with FACS buffer (200µL per well). The cells were resuspended in 100µL of Alexa 488-labeled anti-human IgG (Life Technologies, catalog no. 11013, 2µg / mL) and incubated in the dark at 4°C for 1 hour. The supernatant was then removed and the cells were washed twice with 100µL of PBS, each time by centrifugation at 400g for 5 minutes.

[0710] Results: 290 mg of protein was obtained from the first-stage affinity purification of 6 L of culture medium using Mabselect Prism. After purification using Superdex S-200 column chromatography, 215 mg of Fab protein was obtained. After the conjugation and dialysis procedures, 112 mg of conjugate was obtained. The drug-to-antibody ratio (DAR) determined by LC-MS was 2.28 (Figure 6).

[0711] FACS binding assay of trastuzumab Fab, trastuzumab-Me-Tet-PEG9 ADC to HER2-positive cell lines: The binding profile (Figure 7) shows similar or slightly reduced binding activity of trastuzumab Fab conjugated to Me-Tet-PEG9 compared to unconjugated Fab.

[0712] Example 25: Efficacy study of trastuzumab Fab-Tz (from Example 24) with Compound A in the treatment of a subcutaneous NCI-N87 xenograft model in CB17 SCID mice In this study, the antitumor efficacy of MMAE, Tz-Fab, Tz-Fab + Compound A, Compound A, SQL70 + Compound A, and T-DM1 in treating the NCI-N87 xenograft model was evaluated. The objective was to evaluate the ability of Fab-tetrazine conjugates to localize the corresponding TCO-MMAE protodrug in efficacy studies for the treatment of the subcutaneous NCI-N87 xenograft model in female CB17 SCID mice.

[0713] TIFF2024542021000189.tif69170

[0714] Methods: One hundred and ten CB17 SCID female mice (Mus musculus, age: 6-8 weeks, weight: 18-22 g, Shanghai LingChang Biotech Co., LTD) were subcutaneously inoculated with NCI-N87 cells (3 × 10 6 The cells were suspended in 0.2 mL of a mixture of 1640 and BD Matrigel (1640:Matrigel = 1:1) to allow tumor development. Seven days after inoculation, 70 animals were divided into eight groups using stratified randomization based on their body weight and tumor volume, with groups 1, 2, 5, 6, 7, and 8 each containing 10 animals, and groups 3 and 4 each containing 5 animals. For these 70 mice, the average tumor size was 158 mm. 3 (106mm 3 ~221mm3 Treatment was initiated once the patient reached the 500mg / kg / day (500mg / kg / day) daily dose. Group 1: Vehicle, intravenously, QD x 3 x 2 weeks. Group 2: MMAE, 0.25 mpk, intravenously, QW x 2 weeks. Group 3: Tz-Fab, 50 mpk, intravenously, QW x 2 weeks. Group 4: Compound A, 20 mpk, intravenously, QD x 3 x 2 weeks. Group 5: Tz-Fab, 50 mpk, intravenously, QW x 2 weeks + Compound A, 20 mpk, intravenously, QD x 3 x 2 weeks. Group 6: Tz-Fab, 50 mpk, intravenously, QW x 2 weeks + Compound A, 5 mpk, intravenously, QD x 3 x 2 weeks. Group 7: SQL70, 100 μL, intrathecal x 1 dose + Compound A, 1.9 mpk, intravenously, QD x 5. Group 8: T-DM1, 3 mpk, intravenously, QW x 2 weeks. During treatment, tumor size and body weight were measured twice a week. The dosing schedule ended on PG-D10. The entire study ended on PG-D37, with all tumor samples collected for Groups 5 and 6, and three tumors per group for Groups 1 and 2.

[0715] TIFF2024542021000190.tif47170

[0716] TIFF2024542021000191.tif72170

[0717] Experimental Methods and Procedures: Cell Preparation and Implantation: NCI-N87 (gastric carcinoma, ATCC™ CRL-5822™, Lot No. 7686255) cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely subcultured every 5 days by trypsin-EDTA treatment. Cultured NCI-N87 cells were harvested and cultured at 1.5 x 10 per mL. 7 The right flank of each mouse was inoculated with 3 × 10 cells in 0.2 mL of a mixture of 1640 and BD Matrigel (1:1). 6 The mice were inoculated subcutaneously and allowed to develop tumors.

[0718] Randomization and treatment: Mice were randomized to a group with a mean tumor size of 158 mm 3 (106mm 3 ~221mm 3 Treatment began with dividing the mice into groups 1 to 8 based on the number of mice reached. Each group consisted of 10 or 5 mice. The test articles were administered to the mice according to the regimen shown in the experimental design table (Table 4).

[0719] TIFF2024542021000192.tif126170

[0720] The formulation preparation is shown in Table 5.

[0721] TIFF2024542021000193.tif232170

[0722] Tumor measurements and endpoints: The primary endpoint is to determine whether tumor growth can be slowed or whether the mice can be cured. Tumor size is measured in two dimensions using calipers and this volume is calculated using the formula: V = 0.5a x b 2 (where a and b are the long and short diameters of the tumor, respectively) 3 The tumor size was then used to calculate both tumor growth inhibition (TGI) values.

[0723] TGI was calculated for each group using the following formula: TGI (%) = [1-(TV 処置N日目 -TV 処置0日目 ) / (TV ビヒクルN日目 -TV ビヒクル0日目 )]×100%;TV 処置N日目 is the mean tumor volume of the treatment group on a given day, and TV 処置0日目 is the mean tumor volume of the treatment group on the first day of treatment, and TV ビヒクルN日目 is the mean tumor volume of the vehicle control group on a given day, and TV ビヒクル0日目 is the mean tumor volume in the vehicle group on the first day of treatment.

[0724] The T / C value (percent) is an index of antitumor efficacy. T / C (%) = RTV処置 / RTV コントロール ×100%(RTV 処置 : Mean RTV of treatment group, RTV コントロール : Mean RTV of vehicle-treated group). RTV (relative tumor volume) = TV N日目 / TV 0日目 TV N日目 and TV 0日目 are the tumor volumes on day N and day 0, respectively. According to the criteria of the National Cancer Institute, T / C (%) ≦ 42% is considered to be significant antitumor activity, and T / C (%) < 10% is considered to be highly significant antitumor activity.

[0725] The relative change in body weight (RCBW) of each mouse was calculated according to the following formula: RCBW (%) = (BW 処置N日目 -BW 処置0日目 ) / BW 処置0日目 ×100%.

[0726] Statistical analysis: Tumor volumes between different groups were analyzed by two-way repeated measures ANOVA followed by Tukey's post-hoc test. All data were analyzed using GraphPad Prism 6.0. P<0.05 was considered statistically significant.

[0727] result Body weight changes: Animal body weight was monitored as an indirect measure of toxicity. After grouping, all treatments were well tolerated. No significant changes in body weight were observed. No other obvious abnormalities were observed in these mice.

[0728] Tumor Volume and Tumor Growth Curves: Tumor growth curves (shown as mean tumor volume) over time in NCI-N87 tumor-bearing female CB17 SCID mice dosed with MMAE, Tz-Fab, Tz-Fab + Compound A, Compound A, SQL70 + Compound A, and T-DM1 are shown in Figure 8 (data points represent group means, error bars represent standard error of the mean (SEM)).

[0729] The tumor growth inhibition curves are shown in FIG.

[0730] The results of tumor size in different groups at different time points are shown in Figure 3. The average tumor size in vehicle control mice was 1009 mm on day 35 after group division. 3 On day 35, the mean tumor size for each treatment group was: 586 mm 3 (Group 2), 1043mm 3 (Group 3), 653mm 3 (Group 4), 8mm 3 (Group 5), 361mm 3 (Group 6), 871mm 3 (Group 7), 873mm 3 (Group 8). Compared with the vehicle control group (Group 1), treatment groups 2, 4, 5, and 6 showed statistically significant differences (P<0.05, TGI was 49.68%, 41.74%, 117.52%, and 76.30%, respectively; T / C was 57.50%, 62.12%, 0.82%, and 35.44%, respectively).

[0731] At day 35, 5 of 10 animals in group 5 were tumor-free (complete response). No animals in the other groups were tumor-free at day 35. All treatments were well tolerated by NCI-N87 tumor-bearing female CB17 SCID mice, and no other serious side effects were observed.

[0732] Example 26: Antibody fragment moiety Additional targeting moieties can be prepared using the sequences shown below, for example as in Example 6.

[0733] a) Synthesis of Fab of L19 that binds to FN-1 (gene ID 2335) Vector Construction: The coding sequences (listed below) are synthesized and subcloned into an expression vector. The constructed plasmids are transformed and propagated in E. coli. Large-scale plasmid production is performed using the NucleoBond Xtra Maxi Plus EF kit. The purified plasmids are verified by agarose gel and confirmed by sequencing.

[0734] L19-Fab HC sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHT (SEQ ID NO: 5).

[0735] L19-Fab LC sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTKSFNRGEC (SEQ ID NO: 6).

[0736] Protein expression: Constructs containing the heavy and light chains of Fab are co-transfected into HEK293 cells using PEI. Culture medium is harvested 6-7 days after transfection.

[0737] Protein purification: The conditioned medium expressing the target Fab is collected by centrifugation and filtration and then loaded onto a KappaSelect affinity column (Mabselect Prism). The loading buffer is 25 mM Tris (pH 8.0) containing 150 mM NaCl, the wash buffer is 25 mM Tris buffer (pH 8.0) containing 150 mM NaCl and 0.2% Triton X-100 / 114, and the elution buffer is 100 mM sodium citrate buffer (pH 2.5) containing 150 mM NaCl. The collected solution is neutralized with 1 M arginine, 400 mM succinate buffer (pH 9.0). The affinity-purified protein is further purified by gel filtration using Superdex S-200 column chromatography. The purified Fab is analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0738] Conjugate preparation: 120 mg of Fab protein is dialyzed overnight against PBS (pH 7.4) with one buffer change approximately 4 hours after initiation. 10 mM Me-Tet-PEG9-NHS is prepared in DMSO. The two components are reacted at a drug-to-protein molar ratio of 3:1 at 25°C for 2 hours, and the reaction mixture is dialyzed against PBS (pH 7.4) to remove excess Me-Tet-PEG9-NHS compound from the protein component.

[0739] b) Synthesis of Fab of F16 that binds to TNC (gene ID 3371) Vector Construction: The coding sequences (listed below) are synthesized and subcloned into an expression vector. The constructed plasmids are transformed and propagated in E. coli. Large-scale plasmid production is performed using the NucleoBond Xtra Maxi Plus EF kit. The purified plasmids are verified by agarose gel and confirmed by sequencing.

[0740] F16-Fab HC sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHT (SEQ ID NO: 7).

[0741] F16-Fab LC sequence: SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 8).

[0742] Protein expression: Constructs containing the heavy and light chains of Fab are co-transfected into HEK293 cells using PEI. Culture medium is harvested 6-7 days after transfection.

[0743] Protein purification: The conditioned medium expressing the target Fab is collected by centrifugation and filtration and then loaded onto a KappaSelect affinity column (Mabselect Prism). The loading buffer is 25 mM Tris (pH 8.0) containing 150 mM NaCl, the wash buffer is 25 mM Tris buffer (pH 8.0) containing 150 mM NaCl and 0.2% Triton X-100 / 114, and the elution buffer is 100 mM sodium citrate buffer (pH 2.5) containing 150 mM NaCl. The collected solution is neutralized with 1 M arginine, 400 mM succinate buffer (pH 9.0). The affinity-purified protein is further purified by gel filtration using Superdex S-200 column chromatography. The purified Fab is analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0744] Conjugate preparation: 120 mg of Fab protein is dialyzed overnight against PBS (pH 7.4) with one buffer change approximately 4 hours after initiation. 10 mM Me-Tet-PEG9-NHS is prepared in DMSO. The two components are reacted at a drug-to-protein molar ratio of 3:1 at 25°C for 2 hours, and the reaction mixture is dialyzed against PBS (pH 7.4) to remove excess Me-Tet-PEG9-NHS compound from the protein component.

[0745] c) Synthesis of NJB2 construct (ECM targeting sequence) Vector Construction: The coding sequences (listed below) are synthesized and subcloned into an expression vector. The constructed plasmids are transformed and propagated in E. coli. Large-scale plasmid production is performed using the NucleoBond Xtra Maxi Plus EF kit. The purified plasmids are verified by agarose gel and confirmed by sequencing.

[0746] NJB2 construct sequence: Monovalent 1-VHH Fibronectin-His6 QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGGHHHHHH (SEQ ID NO: 9).

[0747] Monovalent 2-VHH Fibronectin-His6-Cys QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGGHHHHHHC (SEQ ID NO: 10).

[0748] Bivalent 1-VHH-6GS-VHH-His6 QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPET GGGSGGGSGGGSGGGSGGGSGGGSGGGSQVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGGHHHHHH (SEQ ID NO: 11).

[0749] Bivalent 2-VHH-His6-cys-6GS-VHH QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGGHHHHHHCGGGSGGGSGGGSGGGSGGGSGGGSQVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGG(SEQ ID NO: 12)

[0750] Bivalent 3-VHH-6GS-cys-His6-VHH QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGGGGGSGGGSGGGSGGGSGGGSGGGSCHHHHHHQVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGG(SEQ ID NO: 13)

[0751] Bivalent 4-VHH-30GS-VHH-His6 QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSQVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGGHHHHHH (SEQ ID NO: 14)

[0752] Bivalent 5-VHH-His6-cys-30GS-VHH QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGGHHHHHHCGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSQVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGG (SEQ ID NO: 15)

[0753] Bivalent 6-VHH-30GS-cys-His6-VHH QVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSCHHHHHHQVQLVETGGGLVQAGGSLRLSCAASGSTFSHNAGGWYRQAPEKQRELVAGISSDGNINYADSVKDRFTISRDNASNTMYLQMNLKPEDTAVYVCNIRGSYGNTYYSRWGQGTQVTVSSGGLPETGG (SEQ ID NO: 16)

[0754] Protein expression: For each construct, transfect HEK293 cells with an expression vector containing the gene for the target protein using PEI. Collect the culture medium 6-7 days after transfection.

[0755] Protein purification: The conditioned medium expressing the target protein can be collected by centrifugation and filtration and then loaded onto an IMAC column (GE). The loading buffer is 25 mM Tris (pH 8.0) containing 150 mM NaCl and 10 mM histidine. The washing buffer is 25 mM Tris buffer (pH 8.0) containing 150 mM NaCl, 20 mM histidine, and 0.2% Triton X-100 / 114. The elution buffer is 25 mM Tris buffer (pH 8.0) containing 150 mM NaCl and 1 M histidine. The affinity-purified protein is further purified by gel filtration using a Superdex S-200 column. The purified protein is analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0756] Conjugate preparation: 120 mg of protein is dialyzed overnight against PBS (pH 7.4) with one buffer change approximately 4 hours after initiation. 10 mM Me-Tet-PEG9-NHS is prepared in DMSO. The two components are reacted at a drug-to-protein molar ratio of 3:1 at 25°C for 2 hours, and the reaction mixture is dialyzed against PBS (pH 7.4) to remove excess Me-Tet-PEG9-NHS compound from the protein component.

[0757] Preparation of the alternative conjugate: 120 mg of protein is dialyzed overnight against Tris (pH 8.0) with one buffer change at approximately 4 hours from the start. 10 mM Me-Tet-PEG9-maleimide is prepared in DMSO. The two components are reacted at a 3:1 drug-to-protein molar ratio for 2 hours at 25°C, and the reaction mixture is dialyzed against PBS (pH 7.4) to remove excess Me-Tet-PEG9-maleimide compound from the protein component.

[0758] Example 27: Efficacy study of Ab-Tz (prepared as in Example 6) with Compound B in the treatment of a subcutaneous NCI-N87 xenograft model in CB17 SCID mice In this study, the antitumor efficacy of MMAE, HER2 Tz-nanophytin, HER2 Tz-nanophytin + Compound B, isotype Tz-nanophytin + Compound B, Compound B, and trastuzumab emtansine was tested in the NCI-N87 xenograft model of gastric cancer in SCID mice.

[0759] Animal welfare for this study complies with the US Department of Agriculture's Animal Welfare Act (9 CFR Parts 1, 2, and 3), as applicable.

[0760] Materials and Methods: Female CB-17 / SCID mice (6-9 weeks old, approximately 20 g) were inoculated with tumors in the flank by injection of 5 million viable cells suspended in serum-free medium and Cultrex ECM. The average tumor size was approximately 100 mm. 3 Groups were randomized when ≥100 mg / kg was reached.

[0761] Treatment was carried out as follows:

[0762] TIFF2024542021000194.tif97170

[0763] Animals were monitored weekly for palpable tumors or any changes in appearance or behavior. Once tumors were palpable, they were measured at least once a week using calipers. Tumor volume was calculated using the following formula: (longest diameter x shortest diameter) 2 ) / 2. Once tumors reached an appropriate size to begin the study, tumors and body weights were measured at least twice a week for the duration of the study. One person was responsible for tumor measurements throughout the study.

[0764] After randomization and initiation of treatment, body weights were measured at least twice weekly. Animals may be given Hydrogel / DietGel and / or a washout period due to weight loss, and weight loss was calculated based on the body weight (BW) of the mice on the first day of treatment.

[0765] Clinical observations were performed at least twice a week with tumor and body weight measurements. The results of tumor volume measurements and body weight are shown in Figures 10A and 10B, respectively.

[0766] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this di...

Claims

1. Formula I, Formula II, or Formula V: 【Chemistry 1】 (In the formula, Ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; The dotted line is R ３ and R ４ represent an additional bond forming a tetrazine when both are absent, or R ３ and R ４ when both are present, represent an additional bond to form a dihydrotetrazine, provided that when ring A is aryl, R ３ and R ４ Both exist, X is a biocompatible support, an antibody, or an antibody fragment moiety, with the proviso that for Formula I and Formula II, X is not a biocompatible support; p is 1 to 150; L, independently at each occurrence, is a linker; R １ is independently at each occurrence hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ NR'R'', C(=O)OR', C(=O)SR', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(= O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', and NR'C(=S)NR''R'', wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally selected from the group consisting of 1 to 3 Z １ is replaced by R ２ is independently at each occurrence halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; and １ is replaced by R ３ and R ４ are both absent or R ３ and R ４ are each independently hydrogen or a group that is removable after a triggering event; R ２０ is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF ３ , C.F. ２ -R', NO ２ , OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ is selected from the group consisting of NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R'", SC(=S)R'R", NR'C(=O)NR''R", and NR'C(=S)NR''R'', R ２２ is, in each occurrence, independently a linker of 1 to 100 linked atoms optionally containing one or more ethyleneoxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups; R ３０ is independently at each occurrence halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; R ａ , R ３１ａ , and R ３１ｂ are each independently hydrogen, C １ ~C ６ - alkyl, or C １ ~C ６ -haloalkyl, Each Z １ are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ selected from NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R'", SC(=S)R'R", NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl, and t at each occurrence is independently 0, 1, 2, 3, or 4.

2. 2. The targeting moiety of claim 1, wherein p is 1-16, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2.

3. Formula I or Formula II: 【Chemistry 2】 (In the formula, X is an antibody or antibody fragment moiety; p is 1 to 16; L, independently at each occurrence, is a linker; R ２０ is independently, in each occurrence, hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF ３ , C.F. ２ -R', NO ２ , OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ is selected from the group consisting of NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R'", SC(=S)R'R", NR'C(=O)NR''R", and NR'C(=S)NR''R'', R ２２ is, in each occurrence, independently a linker of 1 to 100 linked atoms optionally containing one or more ethyleneoxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups; R ３０ is independently at each occurrence halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; R ａ , R ３１ａ , and R ３１ｂ are each independently hydrogen, C １ ~C ６ - alkyl, or C １ ~C ６ -haloalkyl, R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl, and t at each occurrence is independently 0, 1, 2, 3, or 4.

4. Formula IIA: 【Transformation 3】 4. The targeting moiety of claim 3, represented by:

5. Formula IIB, or Formula IIC: 【Chemistry 4】 4. The targeting moiety of claim 3, represented by:

6. Formula V: 【Transformation 5】 (In the formula, Ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; The dotted line is R ３ and R ４ represent an additional bond forming a tetrazine when both are absent, or R ３ and R ４ when both are present, represent an additional bond to form a dihydrotetrazine, provided that when ring A is aryl, R ３ and R ４ Both exist, X is a biocompatible support, an antibody, or an antibody fragment moiety; p is 1 to 150; L, independently at each occurrence, is a linker; R １ is independently at each occurrence hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ NR'R'', C(=O)OR', C(=O)SR', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(= O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', and NR'C(=S)NR''R'', wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally selected from the group consisting of 1 to 3 Z １ is replaced by R ２ is independently at each occurrence halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; and １ is replaced by R ３ and R ４ are both absent or R ３ and R ４ are each independently hydrogen or a group that is removable after a triggering event; Each Z １ are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ selected from NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R'", SC(=S)R'R", NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R′ and R″ at each occurrence are independently selected from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl, and t at each occurrence is independently 0, 1, 2, 3, or 4.

7. The portion: 【Transformation 6】 At least one of 【Transformation 7】 【change】 7. The targeting moiety of claim 6, represented by a formula selected from:

8. 7. The targeting moiety of claim 6, wherein X further comprises an imaging contrast agent.

9. L is a group of the formula: -Y １０ —(CHR １３０ ( ｎ’ -Y ２０ —(CHR １４０ ( ｎ’’ -Y ３０ —(CHR １５０ ( ｍ’’ -Y ４０ — (In the formula, Y １０ , Y ２０ , Y ３０ , and Y ４０ each independently represents a bond, —NR １１０ -, -O-, -S(O) ０～２ -, -NR １１０ C(O)-, -C(O)NR １１０ -, -NR １１０ S (O) ２ -, -S(O) ２ NR １１０ -, -CR １２０ = N-NR １１０ -, -NR １１０ -N=CR １２０ -, -C(O)-, -OC(O)-, -OC(O)O-, -(CH ２ CH ２ O) １～５ -, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene, wherein each alkylene, alkenylene, alkynylene, arylene, or heteroarylene independently is oxo, halo, C １～４ Alkyl, C １～４ Alkoxy, and C １～４ optionally substituted with 1 to 5 substituents independently selected from haloalkyl; Each R １１０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R １２０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R １３０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain, each R １４０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain, each R １５０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; and 2. The targeting moiety of claim 1, wherein n', n'', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

10. L is a group of the formula: -Y １０ —(CH ２ ( ｎ’ -Y ２０ —(CH ２ ( ｍ’’ -Y ３０ — (In the formula, Y １０ , Y ２０ , and Y ３０ each independently represents a bond, —NR １１０ -, -O-, -S(O) ０～２ -, -NR １１０ C(O)-, -C(O)NR １１０ -, -NR １１０ S (O) ２ -, -S(O) ２ NR １１０ -, -CR １２０ = N-NR １１０ -, -NR １１０ -N=CR １２０ -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene independently is oxo, halo, C １～４ Alkyl, C １～４ Alkoxy, and C １～４ and optionally substituted with 1 to 5 substituents independently selected from haloalkyl, and each R １１０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R １２０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and 2. The targeting moiety of claim 1, wherein n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

11. L is, 【Transformation 8】 【change】 【change】 【change】 【change】 2. The targeting moiety of claim 1, wherein:

12. X is an antibody or antibody fragment moiety that targets HER2, TROP2, nectin-4, claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5; and L is independently in each occurrence: 【Chemistry 9】 2. The targeting moiety of claim 1, wherein the linker is selected from the group consisting of:

13. Formula IID: 【Chemistry 10】 2. The targeting moiety of claim 1, having the formula:

14. Formula IIE: 【Chemistry 11】 10. The targeting moiety of claim 1 having the formula: wherein p is 1-20 and X is an antibody or antibody fragment moiety.

15. A pharmaceutical composition comprising the targeting moiety of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

16. 15. A pharmaceutical composition for treating cancer or enhancing or inducing an immune response, comprising a targeting moiety or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, administered in combination with a conjugate comprising a payload linked to one or more trans-cyclooctene moieties, or a pharmaceutically acceptable salt thereof.

17. The conjugate has the formula X: 【Chemistry 12】 (In the formula, G, independently in each occurrence, 【Chemistry 13】 and L １ is, independently in each occurrence, a linker, m is an integer from 1 to 150, D is the payload, R １Ａ is, in each instance, independently １～４ Alkyl, C １～４ Haloalkyl, and C １～４ alkoxy; q is 0, 1, or 2; q1 is 0 or 1; R １Ｂ is, in each case independently, G １ , OH, -NR １ｃ -C １～４ Alkylene-G １ , -NR １ｃ -C １～４ Alkylene-N(R １ｄ ) ２ , -NR １ｃ -C １～６ Alkylene-N(C １～４ alkyl) ３ ＋ , -N(R １ｃ ) CHR １ｅ CO ２ H, -N(R １ｃ )-C １～６ Alkylene-CO ２ H, -N(R １ｆ )-C ２～４ Alkylene-(N(C １～４ Alkylene-CO ２ H)-C ２～４ alkylene) ｎ -N(C １～４ Alkylene-CO ２ H) ２ , -N(R １ｃ ) CHR １ｅ C(O)OC １～６ Alkyl, —N(R １ｃ )-C １～６ Alkylene-C(O)OC １～６ Alkyl, —N(R １ｆ )-C ２～４ Alkylene-(N(C １～４ Alkylene-C(O)OC １～６ alkyl)-C ２～４ alkylene) ｎ -N(C １～４ Alkylene-C(O)OC １～６ alkyl) ２ , -N(R １ｃ )-C １～６ Alkylene-SO ３ H, -N(R １ｃ )-(CH ２ CH ２ O) １～３ -CH ２ CH ２ N ((CH ２ CH ２ O) １～３ -C １～６ Alkylene-CO ２ H) ２ , and −N(R １ｃ )-CH(CH ２ O-(CH ２ CH ２ O) ０～２ -C １～６ Alkylene-CO ２ H) ２ is selected from the group consisting of R １ｃ and R １ｄ is, in each occurrence, independently hydrogen or C １～４ alkyl, and R １ｅ is, independently in each occurrence, -C １～４ Alkylene-CO ２ H, -C １～４ Alkylene -CONH ２ , or -C １～４ alkylene-OH, R １ｆ is, in each occurrence, independently hydrogen, C １～６ Alkyl, or C １～４ Alkylene-CO ２ H, n is independently 0, 1, 2, or 3 at each occurrence; L ２ is, in each occurrence, independently —C(O)— and C １～３ alkylene; and G １ and R is independently at each occurrence an optionally substituted heterocyclyl, or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical composition of claim 16 for enhancing or inducing an immune response.

19. 19. The pharmaceutical composition of claim 18, wherein the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

20. The pharmaceutical composition of claim 16, further administered in combination with an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, or a trans-cyclooctene prodrug thereof.

Images