Trans-cyclooctene-modified targeted protein degradation agent conjugates

Abstract

The present disclosure relates generally to trans-cyclooctene modified TPD conjugates comprising a targeting protein degrading agent (TPD) optionally covalently bound to at least one trans-cyclooctene moiety via a linker, for use in, for example, cancer therapy, tumor growth, and immunotherapy.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application 63 / 503,126, filed May 18, 2023, pursuant to 35 USC §119(e), which is hereby incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure generally relates to trans-cyclooctene-modified targeted protein degrader (TPD) conjugates comprising a targeted protein degrader (TPD) optionally covalently linked to at least one trans-cyclooctene moiety via a linker, said conjugates being used, for example, in cancer treatment, tumor growth, and immunotherapy. Background Technology

[0003] Targeted protein degraders (TPDs), sometimes called protein hydrolysis-targeted chimeras (PROTACs), are engineered targeted protein degradation technologies and have emerged as a promising approach for targeted therapies for various diseases, particularly cancer. Bifunctional TPD molecules typically consist of two covalently linked ligands and are used to recruit the target protein together with an E3 ubiquitin ligase to trigger proteasome degradation of the target protein via the ubiquitin-proteasome system (UPS).

[0004] To function properly, TPDs need to enter cells to mobilize intracellular UPS, thus their membrane permeability is crucial for TPD function. However, the large molecular weight and large exposed polar surface area of ​​TPDs may result in low cell / tissue permeability. Furthermore, some E3 ligands exhibit low specificity, leading to off-target effects in such TPDs.

[0005] Therefore, there is a need to provide modified TPDs for efficient and safe delivery in vivo. Summary of the Invention

[0006] Bioorthogonal coupling or click reactions are selective and orthogonal (non-interacting) functions found in biological systems and have been used in a variety of applications in chemistry, chemical biology, molecular diagnostics, and medicine, where they can facilitate the selective manipulation of molecules, cells, particles, and surfaces, as well as the labeling and tracking of biomolecules in vitro and in vivo.

[0007] This document provides a trans-cyclooctene-modified targeted protein degrader (TPD) (or PROTAC) conjugate comprising a targeted protein degrader (TPD) covalently bound to at least one trans-cyclooctene moiety via a linker. The trans-cyclooctene-modified TPD conjugate described herein is designed for use with a tetrazine activator, such that the TPD is released in vivo upon contact between the tetrazine activator and the trans-cyclooctene-modified TPD conjugate.

[0008] It is envisioned that the trans-cyclooctene-modified TPD conjugates described herein could offer enhanced benefits, such as, but not limited to, improved TPD permeability and masking TPD activity to enhance safety properties. Further envisioning, the cell permeability of TPD could be improved by: using a linker between the TPD and the trans-cyclooctene moiety; or by derivatizing one or more trans-cyclooctene moieties to form partially reduced intramolecular hydrogen bonds; or by attaching cell-permeable peptides such as poly-D-arginine sequences thereto.

[0009] In some embodiments, a conjugate as shown in Table 1 is provided.

[0010] In some embodiments, a method for treating cancer is provided, the method comprising administering to a subject in need a conjugate as described herein, and administering to the subject a tetrazine activator as described herein.

[0011] Additionally, this document provides compounds comprising precursors to TPD, which react in vivo to produce TPD when administered alone. Therefore, in some embodiments, a system is provided comprising a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand. A system is also provided comprising a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand.

[0012] In some embodiments, a method for treating cancer or enhancing or inducing an immune response is provided, the method comprising administering to a subject in need a therapeutically effective amount of a trans-cyclooctene-modified E3 ubiquitin ligand ligand and a tetrazine-modified target protein ligand, or a tetrazine-modified E3 ubiquitin ligand ligand and a trans-cyclooctene-modified target protein ligand.

[0013] In some embodiments, a compound from Table 2 is provided. In some embodiments, a kit is provided that comprises two or more compounds from Table 2.

[0014] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, head / neck squamous cell carcinoma, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, 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.

[0015] In some embodiments, the cancer is melanoma, kidney 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 hematologic malignancy. Detailed Implementation

[0016] The following description illustrates exemplary embodiments of the present technology. However, it should be understood that such description is not intended to limit the scope of this disclosure, but is provided as a description of exemplary embodiments.

[0017] 1. Definition For clarity, certain features of this disclosure described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for simplicity, various features of this disclosure described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to this disclosure are specifically covered and disclosed herein, just as each and every combination is disclosed individually and explicitly. To some extent, such combinations cover, for example, the subject matter of compounds as stable compounds (i.e., compounds that can be prepared, isolated, characterized, and tested for biological activity). In addition, this disclosure also specifically covers and discloses all sub-combinations of various embodiments and their elements (e.g., elements of chemical groups listed in embodiments describing such variables) as if each and every such sub-combination were disclosed individually and explicitly herein.

[0018] A. Definition 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. In case of conflict, this document (including the definitions) shall prevail. Although similar or equivalent methods and materials to those described and materials herein may be used to practice or test this disclosure, preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0019] As used herein, the terms “comprise(s)”, “include(s)”, “having”, “has”, “can”, “contain(s)”, and variations thereof are intended as open-ended transitional phrases, terms, or words that do not exclude the possibility of additional behaviors or structures. Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural indicators. This disclosure also contemplates other embodiments, whether explicitly listed or not, that “comprise”, “consist of”, and “substantially constitute” the embodiments or elements presented herein.

[0020] The modifier “about” used in conjunction with a quantity includes the stated value and has a meaning specified by the context (e.g., it includes at least the degree of error associated with a particular quantity of measurement). The modifier “about” should also be considered to disclose a range defined by the absolute values ​​of its two endpoints. For example, expressing “about 2 to about 4” also discloses the range “2 to 4”. The term “about” can refer to the indicated number plus or minus 10%. 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” may be apparent from the context, such as rounding, so for example, “about 1” can also mean 0.5 to 1.4.

[0021] The connecting term "or" includes any and all combinations of one or more of the listed elements associated with the connecting term. For example, the phrase "device containing A or B" can refer to a device that includes A but does not contain B, a device that includes B but does not contain A, or a device that contains both A and B. The phrase "at least one of A, B, ... and N" or "at least one of A, B, ... and N or a combination thereof" is defined in the broadest sense as meaning one or more elements selected from the group containing A, B, ... and N. That is, any combination of one or more of elements A, B, ... and N includes any single element or a combination with one or more of the other elements, and may also include additional unlisted elements in combination.

[0022] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS edition, Handbook of Chemistry and Physics, 75th edition, inner cover, and specific functional groups are generally defined as described herein. Furthermore, the general principles of organic chemistry, as well as specific functional groups and reactivity, are described in the Handbook of Chemistry and Physics. Organic Chemistry Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, *March's Advanced Organic Chemistry*, 1999. March's Advanced Organic Chemistry ), 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformation ( Comprehensive Organic Transformations ), VCH Publishers, Inc., New York, 1989; Carruthers, Several Modern Methods of Organic Synthesis ( Some Modern Methods of Organic Synthesis In the references cited in the 3rd edition of Cambridge University Press, Cambridge, 1987, the entire contents of each of these references are incorporated herein by reference.

[0023] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. The terms "lower alkyl" or "C1-C6 alkyl" refer to a straight-chain or branched hydrocarbon containing 1 to 6 carbon atoms. The term "C1-C3 alkyl" refers to a straight-chain or branched hydrocarbon containing 1 to 3 carbon atoms. Representative examples of alkyl groups 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.

[0024] As used herein, the term "alkoxy" refers to an alkyl group as defined herein, which is attached to a parent molecule via an oxygen atom. Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0025] As used herein, the term "alkenyl" refers to a hydrocarbon chain containing 2 to 30 carbon atoms, wherein the hydrocarbon chain has at least one carbon-carbon double bond. Alkenyl groups can be substituted or unsubstituted. For example, an alkenyl group can be substituted with an aryl group such as phenyl.

[0026] As used herein, the term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group having 2 to 30 carbon atoms (e.g., 2 to 20 or 2 to 10 carbon atoms) and at least one triple bond unsaturated site. The term "alkynyl" also includes non-aromatic cycloalkyl groups having 5 to 20 carbon atoms (e.g., 5 to 10 carbon atoms) having a monocyclic or polycyclic structure and at least one triple bond. Examples of such alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and propynyl (-CH₂C≡CH), as well as cycloalkynyl moieties, such as, but not limited to, substituted or unsubstituted cyclooctyne moieties.

[0027] As used herein, the term "alkoxyalkyl" means an alkoxy group attached to a parent molecule moiety by an alkyl group as defined herein.

[0028] As used herein, the term "alkylene" refers to a divalent group derived from a straight-chain or branched hydrocarbon having 1 to 30 carbon atoms (e.g., 2 to 10 carbon atoms). Representative examples of alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH3)CH2-, -C(CH3)2CH2-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-.

[0029] The term "amino acid" refers to both natural and non-natural amino acids, protected natural and non-natural amino acids, and amino acid analogs and amino acid simulants that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids include 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 pyrrolidine and selenocysteine. Non-natural amino acids are amino acid analogs that have the same basic chemical structure as naturally occurring amino acids; that is, by way of example, the α-carbon is linked to a hydrogen, carboxyl, amino, and R group. Such analogs may have a modified R group (e.g., leucine as an example) or retain a modified peptide backbone while retaining the same basic chemical structure as natural amino acids. Non-limiting examples of non-natural amino acids or amino acid analogues include citrulline, homoserine, ortholeucine, methionine sulfoxide, methionine methyl thiocation, homophenylalanine, ornithine, formylglycine, phenylglycine, p-azidophenylglycine, p-azidophenylalanine, p-acetylphenylalanine, 4-(3-methyl-(1,2,4,5-tetraazine))-phenylglycine, and 4-(3-methyl-(1,2,4,5-tetraazine))-phenylalanine.

[0030] As used herein, the term "aryl" 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 groups include, but are not limited to, phenyl, naphthyl, and anthracene. Monocyclic, bicyclic, and tricyclic aryl groups are partially attached to the parent molecule by any carbon atom contained within the ring and can be unsubstituted or substituted. Aromatic bicyclic or tricyclic systems do not contain non-aromatic rings. Therefore, if a bicyclic or tricyclic system contains a non-aromatic ring, the ring system is either cycloalkyl or heterocyclic, depending on the presence of a heteroatom in the non-aromatic ring and regardless of the attachment point of the remaining portion of the molecule.

[0031] In some embodiments, as used herein, the term "aryl" refers to a phenyl or a bicyclic aryl or tricyclic aryl fused-ring system. An example of a bicyclic fused-ring system is a phenyl group attached to a parent molecule moiety and fused with a phenyl group. An example of a tricyclic fused-ring system is a phenyl group attached to a parent molecule moiety and fused with two other phenyl groups. Representative examples of bicyclic aryl groups include, but are not limited to, naphthyl. Representative examples of tricyclic aryl groups include, but are not limited to, anthraceneyl. Monocyclic, bicyclic, and tricyclic aryl groups are attached to the parent molecule moiety by any carbon atom contained within the ring and can be unsubstituted or substituted.

[0032] As used herein, the term "azide" refers to the functional group –N3.

[0033] As used herein, the term "cycloalkyl" refers to a non-aromatic carbocyclic system containing 3 to 10, 3 to 8, 3 to 6, or 5 to 10 carbon atoms and zero heteroatoms. A cycloalkyl ring system may contain one or more double bonds, provided the ring is not aromatic; and 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 systems in which the cycloalkyl group is fused with an aryl or heteroaryl group as defined herein, regardless of the connection point of the remainder of the molecule.

[0034] In some embodiments, as used herein, the term "cycloalkyl" refers to a carbocyclic system containing three to ten carbon atoms, zero heteroatoms, and zero double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. "Cycloalkyl" also includes carbocyclic systems in which the cycloalkyl group is attached to a parent molecule moiety and fused with an aryl group, a heteroaryl group, or a heterocycle as defined herein.

[0035] As used herein, the term "cycloalkenyl" means a non-aromatic monocyclic or polycyclic system containing at least one carbon-carbon double bond and preferably having 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0036] As used herein, the term "cyclooctene" refers to a non-aromatic cyclic alkyl group having eight carbon atoms, either substituted or unsubstituted. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).

[0037] As used herein, the term “fluoroalkyl” means an alkyl group in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by fluorine, as defined herein. Representative examples of fluoroalkyl groups include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl (such as 3,3,3-trifluoropropyl).

[0038] As used herein, the term "alkoxyfluoroalkyl" refers to an alkoxy group attached to a parent molecule moiety by a fluoroalkyl group as defined herein.

[0039] As used herein, the term "fluoroalkoxy" means at least one fluoroalkyl group as defined herein attached to a parent molecule moiety by an oxygen atom. Representative examples of fluoroalkoxy groups include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0040] As used herein, the term "halogen" or "halogenated" refers to Cl, Br, I, or F.

[0041] As used herein, the term “halogenated alkyl” means an alkyl group in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by a halogen, as defined herein.

[0042] As used herein, the term “haloalkoxy” means at least one haloalkyl group as defined herein attached to a parent molecule moiety by an oxygen atom.

[0043] As used herein, the term "heteroalkyl" means, as defined herein, an alkyl group in which one or more carbon atoms have been replaced by a heteroatom selected from S, Si, O, P, and N. The heteroatom may be oxidized. Representative examples of heteroalkyl groups include, but are not limited to, alkyl ethers, secondary alkylamines, and tertiary alkylamines, as well as alkyl sulfides.

[0044] As used herein, the term "heteroaryl" refers to an aromatic group having a single ring, multiple rings, or multiple fused rings, wherein one or more ring heteroatoms are independently selected from nitrogen, oxygen, and sulfur. In some embodiments, as used herein, the term "heteroaryl" refers to an aromatic monocyclic, aromatic bicyclic, or aromatic tricyclic system. An aromatic monocyclic ring is a five- or six-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., one, two, three, or four heteroatoms independently selected from O, S, and N). A five-membered aromatic monocyclic ring has two double bonds, and a six-membered aromatic monocyclic ring has three double bonds. Representative examples of monocyclic heteroaryl groups include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, thiopheneyl, furanyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl. Representative examples of bicyclic heteroaryl groups include, but are not limited to, chromenyl, benzothiopheneyl, benzodioxacyclopentenyl, benzotriazolyl, quinolinyl, thienopyrroleyl, thienothiopheneyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryl groups include, but are not limited to, dibenzofuranyl and dibenzothiopheneyl. Monocyclic, bicyclic, and tricyclic heteroaryl groups are partially linked to the parent molecule via any carbon or nitrogen atom contained within the ring and can be unsubstituted or substituted. In some embodiments, the aromatic bicyclic or tricyclic system does not contain a non-aromatic ring. Therefore, if the bicyclic or tricyclic system contains a non-aromatic ring, the ring system is a cycloalkyl or heterocyclic group, depending on the presence of a heteroatom in the non-aromatic ring and independent of the connection point of the remaining portion of the molecule.

[0045] In some embodiments, a five-membered aromatic monocyclic ring has two double bonds, and a six-membered aromatic monocyclic ring has three double bonds. In some embodiments, an exemplary bicyclic heteroaryl is exemplified as a monocyclic heteroaryl ring attached to a parent molecule moiety and fused with a monocyclic cycloalkyl, a monocyclic aryl, a monocyclic heteroaryl, or a monocyclic heterocycle as defined herein. In some embodiments, a tricyclic heteroaryl is exemplified as a monocyclic heteroaryl ring attached to a parent molecule moiety and fused with both a monocyclic cycloalkyl, a monocyclic aryl, a monocyclic heteroaryl, or a monocyclic heterocycle as defined herein.

[0046] As used herein, “heterocyclic group,” “heterocyclic,” or “heterocyclic” refers to a non-aromatic ring system containing 3 to 10, 3 to 8, 3 to 6, or 5 to 10 carbon atoms and at least one (e.g., 1-5, 1-4, 1-3, 1-2, or 1) heteroatom, and optionally one or more oxolated and / or double bonds. The terms “heterocyclic group,” “heterocyclic,” 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, a monocyclic heterocycle is a ternary, quaternary, pentaneary, hexavalent, septaneous, or octaneous ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, a ternary or quaternary ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. In some embodiments, a pentaneous ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the six-membered ring contains zero, 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 seven- and eight-membered rings contain zero, 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, aziridine, aziridine, diaziridine, 1,3-dioxane, 1,3-dioxolane, 1,3-dioxolane, 1,3-dithiocyclopentane, 1,3-dithiane, 1,3-dimethylpyrimidin-2,4(1H,3H)-dione, imidazolinyl, imidazolinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, and oxadiazole. Alkyl, oxazolinyl, oxazolinyl, oxazolinyl, oxazolinyl, piperazine, piperidinyl, pyranyl, pyrazolinyl, pyrazolinyl, pyrrololinyl, pyrrolylyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl, thiadiazolinyl, thiadiazolinyl, 1,2-thiazinicyl, 1,3-thiazinicyl, thiazolinyl, thiazolinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl (thiomorpholinone), thiopyranyl, and trithiaalkyl. Bicyclic heterocycles are monocyclic heterocycles fused with phenyl groups, or monocyclic heterocycles fused with monocyclic cycloalkyl groups, or monocyclic heterocycles fused with monocyclic cycloalkenyl groups, or monocyclic heterocycles fused with monocyclic heterocycles, or spirocyclic heterocycle groups, or bridged monocyclic heterocycle ring systems in which two non-adjacent atoms on the ring are connected by an alkylene bridge having one, two, three or four carbon atoms or an alkenyl bridge having two, three or four carbon atoms.Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, benzodihydropyranyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophene, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]hept-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), and 2,3-dihydro-1-yl. H -Indoleyl, isoindolinyl, octahydrocyclopentano[ c Pyrrole, octahydropyrrolopyridyl, and tetrahydroisoquinolinyl. Examples of tricyclic heterocycles are bicyclic heterocycles fused to phenyl groups, or bicyclic heterocycles fused to monocyclic cycloalkyl groups, or bicyclic heterocycles fused to monocyclic cycloalkenyl groups, or bicyclic heterocycles fused to monocyclic heterocycles, or bicyclic heterocycles wherein two non-adjacent atoms of the bicyclic ring are connected by an alkylene bridge having one, two, three, or four carbon atoms, or an alkenyl bridge having two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxycyclopentadiene, hexahydro-2... H -2,5-methanecyclopentane[ b Furan, hexahydro-1 H -1,4-methanecyclopentane[ c Furan, aza-adamantane (1-azatricyclic [3.3.1.1]) 3,7 [decane] and oxa-adamantane (2-oxatricyclo[3.3.1.1]) 3,7 [Decane]. Monocyclic, bicyclic, and tricyclic heterocycles are partially connected to the parent molecule via any carbon or nitrogen atom contained within the ring, and can be unsubstituted or substituted.

[0047] As used herein, the term "hydroxyl group" refers to the -OH group.

[0048] As used herein, the term “hydroxyalkyl” means an alkyl group in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by hydroxyl groups, as defined herein.

[0049] The term "substituted" refers to a group that can be further substituted by one or more non-hydrogen substituents. Substituents include, but are not limited to, halogens, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketones, amides, carbamates, and acyl groups.

[0050] The term "tetraazine" refers to a substituted or unsubstituted aromatic cyclic group having a monocyclic ring with three double bonds, two carbon atoms, and four nitrogen atoms. Examples of tetraazine groups include 1,2,3,4-tetraazine and 1,2,4,5-tetraazine. As used herein, 1,2,4,5-tetraazine is referred to as a "Tz" group. The term "tetraazine-based" generally refers to a tetraazine as defined herein or a moiety that can be converted to tetraazine, such as optionally substituted dihydrotetraazine.

[0051] The term "selective delivery" refers to delivering a drug agent (e.g., a TPD) to an organ or tissue (or part thereof) requiring treatment or diagnosis without significant binding to other non-target organs or tissues (or parts thereof). In some embodiments, the trans-cyclooctene-modified TPD conjugates described herein do not have therapeutic effects themselves but are designed to allow selective or targeted delivery of the TPD. However, tetrazine-based targeting agents may have therapeutic effects, and therefore, this disclosure does not exclude such constructs.

[0052] The term "diagnostic agent" refers to a drug that helps diagnose symptoms or diseases. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, and radionuclides. 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 at one wavelength of radiation and detection at a second, different wavelength of radiation. Optical probes disclosed herein include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-octadecyl-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate), and DiR (1,1'-octadecyl-3,3,3',3'-tetramethylindole tricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo detectable radioactive decay. Radionuclides used in embodiments of this disclosure include, but are not limited to, those that exhibit radioactive decay. 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.

[0053] As used herein, the term "ligand" defines a binding partner for a biological target molecule (e.g., an enzyme or receptor) in vivo. The ligands used according to the invention are typically small molecules that can be functionalized by adding a TCO moiety via a linker.

[0054] The term "target agent" refers to a chemical or biological agent that specifically binds to a target (e.g., a target organ or tissue), thereby forming a stable binding between the target agent and the specific target. "Stable binding" means that the compound binds to another part or structure under standard physiological conditions, such as covalently or non-covalently, or otherwise. Stable binding can include covalent and 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, etc. Target agents include ligands that specifically bind (or substantially specifically bind) to specific clinically relevant target receptors or cell surface targets. Specific target agents are described in this article.

[0055] The term "targeted organ or tissue" refers to the organ or tissue targeted for TPD delivery. Representative targeted organs and tissues include those that can be targeted by chemical or biological agents, as well as those that cannot be targeted by chemical or biological agents.

[0056] The term "implant" refers to something that is surgically inserted into a subject's body.

[0057] The term "contacting" refers to the process of bringing at least two different species into contact so that they can interact with each other, such as in non-covalent or covalent interactions or binding reactions. However, it should be understood that the resulting complex or reaction product can be produced directly from the interaction or reaction between the added reagents, or from one or more of the added reagents or intermediates that can be produced in the contact mixture.

[0058] The term "binding agent" refers to an agent having functional groups capable of forming covalent bonds with complementary functional groups of another binding agent in the biological environment. Binding between binding agents in the biological environment can also be referred to as bioconjugation. Binding agents include bioorthogonal binding agents, which are binding agents having bioorthogonal functional groups. The bioorthogonal functional groups of a bioorthogonal binding agent selectively react with complementary bioorthogonal functional groups of another bioorthogonal binding partner. This selective reaction between bioorthogonal binding partners minimizes side reactions with other binding agents, biological compounds, or other non-complementary bioorthogonal binding agents or non-complementary bioorthogonal functional groups. The functional groups of the bioorthogonal portion or bioorthogonal binding agent 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-tetraazine). The binding agents used in this disclosure can be highly reactive with their corresponding binding agents, resulting in rapid reactions.

[0059] The term “functionalization” refers to a portion having functional groups attached to it, such as a portion having binding functional groups attached to it (e.g., bioorthogonal functional groups).

[0060] The term “administration” refers to any suitable route of administration to a subject, such as, but not limited to, oral administration, administration as a suppository, local 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 microosmotic pump) into the subject.

[0061] As used herein, the term “parenteral” refers to the method of administration, which includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0062] The term "therapeutic effective amount" refers to an amount of compound sufficient to treat a particular condition or disease or one or more symptoms thereof and / or to prevent or reduce the risk of the occurrence or recurrence of the disease or condition or its symptoms. In the case of tumorigenic proliferative diseases, among other things, a pharmaceutically or therapeutically effective amount includes an amount sufficient to cause tumor shrinkage or reduce the rate of tumor growth.

[0063] As used herein, the terms “subject” or “patient” include humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to whom the agents of this disclosure may be administered may include mammals, particularly primates, especially humans. For veterinary applications, suitable subjects may include, for example, livestock (e.g., cattle, sheep, goats, dairy cows, pigs, etc.); poultry (e.g., chickens, ducks, geese, turkeys, etc.); and domesticated animals (especially pets, such as dogs and cats). For diagnostic or research applications, suitable subjects may include mammals such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs (e.g., inbred pigs).

[0064] As used herein, the term “treating” means treating a disease or medical condition or its symptoms in a patient (such as a mammal, especially a human), including: (a) improving the disease or medical condition or its symptoms, such as eliminating the patient’s disease or medical condition or its symptoms or causing its remission; (b) suppressing the disease or medical condition or its symptoms, such as by slowing or preventing the development of the patient’s disease or medical condition or its symptoms; or (c) alleviating the symptoms of the patient’s disease or medical condition or its symptoms.

[0065] The term "physiological conditions" is intended to encompass those conditions compatible with living cells, such as the primary aqueous conditions compatible with living cells, such as temperature, pH, and salinity.

[0066] For the compounds described herein, the groups and substituents can be selected according to the permissible valences of the atoms and substituents, such that selection and substitution produce stable compounds, for example, stable compounds that do not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc.

[0067] Where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (unless otherwise explicitly stated by the context, one-tenth of the unit to the lower limit) and any other stated or intermediate values ​​within the range are included within this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also included within this disclosure, subject to any explicitly excluded limits within the range. Where the range includes one or both limits, the range excluding any one or both of the included limits is also included within this disclosure.

[0068] For the numerical ranges listed in this article, each intermediate number with the same degree of precision between them is explicitly envisioned. For example, for the range of 6-9, the numbers 7 and 8 are envisioned in addition to 6 and 9; and for the range of 6.0-7.0, 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 explicitly envisioned.

[0069] Compounds can exist as stereoisomers, containing either asymmetric or chiral centers. The stereoisomer is determined by the configuration of the substituents surrounding the chiral carbon atom. R "or" S The term "" as used in this article R "and" S"The configuration is as defined in the IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry", Pure Appl. Chem., 1976, 45:13-30. This disclosure contemplates various stereoisomers and mixtures thereof, and these are explicitly included within the scope of this disclosure. Stereoisomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of a compound may be prepared synthetically from commercially available starting materials containing an asymmetric or chiral center, or by preparing a racemic mixture followed by decomposition methods well known to those skilled in the art. Examples of such decomposition methods include: (1) linking a mixture of enantiomers to a chiral auxiliary, separating the resulting diastereomer mixture by recrystallization or chromatography, and as Furniss, Hannaford, Smith, and Tatchell, The optional methods described in “Vogel’s Textbook of Practical Organic Chemistry”, 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, are: (1) releasing the optically pure product from the auxiliaries; (2) directly separating the optically enantiomer mixture on a chiral chromatographic column; or (3) stepwise recrystallization.

[0070] This disclosure also includes isotopically labeled compounds, identical to those described herein, but with the distinction that one or more atoms are replaced by atoms with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes suitable for inclusion in compounds of this disclosure are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, 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. Used as in deuterium (i.e., 2Substitution with heavier isotopes such as H can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dose requirement, and is therefore preferred in some cases. Compounds can be incorporated with positron emission isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron emission isotopes that can be incorporated include... 11 C 13 N、 15 O and 18 F. The isotopically labeled compounds disclosed herein can generally be prepared by those skilled in the art using known conventional techniques, or by methods similar to those described in the appended examples, using appropriate isotopically labeled reagents instead of non-isotopically labeled reagents.

[0071] B. Trans-cyclooctene-modified TPD conjugates The trans-cyclooctene-modified TPD conjugates described herein are designed to target specific sites in the body of a subject upon administration. The trans-cyclooctene-modified TPD conjugates can be administered locally or systemically. During administration, a tetrazine activator can be administered, which, upon contact with the trans-cyclooctene-modified TPD conjugate in vivo, enables targeted drug delivery of the TPD.

[0072] This article provides a trans-cyclooctene-modified TPD conjugate comprising a targeted protein degrader (TPD) optionally covalently linked to at least one trans-cyclooctene moiety via a linker.

[0073] In some embodiments, the trans-cyclooctene-modified TPD conjugate has the formula XL-TPD; wherein X is the trans-cyclooctene moiety; L is the linker; and TPD is a targeted protein degrader.

[0074] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the TPD comprises an E3 ubiquitin ligand selected from the group consisting of: Von Hippel–Lindau (VHL) ligand, cereblon (CRBN) ligand, mouse dual microsome 2 homologue (MDM2) ligand, and a β-transduction protein repeat sequence protein (β-TrCP) ligand.

[0075] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of thalidomide, lenalidomide, pomalidomide, MLN4924 (Pevonedistat), Nutlin-3, and curcumin.

[0076] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the TPD comprises an E3 ubiquitin ligase ligand selected from Table A, wherein the wavy line indicates the connection point with the remainder of the TPD.

[0077] Table A In the embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the TPD comprises a target protein ligand selected from the group consisting of: receptor tyrosine kinase (RAF) kinase ligands (e.g., BRAF kinase ligands), steroid receptor ligands (e.g., androgen receptor (AR) ligands, estrogen receptor (ER) ligands, or progesterone receptor (PR) ligands), cyclin-dependent kinase (CDK) ligands, BTK ligands, BET ligands, and inhibitors of apoptosis proteins (IAP) ligands.

[0078] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the TPD contains a target protein ligand selected from Table B, wherein the wavy line indicates the connection point with the remainder of the TPD.

[0079] Table B In some examples of the trans-cyclooctene-modified TPD conjugates disclosed herein, the TPD is selected from Table C: Table C In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the trans-cyclooctene moiety has the formula X: X in: R 1A Each time it appears, independently select a group consisting of the following items: C 1-4 Alkyl, C 1-4 Halogenated alkyl and C 1-4 Alkoxy; q is 0, 1, or 2; q1 is either 0 or 1; R 1B Each time it appears, independently select a group consisting of the following items: G 1 OH, –NR1c –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 –CO2H, –N(R) 1f )–C 2-4 Alkylene–(N(C) 1-4 alkylene (–CO2H)–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 (–CO2H)2 and –N(R) 1c )–CH(CH2O–(CH2CH2O) 0-2 –C 1-6 Alkylene (CO2H)2; R 1c and R 1d It is either hydrogen or C each time it appears. 1-4 alkyl; R 1e It is independently set to –C each time it appears. 1-4 Alkylene –CO2H, –C1-4 Alkylene –CONH2 or –C 1-4 alkylene–OH; R 1f Each time it appears, it is independently hydrogen or C. 1-6 Alkyl or C 1-4 alkylene –CO2H; n is 0, 1, 2 or 3 independently each time it appears; L 2 Each time it appears, independently select the group consisting of the following items: –C(O) – and C 1-3 Alkylene; and G 1 Each time it appears, it is independently a heterocyclic group with optional substitution.

[0080] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the trans-cyclooctene moiety has the formula XI: XI in: R 2 It is -OH, 2-aminoethanesulfonic acid, N-linked natural or non-natural amino acid, or optionally substituted ethylenediamine; wherein R 2 It can optionally be further replaced by polyether.

[0081] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the trans-cyclooctene moiety has the formula: , , , , , , , or .

[0082] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, at least one trans-cyclooctene moiety is .

[0083] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, at least one trans-cyclooctene moiety is .

[0084] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, at least one trans-cyclooctene moiety is Where A is an amino acid side chain (e.g., C). 1-4Alkyl, aryl (e.g., phenyl), etc., wherein each is optionally substituted with the following: -OH, -SH, -SCH3, -C(O)OH, -NH2, -NHNH2NH, -C(O)NH2, etc.

[0085] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, at least one trans-cyclooctene moiety is .

[0086] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the trans-cyclooctene moiety is covalently linked to the TPD via a linker. Suitable linkers are described below.

[0087] In some embodiments of the trans-cyclooctene-modified TPD conjugates disclosed herein, the linker is a cleavable linker.

[0088] In some embodiments, the linker comprises one or more of the following: carbamate (-NHC(O)O-), ester and amide, hydrazone, hydrazide, disulfide, N-succinimide-4-(2-pyridinedithio)valerate (SPP), N-succinimide-4-(2-pyridinedithio)butyrate (SPDB), 4-(4'-acetylphenoxy)butyric acid (AcBut), one or more linear or branched natural or non-natural amino acids, valine-citrulline (Val-Cit) moiety, or phenylalanine-lysine (Phe-Lys) moiety.

[0089] In some embodiments, the connector comprises 1 to 100 connecting atoms, 1 to 50 connecting atoms, or 5 to 50 connecting atoms, or 10 to 50 connecting atoms, or 1 to 40 connecting atoms, or 1 to 30 connecting atoms, or 1 to 20 connecting atoms, or 1 to 10 connecting atoms, or 1 to 5 connecting atoms, or 5 to 30 connecting atoms, or 10 to 30 connecting atoms, or 5 to 40 connecting atoms, or 5 to 50 connecting atoms, or 10 to 50 connecting atoms.

[0090] In some embodiments, the linker comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkyneene, arylene, heteroarylene, cycloalkylene, or heterocyclic moieties; wherein the alkylene, alkenylene, alkyneene, arylene, heteroarylene, cycloalkylene, or heterocyclic moieties may each be optionally and independently substituted by one to five substituents independently selected from the following: oxo, halogenated, C 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups.

[0091] In some embodiments, the connector is a non-cuttable connector.

[0092] In some embodiments, the connector is a cuttable connector.

[0093] In some embodiments, the linker comprises one or more amino acids.

[0094] In some embodiments, the linker comprises a polypeptide.

[0095] In some embodiments, the linker is an alkylene linker that optionally contains one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thiosuccinimide, or ketone functional groups.

[0096] In some embodiments, the connector has the following formula: -Y 10 -(CHR 130 ) n' -Y 20 -(CHR 140 ) n'' -Y 30 -(CHR 150 ) m'' -Y 40 - in: Y 10 Y 20 Y 30 and Y 40 Each of them is an independent key, -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, ynylene, arylene, or heteroarylene; wherein each of the alkylene, alkenylene, ynylene, arylene, or heteroarylene groups is independently and optionally substituted by one to five substituents selected independently from the following: oxo, halogenated, C 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups; Each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4Halogenated alkyl, aryl, heteroaryl, cycloalkyl or heterocyclic; Each R 120 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl or heterocyclic; Each R 130 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic or amino acid side chain; Each R 140 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic or amino acid side chain; Each R 150 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic, or amino acid side chains; and n', n'' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0097] In some embodiments, the connector has the following formula: -Y 10 -(CH2) n' -Y 20 -(CH2) m'' -Y 30 - in: Y 10 Y 20 and Y 30 Each of them is an independent key, -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, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkylene; wherein each of the alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkylene is independently and optionally substituted by one to five substituents independently selected from the following: oxo, halogenated, C 1-4 Alkyl, C1-4 Alkoxy and C 1-4 Halogenated alkyl groups; Each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl or heterocyclic; Each R 120 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclic groups; and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0098] In some embodiments, each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloaryl, or heterocyclic; and each R 120 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclic groups. In some embodiments, the linker is not a bond.

[0099] Linkers may include one or more of the following: polyethylene glycol (e.g., PEG with an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-dimethylbis(methyl carbamate), arylene (e.g., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.

[0100] In some embodiments, the connector includes one or more of the following: , , , , , or .

[0101] In some embodiments, the connector includes one or more of the following: , , , , , , or .

[0102] In some embodiments, the connector includes one or more of the following: , , , , or .

[0103] In some embodiments, the connector comprises one or more In some embodiments, the connector comprises one or more .

[0104] In some embodiments, the connector is or includes one or more of the following: , , , , , , , , , , , , , , , or .

[0105] In some embodiments, the connector is or includes one or more of the following: , , , , or .

[0106] In some embodiments, the linker is or comprises one or more natural or non-natural amino acids, which may be referred to as a peptide linker. The linker may be a peptide linker composed of a carboxyl acyl unit and one or more amino acids that make up a protein or peptide sequence. The linker may also contain a self-sacrificing spacer that separates the drug from the protein peptide sequence.

[0107] In some embodiments, the connector is or includes the following: "A—Y—Z—X" 2 "-W" indicates a peptide linker, where "A" is a carboxyl acyl unit, "Y" and "Z" are each one or more natural or non-natural amino acids, which together form the peptide sequence, and "X" represents a linker containing a peptide. 2 "" and "W" are optional additional linkers having 1 to 50 linking atoms, or 5 to 10 linking atoms, or 1 to 10 linking atoms. In some embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0108] 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.

[0109] In some embodiments, Z may be at least one amino acid selected from the group consisting of: alanine, lysine, lysine protected with an acetyl or formyl group, arginine, arginine protected with a toluenesulfonyl or nitro group, histidine, ornithine, ornithine protected with an acetyl or formyl group, and citrulline. In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0110] Exemplary YZ combinations include valine-citrulline; valine-alanine; and alanine-alanine.

[0111] In some embodiments, A is -OC(O)-.

[0112] In some embodiments, X 2 It is -OC(O)-.

[0113] In some embodiments, W is -OC(O)-. In some embodiments, X 2 It does not exist and W is -OC(O)-.

[0114] In some embodiments, part —X 2 —W includes In some embodiments, part —X 2 for .

[0115] In some embodiments, —X—W is .

[0116] In some embodiments, —X—W is .

[0117] In some embodiments, the peptide linker is specifically tailored such that it will be selectively cleaved (e.g., enzymatically) to release a drug, such as via one or more tumor-associated proteases.

[0118] In some embodiments, the peptide linker has a chain length of two to four amino acid residues (i.e., a dipeptide, tripeptide, or tetrapeptide). However, it will be understood that peptide linkers with up to five, six, seven, or eight amino acid residues may also be suitably employed.

[0119] In some embodiments, the peptide linker is 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 -Toluenesulfonyl-Arg or Phe-N 9 -Nitro-Arg. In some embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In some embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0120] In some embodiments, L or the connector is or includes one or more of the following: , , , , , , , , , , , , , , , , , , , , , , , , or .

[0121] In some embodiments, the connector includes one or more of the following: (For example, ), (For example, ), , , , , , , , , or .

[0122] In some embodiments, the connector includes one or more of the following: (For example, ), , , , , , , , , , or .

[0123] The aforementioned linker can bind to an amino acid side chain present on X, such as lysine or cysteine ​​(e.g., , , , , , ).

[0124] In some embodiments, the connector is –C(O)L 4 –or–C(O)C 1-6 Alkylene C(O)L 4 –; L 4 For key, –N(R) 12 )–C 2-3 alkylene–N(R) 13 )C(O)–、-CH(NHC(O)R 14 C 1-4 Alkylene–S–S–C 1-4 Alkylene –OC(O)–, –NHNHC(O)CH(NHC(O)R 15 CH2C(O)–、–C 1-6 Alkylene – CH(G) x )OC(O)–、 , , , or ; R 12 R 13 R 14 R 15 and R 19 Each is independently hydrogen or C 1-4 alkyl; R 16 For hydrogen, C 1-4 Alkyl, –C 1-4 alkylene –OH, –C 1-4 Alkylene–OC 1-4 Alkyl, –C 1-4 Alkylene –CO2H or –C 1-4 alkylene –CONH2; and G x A phenyl group optionally substituted with 1 to 5 substituents independently selected from the group consisting of: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, cyano, and nitro groups.

[0125] In some embodiments, the linker comprises a carbonyl moiety for conjugating at least one trans-cyclooctene moiety to the TPD. 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 additional lysine or other amino acid side chains conjugated to the carbonyl moiety. In some embodiments, the linker may comprise... .

[0126] In some embodiments, the connector is or includes one or more of the following: , , , , , , , , , , , , , , or .

[0127] In some embodiments, the connector is: , , , , , or .

[0128] In some embodiments, the connector is or includes one or more , , , , , , , , , , or .

[0129] In some embodiments, the connector is or includes: .

[0130] In some embodiments, a trans-cyclooctene-modified TPD conjugate selected from Table 1 is provided.

[0131] Table 1 C. Tetraazine activator In some embodiments, a system is provided comprising a trans-cyclooctene-modified TPD conjugate as described herein and a tetrazine activator. The tetrazine activator may be formulated as a free tetrazine compound or optionally administered systemically via a linker covalently bound to an antibody or antibody fragment portion of a tetrazine-containing group, or administered via a therapeutic support composition comprising a biocompatible support and a tetrazine-containing group.

[0132] In some embodiments, the trans-cyclooctene-modified TPD conjugate can target specific proteins at the treatment site or on subject cells. In some embodiments, the trans-cyclooctene moiety substantially blocks or inhibits the efficient binding of a portion of the TPD to its target. Once the trans-cyclooctene-modified TPD conjugate is bound to the treatment site, contact with a tetrazine activator releases the trans-cyclooctene moiety, thereby exposing the TPD and allowing it to bind to its target.

[0133] In some embodiments, the tetrazine activator is formulated for systemic administration.

[0134] In some embodiments, the tetrazine activator has formula IV: IV in: Each R 20Independently select from the group consisting of: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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'', 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, heterocyclic or cycloalkyl is optionally surrounded by one to three Z 1 replace; Each Z 1 Independently selected from halogenated, oxo-, cyano-, nitro-, hydroxyl-, alkyl-, haloalkyl-, alkenyl-, alkoxy-, haloalkoxy-, heteroalkyl-, aryl-, heteroaryl-, heterocyclic-, 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)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'' are independently selected from hydrogen, aryl, and alkyl groups each time they appear; and R''' is selected independently from aryl and alkyl groups each time it appears.

[0135] In some embodiments, each R 20Independently, it is phenyl, pyrimidinyl, triazinyl, oxazolyl, isoxazolyl, 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; wherein each is optionally independently surrounded by one to three Z 1 replace.

[0136] In some embodiments, the tetrazine activator is: , , , , , , , , , or .

[0137] In some embodiments, the tetrazine activator is: , , , , , , , , , or .

[0138] In some embodiments, the tetrazine activator comprises a biocompatible support, an antibody, or an antibody fragment portion, or in some embodiments, the antibody or antibody fragment portion is covalently bound to one or more tetrazine portions.

[0139] In some embodiments, the tetrazine activator has formula I, formula II, or formula V: I II V in: Ring A is aryl, cycloalkyl, heterocyclic, or heteroaryl; When R 3 and R 4 When neither of these conditions is present, the dashed line represents the additional bond forming the tetrazine; or when R... 3 and R 4 When both are present, the dashed line represents the additional bond forming the dihydrotetraazine; the condition is that when ring A is aryl, then R... 3 and R 4 Both exist; X represents a biocompatible support, antibody, or antibody fragment. p is 1-150; L is an independent connector each time it appears; R 1 Each time it appears, it is independently selected from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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'', 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, heterocyclic or cycloalkyl is optionally surrounded by one to three Z 1 replace; R 2 Each of the following is independently halogenated, cyano, nitro, hydroxyl, alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclic or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic or cycloalkyl is optionally marked by one to three Z 1 replace; R 3 and R 4 Neither exists; or R 3 and R 4 Each is independently hydrogen or a group that can be removed after a triggering event; R 20Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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 Each time it appears, it is an independent linker of 1 to 100 linking atoms, wherein the linking atoms optionally contain one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate or ketone functional groups; R 30 Each time it appears, it is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl. R a R 31a and R 31b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; Each Z 1Independently selected from halogenated, oxo-, cyano-, nitro-, hydroxyl-, alkyl-, haloalkyl-, alkenyl-, alkoxy-, haloalkoxy-, heteroalkyl-, aryl-, heteroaryl-, heterocyclic-, 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)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'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; and t is independently 0, 1, 2, 3 or 4 each time it appears.

[0140] In some embodiments, the tetrazine activator has Formula I: I in: X represents an antibody or a fragment of an antibody. p is 1-16; L is an independent connector each time it appears; R 20Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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 Each time it appears, it is an independent linker of 1 to 100 linking atoms, wherein the linking atoms optionally contain one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate or ketone functional groups; R' and R'' are independently selected from hydrogen, aryl, and alkyl groups each time they appear; and R''' is selected independently from aryl and alkyl groups each time it appears.

[0141] In one embodiment, the tetrazine activator has Formula II: II in: X represents an antibody or a fragment of an antibody. p is 1-16; L is an independent connector each time it appears; R 20Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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 Each time it appears, it is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl. R a R 31a and R 31b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; and t can be 0, 1, 2, 3 or 4 independently.

[0142] In one embodiment, R 22 Each time it appears, it is an independent linker with 1 to 100 connecting atoms, and may include ethylene-oxygen, amine, ester, amide, carbamate, carbonate and ketone functional groups.

[0143] In one embodiment, the tetrazine activator has formula IIA: IIA Among them, L, p, X, and R 20 Each is defined independently as described in this article.

[0144] In one embodiment, the tetrazine activator has formula IIB: IIB L, p, and X are each defined independently as described in this paper.

[0145] In one embodiment, the tetrazine activator has the formula IIC: IIC L, p, and X are each defined independently as described in this paper.

[0146] In one embodiment, the tetrazine activator has Formula III: III in: X represents an antibody or a fragment of an antibody. p is 1-16; L is an independent connector each time it appears; R 20 Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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 Each time it appears, it is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl. R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; and t can be 0, 1, 2, 3 or 4 independently.

[0147] In some embodiments, the tetrazine activator is represented by formula IID: IID Where X and R 20 Each is independent as defined herein. In some embodiments, R 20 X is a methyl group. In some embodiments, X is an antigen-binding protein. In some embodiments, X is an antigen-binding protein that targets HER2.

[0148] In some embodiments, the tetrazine activator has formula IIE: IIE p and X are each defined independently as described in this paper.

[0149] In some embodiments, the tetrazine activator has the formula IIF: IIF p and X are each defined independently as described in this paper.

[0150] In some embodiments, the tetrazine activator has the formula IIG: IIG p and X are each defined independently as described in this paper.

[0151] In some embodiments of Formula IIA, at least one for: , , , , , , , or .

[0152] In some embodiments of Formula IIA, at least one for , where R 20 As defined in this article.

[0153] In some embodiments of Formula IIA, at least one for .

[0154] In some embodiments of Formula IIA, at least one for , where R 20 As defined in this article.

[0155] In some embodiments of Formula IIA, at least one for .

[0156] In some embodiments, P is 1-12. In some embodiments, X is an antibody. In some embodiments, p is 1-6 or 5-6. 2. In some embodiments, 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. In some embodiments, X is an antibody fragment portion (e.g., Fab).

[0157] In one embodiment, the tetrazine activator has formula V: V in: Ring A is aryl, cycloalkyl, heterocyclic, or heteroaryl; When R 3 and R 4 When neither of these conditions is present, the dashed line represents the additional bond forming the tetrazine; or when R... 3 and R 4 When both are present, the dashed line represents the additional bond forming the dihydrotetraazine; the condition is that when ring A is aryl, then R... 3 and R 4 Both exist; X represents a biocompatible support, antibody, or antibody fragment. p is 1-150; L is an independent connector each time it appears; R 1Each time it appears, it is independently selected from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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'', 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, heterocyclic or cycloalkyl is optionally surrounded by one to three Z 1 replace; R 2 Each of the following is independently halogenated, cyano, nitro, hydroxyl, alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclic or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic or cycloalkyl is optionally marked by one to three Z 1 replace; R 3 and R 4 Neither exists; or R 3 and R 4 Each is independently hydrogen or a group that can be removed after a triggering event; Each Z 1Independently selected from halogenated, oxo-, cyano-, nitro-, hydroxyl-, alkyl-, haloalkyl-, alkenyl-, alkoxy-, haloalkoxy-, heteroalkyl-, aryl-, heteroaryl-, heterocyclic-, 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)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'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; and t is independently 0, 1, 2, 3 or 4 each time it appears.

[0158] In some embodiments, the tetrazine activator has formula V: V in: Ring A is a cycloalkyl, heterocyclic, or heteroaryl group; When R 3 and R 4 When neither of these conditions is present, the dashed line represents the additional bond forming the tetrazine; or when R... 3 and R 4 When both are present, the dashed line represents the additional bond that forms the dihydrotetraazine; X represents a biocompatible support, antibody, or antibody fragment. p is 1-150; L is an independent connector each time it appears; R 1Each time it appears, it is independently selected from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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'', 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, heterocyclic or cycloalkyl is optionally surrounded by one to three Z 1 replace; R 2 Each of the following is independently halogenated, cyano, nitro, hydroxyl, alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclic or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic or cycloalkyl is optionally marked by one to three Z 1 replace; R 3 and R 4 Neither exists; or R 3 These are groups that can be removed after the event is triggered; R 4 For hydrogen or R 3 ; Each Z 1Independently selected from halogenated, oxo-, cyano-, nitro-, hydroxyl-, alkyl-, haloalkyl-, alkenyl-, alkoxy-, haloalkoxy-, heteroalkyl-, aryl-, heteroaryl-, heterocyclic-, 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)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'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; and t is independently 0, 1, 2, 3 or 4 each time it appears.

[0159] In some embodiments, the tetrazine activator has formula VI: VI Where R 1 R 2 R 3 R 4 Each of rings A, L, p, t, and X is independently as defined herein.

[0160] In some embodiments, R 4 It is hydrogen.

[0161] In some embodiments, R 3 This refers to a group that can be removed after a triggering event. In some embodiments, the triggering event occurs within the body. Once the triggering event occurs and R... 3 When removed, the dihydrotetraazine-containing group is oxidized, resulting in a tetraazine as shown in formula VII: VII Where R 1 R 2 Each of rings A, L, p, t, and X is independently as defined herein.

[0162] The triggering event is initiated upon administration of the tetrazine activator to the subject and can be initiated in any manner, such as internal (e.g., by enzymatic cleavage of functional groups, optionally followed by degradation) or external (e.g., by photocleavage of linkers). In some embodiments, R 3 It includes a targeting component, such as an antibody or antibody fragment as described herein.

[0163] In some embodiments, R 3 It contains a specific amino acid sequence that can be cleaved by proteases or esterases.

[0164] In some embodiments, R 3 It contains specific amino acid sequences that can be cleaved by proteases as shown in Table 1A.

[0165] Table 1A In some embodiments, R 3 It contains a specific amino acid sequence that can be cleaved by cathepsins, matrix metalloproteinases (MMPs), or PSMA. For example, in some embodiments, R 3 Contains Val-Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly cleaved by cathepsins. In some embodiments, R 3 Contains Ac-γE-PLG-S(OBn)YL or Ac-PLG–HofOrnL, where Hof is homophenylalanine and Orn is ornithine cleaved by MMP. In some embodiments, R 3 It contains the amino acid sequence shown in Table 1B.

[0166] Table 1B ↓ Indicates the cutting site Special amino acid abbreviations: Cit: Citrulline; Cha: β-Cyclohexylalanine; Hof: Homophenylalanine; Nva: Azelaic acid; Dpa: D-phenylalanine; Nle: Leucine; Smc: S-cysteine ​​methyl ester Multiple amino acids listed before, between, or after the slash indicate that alternative amino acids can be substituted at the stated position; a "-" indicates that any amino acid can be substituted with the corresponding amino acid indicated in the middle column. Any L-amino acid except proline Hy represents any hydrophobic L-amino acid. γ indicates that this bond is a γ-carboxyl group linkage. The other cleavage groups are described in Choi et al., Theranostics. 2012; 2(2):156-178, of which Table 2 is hereby incorporated by reference.

[0167] In some embodiments, R 3 It is photoinstable. In some embodiments, the photoinstable group will become unstable or decompose when exposed to light with a wavelength matching the absorbance profile of the photoinstable group.

[0168] In some embodiments, R 3 for ; L 5 For direct keys or connectors; and X 1 It is -NO2, an optionally substituted sugar moiety, or an optionally substituted peptide unit containing one or more natural or non-natural amino acids.

[0169] In some embodiments, at least one portion The expression is selected from the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , as well as ;where R 1 R 2 R 3 and R 4 Each of them is independent as defined herein, and optionally the ring A portion can be one or more R... 2 Partial replacement.

[0170] In some embodiments, at least one portion The expression is selected from the following: , , , as well as ;where X 2 Alkyl groups optionally substituted with the following (e.g., methyl): PEG, amino acids, esters, amides, amines, -C(O)OH, -SO2, -SO3, -PO3, -PO4, or other solubility-enhancing substituents; and L, cyclic A, R 1 R 2 Each of t, p, and X is independently as defined in this document.

[0171] In some embodiments, ring A is a cycloalkyl group. In some embodiments, ring A is a heterocyclic group. In some embodiments, ring A is a heteroaryl group. In some embodiments, ring A is an aryl group.

[0172] In some embodiments, ring A is pyrimidinyl, triazineyl, oxazolyl, isoxazolyl, 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.

[0173] In some embodiments, ring A is a phenyl group.

[0174] In some embodiments, R 1 Each time it appears, it is independently hydrogen, alkyl, alkenyl, ynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, or cycloalkyl; wherein each alkyl, alkenyl, ynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, or cycloalkyl is optionally marked by one to three Z. 1 replace.

[0175] In some embodiments, R 1 Each time it appears, it is independently and arbitrarily assigned to one to three Z. 1 Substituted hydrogen or alkyl group.

[0176] In some embodiments, Z 1 Each time it appears, it is independently selected from halogenated, hydroxylated, alkoxylated, and OC(=O)OR'.

[0177] In some embodiments, R 2 Each time it appears, it is independently halogenated, cyano, nitro, hydroxyl, alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, or cycloalkyl. In some embodiments, R 2 Each time it appears, it is independently a halo, alkyl, or haloalkyl. In some embodiments, R 2 Each time it appears, it is independently either halogenated or alkyl.

[0178] In some embodiments, t is 0 each time it occurs.

[0179] In some embodiments, the tetrazine activator has the formula: Where p and X are each independently as defined herein. In some embodiments, the tetrazine activator has the formula: p and X are each defined independently as described in this paper.

[0180] In some embodiments, X is a biocompatible support.

[0181] In some embodiments, ring A is not pyridyl. In some embodiments, ring A is not aryl. In some embodiments, ring A is not phenyl.

[0182] In some embodiments, X is a biocompatible support comprising particles, polymers, viscous or non-viscous liquid materials, gels, hydrogels, cross-linked polymer matrices, metals, ceramics, plastics, bone graft materials, or proteins.

[0183] In some embodiments, X is a biocompatible support comprising a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid, chitosan, chitosan polysaccharide, chitin, hyaluronic acid, chondroitin sulfate, heparin, suitable glycosyl biomaterials, polyphosphazene, polyanhydride, polyacetal, poly(orthoester), polyphosphate, polycaprolactone, polyurethane, polylactide, polycarbonate, polyamide, polyether, blends / composites / or copolymers thereof, collagen, gelatin, elastin, elastin-like peptides, 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 with a molecular weight of about 5-25 kD, or 26-75 kD, or 76-200 kD, or >201 kD.

[0184] In some embodiments, X is an antibody or an antibody fragment portion.

[0185] Antibodies and antibody fragments In some embodiments, the target agent or X is an antibody or antibody fragment that targets one or more of the following: 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), cohesin 4 (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 930), CD22 (NCBI gene ID 933), CD20 (NCBI gene ID 920), CD19 (NCBI gene ID 930), CD79b (NCBI gene ID 933), CD20 (NCBI gene ID 930), CD22 (NCBI gene ID 933), CD20 ... 974), CD38 (NCBI gene ID 952), CD45 (NCBI gene ID 5788), endothelial glycoprotein (NCBI gene ID 2022), FGFR2 (NCBI gene ID 14183), C4.4A (NCBI gene ID 27076), Claudin-18.2 (NCBI gene ID 51208), MMP9 (NCBI gene ID 4318), folic acid 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 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 51208), IL-6R (NCBI gene ID 431 ... 945), CD123 (NCBI gene ID 3563), GPNMB (NCBI gene ID 10457), cMET (NCBI gene ID 4233), 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), 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 3563), CD123 (NCBI gene ID 3563), CD123 (NCBI gene ID 10457), CD123 (NCBI gene ID 4233), 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), 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 3563), CD123 (NC 7852) or TfR1 (NCBI gene ID 7037).

[0186] In some embodiments, the target agent or X is an antibody or antibody fragment that targets the following: HER2 (NCBI gene ID 2064), 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), GPNMB (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), ROR2 (NCBI gene ID 20531), and CLDN4 (NCBI gene ID 2064). 4920), ANTXR1 (NCBI gene ID 84168) or FAP (NCBI gene ID 2191).

[0187] In some embodiments, the target or X is an antibody or antibody fragment that targets the following: CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

[0188] In some embodiments, the target or X is an antibody or antibody fragment that targets the following: HER2, TROP2, cohesin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC or ECM, EPCAM, CEA or CEACAM5.

[0189] In some embodiments, the target or X is an antibody or antibody fragment that targets the following: CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, or CD25.

[0190] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD25, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment portion derived therefrom.

[0191] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CEA, such as labetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment portion derived therefrom.

[0192] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CEACAM5, such as tusamitiamab or CC4, or an antibody fragment portion derived therefrom.

[0193] In some embodiments, X is an antibody or antibody fragment portion that targets ASPH, such as PAN-622, or an antibody fragment portion derived therefrom.

[0194] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets EGFR, such as cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, duligotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, or laprituximab, or an antibody fragment portion derived therefrom.

[0195] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets EPCAM, such as oportuzumab, citatuzumab, tucotuzumab, catumaxomab, edrecolomab, or adecatumumab, or an antibody fragment portion derived therefrom.

[0196] In some embodiments, the target or X is an antibody or antibody fragment portion that targets VEGFR, such as ramucizumab, ramucirumab, or vulinacimab, or an antibody fragment portion derived therefrom.

[0197] In some embodiments, the target or X is an antibody or antibody fragment portion that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment portion derived therefrom.

[0198] In some embodiments, the target or X is an antibody or antibody fragment portion that targets TROP2, such as sacituzumab or Pr1E11, or an antibody fragment portion derived therefrom.

[0199] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets cohesin 4, such as enfortumab, or an antibody fragment portion derived therefrom.

[0200] In some embodiments, the target or X is an antibody or antibody fragment portion that targets PSMA, such as J591 or MLN591, or an antibody fragment portion derived therefrom.

[0201] In some embodiments, the target or X is an antibody or antibody fragment portion that targets BCMA, such as belantamab, or an antibody fragment portion derived therefrom.

[0202] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets CD22, such as moxetumomab, inotuzumab, epratuzumab, or pinatuzumab, or an antibody fragment portion derived therefrom.

[0203] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets CD20, such as ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, or ibritumomab, or an antibody fragment portion derived therefrom.

[0204] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD19, such as loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, or MDX-1342, or an antibody fragment portion derived therefrom.

[0205] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets CD79b, such as polatuzumab, or an antibody fragment portion derived therefrom.

[0206] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD38, such as isatuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment portion derived therefrom.

[0207] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD45, such as I-131-BC8 or Iomab-B, or an antibody fragment portion derived therefrom.

[0208] In some embodiments, the target agent or X is an antibody or antibody fragment that targets an endothelial glycoprotein, such as carotuximab, or an antibody fragment derived therefrom.

[0209] In some embodiments, the target or X is an antibody or antibody fragment portion that targets FGFR2, such as bemarituzumab or aprutumab, or an antibody fragment portion derived therefrom.

[0210] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets C4.4A, such as lupartumab, or an antibody fragment portion derived therefrom.

[0211] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets Claudin-18.2, such as zolbetuximab or claudiximab, or an antibody fragment portion derived therefrom.

[0212] In some embodiments, the target or X is an antibody or antibody fragment portion that targets MMP9, such as andecaliximab, or an antibody fragment portion derived therefrom.

[0213] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets the folate receptor, such as mirvetuximab, farletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment portion derived therefrom.

[0214] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets DLL3, such as lovatouzumab, or an antibody fragment portion derived therefrom.

[0215] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD138, such as indatuximab, or an antibody fragment portion derived therefrom.

[0216] In some embodiments, the target or X is an antibody or antibody fragment that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment derived therefrom.

[0217] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD37, such as BI 836826, otlertuzumab, or naratuximab, or an antibody fragment portion derived therefrom.

[0218] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD74, such as milatuzumab, or an antibody fragment portion derived therefrom.

[0219] In some embodiments, the target or X is an antibody or antibody fragment portion that targets mesothelin, such as anetumab, amatuximab, or MMOT-0530A, or an antibody fragment portion derived therefrom.

[0220] In some embodiments, the target or X is an antibody or antibody fragment portion that targets IL-6R, such as tocilizumab or sarilumab, or an antibody fragment portion derived therefrom.

[0221] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets SLAMF7, such as elotuzumab, or an antibody fragment portion derived therefrom.

[0222] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets BAFF, such as belimumab, or an antibody fragment portion derived therefrom.

[0223] In some embodiments, the target or 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.

[0224] In some embodiments, the target or X is an antibody or antibody fragment portion that targets GPC3, such as codrituzumab, ECT204, or MDX-1414, or an antibody fragment portion derived therefrom.

[0225] In some embodiments, the target or X is an antibody or antibody fragment portion that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment portion derived therefrom.

[0226] In some embodiments, the target or X is an antibody or antibody fragment portion that targets HER3, such as patritumab, seribantumab, lumretuzumab, elgemtumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment portion derived therefrom.

[0227] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD30, such as brentuximab, or an antibody fragment portion derived therefrom.

[0228] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD33, such as gemtuzumab, BI 835858, vadastuximab, or lintuzumab, or an antibody fragment portion derived therefrom.

[0229] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment portion derived therefrom.

[0230] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets GPNMB, such as glembatumumab, or an antibody fragment portion derived therefrom.

[0231] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment portion derived therefrom.

[0232] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD142, such as tisotumab, or an antibody fragment portion derived therefrom.

[0233] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets NaPi2B, such as lifastuzumab, or an antibody fragment portion derived therefrom.

[0234] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets GCC, such as indusatumab, or an antibody fragment portion derived therefrom.

[0235] In some embodiments, the target or X is an antibody or antibody fragment portion that targets STEAP1, such as vandortuzumab, or an antibody fragment portion derived therefrom.

[0236] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets MUC16, such as sofituzumab, or an antibody fragment portion derived therefrom.

[0237] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD70, such as vorsetuzumab, or an antibody fragment portion derived therefrom.

[0238] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CD44, such as bivatuzumab, or an antibody fragment portion derived therefrom.

[0239] In some embodiments, the target or X is an antibody or antibody fragment portion that targets vWF, such as caplacizumab, or an antibody fragment portion derived therefrom.

[0240] In some embodiments, the target or X is an antibody or antibody fragment portion that targets TNF, such as ozoralizumab, V565, or PF-05230905, or an antibody fragment portion derived therefrom.

[0241] In some embodiments, the target or X is an antibody or antibody fragment portion that targets IL-6R, such as vobarilizumab, or an antibody fragment portion derived therefrom.

[0242] In some embodiments, the target or X is an antibody or antibody fragment portion that targets BCMA, such as LCAR-B38M, or an antibody fragment portion derived therefrom.

[0243] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets ADAMTS5, such as M6495, or an antibody fragment portion derived therefrom.

[0244] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CX3CR1, such as BI 655088, or an antibody fragment portion derived therefrom.

[0245] In some embodiments, the target or X is an antibody or antibody fragment portion that targets CXCR4, such as AD-214 or ALX-0651, or an antibody fragment portion derived therefrom.

[0246] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets TfR1, such as TXB4, or an antibody fragment portion derived therefrom.

[0247] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets VEGFR, such as CDP791, or an antibody fragment portion derived therefrom.

[0248] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets PSMA, such as GY1, or an antibody fragment portion derived therefrom.

[0249] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets FN1, such as L19 or NJB2, or an antibody fragment portion derived therefrom.

[0250] In some embodiments, the target or X is an antibody or antibody fragment portion that targets FAP, such as F19, OMTX005, or sibrotuzumab, or an antibody fragment portion derived therefrom.

[0251] In some embodiments, the target agent or X is an antibody or antibody fragment portion that targets TNC, such as F16 or R6N, or an antibody fragment portion derived therefrom.

[0252] In some embodiments, the target agent or X is an antibody.

[0253] In some embodiments, the antibody is daklizumab, RG6292, baliximab, HuMax-TAC, labezizumab, 15-1-32, PR1A3, cT84.66, tasicitumab, CC4, PAN-622, cetuximab, nimotuzumab, mateuzumab, AMG595, depertoxigenics, dapertoxigenics, dugoutuzumab, vortex Toxotuzumab, GC1118, Imatrozumab, Panitumumab, Alutusumab, Toltuzumab, Latoxicumab, Moozumab, Sitaturazumab, Tocozicumab, Caputoxumab, Ezekielumab, Ademumab, Ramoximumab, Ramorumab, Vulinacitabine, Olatuzumab, Ramorumab, Saxitoxicumab, Pr1E11, Envertuzumab, J591, MLN591, Belan Tarituximab, Mosetuximab, Intotuzumab, Ipratuzumab, Pinatuximab, Utoximab, Ofamumab, Rituximab, Obinutuximab, Tosimomab, Tiimumab, Rantuximab, XMAB-5574, MOR208, Cortuximab, Dietuximab, Tarituximab, MDX-1342, Polazumab, Isatuximab, Daremumab, MOR2 02, TAK-079, I-131-BC8, Iomab-B, Caputuximab, Bematocilizumab, Apulutuzumab, Lupatumab, Zoltocilizumab, Claudiciximab, Andrexiximab, Mituxumab, Falletuzumab, MORAb-202, MORAb-003, SP8166, Lovatocilizumab, Indanetocilizumab, Lovotocilizumab, Promezab, BI 836826, Oletocilizumab, Naltoxicumab, Milazolizumab, Anametuzumab, Ametuzumab, MMOT-0530A, Thalidomide, Erotocilizumab, Belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, Crituzumab, 8HuDS6, Gatuzumab, AR20.5. Cantuzumab, Trastuzumab, ECT204, MDX-1414, Pertuzumab, Trastuzumab, Magutuximab, Pertrastuzumab, Seretuzumab, Lutuzumab, Eganutuzumab, AV-203, CDX-3379, GSK284933, Brentuximab, Gelatuzumab, BI 835858, Vardatocizumab, Lintoxumab, KHK2823, Tacrolimus, G4723A, Gbatocizumab, Teritoluzumab, Onatuzumab, SAIT301, Texotuzumab, Lifatocizumab, Indrutuzumab, Vantocizumab, Sofostocizumab, Voseltuzumab, Bivaruzumab, Calacimab, Oriziluzumab, V565, PF-05230905, Wobbalizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, Sirolizumab, F16 or R6N.

[0254] In some embodiments, the target agent or X is an antibody selected from the following: atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, erlotuzumab, ipilimumab, esatuzumab, mogamulizumab, nexituzumab, nivolumab, oxalitumumab, ofamumab, olatotumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, and trastuzumab.

[0255] In some embodiments, the target agent or X is an antibody fragment portion.

[0256] In some embodiments, the target agent or X or antibody fragment portion is selected from the group consisting of: single-chain variable fragments (scFv), divalent or bivalent single-chain variable fragments (di-scFvs, bi-scFvs), antigen-binding fragments (Fab), single-domain antibodies (sdAb), single-domain antibodies (sdAb), antigen-binding proteins, DotBody, affibody, DARPin, DART, TandAb, biantibody, ribobody, centyrin, knottin, affilin, affimer, alphabody, antiicalin, atrimer, avimer, fynomer, kunitz domain, obody, pronectin, repeatbody, and bicyclic peptides or human bodies.

[0257] In some embodiments, the target or X is an antibody fragment portion selected from the group consisting of: single-chain variable fragments (scFv), divalent or bivalent single-chain variable fragments (di-scFvs, bi-scFvs), antigen-binding fragments (Fab), single-domain antibodies (sdAb), and divalent or bivalent single-domain antibodies (sdAb).

[0258] In some embodiments, the antibody fragment portion is an antigen-binding protein, DotBody, affinity protein, DARPin, DART, TandAb, biantibody, ribobody, centyrin, knotting protein, affilin, affimer, alpha body, anticalin, atrimer, avimer, fynomer, kunitz domain, obody, pronectin, repeat body, bicyclic peptide, or human-derived protein.

[0259] In some embodiments, the target agent or X or antibody fragment portion is an antigen-binding fragment (Fab). Fab is the antigen-binding region on the antibody and consists of a constant domain and a variable domain on each of the heavy and light chains. In some embodiments, Fab comprises four domains: VH, CH1, VL, and CL1. In some embodiments, Fab comprises 400-500 amino acids or 440-480 amino acids. In some embodiments, the molecular weight of Fab is about 50 kDa, or 40-55 kDa, or 45-50 kDa, or 45-55 kDa.

[0260] In some embodiments, trastuzumab, entaftuzumab, brentuximab, sacituzumab, and the Fab of L19 bind to FN-1 (gene ID 2335); F16 binds to TNC (gene ID 3371) or NJB2 (ECM targeting sequence).

[0261] In some embodiments, the antibody fragment portion includes one or more PEG units, which can extend cycle life.

[0262] In some embodiments, the antibody fragment portion is an antigen-binding protein. An antigen-binding protein is a protein engineered as an antibody mimic that exhibits high affinity and specificity for a given target. In some embodiments, the antigen-binding protein is a single-chain antigen-binding protein, a novel recombinant polypeptide, composed of a variable light chain amino acid sequence (VL) and a variable heavy chain sequence (VH) of an antibody connected by a peptide tether designed to link the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence.

[0263] In some embodiments, the antigen-binding protein is about 5-10 kDa or about 7 kDa. In some embodiments, the length of the antigen-binding protein is about 50-80, 60-70, or 66 amino acids. In some embodiments, the antigen-binding protein contains cysteine ​​only at the N-terminus or C-terminus. In some embodiments, the antigen-binding protein contains cysteine ​​only at the N-terminus. In some embodiments, the antigen-binding protein contains cysteine ​​only at the C-terminus.

[0264] In some embodiments, the antibody fragment portion is an antigen-binding protein targeting the following: TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, or CD25. In some embodiments, the antibody fragment portion is an antigen-binding protein targeting HER2. The antigen-binding protein can be prepared and tested according to standard methods or purchased from commercial sources (e.g., Affilogic, Inc.).

[0265] In some embodiments, the antibody fragment is partially derived from the following: daklizumab, RG6292, baliximab, HuMax-TAC, labezizumab, 15-1-32, PR1A3, cT84.66, tasicitumab, CC4, PAN-622, cetuximab, nimotuzumab, mateuzumab, AMG595, depertuximab, dapertuximab. Dugutuzumab, Votoxicumab, GC1118, Imatrozumab, Panitumumab, Alutusumab, Toltuzumab, Latoxicumab, Moozumab, Sitaturuzumab, Tocozicumab, Caputoxumab, Ezekielumab, Ademumab, Ramoximumab, Ramorumab, Vulinacicumab, Olatuzumab, Ramorumab, Saxitouzumab, Pr1E11, Envertuzumab, J591, MLN 591, Belantuzumab, Mosetuzumab, Intozumab, Iprazumab, Pinatuzumab, Utoximab, Ofamumab, Rituximab, Obinutuzumab, Tosimomab, Tiimumab, Rantuzumab, XMAB-5574, MOR208, Cortuzumab, Dietuzumab, Taritumab, MDX-1342, Polazumab, Isaltuzumab, Daremumab, M OR202, TAK-079, I-131-BC8, Iomab-B, Caputuximab, Bematocilizumab, Apulutuzumab, Lupatumab, Zoltocilizumab, Claudiciximab, Andrexiximab, Mitocilizumab, Falletuzumab, MORAb-202, MORAb-003, SP8166, Lovatocilizumab, Indanetocilizumab, Lovotocilizumab, Promezab, BI 836826, Oletocilizumab, Naltoxicumab, Milazolizumab, Anametuzumab, Ametuzumab, MMOT-0530A, Thalidomide, Erotocilizumab, Belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, Crituzumab, 8HuDS6, Gatuzumab, AR20.5. Cantuzumab, Cortrastuzumab, ECT204, MDX-1414, Pertuzumab, Trastuzumab, Magutuximab, Pertrastuzumab, Seretuximab, Lutuzumab, Egantuzumab, AV-203, CDX-3379, GSK284933, Brentuximab, Gelatuzumab, BI835858, Vardatuzumab, Lintuzumab, KHK28 23. Tacrolimus, G4723A, Gabatumumab, Teritolumab, Onatuzumab, Sait301, Texotuzumab, Lifatuzumab, Indotumumab, Vantotuzumab, Sofostotuzumab, Voseltuzumab, Bivalizumab, Calacimab, Orizumab, V565, PF-05230905, Wobbalizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, Sirolizumab, F16 or R6N.

[0266] In some embodiments, X is an antibody fragment derived from: atuzumab, averuzumab, bevacizumab, simipremab, cetuximab, daratumumab, denutoximab, durvalumab, erlotuzumab, ipilimumab, esatuzumab, moglizumab, nexitoximab, nivolumab, olbinutuzumab, ofamumab, olatoximab, panitumumab, pembrolizumab, pertuzumab, ramorumab, rituximab, or trastuzumab.

[0267] In some embodiments, X is an antibody or antibody fragment that targets vWF, such as caralacimab.

[0268] In some embodiments, X is an antibody or antibody fragment that targets TNF, such as orizumab, V565, or PF-05230905.

[0269] In some embodiments, X is an antibody or antibody fragment that targets IL-6R, such as vobalizumab.

[0270] In some embodiments, X is an antibody or antibody fragment that targets BCMA, such as LCAR-B38M.

[0271] In some embodiments, X is an antibody or antibody fragment portion that targets ADAMTS5, such as M6495.

[0272] In some embodiments, X is an antibody or antibody fragment targeting CX3CR1, such as BI 655088.

[0273] In some embodiments, X is an antibody or antibody fragment that targets CXCR4, such as AD-214 or ALX-0651.

[0274] In some embodiments, X is an antibody or antibody fragment portion that targets TfR1, such as TXB4.

[0275] In some embodiments, X is an antibody or antibody fragment that targets VEGFR, such as CDP791.

[0276] In some embodiments, X is an antibody or antibody fragment portion that targets PSMA, such as GY1.

[0277] In some embodiments, the antibody fragment portion is carracizumab, orizizumab, V565, PF-05230905, vobalizumab, LCAR-B38M, M6495, BI 655088, AD-214, ALX-0651, TXB4, CDP791, or GY1.

[0278] In some embodiments, X further comprises an imaging developer. In some embodiments, the imaging developer is a protein.

[0279] In some embodiments, X is a support composition. The support composition comprises a support. The support can 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 substantially does not react with the subject's tissues or biological compounds. For example, the support can be a hydrogel, etc. The support is implantable into the subject and supports the binder (e.g., a tetrazine-containing group) and the binder-conjugated TPD. Representative supports include, but are not limited to, polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, cross-linked polymer matrices, metals, ceramics, plastics, bone graft materials, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, etc. Supports also include particles, such as nanoparticles, microparticles, etc.

[0280] Hydrogels can be polysaccharide hydrogels, alginate, cellulose, hyaluronic acid, chitosan, chitosan polysaccharide, chitin, hyaluronic acid, chondroitin sulfate, heparin, etc. Other suitable sugar-based biomaterials include those described in "Advanced Polymer Technology" (…). Polymer Advanced TechnologyBiomaterials described in [reference needed], 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 their blends / composite materials / copolymers. Representative polyethers include, but are not limited to, poly(ethylene glycol) (PEG), polypropylene glycol (PPG), and triblock Pluronic ([PEG]). n -[PPG] m -[PEG] n PEG diacrylate (PEGDA) and PEG dimethacrylate (PEGDMA), where n and m are each independent integers from 1 to 100. Supports may also include proteins and other poly(amino acids), such as collagen, gelatin, elastin and elastin-like peptides, albumin, fibrin, poly(γ-glutamic acid), poly(L-lysine), poly(L-glutamic acid), poly(aspartic acid), etc.

[0281] 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 that is substantially uncrosslinked). In some embodiments, the support is chitosan. In some embodiments, the support is chitosan.

[0282] In some embodiments, the support is a particle. The diameter of the particles disclosed herein may be 2 cm or smaller, such as 1.5 cm or smaller, or 1 cm or smaller, or 0.5 cm or smaller. For example, the particles may be nanoparticles or microparticles. Nanoparticles include particles with an average size in the nanometer range (e.g., 1000 nm or smaller). Microparticles are particles with an average size in the micrometer range (e.g., 1000 μm or smaller). “Average” means arithmetic mean. In some embodiments, the diameter of the nanoparticles ranges from 1 nm to 1 μm, such as 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 diameter of the particles ranges from 1 μm to 1 mm, such as 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 with diameters on the order of 10-100 nm can assemble to form larger complexes, such as clusters or assemblies on the order of 1-10 μm. The particles of this disclosure can be substantially spherical, such that the particles have a substantially circular cross-section. Other particle shapes can also be used, such as, but not limited to, elliptical, cubic, cylindrical, conical, needle-shaped, or other irregular shapes.

[0283] "Particles" can take the form of any manufacturing material, molecule, cryptopharma, virus, bacteriophage, etc. Particles can be composed of materials such as, but not limited to, metals, ceramics, plastics, glass, composites, polymers, hydrogels, etc. For example, particles can be made of inert materials such as alginate or iron oxide. In some instances, particles can be magnetic and can be formed from paramagnetic, superparamagnetic, or ferromagnetic materials or other materials that respond to magnetic fields. Furthermore, particles can be of any shape, such as spheres, rods, asymmetric shapes, etc. A group of particles or a complex can be functionalized with receptors having specific affinity to bind to or interact with clinically relevant substrates. The receptor may be inherent to the particle itself. For example, the particle itself may be a virus or bacteriophage with an inherent affinity for certain substrates. Alternatively or alternatively, particles can be functionalized by covalently or otherwise linking or binding to receptors that specifically bind to or otherwise recognize specific clinically relevant substrates. Functionalized receptors can be antibodies, peptides, nucleic acids, bacteriophages, bacteria, viruses, or any other molecules with a defined affinity for the target substrate. Examples of materials that can be used for “particles” and / or “carriers” include polylactic acid, polyglycolic acid, PLGA polymers, alginate and alginate derivatives, gelatin, collagen, fibroin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, peptides, polyesters, polyanhydrides, polyphosphonazines, poly(vinyl alcohol), poly(epoxyalkane), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pronic acid polyols, polyoxamer, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of any of the above. These examples do not limit their concentration, crosslinking with different agents, methods of administration, customized degradation characteristics, or other properties known to those skilled in the art.

[0284] A group of particles or complexes may be functionalized with a targeting agent (e.g., a ligand or antibody) that specifically (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 can be an antibody, peptide, nucleic acid, bacteriophage, bacteria, virus, or any other molecule with a specific affinity for a 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, CEACAM5, or other cell receptors or cell surface targets.

[0285] In some embodiments, the target is a monoclonal antibody. The monoclonal antibody can be a complete monoclonal antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In some embodiments, the target is an antibody or antibody fragment that targets one or more of the following: 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), cohesin 4 (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 933), CD20 (NCBI gene ID 920), CD19 (NCBI gene ID 930), CD79b (NCBI gene ID 974), CD38 (NCBI gene ID 933), CD20 (NCBI gene ID 930), CD2 ...34), CD20 (NCBI gene ID 934), CD20 (NCBI gene ID 934), CD20 (NCBI gene ID 934), CD20 (NCBI gene ID 934), CD20 (NCBI gene ID 934), CD20 (NCBI gene ID 934), CD20 (NCBI gene 952), CD45 (NCBI gene ID 5788), endothelial glycoprotein (NCBI gene ID 2022), FGFR2 (NCBI gene ID 14183), C4.4A (NCBI gene ID 27076), Claudin-18.2 (NCBI gene ID 51208), MMP9 (NCBI gene ID 4318), folic acid 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 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 51208), IL-6R (NCBI gene ID 431 ... 945), CD123 (NCBI gene ID 3563), GPNMB (NCBI gene ID 10457), cMET (NCBI gene ID 4233), 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), 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 3563), CD123 (NCBI gene ID 3563), CD123 (NCBI gene ID 10457), CD123 (NCBI gene ID 4233), 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), 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 3563), CD123 (NC 7852) or TfR1 (NCBI gene ID 7037).

[0286] In some embodiments, the target is a monoclonal antibody. The monoclonal antibody can be a complete monoclonal antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In some embodiments, the target is an antibody or antibody fragment that targets one or more of the following: HER2 (NCBI gene ID 2064), 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), GPNMB (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 20531), ZIP6 (NCBI gene ID 25800), and ROR1 (NCBI gene ID 20531). 4919), ROR2 (NCBI gene ID 4920), ANTXR1 (NCBI gene ID 84168), or FAP (NCBI gene ID 2191) In some embodiments, the target is an antibody or antibody fragment that targets CD25, such as daklizumab, RG6292, baliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0287] In some embodiments, the target is an antibody or antibody fragment that targets CEA, such as labezizumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment derived therefrom.

[0288] In some embodiments, the target is an antibody or antibody fragment that targets CEACAM5, such as tusamitamab or CC4, or an antibody fragment derived therefrom.

[0289] In some embodiments, the target is an antibody or antibody fragment that targets ASPH, such as PAN-622, or an antibody fragment derived therefrom.

[0290] In some embodiments, the target is an antibody or antibody fragment that targets EGFR, such as cetuximab, nimotuzumab, mateuzumab, AMG595, depertuzumab, dapertuzumab, dugoutuzumab, vortuzumab, GC1118, imatroximab, panitumumab, alutumumab, toltuximab, or latuximab, or an antibody fragment derived therefrom.

[0291] In some embodiments, the target agent is an antibody or antibody fragment that targets EPCAM, such as moozizumab, sitatuzumab, tocotrienzab, caputuzumab, ezetoxumab, or adenomyumab, or an antibody fragment derived therefrom.

[0292] In some embodiments, the target is an antibody or antibody fragment that targets VEGFR, such as ramoximab, ramorumab, or volinacizumab, or an antibody fragment derived therefrom.

[0293] In some embodiments, the target is an antibody or antibody fragment that targets PDGFR, such as olatuzumab or ramucirumab, or an antibody fragment derived therefrom.

[0294] In some embodiments, the target is an antibody or antibody fragment that targets TROP2, such as sacitrus or Pr1E11, or an antibody fragment derived therefrom.

[0295] In some embodiments, the target is an antibody or antibody fragment that targets cohesin 4, such as entaftuzumab, or an antibody fragment derived therefrom.

[0296] In some embodiments, the target is an antibody or antibody fragment that targets PSMA, such as J591 or MLN591, or an antibody fragment derived therefrom.

[0297] In some embodiments, the target agent is an antibody or antibody fragment that targets BCMA, such as belantuzumab, or an antibody fragment derived therefrom.

[0298] In some embodiments, the target is an antibody or antibody fragment that targets CD22, such as mosetumumab, intuzumab, epazolizumab, or pinatumumab, or an antibody fragment derived therefrom.

[0299] In some embodiments, the target agent is an antibody or antibody fragment that targets CD20, such as utuximab, ofamumab, rituximab, oxetuzumab, tosimomumab, or tiimumab, or an antibody fragment derived therefrom.

[0300] In some embodiments, the target is an antibody or antibody fragment that targets CD19, such as rontoxicumab, XMAB-5574, MOR208, cotoxicumab, dinetuzumab, taritumab, or MDX-1342, or an antibody fragment derived therefrom.

[0301] In some embodiments, the target is an antibody or antibody fragment that targets CD79b, such as polacillinumab, or an antibody fragment derived therefrom.

[0302] In some embodiments, the target is an antibody or antibody fragment that targets CD38, such as esazotuximab, daratumumab, MOR202 or TAK-079, or an antibody fragment derived therefrom.

[0303] In some embodiments, the target is an antibody or antibody fragment that targets CD45, such as I-131-BC8 or Iomab-B, or an antibody fragment derived therefrom.

[0304] In some embodiments, the target is an antibody or antibody fragment that targets an endothelial glycoprotein, such as capuximab, or an antibody fragment derived therefrom.

[0305] In some embodiments, the target is an antibody or antibody fragment that targets FGFR2, such as bematuzumab or apulutuzumab, or an antibody fragment derived therefrom.

[0306] In some embodiments, the target is an antibody or antibody fragment that targets C4.4A, such as rupatumab, or an antibody fragment derived therefrom.

[0307] In some embodiments, the target is an antibody or antibody fragment that targets Claudin-18.2, such as zotocimab or clodicimab, or an antibody fragment derived therefrom.

[0308] In some embodiments, the target is an antibody or antibody fragment that targets MMP9, such as adeliximab, or an antibody fragment derived therefrom.

[0309] In some embodiments, the target is an antibody or antibody fragment that targets the folic acid receptor, such as mitoximab, faletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment derived therefrom.

[0310] In some embodiments, the target is an antibody or antibody fragment that targets DLL3, such as lovatozumab, or an antibody fragment derived therefrom.

[0311] In some embodiments, the target is an antibody or antibody fragment that targets CD138, such as indextrin, or an antibody fragment derived therefrom.

[0312] In some embodiments, the target is an antibody or antibody fragment that targets CD56, such as lovotozumab, propimumab, or an antibody fragment derived therefrom.

[0313] In some embodiments, the target is an antibody or antibody fragment that targets CD37, such as BI 836826, olletuzumab, or naltuximab, or an antibody fragment derived therefrom.

[0314] In some embodiments, the target is an antibody or antibody fragment that targets CD74, such as mirtizumab, or an antibody fragment derived therefrom.

[0315] In some embodiments, the target is an antibody or antibody fragment that targets mesothelin, such as annatuzumab, amatotuzumab, or MMOT-0530A, or an antibody fragment derived therefrom.

[0316] In some embodiments, the target is an antibody or antibody fragment that targets IL-6R, such as tocilizumab or thalidomide, or an antibody fragment derived therefrom.

[0317] In some embodiments, the target is an antibody or antibody fragment that targets SLAMF7, such as erlotinumab, or an antibody fragment derived therefrom.

[0318] In some embodiments, the target is an antibody or antibody fragment that targets BAFF, such as belimumab, or an antibody fragment derived therefrom.

[0319] In some embodiments, the target is an antibody or antibody fragment that targets MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, cristatuzumab, 8HuDS6, gatuzumab, AR20.5, or cantuzumab, or an antibody fragment derived therefrom.

[0320] In some embodiments, the target is an antibody or antibody fragment that targets GPC3, such as cotrastuzumab, ECT204, or MDX-1414, or an antibody fragment derived therefrom.

[0321] In some embodiments, the target is an antibody or antibody fragment that targets HER2, such as pertuzumab, trastuzumab, or magnituximab, or an antibody fragment derived therefrom.

[0322] In some embodiments, the target agent is an antibody or antibody fragment that targets HER3, such as pertratuzumab, seretuzumab, rutozumab, edentulumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment derived therefrom.

[0323] In some embodiments, the target is an antibody or antibody fragment that targets CD30, such as brentuximab, or an antibody fragment derived therefrom.

[0324] In some embodiments, the target is an antibody or antibody fragment that targets CD33, such as gemtuzumab, BI835858, varatustaxi, or lintuzumab, or an antibody fragment derived therefrom.

[0325] In some embodiments, the target is an antibody or antibody fragment that targets CD123, such as KHK2823, tacrolimus, or G4723A, or an antibody fragment derived therefrom.

[0326] In some embodiments, the target agent is an antibody or antibody fragment that targets GPNMB, such as grabartimumab, or an antibody fragment derived therefrom.

[0327] In some embodiments, the target is an antibody or antibody fragment that targets cMET, such as terituzumab, onatuzumab, or SAIT301, or an antibody fragment derived therefrom.

[0328] In some embodiments, the target is an antibody or antibody fragment that targets CD142, such as tesutumab, or an antibody fragment derived therefrom.

[0329] In some embodiments, the target is an antibody or antibody fragment that targets NaPi2B, such as lifatuzumab, or an antibody fragment derived therefrom.

[0330] In some embodiments, the target agent is an antibody or antibody fragment that targets GCC, such as indultatumab, or an antibody fragment derived therefrom.

[0331] In some embodiments, the target agent is an antibody or antibody fragment that targets STEAP1, such as vandotusumab, or an antibody fragment derived therefrom.

[0332] In some embodiments, the target agent is an antibody or antibody fragment that targets MUC16, such as sofitetuzumab, or an antibody fragment derived therefrom.

[0333] In some embodiments, the target is an antibody or antibody fragment that targets CD70, such as vortexumab, or an antibody fragment derived therefrom.

[0334] In some embodiments, the target is an antibody or antibody fragment that targets CD44, such as bivalizumab, or an antibody fragment derived therefrom.

[0335] In some embodiments, the target is an antibody or antibody fragment that targets vWF, such as caralacimab, or an antibody fragment derived therefrom.

[0336] In some embodiments, the target is an antibody or antibody fragment that targets TNF, such as orizumab, V565, or PF-05230905, or an antibody fragment derived therefrom.

[0337] In some embodiments, the target is an antibody or antibody fragment that targets IL-6R, such as vobalizumab, or an antibody fragment derived therefrom.

[0338] In some embodiments, the target is an antibody or antibody fragment that targets BCMA, such as LCAR-B38M, or an antibody fragment derived therefrom.

[0339] In some embodiments, the target is an antibody or antibody fragment that targets ADAMTS5, such as M6495, or an antibody fragment derived therefrom.

[0340] In some embodiments, the target is an antibody or antibody fragment that targets CX3CR1, such as BI 655088, or an antibody fragment derived therefrom.

[0341] In some embodiments, the target is an antibody or antibody fragment that targets CXCR4, such as AD-214 or ALX-0651, or an antibody fragment derived therefrom.

[0342] In some embodiments, the target is an antibody or antibody fragment that targets TfR1, such as TXB4, or an antibody fragment derived therefrom.

[0343] In some embodiments, the target is an antibody or antibody fragment that targets VEGFR, such as CDP791, or an antibody fragment derived therefrom.

[0344] In some embodiments, the target is an antibody or antibody fragment that targets PSMA, such as GY1, or an antibody fragment derived therefrom.

[0345] Other compounds or molecules that may aid in particle detection (e.g., in vivo detection), such as fluorophores or autofluorescent or luminescent markers, can also be attached to the particles. Ligands and / or detectable tags can be directly attached to the particles or attached via bioorthogonal functional groups as described herein.

[0346] In some embodiments, the support is a bone graft material, such as a bone graft substitute. The bone graft substitute is a material with a structure similar to bone. In some cases, the bone graft substitute is bioresorbable, allowing it to dissolve or be absorbed by the body over time. The bone graft substitute may be osteoconductive, thus promoting angiogenesis and new bone formation within it. In some cases, the bone graft substitute is osteoinducible, thereby promoting new bone formation by actively recruiting mesenchymal stem cells from surrounding tissues. For example, the bone graft substitute may include growth factors such as bone morphogenetic proteins. Bone graft substitutes include, but are not limited to, hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone fragments, and combinations thereof.

[0347] In some embodiments, the tetrazine activator of this disclosure comprises a support and a tetrazine-containing group covalently linked to the support. The tetrazine-containing group may be attached to the support surface, such as a solvent-accessible surface of the support (e.g., a surface of the support in contact with surrounding solvents). In some cases, the tetrazine-containing group is directly attached to the support. For example, the tetrazine-containing group may be covalently attached to the surface of the support, for example, via covalent bonds (such as amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, thioureas, etc.). In some cases, the tetrazine-containing group is covalently attached to the support via an amide bond. In other cases, the tetrazine-containing group may be attached to the support via a linker. Any suitable linker may be used to attach the tetrazine-containing group to the support. Representative linkers may have 1 to 100 linking atoms and may include ethylene-oxygen, amine, ester, amide, carbamate, carbonate, and ketone functional groups. For example, a linker can have 1 to 50 linking atoms, or 5 to 50 linking atoms, or 10 to 50 linking atoms. Representative linkers include, but are not limited to, those shown below: In some embodiments, the tetrazine activator comprises a support and a tetrazine-containing group having the following formulas: ; ;or ; Where R 20Choose from the following groups: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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'' are independently selected from hydrogen, aryl and alkyl in each occurrence; and R''' is independently selected from aryl and alkyl in each occurrence; R 30 Halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl or cycloalkenyl; R a R 31a and R 31b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; and t is 0, 1, 2, 3, or 4.

[0348] In some embodiments, the tetrazine activator has the formula: in R 20Choose from the following groups: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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'' are independently selected from hydrogen, aryl and alkyl in each occurrence; R''' is independently selected from aryl and alkyl in each occurrence; and R 22 The linker has 1 to 100 linking atoms and may include ethylene-oxy, 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.

[0349] In some embodiments, the tetrazine activator has the formula: ;or ; in R 20Choose from the following groups: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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'' are independently selected from hydrogen, aryl and alkyl in each occurrence; R''' is independently selected from aryl and alkyl in each occurrence; R 30 Halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl or cycloalkenyl; R a R 31a and R 31b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; and t is 0, 1, 2, 3, or 4.

[0350] In some embodiments, the tetrazine activator comprises a substituted alginate having units of the following formulas: and / or or its salt, Where R 20Choose from the following groups: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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'' are independently selected from hydrogen, aryl and alkyl in each occurrence; and R''' is independently selected from aryl and alkyl in each occurrence.

[0351] In some embodiments, the tetrazine activator comprises units of the following formulas: or .

[0352] In some embodiments, the tetrazine activator comprises units of the following formulas: .

[0353] In some embodiments, the tetrazine activator comprises units of the following formulas: and .

[0354] In some embodiments, the tetrazine activator comprises substituted hyaluronic acid having units of the following formulas: Among them G 2 for ;R 22 For connectors having 1 to 100 connecting atoms; and R 20 As defined in this article.

[0355] In another embodiment, G 2 for or In yet another embodiment, G 2for And R 20 It is hydrogen or C 1-4 alkyl.

[0356] In some embodiments, the tetrazine activator comprises units of the following formulas: Where R 20 Choose from the following groups: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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'' are independently selected from hydrogen, aryl, and alkyl in each occurrence; and R''' is independently selected from aryl and alkyl in each occurrence. In some embodiments, R 20 It is hydrogen or C 1-4 alkyl.

[0357] In some embodiments, the tetrazine activator comprises units of the following formulas: ; ;or .

[0358] Other tetrazine activators are exemplified in WO2017 / 044983, WO / 2015 / 139025A1 and WO / 2014 / 205126A1, the entire contents of each of which are incorporated herein by reference in their entirety.

[0359] Hyaluronic acid derivatives include hyaluronic acid having multiple glucuronic acid units and a tetrazine-containing group attached to or directly bonded to the glucuronic acid units of the hyaluronic acid. Hyaluronic acid may also have multiple N-acetylglucosamine units. In some embodiments, the N-acetylglucosamine units of the hyaluronic acid are not attached to or conjugated to the tetrazine-containing group.

[0360] The tetrazine-containing group can be linked to or directly bonded to a carboxylic acid unit of glucuronic acid. The tetrazine-containing group can be incorporated into hyaluronic acid at a rate of about 0.1% to about 80%, as measured in percentages of the carboxylic acid to which the tetrazine-containing group is attached or conjugated, such as about 1% to about 75%, about 5% to about 75%, about 10% to about 50%, or about 40% to about 75%, as measured in percentages of the carboxylic acid to which the tetrazine-containing group is attached or conjugated.

[0361] In the tetrazine activators disclosed herein, the linker can be linear or branched (including but not limited to divalent, trivalent, and tetravalent branched branches). Therefore, in a tetrazine-based targeting agent, one or more targeting moieties can be covalently bonded to one or more tetrazine-containing groups. In the formulas disclosed herein, "L" can be inside or outside the name "p," however, the linker is intended to be linear, having one bond with a tetrazine-containing group and one bond with the targeting moieties, wherein more than one [tetrazine-L]- group is bonded to a single targeting moieties; or branched, such that the linker has bonds with two tetrazine moieties and one bond with the targeting moieties.

[0362] In some embodiments, L or the linker comprises one or more of the following: hydrazone, hydrazide, disulfide, N-succinimide-4-(2-pyridinedithio)valerate (SPP), N-succinimide-4-(2-pyridinedithio)butyrate (SPDB), 4-(4'-acetylphenoxy)butyric acid (AcBut), one or more linear or branched natural or non-natural amino acids, valine-citrulline (Val-Cit) moiety, or phenylalanine-lysine (Phe-Lys) moiety.

[0363] In some embodiments, L or a connector comprises 1 to 100 connecting atoms, 1 to 50 connecting atoms, or 5 to 50 connecting atoms, or 10 to 50 connecting atoms, or 1 to 40 connecting atoms, or 1 to 30 connecting atoms, or 1 to 20 connecting atoms, or 1 to 10 connecting atoms, or 1 to 5 connecting atoms, or 5 to 30 connecting atoms, or 10 to 30 connecting atoms, or 5 to 40 connecting atoms, or 5 to 50 connecting atoms, or 10 to 50 connecting atoms.

[0364] In some embodiments, L or the linker comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkyneene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkyl moieties; wherein the alkylene, alkenylene, alkyneene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkyl moieties may each be optionally substituted independently by one to five substituents independently selected from the following: oxo, halogenated, C-substituted. 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups.

[0365] In some embodiments, L or the linker binds to X via a lysine residue on cysteine ​​or X.

[0366] In some embodiments, L or the connector is a non-cuttable connector.

[0367] In some embodiments, L or the connector is a cuttable connector.

[0368] In some embodiments, L or the linker comprises one or more amino acids.

[0369] In some embodiments, L or the linker comprises a polypeptide.

[0370] In some embodiments, L or the linker is an alkylene linker that optionally contains one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thiosuccinimide, or ketone functional groups.

[0371] In some embodiments, L or the connector has the following formula: -Y 10 -(CHR 130 ) n' -Y 20 -(CHR 140 ) n'' -Y 30 -(CHR 150 ) m'' -Y 40 - in: Y 10 Y 20 Y 30 and Y 40 Each of them is an independent key, -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-NR110 -、-NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, -(CH2CH2O) 1-5 -, -C(O)O-, alkylene, alkenylene, ynylene, arylene, or heteroarylene; wherein each of the alkylene, alkenylene, ynylene, arylene, or heteroarylene groups is independently and optionally substituted by one to five substituents selected independently from the following: oxo, halogenated, C 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups; Each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl or heterocyclic; Each R 120 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl or heterocyclic; Each R 130 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic or amino acid side chain; Each R 140 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic or amino acid side chain; Each R 150 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic, or amino acid side chains; and n', n'' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0372] In some embodiments, L or the connector has the following formula: -Y 10 -(CH2) n' -Y 20 -(CH2) m'' -Y 30 - in: Y 10 Y 20 and Y 30 Each of them is an independent key, -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, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkylene; wherein each of the alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkylene is independently and optionally substituted by one to five substituents independently selected from the following: oxo, halogenated, C 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups; Each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl or heterocyclic; Each R 120 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclic groups; and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0373] In some embodiments, each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloaryl, or heterocyclic; and each R 120 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclic groups. In some embodiments, the linker is not a bond.

[0374] L or linker may include one or more of the following: polyethylene glycol (e.g., PEG with an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-dimethylbis(methyl carbamate), arylene (e.g., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.

[0375] In some embodiments, the connector includes one or more of the following: , , , , , or .

[0376] In some embodiments, the connector includes one or more of the following: , , , , , , or .

[0377] In some embodiments, the connector includes one or more of the following: , , , , or .

[0378] In some embodiments, the connector comprises one or more In some embodiments, the connector comprises one or more .

[0379] In some embodiments, the connector is or includes one or more of the following: , , , , , , , , , , , , , , , or .

[0380] In some embodiments, the connector is or includes one or more of the following: , , , , or .

[0381] In some embodiments, the linker is or comprises one or more natural or non-natural amino acids, which may be referred to as a peptide linker. The linker may be a peptide linker composed of a carboxyl acyl unit and one or more amino acids that make up a protein or peptide sequence. The linker may also contain a self-sacrificing spacer that separates the drug from the protein peptide sequence.

[0382] In some embodiments, the connector is or includes the following: "A—Y—Z—X" 2 "-W" indicates a peptide linker, where "A" is a carboxyl acyl unit, "Y" and "Z" are each one or more natural or non-natural amino acids, which together form the peptide sequence, and "X" represents a linker containing a peptide. 2 "" and "W" are optional additional linkers having 1 to 50 linking atoms, or 5 to 10 linking atoms, or 1 to 10 linking atoms. In some embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0383] 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.

[0384] In some embodiments, Z may be at least one amino acid selected from the group consisting of: alanine, lysine, lysine protected with an acetyl or formyl group, arginine, arginine protected with a toluenesulfonyl or nitro group, histidine, ornithine, ornithine protected with an acetyl or formyl group, and citrulline. In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0385] Exemplary YZ combinations include valine-citrulline; valine-alanine; and alanine-alanine.

[0386] In some embodiments, A is -OC(O)-.

[0387] In some embodiments, X 2 It is -OC(O)-.

[0388] In some embodiments, W is -OC(O)-. In some embodiments, X 2 It does not exist and W is -OC(O)-.

[0389] In some embodiments, part —X 2 —W includes In some embodiments, part —X 2 for .

[0390] In some embodiments, —X—W is .

[0391] In some embodiments, —X—W is .

[0392] In some embodiments, the peptide linker is specifically tailored such that it will be selectively cleaved (e.g., enzymatically) to release a drug, such as via one or more tumor-associated proteases.

[0393] In some embodiments, the peptide linker has a chain length of two to four amino acid residues (i.e., a dipeptide, tripeptide, or tetrapeptide). However, it will be understood that peptide linkers with up to five, six, seven, or eight amino acid residues may also be suitably employed.

[0394] In some embodiments, the peptide linker is 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 -Toluenesulfonyl-Arg or Phe-N 9 -Nitro-Arg. In some embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In some embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0395] In some embodiments, L or the connector is or includes one or more of the following: , , , , , , , , , , , , , , , , , , , , , , , , or .

[0396] In some embodiments, the connector includes one or more of the following: (For example, ), (For example, ), , , , , , , , , , or .

[0397] In some embodiments, the connector includes one or more of the following: (For example, ), , , , , , , , , , or .

[0398] The aforementioned linker can bind to an amino acid side chain present on X, such as lysine or cysteine ​​(e.g., , , , , , ).

[0399] In some embodiments, the connector is –C(O)L 4 –or–C(O)C 1-6 Alkylene C(O)L 4 –; L 4 For key, –N(R) 12 )–C 2-3 alkylene–N(R) 13)C(O)–、-CH(NHC(O)R 14 C 1-4 Alkylene–S–S–C 1-4 Alkylene –OC(O)–, –NHNHC(O)CH(NHC(O)R 15 CH2C(O)–、–C 1-6 Alkylene – CH(G) x )OC(O)–、 , , , or ; R 12 R 13 R 14 R 15 and R 19 Each is independently hydrogen or C 1-4 alkyl; R 16 For hydrogen, C 1-4 Alkyl, –C 1-4 alkylene –OH, –C 1-4 Alkylene–OC 1-4 Alkyl, –C 1-4 Alkylene –CO2H or –C 1-4 alkylene –CONH2; and G x A phenyl group optionally substituted with 1 to 5 substituents independently selected from the group consisting of: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, cyano, and nitro groups.

[0400] In some embodiments, the linker comprises a carbonyl moiety conjugated to the linker or X with a tetrazine-containing group. 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 additional lysine or other amino acid side chains conjugated to the carbonyl moiety. In some embodiments, the linker may comprise .

[0401] In some embodiments, the connector is or includes one or more of the following: , , , , , , , , , , , , , , or .

[0402] In some embodiments, the connector is: , , , , , or .

[0403] In some embodiments, the connector is or includes one or more of the following: , , , , , , , , , , or .

[0404] D. TPD precursor In some embodiments, this document provides compounds comprising precursors to TPD, which react in vivo upon administration alone to produce TPD. Accordingly, in some embodiments, a system is provided comprising a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand. A system is also provided comprising a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand.

[0405] In some embodiments, a method for preparing a TPD at a target site in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a trans-cyclooctene-modified E3 ubiquitin ligand ligand and a tetrazine-modified target protein ligand.

[0406] In some embodiments, a method is provided for preparing a TPD at a target site in a subject, the method comprising administering to the subject a therapeutically effective amount of a tetrazine-modified E3 ubiquitin ligand ligand and a trans-cyclooctene-modified target protein ligand.

[0407] In some embodiments, a method is provided for delivering TPD to a target site in a subject, the method comprising administering to the subject a therapeutically effective amount of a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand.

[0408] In some embodiments, a method is provided for delivering TPD to a target site in a subject, the method comprising administering to the subject a therapeutically effective amount of a tetrazine-modified E3 ubiquitin ligand ligand and a trans-cyclooctene-modified target protein ligand.

[0409] In some embodiments, a method for treating cancer or enhancing or inducing an immune response is provided, the method comprising administering to a subject in need a therapeutically effective amount of a trans-cyclooctene-modified E3 ubiquitin ligand ligand and a tetrazine-modified target protein ligand, or a tetrazine-modified E3 ubiquitin ligand ligand and a trans-cyclooctene-modified target protein ligand.

[0410] In some embodiments, a compound from Table 2 is provided. In some embodiments, a kit is provided that comprises two or more compounds from Table 2.

[0411] Table 2 E. Treatment methods Various aspects of this disclosure include methods for delivering a TPD to a target site in a subject. In some embodiments, the method includes selectively delivering the TPD to a target site in the subject. Selective delivery of the TPD can be achieved by using a tetrazine activator as described herein.

[0412] In some cases, the tetrazine activator of this disclosure can be localized to a desired target site in a subject. For example, the methods of this disclosure may include administering a tetrazine activator as described herein to a subject. The tetrazine activator can be administered to the subject at a desired target site. In some cases, the tetrazine activator can be locally injected into the subject at a desired target site. In some embodiments, the tetrazine activator is administered systemically. In these embodiments, the tetrazine activator can be localized to a desired target site in the subject by specifically binding to its target (e.g., antibody-antigen interaction, etc.) or by specifically binding to its target (e.g., antibody-antigen interaction, etc.) on the surface of a desired target (e.g., cell surface).

[0413] As described herein, selective binding can occur between bioorthogonal binding partners (e.g., between the tetrazine of a tetrazine activator and its complementary trans-cyclooctene of a trans-cyclooctene-modified TPD conjugate). Because the tetrazine activator is administered and directed to the desired location in the subject, the selective binding between the tetrazine and its complementary trans-cyclooctene binder on the trans-cyclooctene-modified TPD conjugate will allow the TPD to be directed to the desired target site.

[0414] This article provides a method for treating cancer, the method comprising administering to a subject in need a therapeutically effective amount of a tetrazine activator as described herein or a pharmaceutically acceptable salt thereof, and a trans-cyclooctene-modified TPD conjugate as described herein.

[0415] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, head / neck squamous cell carcinoma, 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.

[0416] In some embodiments, the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer.

[0417] In some embodiments, the cancer is a solid tumor.

[0418] In some embodiments, the cancer is a soft tissue sarcoma.

[0419] In some embodiments, the soft tissue sarcoma is fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0420] In some embodiments, the method further comprises enhancing or inducing an immune response. In some embodiments, the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0421] In some embodiments, the method further comprises administering a therapeutically effective amount of another therapeutic agent selected from the group consisting of anticancer agents, immunomodulators, or their trans-cyclooctene prodrugs. Anticancer agents, immunomodulators, and their trans-cyclooctene prodrugs are known in the art.

[0422] The indications for this method include both cancer, hematologic malignancies, and solid tumors. In some embodiments, the method can be used to treat and / or diagnose soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma, and all different subtypes of soft tissue sarcoma, as well as osteosarcoma. The composition can be used to treat and / or diagnose pigmented villonodular synovitis.

[0423] In some embodiments, the method can be used to treat and / or diagnose hematologic malignancies, such as myelodysplastic syndromes, acute myeloid leukemia, multiple myelodysplastic syndromes, chronic myeloid leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classic Hodgkin's 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 lymphocytic predominant Hodgkin's lymphoma, etc.

[0424] "Treatment" means achieving at least improvement in the symptoms associated with a symptom troubling a subject, where improvement is broadly used to refer to at least a reduction in the magnitude of a parameter, such as the symptoms associated with the treated symptom. Therefore, treatment also includes the complete suppression (e.g., prevention of occurrence) or cessation (e.g., termination) of the pathological symptom or at least the symptoms associated with it, such that the subject no longer suffers from said symptom or at least no longer suffers from the symptoms characterizing said symptom. Treatment can include suppression, i.e., preventing the development or further development of clinical symptoms, such as alleviating or completely suppressing active disease. Treatment can include remission, i.e., causing the disappearance of clinical symptoms. For example, in the context of cancer, the term "treatment" includes any or all of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing the overall tumor burden, prolonging survival, and improving one or more symptoms associated with cancer.

[0425] Subjects to be treated can be subjects requiring therapy, wherein the subjects to be treated are suitable for treatment with the parent drug. Therefore, a variety of subjects can be treated using the compositions disclosed herein. Typically, such subjects are “mammals,” with humans being the focus. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

[0426] In some embodiments, additional therapeutic agents and methods may be used to treat, prevent, and / or diagnose solid tumors, including but not limited to melanoma (e.g., unresectable metastatic melanoma), renal cell carcinoma (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 (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, head / neck squamous cell carcinoma, 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, cutaneous T-cell lymphoma, etc. The disclosed methods are well-suited as adjuvant / neoadjuvant systems. For example, particles as disclosed herein can be placed during a biopsy, and once the results are obtained, the practitioner can deliver an appropriate mixture to the desired location in the body. This will minimize the size of the tumor, especially in the context of a tumor that can be surgically removed. Then, at the end of the surgery, the surgeon can administer additional tetrazine activator to the subject to target the surgical cavity and treat the patient with further therapeutic doses (e.g., chemotherapy performed using the disclosed method), thereby minimizing the risk of any cancer cells remaining at the surgical margin.

[0427] In some embodiments, a tetrazine activator and a trans-cyclooctene-modified TPD conjugate, as disclosed herein, may be administered, and the practitioner may deliver the appropriate mixture to the desired location in the body. This will minimize the size of the tumor, particularly in the context of a tumor that can be surgically removed. Then, at the end of the surgery, the surgeon may administer additional tetrazine activator or a trans-cyclooctene-modified TPD conjugate to the subject to target the surgical cavity and treat the patient with further therapeutic doses (e.g., chemotherapy performed by the disclosed method), thereby minimizing the risk of any cancer cells remaining at the surgical margin.

[0428] In some embodiments, the disclosed method provides the ability to place particles as disclosed herein during a biopsy. When the results are returned, the practitioner can deliver the TPD to the biopsy site.

[0429] In some embodiments, the disclosed method provides the ability for practitioners to deliver TPD to enhance the immune system with fewer side effects. This method is beneficial to patients. The TPD will treat solid tumors or specific locations.

[0430] cancer The disclosed methods can be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that may include persistent proliferative signaling, evasion of growth inhibitors, resistance to cell death, replication immortalization, induction of angiogenesis, and activation of invasion and metastasis. The disclosed methods can enhance or induce an immune response against cancer in a subject. The immune response may result in an increase in one or more of the following: leukocytes, lymphocytes, monocytes, and eosinophils.

[0431] Cancers that can be treated using 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, brainstem cancer, brainstem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse endogenous pontine glioma, ductal carcinoma, endometrial cancer, ependymoma, Ewing's sarcoma, esophageal cancer, ocular cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, germ cell tumors, gliomas, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, macroglobulinemia, and melanoma. Mesothelioma, 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 tumor, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, laryngeal 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 squamous cell carcinoma (OTSCC).

[0432] In some embodiments, the cancers that can be treated by the disclosed methods are melanoma, renal cell carcinoma, 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 a diffuse endogenous pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0433] In some embodiments, the cancers that can be treated by the disclosed methods are hematologic malignancies, such as myelodysplastic syndromes, acute myeloid leukemia, multiple myelodysplastic syndromes, chronic myeloid 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 disorders (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's lymphoma, etc.

[0434] Therefore, the local release of ICD inducers using the compounds and methods of this disclosure can be advantageously combined with one or more immunomodulators.

[0435] In some embodiments, tetrazine-based targeting agents can be used to treat, prevent, and / or diagnose solid tumors, including but not limited to melanoma (e.g., unresectable metastatic melanoma), renal cell carcinoma (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, etc.

[0436] The disclosed methods are well-suited for use as adjuvant / neoadjuvant systems. For example, a tetrazine activator, as disclosed herein, can be placed during a biopsy, and once research results are obtained, a practitioner can administer an appropriate mixture to deliver treatment to the desired site in the body (such as the compounds disclosed herein and optional additional therapeutic agents). Biopsy results can indicate the amount and type of treatment delivered to the tumor site. For example, chemokines (agents that attract cancer cells and / or immune cells) and adjuvants can be delivered and combined with chemotherapeutic agents to enhance the immune system with fewer side effects and fewer chemotherapeutic agents.

[0437] The disclosed methods may include systemic or local administration. In some embodiments, the tetrazine-based targeting agent is delivered systemically. In some embodiments, both the tetrazine activator and the trans-cyclooctene-modified TPD conjugate are delivered systemically. In some embodiments, the tetrazine activator is delivered locally.

[0438] The disclosed compounds and compositions can be administered prior to surgical resection. The disclosed methods can minimize tumor size prior to surgical resection. This minimizes tumor size, particularly in the context of tumors that are surgically resectable. The disclosed conjugates, compounds, and compositions can be administered during surgical resection. The disclosed conjugates, compounds, and compositions can be administered after surgical resection. Tetraazine-based targeting agents can be placed around the surgical cavity at the end of surgical resection, and the subject can then be treated with further therapeutic doses to minimize the risk of any residual cancer cells at the surgical margins.

[0439] The disclosed method may include multiple systemic doses of a trans-cyclooctene-modified TPD conjugate concentrated at one location.

[0440] The disclosed method can be used to treat diffuse endogenous pontine glioma (DIPG). DIPG is a brainstem tumor in children that can be highly malignant and difficult to treat. There is no known curative treatment for DIPG, and survival rates have remained low over the past four decades. The median overall survival for DIPG patients is only 11 months, with a two-year survival rate of less than 10%. DIPG accounts for 75%–80% of childhood brainstem tumors, affecting approximately 200–300 children annually in the United States. The rarity of this devastating disease and the previous lack of systematic experimental models have hampered research, and survival rates have remained unchanged over the past four decades. Diagnosis of DIPG can begin with clinical symptoms and can be confirmed by MRI. The disease may begin with systemic symptoms over several months, including behavioral changes and learning difficulties, diplopia, abnormal or limited eye movements, asymmetrical smiling, loss of balance, and weakness. Alternatively, severe neurological deterioration may occur much faster, with symptoms appearing less than a month before diagnosis. Clinical examination may reveal a triad of various cranial nerve disorders, long tract signs (such as hyperreflexia and clonic reflexes), and ataxia. Dilatation of the pons portion of the brainstem may cause obstructive hydrocephalus and increased intracranial pressure.

[0441] Cell nuclei, which are essential for life-sustaining functions such as breathing and heartbeat, are located in the pons, and breathing and heartbeat can be impaired by DIPG if left untreated.

[0442] The disclosed methods may include multiple systemic doses of a trans-cyclooctene-modified TPD conjugate or a tetrazine activator concentrated at one location.

[0443] Application method Treatment methods may include any manner of administering the disclosed conjugates, compounds, or compositions. Methods of administration may include tablets; pills; sugar-coated pills; hard capsules and soft capsules; granules; pellets; skin patches; skin creams; skin gels; aqueous, lipid, oily, or other solutions; emulsions (such as oil-in-water emulsions); liposomes, aqueous or oily suspensions; syrups; elixirs; solid emulsions; solid dispersions; or dispersible powders. In pharmaceutical compositions, the conjugates, compounds, or compositions disclosed herein may also be dispersed in microparticles, such as nanoparticle compositions.

[0444] For parenteral application, the conjugates, compounds, or compositions disclosed herein may be dissolved or suspended in physiologically acceptable diluents, such as water; buffer solutions; or oils with or without solubilizers, surfactants, dispersants, or emulsifiers. Suitable oils may include, for example, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil, and sesame oil. For parenteral application, the conjugates, compounds, or compositions disclosed herein may be applied as aqueous, lipid-based, oily, or other types of solutions or suspensions, or even as liposomes or nanosuspensions.

[0445] The amount of the composition administered to a subject may initially be determined based on guidance of the parent drug's dosage and / or dosing regimen. Typically, the composition may provide targeted delivery and / or enhance the serum half-life of the bound drug, thereby providing at least one of a reduced dose or reduced administration in the dosing regimen. Thus, relative to the parent drug prior to conjugation in the composition disclosed herein, the composition may provide a reduced dose and / or reduced administration in the dosing regimen.

[0446] Pharmaceutical formulations can be provided in unit dosage forms. In such forms, pharmaceutical formulations can be subdivided into unit doses containing appropriate amounts of the compositions of this disclosure. Unit dosage forms can be packaged formulations containing discrete amounts of the formulation, such as packaged tablets, capsules, and powders in bags, vials, or ampoules.

[0447] In some embodiments, a kit is provided comprising a tetrazine-based targeting agent or a pharmaceutically acceptable salt thereof as described herein, or comprising the aforementioned pharmaceutical composition, along with instructions for use thereof.

[0448] In some embodiments, the kit further comprises a prodrug.

[0449] The compositions disclosed herein may be present in any suitable amount and may depend on various factors, including but not limited to the subject's weight and age, disease state, etc. Suitable dosage ranges for the compositions disclosed herein 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 of the compositions disclosed herein 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 1,000 mg.

[0450] In some embodiments, multiple doses of the composition are administered. The frequency of administration of the composition can vary based on any of a variety of factors, such as the severity of symptoms, the condition of the subject, etc. For example, in some embodiments, the composition may be 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).

[0451] The compositions of this disclosure can be administered at any suitable frequency, interval, and duration. For example, the compositions of this disclosure can be administered once per hour, or twice, three or more times per hour, once daily, or twice, three or more times daily, or once every 2, 3, 4, 5, 6, or 7 days to provide the desired dose level to the subject. When the compositions of this disclosure are administered more than once daily, representative intervals include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes, and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, and 24 hours. The compositions of this disclosure can be administered once, twice, three or more times for one hour, 1 to 6 hours, 1 to 12 hours, 1 to 24 hours, 6 to 12 hours, 12 to 24 hours, one day, 1 to 7 days, one week, 1 to 4 weeks, one month, 1 to 12 months, one year, or longer, or even indefinitely.

[0452] The compositions of this disclosure can be administered co-administered with another active agent. Co-administration includes administering the compositions and active agents of this disclosure at intervals of 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours between each other. Co-administration also includes administering the compositions and active agents of this disclosure simultaneously or approximately simultaneously (e.g., at intervals of about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes between each other) or sequentially in any order. Additionally, the compositions and active agents of this disclosure can be administered once daily, or twice, three times, or more daily, to provide the desired daily dose level.

[0453] Co-administration can be achieved through co-implantation or co-injection.

[0454] In some embodiments, co-administration can be achieved through co-constitution, for example, by preparing a single pharmaceutical formulation comprising both the composition and the active agent of this disclosure. In other embodiments, the composition and active agent of this disclosure can be formulated separately and co-administered to the subject.

[0455] The compositions and active agents disclosed herein may be present in formulations at any suitable weight ratio, such as 1:100 to 100:1 (w / w), or 1:50 to 50:1, or 1:25 to 25:1, or 1:10 to 10:1, or 1:5 to 5:1 (w / w). The compositions and other active agents disclosed herein may be present at any suitable weight ratio, such as 1:100 (w / w), 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 (w / w). Other dosages and dosage ratios of the compositions and active agents disclosed herein are suitable for the formulations and methods described herein.

[0456] Combination therapy On the one hand, a method for treating cancer or enhancing or inducing an immune response is provided, the method comprising administering to a subject in need: a therapeutically effective amount of the tetrazine-based targeted agent of the present disclosure or a pharmaceutically acceptable salt or composition thereof; and a prodrug, as described herein; and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctene prodrugs thereof.

[0457] This disclosure also provides a pharmaceutical combination comprising a tetrazine-based target agent as described herein or a pharmaceutically acceptable salt or composition thereof; a prodrug as described herein; and an additional therapeutic agent optionally selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, for the treatment or prevention of cancer or for enhancing or inducing an immune response.

[0458] This disclosure also provides the use of a pharmaceutical combination comprising a tetrazine-based target agent as described herein or a pharmaceutically acceptable salt or composition thereof; a prodrug, as described herein; and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, for the treatment or prevention of cancer or for enhancing or inducing an immune response.

[0459] In the methods and uses described herein, the components of a pharmaceutical combination may be administered / used simultaneously, individually, or sequentially in any order, and each component may be administered individually or as a fixed combination. For example, according to this disclosure, delaying disease progression or treatment may comprise administering a first active ingredient in a combination therapeutically effective amount or effective amount (e.g., a daily dose corresponding to the amount described herein), simultaneously or sequentially in any order, in the form of a free or pharmaceutically acceptable salt and in the form of a second active ingredient in the form of a free or pharmaceutically acceptable salt. Individual active ingredients in the combination may be administered separately at different times during the course of treatment, or simultaneously in separate or individual dosage forms. Therefore, this disclosure should be understood to cover all such simultaneous or alternating treatment regimens, and the term "administration" should be interpreted accordingly. Thus, as used herein, a pharmaceutical combination is defined as a fixed combination in the form of a dose unit, or a single dosage form for combination administration, wherein combination administration may be performed independently at the same time or at different times. As a further example, a tetrazine-based targeted agent (or a therapeutic tetrazine-based targeted agent) and a prodrug can be administered simultaneously (e.g., by co-injection or co-implantation), alone, or sequentially, followed by the administration of another therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, or a trans-cyclooctene prodrug thereof.

[0460] Methods and uses for treating cancer include the administration / targeting of tetrazine-based agents at the tumor site. In the methods and uses disclosed herein, administration of a prodrug or a pharmaceutically acceptable salt or composition thereof; a tetrazine-based agent; and optionally additional therapeutic agents can inhibit tumor growth.

[0461] Additional therapeutic agents may be administered simultaneously or sequentially with the disclosed conjugates and compositions. Sequential administration includes administration before or after the disclosed conjugates and compositions. Additional therapeutic agents may be administered before the disclosed conjugates and compositions. Additional therapeutic agents may be administered after the disclosed conjugates and compositions. Additional therapeutic agents may be administered simultaneously with the disclosed conjugates and compositions. In some embodiments, one or more additional therapeutic agents may be administered in the same composition as the disclosed conjugate. In other embodiments, a time interval may exist between the administration of the additional therapeutic agent and the disclosed conjugate or composition. In some embodiments, administering additional therapeutic agents together with the disclosed conjugate or composition may allow for lower doses and / or lower frequency of administration of the other therapeutic agents. When used in combination with one or more other active ingredients, the disclosed conjugates or compositions and other active ingredients may be used at lower doses than when used individually. Therefore, the pharmaceutical compositions of this disclosure include pharmaceutical compositions containing one or more other active ingredients in addition to the disclosed conjugates.

[0462] anticancer agents Exemplary anticancer agents include, but are not limited to: abiraterone acetate, abitrexate (methotrexate), albumin-bound paclitaxel (a paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab / vedotin), ADE, ado-trastuzumab / emtansine, and adriamycin (a saline form of paclitaxel). Doxorubicin, Adrucil (Fluorouracil), Afatinib (Afatinib), Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed disodium) Disodium), Aloxi (palonosetron hydrochloride), Ambochlorin (Chlorambucil), Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anatozole), Aromasin (exemestane), Arranon (nelarabine), Arsenic trioxide, Arzerra (Ofatumumab), Erwinia chrysogenum asparaginase Erwiniachrysanthemi), Avastin (bevacizumab), Axitinib, Azacitidine, BEACOPP, Bendamustine hydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (tositumomab and I-131 iodine tositumomab), Bicalutamide, Bleomycin, Bortezomib, Bosulif (bosutinib), Bosutinib, Brentuximab-Vidotine,Busulfan, Busulfex, Cabazitaxel, Cabozantinib S-malate, CAF, Camppath (alemtuzumab), Camptosar (irinotecan hydrochloride), Capecitabine, CAPOX, Carboplatin, Carboplatin-paclitaxel Carfilzomib, Casodex (bicalutamide), CeeNU (lomustine), Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant bivalent HPV vaccine), Cetuximab, Chloramic acid mustard, Chloramic acid mustard-prednisone, CHOP, Cisplatin, Clafen (cyclophosphamide), Chlorfa Clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cometriq (cazozidinib S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Vidarsporin (Cy tarabine), vidarabine liposomes, Cytosar-U (vidarabine), Cytoxan (cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (decitabine), dextrin, Dasatinib, daunomycin hydrochloride, decitabine, degarelix, denileukin-diphtheria toxin Diftitox, Denosumab, DepoCyt (liposomal vidarabine), DepoFoam (liposomal vidarabine), Dexrazoxane hydrochloride, Docetaxel, Doxil (liposomal doxorubicin hydrochloride), Doxorubicin hydrochloride, Dox-SL (liposomal doxorubicin hydrochloride), DTIC-Dome (dacarbazine), Efudex (fluorouracil).Elitek (Rasburicase), Ellence (Epirubicin hydrochloride), Eloxatin (Oxaliplatin), Eltrombopag (ethanolamine), Emend (Aprepitant), Enzalutamide, Epirubicin hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin mesylate, Erivedge (Vismodegib), Erlotinib hydrochloride, Erwinaze (Erwinia) Chrysanthemi (asparaginase), Etopophos (etoposide phosphate), Etoposide, Etoposide phosphate, Evacet (doxorubicin hydrochloride liposome), Everolimus, Evista (raloxifene hydrochloride), Exemestane, Fareston (toremifene), Faslodex (fullvestrant), FEC, Femara (letrozole), Filgrastim, Fludara (fludarabine phosphate), Fludarabine phosphate, Fluoroplex (fluorouracil), Fluoroplex (methotrexate), Foles (methotrexate), Foles PFS (methotrexate) Folfiri, Folfiri-bevacizumab, Folfiri-cetuximab, Folfirinox, Folfox (leucovorin, fluorouracil, oxaliplatin), Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant quadrivalent HPV vaccine), Gazyva (Aobinyutu). Gemcitabine, Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemtuzumab (Ozogamicin), Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimethylmaleate), Gleevec (Imatinib Mesylate), GlucarpidaseGoserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), bivalent HPV vaccine, quadrivalent recombinant HPV vaccine, Hycamtin (topotecan hydrochloride), enhanced CVAD, tiuxetan (tiemomumab), ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosf), ifosf amide), ifosfamidum (ifosfamidum), imatinib mesylate, imbruvica (ibrutinib), imiquimod, inlyta (axitinib), intron A (recombinant interferon α-2b), iodine-131 tocilizumab and tocilizumab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, istodax (romidepsin), ixabispirone (ixab) epilone, Ixempra (ixaprilone), Jakafi (ruxolitinib phosphate), Jevtana (cabatataxel), Kadcyla (Ado-trastuzumab-emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Kyprolix Is (carfilzomib), Lapatinib (lepidocrolide), Lenalidomide, Letrozole, Leucovorin, Leukeran (chlorambucil), Leuprolide (leuprolide), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), Liposomal doxorubicin, Lomustine, Lupron (acetic acid) Leuprorelin, Lipuanku (leuprorelin acetate), Lipuanku-Ped (leuprorelin acetate), Lipuanku-3 Month (leuprorelin acetate), Lipuanku-4 Month (leuprorelin acetate), Marqibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), Mechlorethamine hydrochloride, Megace (medroxyprogesterone acetate), medroxyprogesterone acetate,Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitozole ytrex (mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (nitrogen mustard hydrochloride), Mutamycin (mitomycin C), Myleran (busulin), Mylosar (azacitidine), Mylotarg (gestuzumab / ozogamycin), paclitaxel nanoparticles (paclitaxel albumin-stabilized nanoparticle formulation), Noviben (N... avelbine (vinorelbine tartrate), nelarabine, neosar (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Novadex (tamoxifen citrate), Nplate (romistim). Romiplostim), Omacetaxine, Omacetaxine, Oncaspar (pegaspargase), Ontak (diphenhydramine diphtheria toxin), OEPA, OPPA, Oxaliplatin, Paclitaxel, Paclitaxel albumin-stabilized nanoparticle formulation, Palovmin, Palonosetron hydrochloride, Pamidronate Disodium, panitumumab, paraplatin (carboplatin), paraplatinin (carboplatin), pazopanib hydrochloride, pegaspargase, pegylated interferon α-2b, PEG-intron (pegylated interferon α-2b), disodium pemetrexed, perjeta (pertuzumab), pertuzumab, platinol (cisplatin), platinol-AQ (cisplatin), prasalifos, pomalidomide, pomalyst (pomalidomide), panatinib hydrochloride, pratuzumab, prednisoneMethylbenzylhydrazine hydrochloride, Proleukin (aldeleukin), Prolia (denosumab), Promacta (eltrombopag), Provenge (Sipuleucel-T), mercaptopurine, radium-223 dichloride, raloxifene hydrochloride, raburicase, R-CHOP, R-CVP, recombinant bivalent HPV vaccine, recombinant quadrivalent HPV vaccine, recombinant interferon alpha-2b, regorafenib, Revlimid. Lenalidomide, Rheumatrex (methotrexate), Rituxan (rituximab), Rituximab, Romidesin, Romistachycin, Rubidomycin (danomycin hydrochloride), Ruxotinib phosphate, Sclerosol intrapleural aerosol (talc), Ciproxetine-T, Sorafenib tosylate, Sprycel (dasatinib), Stanford V, sterile talc powder (talc), sterile talc (Steritalc) (talc), Stevagas ( Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Pegylated Interferon Alpha-2b), Synovir (Thalidomide), Synribo (Homoharringtonine Omaxitine), Tafinlar (Dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (Adaracin), Tarceva (Salt) Erlotinib, Tagratin (Targretin), Tasigna (nilotinib), Paclitaxel, Taxotere (docetaxel), Temodar (temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid, Toposar (etoposide), Topotecan hydrochloride, Toremifene, Torisel (temsirolimus), Tocitumumab and 1 131I-Tocitumumab, Totect (dexrazoxen hydrochloride), Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib dimethylbenzenesulfonate), Vandetanib, VAMPVectibix (panitumumab), VelP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), Vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate liposomes, vinorelbine tartrate, vemodigine, Voraxaze (glucardiase), Vorinostat, Votrient (pazopanib hydrochloride) Wellcovorin (leucovorin), Xalkori (crizotinib), Xeloda (capecitabine), Xelox, Xgeva (denosemab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (teimomab / temozolomide), Zinecard (dexrazoxan hydrochloride), Zoladex (goserelin acetate), Zoledronic acid Acid), Zolinza (Vorinostat), Zometa (zoledronic acid), and Zytiga (abiraterone acetate).

[0463] Anticancer agents may include PBD dimer, calicheamicin, spermycin, tubulolysin B, rhizoxin, dolastatin, didemnin B, camptothecin, CBI, tesiromoxim, actinomycin D, epothilone B, paclitaxel, cryptophycin, SN38, velcade, and bruuceantin. DAVLBH, DM1, Phyllanthoside, Arinidazol, T2 toxin, MMC, Vantalanib, Vinorelbine, Brefeldin, Sunitinib, Daunomycin, Semaxanib, Tasiva, Iressa, Irinotecan, LY-541503, Geldanomycin, Gemcitabine, Methotrexate, Gleevec, Topotecan, Bleomycin, Doxorubicin, Cisplatin, N-Sinapis, Etoposide or 5-FU.

[0464] In some embodiments, the anticancer agent is anthracycline. In some embodiments, the anticancer agent is taxane. In some embodiments, the anticancer agent is gemcitabine. In some embodiments, the anticancer agent is doxorubicin. In some embodiments, the anticancer agent is docetaxel. In some embodiments, the anticancer agent is SN38. In some embodiments, the anticancer agent is auristatin E.

[0465] Synthetic compounds The targeted moieties can be prepared using the methods disclosed herein, as well as their conventional modifications, which will be apparent from the disclosure herein and methods well known in the art. In addition to the teachings herein, conventional and well-known synthetic methods can also be used. The synthesis of typical targeted moieties described herein can be carried out as illustrated in the examples below. Reagents and starting materials can be commercially purchased, if available, from, for example, Sigma Aldrich or other chemical suppliers.

[0466] Additionally, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable 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, a variety of protecting groups are described in Wuts, PGM, Greene, TW, and Greene, TW (2006), *Greene's Protective Groups in Organic Synthesis*, Wiley-Interscience, Hoboken, NJ, and the references cited therein.

[0467] The compounds, conjugates and activators provided herein can be prepared by methods adapted from literature (see, for example, WO2020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, WO2014 / 205126, WO2022 / 032191, WO2021 / 007160, WO2020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025 and WO2014 / 205126, the methods of which are incorporated herein by reference in their entirety).

[0468] For example, the trans-cyclooctene-modified TPD conjugate (P1) as described herein can be prepared under standard coupling conditions according to Scheme I, wherein A is TPD, the linker is as defined herein, TCO is at least one trans-cyclooctene moiety, and Xx is a leaving group and Yy is a nucleophilic moiety, or Xx is a nucleophilic moiety and Yy is a leaving group.

[0469] Option I Example 1: Synthesis of Compound 16 Intermediate 1a (0.50 g, 2.34 mmol), intermediate 2a (1.00 g, 2.34 mmol), and HATU (1.34 g, 3.51 mmol) were combined and dissolved in anhydrous CH2Cl2 (20 mL). Anhydrous DIPEA (1.63 mL, 9.36 mmol) was added, and the mixture was stirred at room temperature under N2 atmosphere for 18 hours. The reaction mixture was directly loaded onto a silica column, and compound 3a was purified by rapid chromatography using a gradient of MeOH / CH2Cl2 (0% to 6%).

[0470] HRMS (ESI) calculated value: C 32 H 44 N5O6S [M + 1] 626.3007; Experimental value: 626.3549 1 H NMR (500 MHz, MeOD) δ 8.89 (s, 1H), 7.47 (t, J = 7.2 Hz, 2H), 7.42(d, J = 8.3 Hz, 2H), 4.67 (d, J = 9.2 Hz, 1H), 4.61 (d, J = 8.4 Hz, 1H), 4.58 –4.56 (m, 1H), 4.52 (d, J = 6.4 Hz, 2H), 4.39 (dd, J = 15.5, 4.5 Hz, 1H), 4.28(dd, J = 12.2, 7.3 Hz, 1H), 3.87 (d, J = 11.1 Hz, 1H), 3.82 (dd, J = 11.0, 3.8 Hz,1H), 2.68 – 2.55 (m, 2H), 2.49 (s, 3H), 2.36 (s, 1H), 2.23 (dd, J = 13.1, 7.7Hz, 1H), 2.10 (ddd, J = 12.9, 8.9, 4.3 Hz, 1H), 1.46 (s, 9H), 1.05 (s, 9H).

[0471] 13C NMR(126 MHz, MeOD) δ 172.99, 171.28, 170.37, 151.77, 147.71,138.85, 132.15, 130.15, 128.82, 127.66, 79.73, 70.73, 69.50, 59.32, 57.31,42.59, 37.71, 35.89, 27.55, 25.54, 14.53.

[0472] Under nitrogen atmosphere, a solution of HCl (4N) in dioxane (6 mL) was cooled in an ice-water bath. A solution of compound 3a in CH₂Cl₂ (6 mL) was added, and the reaction mixture was gradually heated to room temperature and stirred for 1 hour. The solvent was evaporated under reduced pressure. The crude product was used for further synthesis without any purification.

[0473] HRMS (ESI) calculated value: C 27 H 38 ClN5O4S [M + 2] 2+ 281.6161; Experimental value: 282.3049 1 H NMR (500 MHz, MeOD) δ 9.89 (s, 1H), 7.60 – 7.57 (m, 2H), 7.54 (d, J =8.0 Hz, 2H), 4.67 (s, 1H), 4.60 (d, J = 8.7 Hz, 1H), 4.57 (d, J = 8.0 Hz, 1H), 4.53 (s, 1H), 4.43 (d, J = 15.7 Hz, 1H), 4.22 – 4.18 (m, 1H), 3.89 (d, J = 10.8Hz, 1H), 3.83 (dd, J = 10.8, 3.7 Hz, 1H), 3.76 (dd, J = 11.4, 5.5 Hz, 2H), 3.69(d, J = 7.3 Hz, 8H), 3.62 – 3.59 (m, 1H), 3.51 (q, J = 7.0 Hz, 1H), 3.25 (q, J=7.4 Hz, 1H), 2.90 – 2.84 (m, 1H), 2.79 (dd, J = 10.1, 7.5 Hz, 1H), 2.66 (s,1H), 2.62 (s, 3H), 2.26 (dd, J = 12.9, 7.9 Hz, 1H), 2.11 (ddd, J = 13.2, 9.2, 4.3Hz, 1H), 1.40 (t, J = 7.1 Hz, 7H), 1.20 (t, J = 7.0 Hz, 2H), 1.09 (s, 9H).

[0474] 13 C NMR(126 MHz, MeOD) δ 173.14, 170.20, 167.13, 154.87, 141.79,140.88, 135.64, 128.98, 127.94, 75.96, 73.79, 72.17, 71.05, 69.67, 66.79,65.51, 60.80, 59.45, 58.25, 56.59, 54.46, 51.29, 42.38, 42.24, 37.61, 35.19,25.65, 21.07, 17.36, 15.92, 14.06, 11.97, 11.80.

[0475] Intermediate 4a (200 mg, 0.38 mmol), compound 5a (100.05 mg, 0.38 mmol), and HATU (217.24 mg, 0.571 mmol) were combined in anhydrous CH2Cl2 (20 mL). DIPEA (0.264 mL, 1.52 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was loaded onto a silica column, and intermediate 6a was purified by rapid chromatography using a gradient of MeOH / CH2Cl2 (0% to 8%).

[0476] HRMS (ESI) calculated value: C 38 H 55 N6O9S [M + 1] 771.3746; Experimental value: 771.4397 1H NMR(500 MHz, MeOD) δ 8.90 (s, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.44(d, J = 8.1 Hz, 2H), 4.69 (t, J = 6.7 Hz, 1H), 4.66 (s, 1H), 4.60 – 4.55 (m, 2H),4.52 (s, 1H), 4.38 (d, J = 15.5 Hz, 1H), 4.08 (d, J = 2.2 Hz, 2H), 3.87 (d, J =10.8 Hz, 1H), 3.82 (dd, J = 10.9, 3.8 Hz, 1H), 3.74 (dd, J = 5.1, 3.2 Hz, 3H),3.69 – 3.66 (m, 2H), 3.56 (t, J = 5.7 Hz, 2H), 3.37 (s, 2H), 3.25 (dd, J = 11.9,6.3 Hz, 3H), 2.71 (dt, J = 14.5, 5.0 Hz, 2H), 2.50 (s, 4H), 2.41 (s, 1H), 2.22(d, J = 7.8 Hz, 1H), 2.10 (ddd, J = 13.3, 9.0, 4.5 Hz, 1H), 1.45 (s, 9H), 1.38(t, J = 6.0 Hz, 6H), 1.05 (s, 9H)。

[0477] 13 C NMR(126 MHz, MeOD) δ 172.93, 171.51, 170.58, 151.11, 148.14,138.43, 132.19, 130.14, 129.01, 127.52, 78.51, 71.13, 70.57, 69.37, 59.30,57.53, 51.37, 42.23, 37.48, 35.15, 27.71, 25.09, 21.27, 14.18。

[0478] Intermediate 6a (0.150 g, 0.38 mmol) was dissolved in CH2Cl2 (5 mL) and TFA (5 mL). The reaction mixture was stirred at room temperature under N2 atmosphere for 2 hours. The solvent was removed under reduced pressure, and the product was azeotropically treated with CH2Cl2 (5 mL) to remove any residual TFA.

[0479] 1 H NMR (500 MHz, MeOD) δ 9.58 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.48(d, J = 8.1 Hz, 2H), 4.83 (t, J = 6.1 Hz, 1H), 4.66 – 4.61 (m, 2H), 4.58 – 4.53(m, 2H), 4.44 – 4.39 (m, 1H), 4.09 (s, 2H), 3.92 (d, J = 11.0 Hz, 1H), 3.82(dd, J = 11.0, 3.4 Hz, 1H), 3.78 – 3.70 (m, 8H), 3.25 – 3.19 (m, 2H), 2.75 –2.62 (m, 2H), 2.56 (s, 3H), 2.40 (s, 1H), 2.29 (dd, J = 12.6, 7.4 Hz, 1H), 2.16– 2.08 (m, 1H), 1.38 (t, J = 6.2 Hz, 5H), 1.06 (s, 9H).

[0480] Intermediate 8a (0.060 g, 0.15 mmol) and PyBOP (0.117 g, 0.225 mmol) were combined in anhydrous DMF (6 mL). DIPEA (0.104 mL, 0.6 mmol) was added dropwise, and the reaction mixture was stirred at room temperature under N2 atmosphere for 15 min. Intermediate 7a (0.10 g, 0.15 mmol) was added, and the reaction mixture was stirred for 18 h. The reaction mixture was diluted with 1:1 DMSO:H2O and purified by preparative HPLC using a gradient of CH3CN (0.1% formic acid) / H2O (0.1% formic acid) from 30% to 95%.

[0481] HRMS (ESI) calculated value: C52 H 62 ClN 10 O8S2[M + 1] 1053.3877; Experimental value: 1053.2644 1 H NMR(500 MHz, MeOD) δ 8.90 (s, 1H), 8.10 (d, J J = 7.7 Hz, 1H), 7.47(d, J J = 7.9 Hz, 4H), 7.42 (d, J J = 8.4 Hz, 4H), 4.71 (t, J J = 6.7 Hz, 1H), 4.66 (t, J J = 6.4 Hz, 2H), 4.59 (t, J J = 8.2 Hz, 1H), 4.53 (d, J J = 15.3 Hz, 1H), 4.47 (s, 1H),4.37 (d, J J = 15.4 Hz, 1H), 4.07 (d, J J = 3.5 Hz, 2H), 3.87 (d, J J = 10.8 Hz, 1H),3.​​​​​​​​​​​​​C NMR(126 MHz, DMSO) δ 172.32, 170.15, 169.80, 169.74, 169.55,163.57, 163.51, 151.98, 148.18, 139.94, 137.16, 135.74, 132.72, 131.26,130.64, 130.34, 130.13, 130.07, 129.14, 128.94, 127.93, 80.97, 73.45, 70.72,70.25, 69.85, 69.71, 69.32, 59.21, 57.13, 56.82, 54.26, 51.19, 42.14, 40.90, 38.34, 37.91, 36.09, 26.72, 22.17, 16.39, 14.52, 13.16, 11.78.

[0483] Intermediate 9a (0.050 g, 0.047 mmol), compound 10a (9.4 μL, 0.047 mmol), sodium ascorbate (0.002 g, 0.0094 mmol, 20 mol%), and Cu(II) sulfate (0.002 g, 0.0094 mmol, 20 mol%) were combined in THF (1.5 mL). 5–6 drops of H₂O were added, and the reaction mixture was stirred for 16 hours. The solvent was evaporated under reduced pressure, and the mixture was redissolved in 1:1 DMSO:H₂O (1.5 mL each). Intermediate 11a was purified by preparative HPLC using a gradient of CH₃CN (0.1% formic acid) / H₂O (0.1% formic acid) from 30% to 95%.

[0484] HRMS (ESI) calculated value: C 60 H 80 ClN 14 O 11 S²[M + 1] = 1271.5255; Experimental value: 1271.2644 1 H NMR (500 MHz, MeOD) δ 8.89 (s, 1H), 8.19 (s, 3H), 7.75 (s, 1H), 7.46 (d, J = 2.6 Hz, 4H), 7.43 (d, J = 3.7 Hz, 4H), 4.86 – 4.82 (m, 1H), 4.66 (d, J= 3.3 Hz, 1H), 4.62 (s, 1H), 4.53 (d, J = 5.5 Hz, 3H), 4.49 (s, 1H), 4.41 –4.36 (m, 1H), 4.02 (d, J = 5.3 Hz, 2H), 3.91 (t, J = 9.1 Hz, 1H), 3.85 (d, J = 4.1Hz, 2H), 3.80 (dd, J = 10.8, 3.5 Hz, 1H), 3.68 (t, J = 6.1 Hz, 7H), 3.66 – 3.61(m, 6H), 3.58 (d, J = 7.1 Hz, 6H), 3.49 (dd, J = 12.9, 7.1 Hz, 3H), 3.37 (dd, J =15.2, 5.2 Hz, 1H), 3.24 (dd, J = 14.9, 6.0 Hz, 1H), 3.14 (d, J = 5.7 Hz, 3H),2.70 (s, 3H), 2.68 (s, 1H), 2.48 (s, 3H), 2.46 (s, 3H), 2.26 (dd, J = 12.8, 7.3Hz, 1H), 2.12 – 2.06 (m, 1H), 2.05 (s, 1H), 1.71 (s, 3H), 1.02 (s, 9H)。

[0485] 13C NMR(126 MHz, MeOD) δ 173.01, 171.59, 171.22, 170.84, 170.45,164.78, 164.22, 155.64, 151.48, 150.76, 147.65, 142.67, 138.89, 136.74,136.56, 132.15, 131.98, 131.81, 130.63, 130.56, 130.12, 129.97, 128.97,128.39, 127.90, 127.64, 127.58, 123.75, 70.77, 70.05, 69.90, 69.83, 69.67, 69.46, 69.01, 66.50, 59.47, 57.75, 56.76, 53.84, 52.31, 49.88, 42.32, 39.28, 39.16, 39.03, 37.61, 37.42, 35.35, 27.74, 25.61, 14.46, 13.04, 11.57, 10.20.

[0486] Intermediate 11a (0.090 g, 0.071 mmol), TCO-bis-NHS (0.036 g, 0.085 mmol), and DIPEA (37 mL, 0.21 mmol) were combined in DMF (5 mL). The reaction mixture was stirred for 16 hours. The solvent was evaporated under reduced pressure, and the mixture was redissolved in CH2Cl2 (3 mL). Intermediate 12a was purified by preparative TLC using 15% MeOH / CH2Cl2 as the mobile phase.

[0487] HRMS (ESI) calculated value: C 75 H 97 ClN 15 O 17 S2[M + 1] 1578.6311; Observation: 1578.3682 1H NMR(500 MHz, CDCl3) δ 8.76 (s, 1H), 8.08 – 7.98 (m, 1H), 7.89 – 7.85 (m, 1H), 7.67 – 7.59 (m, 1H), 7.34 (s, 1H), 6.09 – 5.76 (m, 1H), 5.71 – 5.47 (m, 1H), 5.19 – 5.13 (m, 1H), 4.94 – 4.80 (m, 1H), 4.75 – 4.62 (m, 1H), 4.61 – 4.42 (m, 1H), 4.40 – 4.31 (m, 1H), 4.07 – 3.98 (m, 1H), 3.84 – 3.75(m, 1H), 3.67 (s, 1H), 3.56 (s, 1H), 3.45 (s, 1H), 3.23 – 3.01 (m, 1H), 2.82(s, 1H), 2.50 (s, 1H), 2.41 (s, 1H), 2.34 – 2.20 (m, 1H), 2.20 – 2.13 (m,1H), 2.12 – 1.79 (m, 1H), 1.66 (s, 1H), 1.25 (s, 1H), 1.07 – 1.02 (m, 1H), 0.94 (s, 1H).

[0488] Intermediate 12a (0.050 g, 0.031 mmol), aspartic acid (0.021 g, 0.15 mmol), DIPEA (108 mL, 0.63 mmol), and BSA (0.063 g, 0.31 mmol) were combined in CH2Cl2 (5 mL). The reaction mixture was stirred for 16 hours. The solvent was evaporated under reduced pressure, and the mixture was redissolved in a 1:1 solution of CH3CN (4 mL) and 5% aqueous citric acid (4 mL, pH 3.3). The mixture was stirred at room temperature for 1 hour. Compound 16 was purified by preparative HPLC using a gradient of CH3CN (0.1% formic acid) / H2O (0.1% formic acid) from 30% to 95%.

[0489] HRMS (ESI) calculated value: C 75 H 99 ClN 15 O 18 S2[M + 1] 1596.6417; Observation: 1596.3116 1H NMR(500 MHz, MeOD) δ 8.88 (s, 1H), 8.64 – 8.59 (m, 1H), 8.51 –8.47 (m, 1H), 8.16 – 7.95 (m, 2H), 7.74 (s, 1H), 7.51 – 7.36 (m, 7H), 5.95 –5.86 (m, 1H), 5.68 – 5.61 (m, 1H), 5.08 (s, 1H), 4.69 – 4.59 (m, 4H), 4.58 –4.44 (m, 4H), 4.42 – 4.34 (m, 1H), 4.06 – 3.98 (m, 2H), 3.93 (d, 1H), 3.89 –3.76 (m, 3H), 3.73 – 3.61 (m, 6H), 3.58 (s, 8H), 3.54 – 3.46 (m, 4H), 3.40 –3.35 (m, 2H), 3.30 – 3.10 (m, 4H), 2.84 (t, 1H), 2.73 – 2.65 (m, 3H), 2.49 (d, 5H), 2.30 – 2.22 (m, 2H), 2.16 – 2.05 (m, 2H), 2.02 – 1.94 (m, 2H), 1.90 – 1.86 (m, 1H), 1.84 – 1.77 (m, 1H), 1.72 – 1.68 (m, 3H), 1.66 – 1.57 (m,1H), 1.12 (s, 2H), 1.02 (s, 8H).

[0490] 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 disclosure pertains.

[0491] The embodiments illustratively described herein may be practiced appropriately in the absence of any one or more elements, or one or more limitations, not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing,” etc., should be interpreted broadly and without limitation. Furthermore, the terminology and expressions used herein have been used as terminology in the specification and are not limiting, and the use of such terminology and expressions is not intended to exclude any equivalents or portions thereof of the features shown and described, but it should be recognized that various modifications may be made within the scope of the claims.

[0492] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety by reference, as if each were individually incorporated by reference. In the event of any conflict, this specification (including definitions) shall prevail.

[0493] It should be understood that although this disclosure has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate and not limit the scope of this disclosure. Other aspects, advantages, and modifications within the scope of this disclosure will be apparent to those skilled in the art to which this disclosure pertains.

Claims

1. A trans-cyclooctene-modified TPD conjugate comprising a targeted protein degrader (TPD) optionally covalently linked to at least one trans-cyclooctene moiety via a linker.

2. The trans-cyclooctene-modified TPD conjugate according to claim 1, wherein the TPD comprises an E3 ubiquitin ligand selected from the group consisting of: Von Hippel–Lindau (VHL) ligand, cereblon (CRBN) ligand, mouse double microsome 2 homologue (MDM2) ligand, and a protein containing β-transduction protein repeat sequence (β-TrCP) ligand.

3. The trans-cyclooctene-modified TPD conjugate according to claim 1 or 2, wherein the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of: thalidomide, lenalidomide, pomalidomide, MLN4924 (Pevonedistat), Nutlin-3, and curcumin.

4. The trans-cyclooctene-modified TPD conjugate according to claim 1 or 2, wherein the TPD comprises an E3 ubiquitin ligase ligand selected from Table A, wherein the wavy line indicates the connection point with the remainder of the TPD.

5. The trans-cyclooctene-modified TPD conjugate according to claim 1, wherein the TPD comprises a target protein ligand selected from the group consisting of: receptor tyrosine kinase (RAF) kinase ligand (e.g., BRAF kinase ligand), steroid receptor ligand (e.g., androgen receptor (AR) ligand, estrogen receptor (ER) ligand, or progesterone receptor (PR) ligand), cyclin-dependent kinase (CDK) ligand, BTK ligand, BET ligand, and inhibitor of apoptosis protein (IAP) ligand.

6. The trans-cyclooctene-modified TPD conjugate according to claim 1, wherein the TPD comprises a target protein ligand selected from Table B.

7. The trans-cyclooctene-modified TPD conjugate according to claim 1, wherein the TPD is selected from Table C.

8. A bispecific antibody modified with trans-cyclooctene according to any one of the preceding claims, wherein the trans-cyclooctene moiety has the formula X: X in: R 1A Each time it appears, independently select a group consisting of the following items: C 1-4 Alkyl, C 1-4 Halogenated alkyl and C 1-4 Alkoxy; q is 0, 1, or 2; q1 is either 0 or 1; R 1B Each time it appears, independently select a group consisting of the following items: 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 –CO2H, –N(R) 1f )–C 2-4 Alkylene–(N(C) 1-4 alkylene (–CO2H)–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 (–CO2H)2 and –N(R) 1c )–CH(CH2O–(CH2CH2O) 0-2 –C 1-6 Alkylene (CO2H)2; R 1c and R 1d It is either hydrogen or C each time it appears. 1-4 alkyl; R 1e It is independently set to –C each time it appears. 1-4 Alkylene –CO2H, –C 1-4 Alkylene –CONH2 or –C 1-4 alkylene–OH; R 1f Each time it appears, it is independently hydrogen or C. 1-6 Alkyl or C 1-4 alkylene –CO2H; n is 0, 1, 2 or 3 independently each time it appears; L 2 Each time it appears, independently select the group consisting of the following items: –C(O) – and C 1-3 Alkylene; and G 1 Each time it appears, it is independently a heterocyclic group with optional substitution.

9. A bispecific antibody modified with trans-cyclooctene according to any one of the preceding claims, wherein the trans-cyclooctene moiety has the formula XI: XI in: R 2 It is -OH, 2-aminoethanesulfonic acid, N-linked natural or non-natural amino acid, or optionally substituted ethylenediamine; wherein R 2 It can optionally be further replaced by polyether.

10. A bispecific antibody modified with trans-cyclooctene according to any one of the preceding claims, wherein the trans-cyclooctene moiety has the formula: , , , , , , , or .

11. The trans-cyclooctene-modified TPD conjugate according to any one of the preceding claims, wherein at least one trans-cyclooctene moiety is .

12. The trans-cyclooctene-modified TPD conjugate according to any one of the preceding claims, wherein at least one trans-cyclooctene moiety is or .

13. A trans-cyclooctene-modified TPD conjugate according to any one of the preceding claims, wherein the trans-cyclooctene moiety is covalently bonded to the TPD via a linker.

14. The trans-cyclooctene-modified TPD conjugate according to any one of the preceding claims, wherein the linker is a cleavable linker.

15. A trans-cyclooctene-modified TPD conjugate selected from those in Table 1.

16. A system comprising a trans-cyclooctene-modified TPD conjugate according to any one of the preceding claims and a tetrazine activator.

17. The system of claim 16, wherein the tetrazine activator is formulated for systemic administration.

18. The system according to claim 16 or 17, wherein the tetrazine activator has formula IV: IV in: Each R 20 Independently select from the group consisting of: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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'', 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, heterocyclic or cycloalkyl is optionally surrounded by one to three Z 1 replace; Each Z 1 Independently selected from halogenated, oxo-, cyano-, nitro-, hydroxyl-, alkyl-, haloalkyl-, alkenyl-, alkoxy-, haloalkoxy-, heteroalkyl-, aryl-, heteroaryl-, heterocyclic-, 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)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'' are independently selected from hydrogen, aryl, and alkyl groups each time they appear; and R''' is selected independently from aryl and alkyl groups each time it appears.

19. The system of claim 18, wherein each R 20 Independently, it is phenyl, pyrimidinyl, triazinyl, oxazolyl, isoxazolyl, 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; wherein each is optionally independently surrounded by one to three Z 1 replace.

20. The system of claim 16, wherein the tetrazine activator comprises a support composition comprising a biocompatible support and a tetrazine-containing group.

21. The system of claim 20, wherein the tetrazine activator comprises a biocompatible support and a tetrazine-containing group of formula IVA, IVB, or IVC: IVA; IVB; or IVC; in: R 20 Choose from the following groups: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; R 30 Halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl or cycloalkenyl; R a R 31a and R 31b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; and t can be 0, 1, 2, 3 or 4.

22. The system of claim 16, wherein the tetrazine activator has formula V: V in: Ring A is aryl, cycloalkyl, heterocyclic, or heteroaryl; When R 3 and R 4 When neither of these conditions is present, the dashed line represents the additional bond forming the tetrazine; or when R... 3 and R 4 When both are present, the dashed line represents the additional bond forming the dihydrotetraazine; the condition is that when ring A is aryl, then R... 3 and R 4 Both exist; X represents a biocompatible support, antibody, or antibody fragment. p is 1-150; L is an independent connector each time it appears; R 1 Each time it appears, it is independently selected from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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'', 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, heterocyclic or cycloalkyl is optionally surrounded by one to three Z 1 replace; R 2 Each of the following is independently halogenated, cyano, nitro, hydroxyl, alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclic or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic or cycloalkyl is optionally marked by one to three Z 1 replace; R 3 and R 4 Neither exists; or R 3 and R 4 Each is independently hydrogen or a group that can be removed after a triggering event; Each Z 1 Independently selected from halogenated, oxo-, cyano-, nitro-, hydroxyl-, alkyl-, haloalkyl-, alkenyl-, alkoxy-, haloalkoxy-, heteroalkyl-, aryl-, heteroaryl-, heterocyclic-, 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' N R'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'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; and t is independently 0, 1, 2, 3 or 4 each time it appears.

23. The system according to any one of claims 20 to 22, wherein the biocompatible support is a polymer, a viscous or non-viscous liquid material, a gel, a hydrogel, a polysaccharide hydrogel, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, particles, nanoparticles, microparticles, alginate, cellulose, hyaluronic acid, chitosan, chitosan polysaccharide, chitin, hyaluronic acid, or chondroitin sulfate.

24. The system of claim 16, wherein the tetrazine activator has formula I: I in: X represents an antibody or antibody fragment. p is 1-16; L is an independent connector each time it appears; R 20 Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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 Each time it appears, it is an independent linker of 1 to 100 linking atoms, wherein the linking atoms optionally contain one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate or ketone functional groups; R' and R'' are independently selected from hydrogen, aryl, and alkyl groups each time they appear; and R''' is selected independently from aryl and alkyl groups each time it appears.

25. The system of claim 16, wherein the tetrazine activator has formula II: II in: X represents an antibody or antibody fragment. p is 1-16; L is an independent connector each time it appears; R 20 Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, 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-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 Each time it appears, it is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl. R a R 31a and R 31b Each is independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl groups each time it appears; and t can be 0, 1, 2, 3 or 4 independently.

26. A method for treating cancer or enhancing or inducing an immune response, the method comprising administering to a subject in need a therapeutically effective amount of a trans-cyclooctene-modified TPD conjugate according to any one of claims 1 to 15 or a system according to any one of claims 16 to 25.

27. A method for treating cancer or enhancing or inducing an immune response, the method comprising administering to a subject in need a therapeutically effective amount of a trans-cyclooctene-modified E3 ubiquitin ligand ligand and a tetrazine-modified target protein ligand, or a tetrazine-modified E3 ubiquitin ligand ligand and a trans-cyclooctene-modified target protein ligand, wherein each is independently selected from Table 2.

28. The method of claim 26 or 27, wherein the cancer is melanoma, renal cell carcinoma, 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 / 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.

29. The method according to claim 26 or 27, wherein the cancer is a solid tumor.

30. The method of claim 26 or 27, wherein the cancer is a soft tissue sarcoma.

31. The method according to claim 26 or 27, wherein the cancer is a hematologic malignancy.

32. The method according to claim 31, wherein the hematologic malignancy is myelodysplastic syndrome, acute myeloid leukemia, multiple myelodysplastic syndromes, chronic myeloid 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, or nodular lymphocyte-predominant Hodgkin lymphoma.

33. The method of claim 32, wherein the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

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