Methods for targeted in vivo delivery of a payload

Abstract

This disclosure generally relates to a method for bioorthogonally delivering a payload to a target site in a subject using an antibody-tetraazine conjugate, said conjugate being used, for example, in the treatment of cancer, tumor growth, and immunotherapy.

Description

Cross-citation of related applications

[0001] This application claims the rights of U.S. Provisional Application No. 63 / 583,189, filed September 15, 2023, 63 / 660,911, filed June 17, 2024, and 63 / 681,755, filed August 9, 2024, all of which are incorporated herein by reference in their entirety, pursuant to 35 USC §119(e). Technical Field

[0002] This disclosure generally relates to methods for the orthogonal delivery of payload bio-conjugates to target sites within a subject using antibody-tetraazine conjugates, which are used for applications such as the treatment of cancer, tumor growth, and immunotherapy. Background Technology

[0003] 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 selective manipulation of molecules, cells, particles, and surfaces. in vitro and in vivo Labeling and tracking of biomolecules. These reactions include Staudinger coordination reactions, azide-cyclooctynyl cycloaddition reactions, and the anti-electron demanding Diels-Alder reactions. Summary of the Invention

[0004] This disclosure relates to a method for delivering a payload to a targeted site within a subject. In one aspect, a method is provided for delivering a payload to a subject in need. in vivo A method for forming antibody-load conjugates, the method comprising: Administering an effective amount of the targeting portion to a subject, wherein the targeting portion comprises at least one antibody or a fragment thereof having at least one tetrazine portion covalently linked thereto; A single dose of a therapeutically effective amount of a payload-TCO conjugate is administered to a subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently linked thereto; The antibody or its fragment has binding affinity to receptors on tumors, and further... in vivo The amount of antibody-payload conjugates formed was greater at the tumor site than in the plasma.

[0005] In one aspect, a method is provided for administering a therapeutically effective amount of a payload to a subject, the method comprising: a) administering an effective amount of the targeted portion to a subject, wherein the targeted portion comprises a Fab having at least one tetrazine moiety covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of a payload-TCO conjugate to a subject, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1 to 3, 1 to 2, or 1) trans-cyclooctene moiety covalently linked thereto. Following administration of the targeted portion to the subject, a single dose of the payload-TCO conjugate is administered to the subject within a timeframe of approximately 2 to 48 hours, or approximately 3 to 48 hours, or approximately 4 to 48 hours.

[0006] In some embodiments, the targeting portion comprises a Fab having 1 to 5, 1 to 4, or 1 to 3 tetrazine moieties covalently linked thereto (i.e., the average ratio of DAR or Fab to tetrazine is 5, 4, 3, or 2).

[0007] The targeting portion used in the methods disclosed herein is designed to be localized to a target site within the subject's body once administered. The targeting portion can be administered locally or systemically. Upon administration, a prodrug comprising a payload and one or more complementary bioorthogonal components (i.e., the trans-cyclooctene moiety) is administered, while... in vivo When in contact with the target portion, the prodrug allows for targeted delivery of the payload or therapeutic agent.

[0008] In one aspect, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine moieties covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of a payload-TCO conjugate to a subject, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1 to 3, 1 to 2, or 1) trans-cyclooctene moiety covalently linked thereto. The subjects were given a single dose of the payload-TCO conjugate more than 1 hour but less than 48 hours after the administration of the targeted portion.

[0009] In one aspect, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine moieties covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of a payload-TCO conjugate to a subject, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1 to 3, 1 to 2, or 1) trans-cyclooctene moiety covalently linked thereto. The subjects were given a single dose of the payload-TCO conjugate more than 4 hours but less than 48 hours after the administration of the targeted portion.

[0010] 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.

[0011] 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 lymphoma or leukemia. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a solid tumor.

[0012] In some embodiments, a targeting portion of the formula IIF is provided: IIF Where p is 1 to 10; and X is an antibody fragment portion comprising SEQ ID NO. 9 and SEQ ID NO. 10. In some embodiments, p is 1 to 5.

[0013] In one aspect, a method for reducing the tumor volume of a subject with a tumor is provided, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine moieties covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of a payload-TCO conjugate to a subject, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1 to 3, 1 to 2, or 1) trans-cyclooctene moiety covalently linked thereto. The subjects were given a single dose of the payload-TCO conjugate more than 1 hour but less than 48 hours after the administration of the targeted portion.

[0014] In one aspect, a method for reducing the tumor volume of a subject with a tumor is provided, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine moieties covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of a payload-TCO conjugate to a subject, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1 to 3, 1 to 2, or 1) trans-cyclooctene moiety covalently linked thereto. The subjects were given a single dose of the payload-TCO conjugate more than 4 hours but less than 48 hours after the administration of the targeted portion. Attached Figure Description

[0015] Figures 1A to 1D The tumor volume of NCI-N87 tumors in SCID CB17 mice is shown after treatment with solvent, compound TM-1 and compound B, and isotype Fab-Tz and compound B. Tumor volumes were observed 4 hours after administration of compound TM-1. Figure 1A ), 8 hours ( Figure 1B ), 24 hours Figure 1C ) or 48 hours ( Figure 1D Compound B was administered. N = 5 mice per group.

[0016] Figure 2A The timeline for drug administration and tumor collection is shown.

[0017] Figure 2B It is shown that 15 minutes after administration, compound TM-1 activates compound B to release MMAE in the tumor.

[0018] Figure 3A This demonstrates the quantification of total Fab in HER2-positive tumors.

[0019] Figure 3B The compound TM-1 is shown to be localized in HER2-positive tumors.

[0020] Figure 4AThe total Fab at the tumor site is shown compared to that detected in the plasma.

[0021] Figure 4B The total tetrazine detected at the tumor site and in the plasma is shown.

[0022] Figure 5 The study showed prolonged Fab exposure in plasma and at the tumor site (Tz levels in plasma decreased rapidly but were prolonged at the tumor site).

[0023] Figure 6 The total tetrazine detected at tumor sites at specific time intervals is shown.

[0024] Figure 7 The total Fab detected at the tumor site at a specific time interval is shown.

[0025] Figure 8 This shows the ratio of tetrazine (Fab-Biotin) at the tumor site to that at the tissue site.

[0026] Figure 9 This shows the total Fab ratio at the tumor site to that at the tissue site.

[0027] Figure 10 and Figure 11 The study showed that compound TM-1 preferentially accumulates at the tumor site compared to plasma. Detailed Implementation

[0028] 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 rather is provided as a description of exemplary embodiments.

[0029] definition It should be understood that, 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 brevity, 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.

[0030] 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. While exemplary methods and materials are described below, similar or equivalent methods and materials may be used in implementations or tests of this disclosure. 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 are not intended to be limiting.

[0031] As used herein, the terms “comprising,” “including,” “having,” “having,” “may,” “containing,” and variations thereof are intended as open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. This disclosure also contemplates other embodiments that “comprise” the embodiments or elements presented herein, “consist of” the embodiments or elements presented herein, and “consist substantially of” the embodiments or elements presented herein, whether or not explicitly stated.

[0032] The modifier “about” used with respect to 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 plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean 0.9 to 1.1. Other meanings of “about” may be apparent from the context, such as rounding, so for example, “about 1” can also mean 0.5 to 1.4.

[0033] 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 comprising A or B" could refer to a device comprising A but not B, a device comprising B but not A, or a device comprising both A and B. The phrase "at least one of A, B, ... and N" or "at least one of A, B, ... N or a combination thereof" is defined in the broadest sense as meaning one or more elements selected from the group comprising A, B, ... and N. That is, any combination of one or more of elements A, B, ... or N includes any single element or combination with one or more of the other elements, and may also include additional unlisted elements in combination.

[0034] 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 following references: Organic Chemistry Thomas Sorrell, University Science Books, Sosalito, 1999; Smith and March March's Advanced Organic Chemistry , 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis , 3rd edition, Cambridge University Press, Cambridge, 1987; the full text of each of these references is incorporated herein by reference.

[0035] 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.

[0036] As used herein, the term "alkoxy" refers to an alkyl group, as defined herein, 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.

[0037] As used herein, the term "alkenyl" refers to a hydrocarbon chain containing 2 to 30 carbon atoms, having 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 a phenyl group).

[0038] As used herein, the term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group having 2 to 30 carbon atoms (such as 2 to 20 or 2 to 10 carbon atoms) and having at least one triple bond unsaturated site. The term "alkynyl" also includes non-aromatic cycloalkyl groups having 5 to 20 carbon atoms (such as 5 to 10 carbon atoms) having a monocyclic or polycyclic structure and having at least one triple bond. Examples of such alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and propynyl (-CH2C≡CH), and cycloalkynyl moieties, such as, but not limited to, substituted or unsubstituted cyclooctyne moieties.

[0039] As used herein, the term "alkoxyalkyl" refers to an alkoxy group attached to a parent molecule portion by an alkyl group as defined herein.

[0040] 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-.

[0041] 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 attached to a hydrogen, carboxyl, amino, and R group. Such analogs may have modified R groups (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 analogs include citrulline, homoserine, ortholeucine, methionine sulfoxide, methionine sulfone, 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.

[0042] 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.

[0043] In some embodiments, as used herein, the term "aryl" refers to a phenyl group 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.

[0044] As used herein, the term "azide" refers to the functional group –N3.

[0045] 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 groups 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 attachment point to the rest of the molecule.

[0046] 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 groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. "Cycloalkyl" also includes carbocyclic systems in which a cycloalkyl group is attached to a parent molecule moiety and fused with an aryl group, a heteroaryl group, or a heterocycle as defined herein.

[0047] As used herein, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic system containing at least one carbon-carbon double bond (e.g., 5-10 carbon atoms per ring). Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0048] As used herein, the term "cyclooctene" refers to a substituted or unsubstituted non-aromatic cyclic alkyl group having eight carbon atoms in a monocyclic ring with a double bond. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).

[0049] 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).

[0050] As used herein, the term "alkoxyfluoroalkyl" refers to an alkoxy group attached to a parent molecule portion by a fluoroalkyl group as defined herein.

[0051] As used herein, the term "fluoroalkoxy" means at least one fluoroalkyl group, as defined herein, attached to a parent molecule moiety via an oxygen atom. Representative examples of fluoroalkoxy groups include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0052] As used herein, the term "halogen" or "halogenated" refers to Cl, Br, I, or F.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 group is exemplified as a monocyclic heteroaryl ring attached to a parent molecule moiety and fused with a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic heterocycle as defined herein. In some embodiments, a tricyclic heteroaryl group is exemplified as a monocyclic heteroaryl ring attached to a parent molecule moiety and fused with a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or both of a monocyclic heterocycle as defined herein.

[0058] As used herein, the terms "heterocyclic group," "heterocyclic," or "heterocyclic" refer 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 to 5, 1 to 4, 1 to 3, 1 to 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 bridging 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 a phenyl group, or monocyclic heterocycles fused with a monocyclic cycloalkyl group, or monocyclic heterocycles fused with a monocyclic cycloalkenyl group, or monocyclic heterocycles fused with a monocyclic heterocycle, 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-dihydrobenzothiophenyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptane-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. Tricyclic heterocycles are exemplified as bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl group, or bicyclic heterocycles fused to a monocyclic cycloalkenyl group, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles wherein two non-adjacent atoms of the bicyclic 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.

[0059] As used herein, the term "hydroxyl group" refers to the -OH group.

[0060] 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.

[0061] In some cases, the number of carbon atoms in a hydrocarbon substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix "C". x -C y -" or "C" x-y The symbol "" indicates that x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms. Therefore, for example, "C1-C3 alkyl" and "C 1-3 "Alkyl" refers to an alkyl substituent containing 1 to 3 carbon atoms. Two conventions apply to "C..." x -C y -" or "C" x-y "They are interchangeable and have the same meaning."

[0062] 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.

[0063] 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 the "Tz" group.

[0064] The term "selective delivery" refers to delivering an agent (e.g., a payload) 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 targeting portion or therapeutic targeting portion described herein does not itself have a therapeutic effect but is designed to allow selective or targeted delivery of therapeutic agents. However, the targeting portion may have a therapeutic effect, and therefore, this disclosure does not exclude such constructs.

[0065] The term "payload" refers to a pharmaceutical agent intended for delivery to a target site on a subject. In some embodiments, the payload is a therapeutic agent. In some embodiments, the payload is a diagnostic agent.

[0066] The term "therapeutic agent" refers to a pharmaceutical agent capable of treating and / or improving a subject's condition or disease or one or more symptoms thereof. Therapeutic agents disclosed herein also include prodrug forms of therapeutic agents and chelating agents (e.g., diagnostic or therapeutic) with or without a radionuclide.

[0067] As used herein, therapeutic “radionucleoside” or “radioligand” refers to a radioactive substance, sometimes called a radiopharmaceutical, used to treat medical conditions, particularly cancer. The radionuclide used in the trans-cyclooctene moiety described herein comprises chelating agents and isotopes; such isotopes selected from the group consisting of: 24 Na、 32 P, 33 P, 47 Sc、 59 Fe、 67 Cu、76 As、 77 As、 80 Br、 82 Br、 89 Sr、 90 Nb, 90 Y、 103 Ru、 105 Rh、 109 Pd, 111 Ag、 111 In、 121 Sn、 127 Te、 131 I, 140 La、 141 Ce、 142 Pr, 143 Pr、 144 Pr, 149 Pm, 149 Tb, 151 Pm, 153 Sm、 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 172 Tm、 175 Yb、 177 Lu、 186 Re、 188 Re、 198 Au、 199 Au、 211 At、 211 Bi、 212 Bi、 212 Pb, 213 Bi、 214 Bi、 223 Ra and 225 Ac. Radionuclides can be delivered via direct conjugation or chelation with a chelating agent. Exemplary radionuclides, chelating agents, and connectors for potential TCO conjugate payloads are described below.

[0068] The term "diagnostic agent" refers to a pharmaceutical agent that helps diagnose a condition or disease. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, and radionuclides. Paramagnetic agents are imaging agents that are magnetic 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 of this disclosure 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 (such as diagnostic radionuclides) include, but are not limited to, […]. 3 H, 11 C 13 N、 18 F, 19 F, 60 Co、 64 Cu、 67 Cu、 68 Ga、 82 Rb、 89 Zr、 90 Sr、 90 Y、 99 Tc, 99m Tc, 111 In、 123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs、 177 Lu、 186 Re、 188 Re、 211 At、 212 Pb, 225 Ac, Rn, Ra, Th, U, Pu and 241 Am.

[0069] 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 association between the target agent and the specific target. "Stable association" or "stable association" means that under standard physiological conditions, one part binds to or otherwise associates with another part or structure. Bonds can include covalent bonds 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.

[0070] As used herein, the term "antibody fragment portion" refers to one or more regions or fragments of an antibody that retain the ability to specifically bind to an antigen, or in other words, substantially retain the antigen-binding function of the antibody. Examples of binding fragments included within the antigen-binding portion of an antibody include, but are not limited to, Fab fragments. In some embodiments, a Fab fragment is a fragment containing at least V L and V H The unit price segment. In some embodiments, the Fab segment is a segment containing V. L V H C L and C H 1. Monovalent fragments of the Fab domain. Fab fragments can be obtained using conventional techniques known to those skilled in the art, and fragments are screened for utility in the same manner as intact antibodies. For example, antibody fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Additional examples are also included in the various embodiments disclosed herein.

[0071] "Specific binding" means that an antibody or antibody fragment portion preferentially associates with a target molecule (e.g., an antigen, such as a peptide, polypeptide, glycoprotein, or any other portion having one or more antigenic determinants) or with a cell or tissue containing a target molecule (e.g., a cell surface antigen, such as a receptor or ligand), rather than with a cell or tissue lacking the target molecule. It should be recognized that some degree of non-specific interaction may occur between the binding portion and non-target molecules (either alone or in combination with cells or tissues). However, specific binding can be identified as being mediated by the specific recognition of the target antigen. Specific binding results in a much stronger association between the target portion (e.g., Fab) and, for example, cells containing the target molecule (e.g., an antigen) than between the binding portion and, for example, cells lacking the target molecule. Specific binding typically results in an increase (per unit time) of the amount of the binding portion (per unit time) binding to, for example, cells or tissues containing the target molecule or marker, greater than 2-fold, greater than 5-fold, greater than 10-fold, or greater than 100-fold compared to cells or tissues lacking the target molecule or marker. In some embodiments, the binding portion is, for example, less than 10 -5M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M or less than 10 -15 The dissociation constant of M binds to target molecules or labels. Various assay formats are suitable for measuring this binding, such as solid-phase ELISA immunoassays.

[0072] The term "target organ or tissue" refers to the organ or tissue that is the target of the payload delivery. Representative organs and tissues used for targeting include those that can be targeted by chemical or biological targeting agents, as well as those that cannot be targeted by chemical or biological targeting agents.

[0073] The term "contacting" or "contact" refers to the process of bringing at least two different kinds of substances 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.

[0074] 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.

[0075] 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.

[0076] The terms "pharmaceutical effective amount" and "therapeutic effective amount" refer to the amount of a compound sufficient to treat a specific disorder 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 disorder or one or more symptoms thereof. For tumorigenic proliferative disorders, pharmaceutically or therapeutically effective amounts include amounts sufficient to cause tumor shrinkage or reduce the rate of tumor growth.

[0077] As used herein, the terms “subject,” “patient,” or “organism” include humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to 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 such as cattle, sheep, goats, dairy cows, pigs, etc.; poultry such as chickens, ducks, geese, turkeys, etc.; and domesticated animals, particularly pets such as dogs and cats. For diagnostic or research applications, suitable subjects may include mammals such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs (such as inbred pigs).

[0078] As used herein, the term “treating” means treating a patient’s disease or medical condition or symptoms thereof, such as in a mammal (e.g., a human), including: (a) improving the patient’s disease or medical condition or symptoms thereof, such as eliminating or causing the patient’s disease or medical condition or symptoms to subside; (b) suppressing the disease or medical condition or symptoms thereof, such as by slowing or preventing the development of the patient’s disease or medical condition or symptoms thereof; or (c) alleviating the symptoms of the patient’s disease or medical condition or symptoms thereof.

[0079] The term "physiological conditions" is intended to encompass conditions compatible with living cells. For example Temperature, pH, and salinity compatible with living cells wait Main water-related conditions.

[0080] For the compounds described herein, the groups and substituents may be selected based on 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, or elimination.

[0081] Where a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (one-tenth of a unit to the lower limit, unless otherwise expressly stated) and any other stated or intermediate values ​​within the range are included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in even smaller ranges and are also included in this disclosure, subject to any expressly excluded limits within the stated ranges. Where a stated range includes one or both limits, the range excluding any one or both of the included limits is also included in this disclosure.

[0082] For the numerical ranges listed in this article, each intermediate number with the same precision 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.

[0083] 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 "is the configuration as defined in IUPAC 1974 recommendation, part E of 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 an enantiomer mixture to a chiral auxiliary agent, separating the resulting diastereomer mixture by recrystallization or chromatography, and as in Furniss, Hannaford, Smith and Tatchell, “Vogel's Textbook of Practical Organic Chemistry,” 5th edition (1989), Longman The method described in Scientific & Technical, Essex CM20 2JE, UK, is to optionally release the optically pure product from the auxiliaries; or (2) directly separate the optically enantiomer mixture on a chiral chromatographic column; or (3) use a stepwise recrystallization method.

[0084] It should be understood that compounds may have tautomer forms and geometric isomers, and these also constitute an aspect of this disclosure.

[0085] 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. Use heavier isotopes, such as deuterium (i.e., 2 Substitution of H) can provide certain therapeutic advantages resulting from greater metabolic stability, such as in vivo An increased half-life or reduced dosage requirement may be preferred in some cases. The compound 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 are... 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.

[0086] Treatment This disclosure relates to a method for delivering a payload to a target location within a subject's body.

[0087] In one aspect, it provides a way for subjects in need to... in vivo A method for forming antibody-load conjugates, the method comprising: Administering an effective amount of the targeting portion to a subject, wherein the targeting portion comprises at least one antibody or a fragment thereof having at least one tetrazine portion covalently linked thereto; A single dose of a therapeutically effective amount of a payload-TCO conjugate is administered to a subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently linked thereto; The antibody or its fragment has binding affinity to receptors on tumors, and further... in vivoThe amount of antibody-payload conjugates formed was greater at the tumor site than in the plasma.

[0088] In one aspect, it provides a way for subjects in need to... in vivo A method for forming antibody-load conjugates, the method comprising: Administering an effective amount of the targeting portion to a subject, wherein the targeting portion comprises at least one antibody or a fragment thereof having at least one tetrazine portion covalently linked thereto; A single dose of a therapeutically effective amount of a payload-TCO conjugate is administered to a subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently linked thereto; The antibody or its fragment has binding affinity to a receptor on the tumor, and further wherein the ratio of the antibody-load conjugate at the tumor site to the antibody-load conjugate in the plasma is greater than about 2:1.

[0089] In some embodiments, the ratio of the antibody-load conjugate at the tumor site to that in the plasma is greater than 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1, or about 11:1, or about 12:1, or about 13:1, or about 14:1, or about 15:1, or about 16:1, or about 17:1, or about 18:1, or about 19:1.

[0090] In some embodiments, the ratio of the antibody-load conjugate at the tumor site to that in the plasma is greater than about 2:1, or greater than about 3:1, or greater than about 4:1, or greater than about 5:1, or greater than about 6:1, or greater than about 7:1, or greater than about 8:1, or greater than about 9:1, or greater than about 10:1.

[0091] In some embodiments, the application is simultaneous.

[0092] In some embodiments, application is sequential. In some embodiments, the targeting portion is applied prior to the payload-TCO conjugate.

[0093] In some embodiments, the first dose of the payload-TCO conjugate is administered to the subject less than 48 hours after the administration of the targeted portion, or more than 4 hours but less than 48 hours after the administration of the targeted portion.

[0094] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject less than 48 hours after the administration of the targeted portion, or more than 4 hours but less than 48 hours after the administration of the targeted portion.

[0095] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject less than 48 hours after the administration of the targeted portion.

[0096] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject more than 4 hours but less than 48 hours after the administration of the targeted portion.

[0097] In some embodiments, the target portion is applied at least 8 to about 24 hours before the application of the payload-TCO conjugate.

[0098] In some embodiments, the targeting portion is administered over a period of 0 to 8 serum half-lives. It is envisioned that the timing of the payload-TCO conjugate administration is calculated based on tumor distribution, which can be determined by the biology of the antigens at the tumor site and the targeting portion. For example, in some embodiments, the payload-TCO conjugate is administered within the serum half-lives shown in the table below when the targeting portion comprises the targeting form shown therein (see also Berland et al., Biomolecules 2021, 11(5), 637).

[0099] In one aspect, a method for administering a payload to a subject is provided, the method comprising: a) administering an effective amount of the targeted portion to a subject, wherein the targeted portion comprises a Fab having at least one tetrazine moiety covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of the payload-TCO conjugate to a subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto; The single dose of the payload-TCO conjugate is administered to the subject at least about 2 hours, or at least about 3 hours, or at least about 4 hours, less than about 48 hours, or between about 4 hours and 48 hours after administration of the targeted portion.

[0100] In one aspect, a method for administering a payload to a subject is provided, the method comprising: a) administering an effective amount of the targeted portion to a subject, wherein the targeted portion comprises a Fab having at least one tetrazine moiety covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of the payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto; The subjects were given a single dose of the payload-TCO conjugate more than 4 hours but less than 48 hours after the administration of the targeted portion.

[0101] Selective binding occurs between bioorthogonal binding partners (e.g., between the tetrazine of the targeting moiety and the complementary trans-cyclooctene of its payload-TCO conjugate). Because the targeting moiety is locally applied to the desired location on the subject as described above, the selective binding between the trans-cyclooctene of the payload-TCO conjugate and the tetrazine of the targeting moiety positions the payload at the desired target site.

[0102] The method disclosed herein allows for selective and safe delivery of the payload, thus reducing side effects or toxicity associated with off-target delivery. Furthermore, therapeutic efficacy is achieved with only a single dose of the payload-TCO conjugate, administered using the two agents described herein.

[0103] As described herein, selective delivery of payloads can be achieved by administering payload-TCO conjugates within a specific therapeutic window following administration of the target agent. The relatively inert, systemically administered payload-TCO conjugates are activated at the target site (e.g., a tumor) via a covalent click chemistry reaction, followed by chemical rearrangement to release the active payload.

[0104] In some embodiments, the subject has cancer. In one aspect, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) administering an effective amount of the targeted portion to a subject, wherein the targeted portion comprises a Fab having at least one tetrazine moiety covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of the payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto; The single dose of the payload-TCO conjugate is administered to the subject at least about 2 hours, or at least about 3 hours, or at least about 4 hours, less than about 48 hours, or between about 4 hours and 48 hours after administration of the targeted portion.

[0105] In one aspect, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) administering an effective amount of the targeted portion to a subject, wherein the targeted portion comprises a Fab having at least one tetrazine moiety covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of the payload-TCO conjugate to a subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto; The subjects were given a single dose of the payload-TCO conjugate more than 4 hours but less than 48 hours after the administration of the targeted portion.

[0106] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, less than 24 hours, or about 24 hours after administration of the targeted portion.

[0107] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject between approximately 5 and approximately 24 hours, or between approximately 6 and approximately 24 hours, or between approximately 7 and approximately 24 hours, or between approximately 8 and approximately 24 hours, or between approximately 9 and approximately 24 hours, or between approximately 10 and approximately 24 hours, or between approximately 11 and approximately 24 hours, or between approximately 12 and approximately 24 hours, or between approximately 13 and approximately 24 hours, or between approximately 14 and approximately 24 hours, or between approximately 15 and approximately 24 hours, or between approximately 16 and approximately 24 hours, or between approximately 17 and approximately 24 hours, or between approximately 18 and approximately 24 hours, or between approximately 19 and approximately 24 hours, or between approximately 20 and approximately 24 hours, or between approximately 21 and approximately 24 hours, or between approximately 22 and approximately 24 hours, or between approximately 23 and approximately 24 hours after administration of the targeted portion.

[0108] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject between approximately 5 and approximately 22 hours, or between approximately 6 and approximately 22 hours, or between approximately 7 and approximately 22 hours, or between approximately 8 and approximately 22 hours, or between approximately 9 and approximately 22 hours, or between approximately 10 and approximately 22 hours, or between approximately 11 and approximately 22 hours, or between approximately 12 and approximately 22 hours, or between approximately 13 and approximately 22 hours, or between approximately 14 and approximately 22 hours, or between approximately 15 and approximately 22 hours, or between approximately 16 and approximately 22 hours, or between approximately 17 and approximately 22 hours, or between approximately 18 and approximately 22 hours, or between approximately 19 and approximately 22 hours, or between approximately 20 and approximately 22 hours, or between approximately 21 and approximately 22 hours after administration of the targeted portion.

[0109] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject between approximately 5 and approximately 20 hours, or approximately 6 and approximately 20 hours, or approximately 7 and approximately 20 hours, or approximately 8 and approximately 20 hours, or approximately 9 and approximately 20 hours, or approximately 10 and approximately 20 hours, or approximately 11 and approximately 20 hours, or approximately 12 and approximately 20 hours, or approximately 13 and approximately 20 hours, or approximately 14 and approximately 20 hours, or approximately 15 and approximately 20 hours, or approximately 16 and approximately 20 hours, or approximately 17 and approximately 20 hours, or approximately 18 and approximately 20 hours, or approximately 19 and approximately 20 hours after administration of the targeted portion.

[0110] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject between approximately 8 and approximately 12 hours, between approximately 8 and approximately 16 hours, between approximately 8 and approximately 22 hours, between approximately 12 and approximately 16 hours, or between approximately 16 and approximately 20 hours after administration of the targeted portion.

[0111] In some embodiments, the payload-TCO conjugate is an MMAE-TCO conjugate. In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 and approximately 12 hours, between approximately 8 and approximately 16 hours, between approximately 8 and approximately 22 hours, between approximately 12 and approximately 16 hours, or between approximately 16 and approximately 20 hours after administration of the targeted portion.

[0112] In some embodiments, a single dose of the payload-TCO conjugate is administered to the subject between approximately 16 and approximately 20 hours after administration of the targeted portion.

[0113] In one aspect, a method is provided for selectively applying a payload to a tumor site in a subject of need, the method comprising: a) administering an effective amount of the targeted portion to a subject, wherein the targeted portion comprises a Fab having at least one tetrazine moiety covalently linked thereto; and b) Administering a single dose of a therapeutically effective amount of the payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto; A single dose of the payload-TCO conjugate was administered to the subject between approximately 8 and 18 hours after the administration of the targeted portion.

[0114] In some embodiments, the accumulation at the tumor site is at least about 10 times, or as high as about 200 times, the accumulation in the kidney, liver, or spleen tissue. In some embodiments, the targeting portion has Formula I, Formula II, or Formula 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 dihydroceramide; the condition is that when ring A is aryl, then R... 3 and R 4 Both exist; X represents an antibody fragment; p ranges from 1 to 20; L is a connector independently 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, ynyl, 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 group is optionally defined by one to three Z... 1 replace; R 2Each time it appears independently of halogen, cyano, nitro, hydroxy, 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 defined 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 20 Each time it appears, it is independently selected from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, -CF3, -CF2R', -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 connecting atoms, which optionally contains one or more ethylene-oxy, amine, ester, amide, urethane, 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 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; 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.

[0115] In some embodiments, the targeting portion has formula I or II: in: X represents an antibody fragment; p ranges from 1 to 16; L is a connector independently 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 connecting atoms, which optionally contains one or more ethylene-oxy, amine, ester, amide, urethane, 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; 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 t is independently 0, 1, 2, 3 or 4 each time it appears.

[0116] In some embodiments, the targeting portion has formula IIA: IIA; where X, p, L, and R 20 Each is independent as defined in this article.

[0117] In some embodiments, each R 20 It can be hydrogen or alkyl independently.

[0118] In some embodiments, the targeting portion has formula VII: VII; where X, p, L, ring A, R 1 R 2 t and t are each defined independently as in this paper.

[0119] 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.

[0120] In some embodiments, ring A is a phenyl group.

[0121] In some embodiments, p is 1 to 16, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2.

[0122] In some embodiments, each R 1 It can be hydrogen or alkyl independently.

[0123] In some embodiments, each R 2 It is independently a halo, alkyl, or haloalkyl group.

[0124] In some embodiments, t is 0 each time it occurs.

[0125] In some embodiments, X is an antibody fragment that targets the following: TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

[0126] In some embodiments, X is an antibody fragment portion “derived from” the antibody. In some embodiments, the term “derived from” refers to an antibody fragment containing the antibody’s V. H and V L This allows antibody fragments or Fab to bind specifically to the antigen.

[0127] In some embodiments, X is an antibody fragment 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. Anti-, Dugoutuzumab, Votoxicillin, GC1118, Imtracumab, Panitumumab, Alutumumab, Toltuzumab, Latoxicillin, Moozumab, Sitaturumab, Tococillin, Caputoxumab, Ezekielumab, Ademumab, Ramoximumab, Ramorumab, Vulinacil, Olatoxicillin, Ramorumab, Saxitoxicillin, Pr1E11, Envertuzumab, J591, ML N591, Belantuzumab, Mosetuzumab, Intocilizumab, Iprazumab, Pinatuzumab, Utoximab, Ofamumab, Rituximab, Obinutuzumab, Tosimomab, Tiimumab, Rontoximab, XMAB-5574, MOR208, Cortuzumab, Dinituzumab, 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, Trastuzumab, ECT204, MDX-1414, Pertuzumab, Trastuzumab, Magutuximab, Pertrastuzumab, Seretuzumab, Lutuzumab, Eganutuzumab, AV-203, CDX-3379, GSK284933, Brentuximab, Gelatuzumab, BI 835858, Vardatocizumab, Lintocizumab, KHK2823, Tacrolimus, G4723A, Gabatocizumab, Teritolus, Onatuzumab, SAIT301, Tesxotuzumab, Lifatocizumab, Indojutumab, Vantocizumab, Sofostocizumab, Voseltuzumab, Bivaliruzumab, Karacizumab, Orizizumab, V565, PF-05230905, Wabalizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, Sirolizumab, F16, R6N, Datopotamab, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, 74Hz.

[0128] In some embodiments, the targeting portion further comprises an imaging contrast agent. In some embodiments, the imaging contrast agent is a protein.

[0129] In some embodiments, L is independently bonded to X via a cysteine ​​or lysine residue on X each time it appears.

[0130] In some embodiments, each L contains one or more amino acids.

[0131] In some embodiments, each L contains a polypeptide.

[0132] In some embodiments, each L independently 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.

[0133] In some embodiments, each L independently comprises 5 to 50 linking atoms; comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkyneene, arylene, or heteroaryl moieties; wherein each alkylene, alkenylene, alkyneene, arylene, or heteroaryl moieties is independently and optionally substituted by one to five substituents independently selected from: oxo, halogenated, C-substituted. 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups.

[0134] In some embodiments, X is an antibody fragment targeting the following: HER2, TROP2, cohesin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC or ECM, EPCAM, CEA or CEACAM5; and each L is independently selected from the group consisting of: , , , , and .

[0135] In some embodiments, X is an antibody fragment targeting HER2; p is 1 to 5; and each L is independently selected from the group consisting of: , , , , and .

[0136] In some embodiments, the targeting portion has the formula IIF: IIF; where X and p are each independently defined as described herein.

[0137] In some embodiments, X is an antibody fragment targeting HER2; and p is 1 to 5.

[0138] In some embodiments, the payload-TCO conjugate has formula VIII or a pharmaceutically acceptable salt thereof: VIII in: G is independent each time it appears. ; L 1 Each time it appears, it is a connector independently; m is an integer from 1 to 150; D represents the payload; R 1AChoose C independently each time it appears. 1-4 Alkyl, C 1-4 Halogenated alkyl groups and C 1-4 The group consisting of alkoxy groups; 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) 1c CHR 1e C(O)OC 1-6 Alkyl, -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) 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, -N(R) 1c )-C 1-6 Alkylene-C(O)OC 1-6 Alkyl 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 an optionally substituted heterocyclic group.

[0139] In some embodiments, the payload is selected from therapeutic agents used to treat cancer (e.g., paclitaxel, doxorubicin, danomycin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixaprilone, partopilon (epotassium class), platinum-based drugs, ixotecan, delutec, regoxetine (saurus 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, carrichomycin, asteroidin, succinate, seco-DUBA, pyromycin, etc.), immunosuppressants (e.g., paclitaxel, doxorubicin, pyromycin, etc.). Examples of antifungal agents include cyclosporine A and rapamycin, antifungal agents (e.g., amphotericin B), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, rubitidine, gademolide, matrix metalloproteinase (MMP) inhibitors, L-DOPA, oseltamivir, cephalexin, 5-aminopyruvic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and c-AMP).

[0140] In some embodiments, the payload is a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, doxorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixaprilone, partoprilone (epotoxins), platinum-based drugs, ixotecan, delutec, reoxetine (tip serotonin 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, carrichomycin, asteroidin, hexasporin, seco-DUBA, pyromycin, etc.) or an immunosuppressant (e.g., cyclosporine A, rapamycin, etc.).

[0141] In some embodiments, the effective payload is paclitaxel, doxorubicin, doxorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixaprilone, partoprilone, platinum-based drugs, eczemaconazole, delutec, semaphorin 10, MMAE, MMAD, MMAF, mitomycin C, bleomycin, carrichomycin, astrospore, hexasporin, seco-DUBA, pyroxine, cyclosporine A, or rapamycin.

[0142] In some embodiments, the payload-TCO conjugate is selected from: , , , , , and .

[0143] In some embodiments, a method is provided for administering a therapeutically effective amount of monomethylolpropionate E (MMAE) to a subject suffering from cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the trastuzumab Fab-tetrazine targeted portion of formula IIF: IIF Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); and p is 1 to 5; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0144] In some embodiments, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the trastuzumab Fab-tetrazine targeted portion of formula IIF: IIF Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); and p is 1 to 5; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 hours and approximately 48 hours or between approximately 8 hours and approximately 24 hours after administration of the targeted portion.

[0145] In some embodiments, a method for reducing the tumor volume of a subject with a tumor is provided, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the trastuzumab Fab-tetrazine targeted portion of formula IIF: IIF Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); and p is 1 to 5; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0146] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 hours and approximately 24 hours after administration of the targeted portion.

[0147] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 and approximately 12 hours, between approximately 8 and approximately 16 hours, between approximately 8 and approximately 22 hours, between approximately 12 and approximately 16 hours, or between approximately 16 and approximately 20 hours after administration of the targeted portion.

[0148] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 and approximately 22 hours or between approximately 16 and approximately 20 hours after administration of the targeted portion.

[0149] In some embodiments, a method is provided for administering a therapeutically effective amount of monomethylolpropionate E (MMAE) to a subject suffering from cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion has the formula IIF: IIF Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); or X is sacitrullab Fab containing (SEQ ID NO.9) and (SEQ ID NO.10); and p is 1 to 5; as well as b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0150] In some embodiments, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion has the formula IIF: IIF Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); or X is sacitrullab Fab containing (SEQ ID NO.9) and (SEQ ID NO.10); and p is 1 to 5; as well as b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0151] In some embodiments, a method for reducing the tumor volume of a subject with a tumor is provided, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion has the formula IIF: IIF Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); or X is sacitrullab Fab containing (SEQ ID NO.9) and (SEQ ID NO.10); and p is 1 to 5; as well as b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0152] In some embodiments, a method is provided for administering a therapeutically effective amount of monomethylolpropionate E (MMAE) to a subject suffering from cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the trastuzumab Fab-tetrazine targeted portion of formula IIFa: IIFa Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); and p is 2 to 3; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0153] In some embodiments, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the trastuzumab Fab-tetrazine targeted portion of formula IIFa: IIFa Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); and p is 2 to 3; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0154] In some embodiments, a method for reducing the tumor volume of a subject with a tumor is provided, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the trastuzumab Fab-tetrazine targeted portion of formula IIFa: IIFa Where X is trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); and p is 2 to 3; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0155] In some embodiments, a method is provided for administering a therapeutically effective amount of monomethylolpropionate E (MMAE) to a subject suffering from cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the sacituzumab Fab-tetrazine targeted portion of formula IIF: IIF Where X is sacitocilizumab Fab containing (SEQ ID NO. 9) and (SEQ ID NO. 10); and p is 1 to 5; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0156] In some embodiments, a method is provided for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the sacituzumab Fab-tetrazine targeted portion of formula IIF: IIF Where X is sacitocilizumab Fab containing (SEQ ID NO. 9) and (SEQ ID NO. 10); and p is 1 to 5; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0157] In some embodiments, a method for reducing the tumor volume of a subject with a tumor is provided, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is the sacituzumab Fab-tetrazine targeted portion of formula IIF: IIF Where X is sacitocilizumab Fab containing (SEQ ID NO. 9) and (SEQ ID NO. 10); and p is 1 to 5; and b) Administer a single therapeutically effective dose of a MMAE-TCO conjugate having the following structure to the subject: ; A single dose of the MMAE-TCO conjugate was administered to the subject between approximately 2 and 48 hours or between approximately 8 and 24 hours after the administration of the targeted portion.

[0158] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 hours and approximately 24 hours after administration of the targeted portion.

[0159] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 and approximately 12 hours, between approximately 8 and approximately 16 hours, between approximately 8 and approximately 22 hours, between approximately 12 and approximately 16 hours, or between approximately 16 and approximately 20 hours after administration of the targeted portion.

[0160] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 and approximately 22 hours or between approximately 16 and approximately 20 hours after administration of the targeted portion.

[0161] In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 10 mg / kg to about 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 10 mg / kg to about 50 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 20 mg / kg to about 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 20 mg / kg to about 50 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 30 mg / kg to about 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 30 mg / kg to about 50 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 40 mg / kg to about 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 40 mg / kg to about 50 mg / kg.

[0162] In some embodiments, the targeted portion is administered as a single dose in amounts of about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, or about 60 mg / kg.

[0163] In some embodiments, the payload-TCO conjugate is administered as a single dose in an amount ranging from 10 mg / kg to about 50 mg / kg. In some embodiments, the payload-TCO conjugate is administered as a single dose in an amount ranging from 20 mg / kg to about 50 mg / kg. In some embodiments, the payload-TCO conjugate is administered as a single dose in an amount ranging from 20 mg / kg to about 40 mg / kg. In some embodiments, the payload-TCO conjugate is administered as a single dose in an amount ranging from 25 mg / kg to about 35 mg / kg.

[0164] In some embodiments, the payload-TCO conjugate is administered as a single dose in amounts of about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg.

[0165] In some embodiments, the subjects had cancer.

[0166] 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.

[0167] 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.

[0168] In some embodiments, the cancer is a solid tumor.

[0169] In some embodiments, the cancer is a soft tissue sarcoma.

[0170] In some embodiments, the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0171] In some embodiments, the method further includes 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.

[0172] In some embodiments, the method further includes administering a therapeutically effective amount of an additional 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.

[0173] 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 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.

[0174] In some embodiments, the method can be used to treat and / or diagnose hematologic malignancies such as myelodysplastic syndrome, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammaglobulinosis, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder (NOS), T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, nodular predominantly Hodgkin lymphoma, and others.

[0175] The compositions disclosed herein can be used to treat and / or diagnose a condition or disease in a subject who is readily treatable or diagnosable by administration of a payload (e.g., a parent drug (i.e., the drug prior to conjugation with the composition)). “Treatment” means at least achieving improvement in symptoms associated with a condition troubling the subject, where improvement is broadly defined as at least a reduction in the magnitude of parameters (e.g., symptoms) associated with the treated condition. Thus, treatment also includes the complete suppression (e.g., prevention of occurrence) or cessation (e.g., termination) of the pathological condition or at least the symptoms associated with it, such that the subject no longer suffers from the condition or at least no longer suffers from the symptoms characterizing the condition. Treatment may include suppression, i.e., preventing the development or further development of clinical symptoms, such as alleviating or completely suppressing active disease. Treatment may 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 a solid tumor, inhibiting the replication of cancer cells, reducing the overall tumor burden, prolonging survival, and improving one or more symptoms associated with cancer.

[0176] Subjects to be treated can be subjects in need of 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. Generally, such subjects are “mammals,” with humans being of interest. 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).

[0177] 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 site 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 targeted portions to the subject to target the surgical cavity and treat the patient with further therapeutic doses (e.g., chemotherapy performed using the disclosed methods), thereby minimizing the risk of any cancer cells remaining at the surgical margin.

[0178] In some embodiments, the targeted portions disclosed herein may be administered, and the practitioner may deliver an appropriate mixture to the desired site 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 targeted portions to the subject to target the surgical cavity and treat the patient with further therapeutic doses (e.g., chemotherapy performed using the disclosed methods), thereby minimizing the risk of any cancer cells remaining at the surgical margins.

[0179] 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 immunomodulator to the biopsy site.

[0180] In some embodiments, the disclosed methods provide practitioners with the ability to deliver immunomodulatory agents, such as TLR agonists, STING agonists, chemokines (agents that attract cancer cells and / or immune cells), and adjuvants, to enhance the immune system and reduce side effects, as well as chemotherapeutic agents in combination with immunotherapeutic agents. This combination approach is beneficial to patients. Chemotherapy drugs can treat solid tumors or specific sites, while the enhanced response to immunotherapy can help treat distant metastatic sites. For example, in some embodiments, the disclosed compositions and methods can be employed or used in conjunction with anthracyclines, taxanes, gemcitabine, and other agents to enhance the efficacy of one or more immunomodulatory agents, such as ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer).

[0181] 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 immortality, 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.

[0182] 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 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, melanoma, and interstitial lung cancer. Skin tumors, oral cancer, multiple myeloma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma, thyroid cancer, trophoblastic tumors, 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).

[0183] In some embodiments, the cancers treatable 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 entropional glioma. In some embodiments, the cancer is a metastatic cancer.

[0184] In some embodiments, the cancers that can be treated by the disclosed methods are hematologic malignancies, such as myelodysplastic syndrome, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal globulinosis, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder (NOS), T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte-predominant Hodgkin lymphoma, and other cancers.

[0185] Unbound by any particular theory, the local release of certain anticancer agents using the compounds and methods disclosed herein can induce or promote immunogenic cell death (ICD). For example, certain anticancer agents (e.g., anthracyclines, cyclophosphamide, oxaliplatin) have been reported to induce ICD. Kroemer et al. Annu. Rev. Immunol. 2013 (31), 51-72. Immunogenic apoptosis of cancer cells can induce a potent antitumor immune response by activating dendritic cells (DCs) and subsequently activating specific T cell responses. ICD is characterized by secretory damage-associated molecular patterns (DAMPs). Calreticulin (CRT) is one of the DAMP molecules, normally located in the endoplasmic reticulum (ER) lumen, which translocates to the surface of dying cells after inducing immunogenic apoptosis, where it acts as a “eat me” signal for specialized phagocytes. Other important surface-exposed DAMPs are heat shock proteins (HSPs), namely HSP70 and HSP90, which also translocate to the plasma membrane under stress conditions. On cell surfaces, they have immunostimulatory effects based on their interactions with many antigen-presenting cell (APC) surface receptors, such as CD91 and CD40, and also promote the cross-presentation of tumor cell-derived antigens on MHC class I molecules, leading to CD8+ T cell responses. Other important DAMPs (characteristics of ICD) are secreted amphipathic protein (HMGB1) and ATP. HMGB1 is considered a marker of late apoptosis, and its release into the extracellular space appears to be essential for the optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) 2 and 4 expressed on APCs. Recently, it has been found that the DAMP released during immunogenic cell death is ATP, which acts as a “find me” signal for monocytes upon secretion and induces them to be attracted to apoptotic sites. Kroemer et al. Curr. Op. Immunol. 2008 (20), 504-511.

[0186] Therefore, the local release of ICD inducers using the compounds and methods of this disclosure can be advantageously combined with one or more immunomodulators.

[0187] In some embodiments, the targeting portion 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), 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.

[0188] The disclosed methods are well-suited as adjuvant / neoadjuvant systems. For example, a targeted portion, as disclosed herein, can be placed during a biopsy, and once the results are available, a physician can administer an appropriate mixture to deliver treatment to the desired site in the body (such as the compounds disclosed herein and one or more 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.

[0189] The disclosed methods can be used to deliver functionalized payloads to these sites via whole-body or local administration. In some embodiments, the target portion is delivered whole-body. In some embodiments, both the target portion and the payload-TCO conjugate are delivered whole-body.

[0190] 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. Targeted portions 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.

[0191] The functionalized payloads disclosed herein can act as adjuvants. This combination approach is beneficial to patients. Chemotherapy agents can treat solid tumors or specific sites and may enhance or elicit an immune response, while the enhanced response of functionalized payloads and / or immunotherapy with a single agent may help treat distant metastatic sites. For example, in some embodiments, the disclosed compositions and methods can be employed or used in conjunction with anthracyclines, regoxetine, vinca alkaloids, taxanes, gemcitabine, camptothecin analogs, and other agents to enhance ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).

[0192] The disclosed method can be used to treat diffuse intrinsic 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.

[0193] 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.

[0194] In some embodiments, the methods disclosed herein include both parenteral administration of the targeted portion and the payload-TCO conjugate. As used herein, the term "parenteral" refers to a mode of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injection and infusion.

[0195] 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.

[0196] The amount of composition administered to a subject can be initially determined based on guidance on the dosage of the parent drug. Typically, the composition can 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 a dosing regimen. Therefore, relative to the parent drug prior to use in the methods of this disclosure, the composition can provide a reduced dose and / or reduced administration in a dosing regimen.

[0197] Pharmaceutical formulations can be supplied 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 tablets, capsules, and powders packaged in sachets, vials, or ampoules.

[0198] In some embodiments, a kit is provided comprising a target portion as described herein or a pharmaceutically acceptable salt thereof, or comprising the aforementioned pharmaceutical composition, along with instructions for use thereof.

[0199] In some embodiments, the kit further comprises a prodrug.

[0200] The compositions disclosed herein may be present in any suitable amount and may depend on a variety of 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.

[0201] The compositions of this disclosure can be 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 min, 5 min, 10 min, 15 min, 20 min, or 30 minutes between each other) or sequentially in any order. Additionally, the additional active agent may be administered once daily, or twice, three times, or more daily to provide the desired daily dose level. Co-administration can be achieved by co-implantation or co-injection.

[0202] 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 the active agent of this disclosure can be formulated separately and co-administered to the subject.

[0203] The compositions and active agents disclosed herein can be present in formulations in 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 can be present in 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.

[0204] Targeted portion This document provides a targeting moiety comprising an antibody fragment portion covalently bonded to one or more tetrazine moieties. The targeting moieties described herein are designed to target a specific site in the subject's body upon administration. The targeting moieties can be administered locally or systemically. In some embodiments, the targeting moieties are therapeutic targeting moieties. Upon administration, a prodrug comprising a complementary bioorthogonal component (i.e., a trans-cyclooctene moiety) may be administered, which, upon contact with the targeting moieties,... in vivo Upon contact, targeted drug delivery of a payload or therapeutic agent is permitted. In some embodiments, the targeted portion described herein comprises a diagnostic agent, such that the targeted portion described herein can be used to diagnose a condition or disease with or without the administration of a payload or therapeutic agent.

[0205] In some embodiments, the antibody fragment portion is selected from the group consisting of: single-chain variable fragments (scFv), divalent or bivalent single-chain variable fragments (di-scFv, bi-scFv), 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.

[0206] In some embodiments, the antibody fragment portion is selected from the group consisting of: Fab2, Fab, scFV, microantibody, biantibody, VHH, V-NAR, or a fragment or polypeptide that targets a tumor by forming a targeting portion (e.g., peptide)-antigen complex.

[0207] In some embodiments, the antibody fragment portion is selected from the group consisting of: single-chain variable fragments (scFv), divalent or bivalent single-chain variable fragments (di-scFv, bi-scFv), antigen-binding fragments (Fab), single-domain antibodies (sdAb), and single-domain antibodies (sdAb).

[0208] 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.

[0209] This article provides the target portion of Equation I, Equation II, or Equation 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 4When 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 an antibody fragment; p ranges from 1 to 20; L is a connector independently each time it appears; R 1 Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, ynyl, 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 group is optionally defined by one to three Z... 1 replace; R 2 Each time it appears independently of halogen, cyano, nitro, hydroxy, 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 defined 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 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 connecting atoms, which optionally contains one or more ethylene-oxy, amine, ester, amide, urethane, 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 groups 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.

[0210] Compounds of formulas I, II, IIA, IIB, IIC, and III: In one embodiment, the targeting portion has Equation I: I in: X represents an antibody fragment; p ranges from 1 to 16; L is a connector independently 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 connecting atoms, which optionally contains one or more ethylene-oxy, amine, ester, amide, urethane, 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.

[0211] In one embodiment, the targeting portion has Formula II: II in: X represents an antibody fragment; p ranges from 1 to 16; L is a connector independently 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 groups 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.

[0212] In one embodiment, R 22 Each time it appears, it is an independent connector of 1 to 100 connecting atoms, and may include ethylene-oxy, amine, ester, amide, carbamate, carbonate and ketone functional groups.

[0213] In one embodiment, the targeting portion has formula IIA: IIA Where L, p, X, and R 20 Each is independent as defined in this article.

[0214] In one embodiment, the targeting portion has Formula IIB: IIB L, p, and X are each defined independently as described in this paper.

[0215] In one embodiment, the targeting portion has formula IIC: IIC L, p, and X are each defined independently as described in this paper.

[0216] In one embodiment, the targeting portion has Formula III: III in: X represents an antibody fragment; p ranges from 1 to 16; L is a connector independently 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 groups 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.

[0217] In some embodiments, the targeting portion has 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.

[0218] In some embodiments, the targeting portion has formula IIE: IIE p and X are each defined independently as described in this paper.

[0219] In some embodiments, the targeting portion has the formula IIF: IIF p and X are each defined independently as described in this paper.

[0220] In some embodiments, the targeting portion has the formula IIF: IIF Where p is 1 to 10; and X is an antibody fragment containing SEQ ID NO. 9 and SEQ ID NO. 10.

[0221] In some embodiments, p is 1 to 5.

[0222] In some embodiments, a method for administering a payload to a subject is provided, the method comprising: a) administering an effective amount of a target portion having formula IIF: IIF Where p is 1 to 10, or p is 1 to 5; and X is an antibody fragment portion comprising SEQ ID NO. 9 and SEQ ID No. 10; and b) administering a single dose of a therapeutically effective amount of the payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto.

[0223] In some embodiments, a method is provided for subjects in need. in vivo A method for forming antibody-load conjugates, the method comprising: Administer an effective amount of the target portion of the formula IIF: IIF Where p is 1 to 10, or p is 1 to 5; and X is an antibody fragment containing SEQ ID NO. 9 and SEQ ID NO. 10; A single dose of a therapeutically effective amount of a payload-TCO conjugate is administered to a subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently linked thereto; The antibody or its fragment has binding affinity to receptors on tumors, and further... in vivo The amount of antibody-payload conjugates formed was greater at the tumor site than in the plasma.

[0224] In some embodiments, the ratio of the antibody-load conjugate at the tumor site to that in the plasma is greater than 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1, or about 11:1, or about 12:1, or about 13:1, or about 14:1, or about 15:1, or about 16:1, or about 17:1, or about 18:1, or about 19:1, or greater than about 2:1, or greater than about 3:1, or greater than about 4:1, or greater than about 5:1, or greater than about 6:1, or greater than about 7:1, or greater than about 8:1, or greater than about 9:1, or greater than about 10:1.

[0225] In some embodiments, the targeting portion has formula IIG: IIG p and X are each defined independently as described in this paper.

[0226] In some embodiments, a compound of formula IIG is provided: IIG Where p is 1 to 5; and X is an antibody fragment portion comprising SEQ ID NO. 9 and SEQ ID NO. 10. In some embodiments, p is 2 to 3.

[0227] In some embodiments of Formula IIA, at least one: for , , , , , , , or .

[0228] In some embodiments of Formula IIA, at least one: for , where R 20 As defined in this article.

[0229] In some embodiments of Formula IIA, at least one: for .

[0230] In some embodiments of Formula IIA, at least one: for , where R 20 As defined in this article.

[0231] In some embodiments of Formula IIA, at least one: for .

[0232] In some embodiments, p is 1 to 12. In some embodiments, X is an antibody. In some embodiments, p is 1 to 6 or 5 to 6. In some embodiments, p is 1 to 16, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In some embodiments, X is an antibody fragment portion (e.g., Fab).

[0233] Compounds of formulas V, VI, and VII: In some embodiments, the targeting portion 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 an antibody fragment; p ranges from 1 to 20; L is a connector independently 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, ynyl, 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 defined by one to three Z 1 replace; R 2 Each time it appears independently of halogen, cyano, nitro, hydroxy, 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 defined 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.

[0234] This article provides the target part of equation 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 an antibody fragment; p ranges from 1 to 20; L is a connector independently each time it appears; R 1Each time it appears, independently select 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 group is optionally defined by one to three Z-terminals. 1 replace; R 2 Each time it appears independently of halogen, cyano, nitro, hydroxy, 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 defined 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 groups 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.

[0235] In some embodiments, the targeting portion 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.

[0236] In some embodiments, R 4 It is hydrogen.

[0237] In the targeted portion described in this article, R 3 This refers to a group that can be removed after the triggering event. In some embodiments, the triggering event occurs... in vivo Once the triggering event occurs and R 3 If removed, the dihydrotetraazine portion is oxidized to provide the tetraazine as in formula VII: VII Where R 1 R 2 Each of rings A, L, p, t, and X is independently as defined herein.

[0238] The triggering event is initiated after the administration of the targeted portion to the subject and can be initiated in any manner, such as internal (e.g., via enzyme cleavage of functional groups, optionally followed by degradation) or external (e.g., photocleavage of the connector). In some embodiments, R 3 It includes a targeting component, such as the antibody fragments described herein.

[0239] In some embodiments, R 3 It contains amino acid sequences that are specific to cleavage by proteases or esterases.

[0240] In some embodiments, R 3 It contains amino acid sequences that are specific to cleavage by proteases as shown in Table 1A.

[0241] Table 1A In some embodiments, R 3 It contains amino acid sequences that are specific for cleavage 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.

[0242] Table 1B ↓ Indicates the cutting site Special amino acid abbreviations: Cit: Citrulline; Cha: β-Cyclohexylalanine; Hof: Homophenylalanine; Nva: Aminooctanoic acid; Dpa: D-phenylalanine; Nle: Leucine; Smc: S-methylcysteine A list of multiple amino acids before, between, or after a slash indicates the substituted amino acids that can be substituted at that position; a "-" indicates that any amino acid can replace the corresponding amino acid shown in the middle column. x is any L-amino acid other than proline. Hy represents any hydrophobic L-amino acid. γ indicates that this bond is a γ-carboxyl group linkage. Other cleavable groups are described in Choi et al., Theranostics. 2012; 2(2): 156–178, in which Table 2 is hereby incorporated by reference.

[0243] 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.

[0244] In some embodiments, R 3 for ; L 5 For direct keys or connectors; and X 1 It is -NO2, optionally substituted sugar moiety, or optionally substituted peptide unit containing one or more natural or non-natural amino acids.

[0245] In some embodiments, a portion: At least one of them is represented by an expression selected from the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or ;where R 1 R 2 R 3 and R 4 Each of them is independent as defined herein, and optionally the ring A portion may be via one or more R... 2 Partial replacement.

[0246] In some embodiments, a portion: At least one of them is represented by an expression selected from the following: , , , and ;where X 2 It is an alkyl group (e.g., methyl) optionally substituted with PEG, amino acid, ester, amide, amine, -C(O)OH, -SO2, -SO3, -PO3, -PO4 or other solubility-enhancing substituents; and L, cyclic A, R 1 R 2 Each of t, p, and X is independently as defined in this document.

[0247] 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.

[0248] 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.

[0249] In some embodiments, ring A is a phenyl group.

[0250] In some embodiments, a portion: At least one of them is represented by an expression selected from the following: , , , , , , , , , , , , , , , , , and ;where R 1 and R 2 Each of them is independent as defined in this article.

[0251] 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 defined by one to three Z-terminals. 1 replace.

[0252] In some embodiments, R 1 Each time it appears, it is independently hydrogen or optionally via one to three Z. 1 Substituted alkyl groups.

[0253] In some embodiments, Z 1 Each time it appears, it is independently selected from halogenated, hydroxylated, alkoxylated, and OC(=O)OR'.

[0254] In some embodiments, R 2Each 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.

[0255] In some embodiments, t is 0 each time it occurs.

[0256] In some embodiments, the targeting portion has the formula VA: VA p and X are each defined independently as described in this paper.

[0257] In some embodiments, the targeting portion has the following formula: VB p and X are each defined independently as described in this paper.

[0258] In some embodiments, ring A is not pyridyl. In some embodiments, ring A is not aryl. In some embodiments, ring A is not phenyl.

[0259] Antibody fragment portion In some embodiments, X is an antibody fragment targeting 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 ...30), 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), CD22 (NCBI gene ID 933), CD20 (NCBI gene ID 934), CD20 (NCBI gene ID 934), CD22 (NCBI gene ID 933), 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 93 952), 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), (NCBI gene ID), antibody fragment (NCBI gene ID), vWF (NCBI gene ID 7450), TNF (NCBI gene ID 7124), IL-6R (NCBI gene ID 3570), BCMA (NCBI gene ID 608), ADAMTS5 (NCBI gene ID 11096), CX3CR1 (NCBI gene ID) 1524), CXCR4 (NCBI gene ID 7852), TfR1 (NCBI gene ID 7037), VEGFR (NCBI gene ID 3791), or PSMA (NCBI gene ID 2346).

[0260] In some embodiments, X is an antibody fragment targeting the following: CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, CD25, CLDN4 (NCBI gene ID 1364), TNC (NCBI gene ID 3371), FN1 (NCBI gene ID 2335), ITGAV (NCBI gene ID 3685), TACSTD2 (NCBI gene ID 4070), CD174 (NCBI gene ID 2525), 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 20531), and CLDN4 (NCBI gene ID 2525). 25800), ROR1 (NCBI gene ID 4919) or ROR2 (NCBI gene ID 4920).

[0261] In some embodiments, X is an antibody fragment targeting 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 ...30), 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), CD22 (NCBI gene ID 933), CD20 (NCBI gene ID 934), CD20 (NCBI gene ID 934), CD22 (NCBI gene ID 933), 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 93 952), 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), (NCBI gene ID), antibody fragment (NCBI gene ID), vWF (NCBI gene ID 7450), TNF (NCBI gene ID 7124), IL-6R (NCBI gene ID 3570), BCMA (NCBI gene ID 608), ADAMTS5 (NCBI gene ID 11096), CX3CR1 (NCBI gene ID) 1524), CXCR4 (NCBI gene ID 7852), TfR1 (NCBI gene ID 7037), VEGFR (NCBI gene ID 3791), PSMA (NCBI gene ID 2346), ANTXR1 (NCBI gene ID 84168), or FAP (NCBI gene ID 2191).

[0262] In some embodiments, X is an antibody fragment that targets the following: CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

[0263] In some embodiments, X is an antibody fragment portion that targets the following: HER2, TROP2, cohesin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC or ECM, EPCAM, CEA or CEACAM5.

[0264] In some embodiments, X is an antibody fragment that targets the following: CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, or CD25.

[0265] In some embodiments, X is an antibody fragment portion targeting CD25, such as daklizumab, RG6292, baliximab, or HuMax-TAC, or an antibody fragment portion derived therefrom.

[0266] In some embodiments, X is an antibody fragment portion that targets CEA, such as labezizumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment portion derived therefrom.

[0267] In some embodiments, X is an antibody fragment portion targeting CEACAM5, such as tascitalumab or CC4, or an antibody fragment portion derived therefrom.

[0268] In some embodiments, X is an antibody fragment portion that targets ASPH, such as PAN-622, or an antibody fragment portion derived therefrom.

[0269] In some embodiments, X is an antibody fragment portion targeting EGFR, such as cetuximab, nimotuzumab, mateuzumab, AMG595, depertuzumab, dapertuzumab, dugoutuzumab, vortuzumab, GC1118, imatrozumab, panitumumab, alutumumab, toltuximab, or latuximab, or an antibody fragment portion derived therefrom.

[0270] In some embodiments, X is an antibody fragment portion targeting EPCAM, such as moozizumab, sitatuzumab, tocotrienumab, caputuzumab, ezetoxumab, or adenomyumab, or an antibody fragment portion derived therefrom.

[0271] In some embodiments, X is an antibody fragment portion that targets VEGFR, such as ramoximab, ramorumab, or volinacizumab, or an antibody fragment portion derived therefrom.

[0272] In some embodiments, X is an antibody fragment portion that targets PDGFR, such as olatuzumab or ramucirumab, or an antibody fragment portion derived therefrom.

[0273] In some embodiments, X is an antibody fragment portion targeting TROP2, such as sacitrus, desmoprozil, or Pr1E11, or an antibody fragment portion derived therefrom.

[0274] In some embodiments, X is an antibody fragment portion targeting cohesin 4, such as entferutumab, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, or 74Hz, or an antibody fragment portion derived therefrom.

[0275] In some embodiments, X is an antibody fragment portion that targets PSMA, such as J591 or MLN591, or an antibody fragment portion derived therefrom.

[0276] In some embodiments, X is an antibody fragment portion that targets BCMA, such as belantuzumab, or an antibody fragment portion derived therefrom.

[0277] In some embodiments, X is an antibody fragment portion targeting CD22, such as mosetumumab, intozumab, epazolizumab, or pinatumumab, or an antibody fragment portion derived therefrom.

[0278] In some embodiments, X is an antibody fragment portion targeting CD20, such as utuximab, ofamumab, rituximab, oxetuzumab, tosimomumab, or tiimumab, or an antibody fragment portion derived therefrom.

[0279] In some embodiments, X is an antibody fragment portion targeting CD19, such as rontoxicumab, XMAB-5574, MOR208, cotoxicumab, dinetuzumab, taritumab, or MDX-1342, or an antibody fragment portion derived therefrom.

[0280] In some embodiments, X is an antibody fragment portion that targets CD79b, such as polacillinumab, or an antibody fragment portion derived therefrom.

[0281] In some embodiments, X is an antibody fragment portion that targets CD38, such as esazotuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment portion derived therefrom.

[0282] In some embodiments, X is an antibody fragment portion that targets CD45, such as I-131-BC8 or Iomab-B, or an antibody fragment portion derived therefrom.

[0283] In some embodiments, X is an antibody fragment portion that targets an endothelial glycoprotein, such as capuximab, or an antibody fragment portion derived therefrom.

[0284] In some embodiments, X is an antibody fragment portion that targets FGFR2, such as bematuzumab or apulutuzumab, or an antibody fragment portion derived therefrom.

[0285] In some embodiments, X is an antibody fragment portion targeting C4.4A, such as rupatumab, or an antibody fragment portion derived therefrom.

[0286] In some embodiments, X is an antibody fragment portion targeting Claudin-18.2, such as zotocinumab or claudinumab, or an antibody fragment portion derived therefrom.

[0287] In some embodiments, X is an antibody fragment portion that targets MMP9, such as adeliximab, or an antibody fragment portion derived therefrom.

[0288] In some embodiments, X is an antibody fragment portion that targets the folate receptor, such as mitoximab, faletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment portion derived therefrom.

[0289] In some embodiments, X is an antibody fragment portion that targets DLL3, such as lovatozumab, or an antibody fragment portion derived therefrom.

[0290] In some embodiments, X is an antibody fragment portion that targets CD138, such as indextrin, or an antibody fragment portion derived therefrom.

[0291] In some embodiments, X is an antibody fragment portion that targets CD56, such as lovotozumab, propimumab, or an antibody fragment portion derived therefrom.

[0292] In some embodiments, X is an antibody fragment portion that targets CD37, such as BI 836826, olletuzumab, or naltuximab, or an antibody fragment portion derived therefrom.

[0293] In some embodiments, X is an antibody fragment portion that targets CD74, such as mirtizumab, or an antibody fragment portion derived therefrom.

[0294] In some embodiments, X is an antibody fragment portion that targets mesothelin, such as annatuzumab, amatotuzumab, or MMOT-0530A, or an antibody fragment portion derived therefrom.

[0295] In some embodiments, X is an antibody fragment portion targeting IL-6R, such as tocilizumab or thalidomide, or an antibody fragment portion derived therefrom.

[0296] In some embodiments, X is an antibody fragment portion that targets SLAMF7, such as erlotinumab, or an antibody fragment portion derived therefrom.

[0297] In some embodiments, X is an antibody fragment portion that targets BAFF, such as belimumab, or an antibody fragment portion derived therefrom.

[0298] In some embodiments, X is an antibody fragment portion targeting MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, cristatumab, 8HuDS6, gatutuzumab, AR20.5, or cantutuzumab, or an antibody fragment portion derived therefrom.

[0299] In some embodiments, X is an antibody fragment portion that targets GPC3, such as cotrastuzumab, ECT204, or MDX-1414, or an antibody fragment portion derived therefrom.

[0300] In some embodiments, X is an antibody fragment portion that targets HER2, such as pertuzumab, trastuzumab, or magtuximab, or an antibody fragment portion derived therefrom.

[0301] In some embodiments, X is an antibody fragment portion targeting HER3, such as pertratuzumab, seretuzumab, rutozumab, edentulumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment portion derived therefrom.

[0302] In some embodiments, X is an antibody fragment portion that targets CD30, such as brentuximab, or an antibody fragment portion derived therefrom.

[0303] In some embodiments, X is an antibody fragment portion targeting CD33, such as gemutuzumab, BI 835858, varatustabolumab, or lintuzumab, or an antibody fragment portion derived therefrom.

[0304] In some embodiments, X is an antibody fragment portion targeting CD123, such as KHK2823, tacrolimus, or G4723A, or an antibody fragment portion derived therefrom.

[0305] In some embodiments, X is an antibody fragment portion that targets GPNMB, such as grabartimumab, or an antibody fragment portion derived therefrom.

[0306] In some embodiments, X is an antibody fragment portion that targets cMET, such as terituzumab, onatuzumab, or SAIT301, or an antibody fragment portion derived therefrom.

[0307] In some embodiments, X is an antibody fragment portion that targets CD142, such as tesutumab, or an antibody fragment portion derived therefrom.

[0308] In some embodiments, X is an antibody fragment portion that targets NaPi2B, such as lifatuzumab, or an antibody fragment portion derived therefrom.

[0309] In some embodiments, X is an antibody fragment portion that targets GCC, such as indulitumarab, or an antibody fragment portion derived therefrom.

[0310] In some embodiments, X is an antibody fragment portion that targets STEAP1, such as vandotusumab, or an antibody fragment portion derived therefrom.

[0311] In some embodiments, X is an antibody fragment portion that targets MUC16, such as sofostouzumab, or an antibody fragment portion derived therefrom.

[0312] In some embodiments, X is an antibody fragment portion that targets CD70, such as vortexumab, or an antibody fragment portion derived therefrom.

[0313] In some embodiments, X is an antibody fragment portion that targets CD44, such as bivalizumab, or an antibody fragment portion derived therefrom.

[0314] In some embodiments, X is an antibody fragment portion that targets vWF, such as caralacimab, or an antibody fragment portion derived therefrom.

[0315] In some embodiments, X is an antibody fragment portion that targets TNF, such as orizizumab, V565, or PF-05230905, or an antibody fragment portion derived therefrom.

[0316] In some embodiments, X is an antibody fragment portion that targets IL-6R, such as vobalizumab, or an antibody fragment portion derived therefrom.

[0317] In some embodiments, X is an antibody fragment portion that targets BCMA, such as LCAR-B38M, or an antibody fragment portion derived therefrom.

[0318] In some embodiments, X is an antibody fragment portion targeting ADAMTS5, such as M6495, or an antibody fragment portion derived therefrom.

[0319] In some embodiments, X is an antibody fragment portion targeting CX3CR1, such as BI 655088, or an antibody fragment portion derived therefrom.

[0320] In some embodiments, X is an antibody fragment portion targeting CXCR4, such as AD-214 or ALX-0651, or an antibody fragment portion derived therefrom.

[0321] In some embodiments, X is an antibody fragment portion that targets TfR1, such as TXB4, or an antibody fragment portion derived therefrom.

[0322] In some embodiments, X is an antibody fragment portion that targets VEGFR, such as CDP791, or an antibody fragment portion derived therefrom.

[0323] In some embodiments, X is an antibody fragment portion that targets PSMA, such as GY1, or an antibody fragment portion derived therefrom.

[0324] In some embodiments, X is an antibody fragment portion targeting FN1, such as L19 or NJB2, or an antibody fragment portion derived therefrom.

[0325] In some embodiments, X is an antibody fragment portion that targets FAP, such as F19, OMTX005, or siroizumab, or an antibody fragment portion derived therefrom.

[0326] In some embodiments, X is an antibody fragment portion targeting TNC, such as F16 or R6N, or an antibody fragment portion derived therefrom.

[0327] In some embodiments, the antibody fragment portion is derived from (e.g., containing at least V) H and V LThe following antibody fragments (Fab) are available: Daklizumab, RG6292, Baliximab, HuMax-TAC, Labezizumab, 15-1-32, PR1A3, cT84.66, Tascitalumab, CC4, PAN-622, Cetuximab, Resistuximab, Nimotuzumab, Matozumab, AMG595, Depertuximab, Dapertuximab, Dugoutuzumab. Monoclonal antibodies, vortoxicum, GC1118, imatroxicum, panitumum, alutusumab, toltuxumab, latuxumab, moozumab, sitatuxumab, tocozozumab, caputuzumab, ezetoxumab, adenomyum, ramoxumab, ramorumab, velinaxicum, olatoxicum, ramorumab, saxitoxicum, Pr1E11, envertuzumab, J591, MLN591 Belantoumab, Mosetumab, Intozumab, Iprazumab, Pinatumab, Utoximab, Ofamumab, Rituximab, Obinutumab, Tosimomab, Tiimumab, Rantuximab, XMAB-5574, MOR208, Cortuximab, Dinituximab, Tarituximab, MDX-1342, Polazumab, Isaltuximab, Daremumab, MOR 202, TAK-079, I-131-BC8, Iomab-B, Caputuximab, Bematocilizumab, Apulutuximab, Lupatuximab, Zolotocilizumab, 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, Trastuzumab, ECT204, MDX-1414, Pertuzumab, Trastuzumab, Magutuximab, Pertrastuzumab, Seretuzumab, Lutuzumab, Eganutuzumab, AV-203, CDX-3379, GSK284933, Brentuximab, Gelatuzumab, BI 835858, Vardatocizumab, Lintocizumab, KHK2823, Tacrolimus, G4723A, Gabatocizumab, Teritolus, Onatuzumab, SAIT301, Tesxotuzumab, Lifatocizumab, Indojutumab, Vantocizumab, Sofostocizumab, Voseltuzumab, Bivaliruzumab, Karacizumab, Orizizumab, V565, PF-05230905, Wabalizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, Sirolizumab, F16, R6N, Datopotamab, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, 74Hz.

[0328] In some embodiments, X is an antibody selected from the following: atuzumab, averumumab, bevacizumab, simipremab, cetuximab, daratumumab, denutoximab, durvalumab, erlotuzumab, ipilimumab, esatuzumab, moglizamab, nexituzumab, nivolumab, oxinutuzumab, ofamumab, olatoximab, panitumumab, pembrolizumab, pertuzumab, ramorumab, rituximab, and trastuzumab.

[0329] In some embodiments, X or the 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.

[0330] In some embodiments, trastuzumab, entaftuzumab, brentuximab, saxituzumab, L19 Fab binds to FN-1 (gene ID 2335); or F16– binds to TNC (gene ID 3371).

[0331] In some embodiments, the antibody fragment portion includes one or more PEG units, which can extend cycle life.

[0332] 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.

[0333] 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.

[0334] 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.).

[0335] 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, Onatolumab, SAIT301, Texotuzumab, Lifatuzumab, Indotumumab, Vantotuzumab, Sofostotuzumab, Voseltuzumab, Bivalizumab, Karacizumab, Orizilumab, V565, PF-05230905, Voebalizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, Sirolizumab, F16, R6N, Dapoxetine, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, 74HZ.

[0336] 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.

[0337] In some embodiments, X is an antibody fragment portion that targets vWF, such as caralacimab.

[0338] In some embodiments, X is an antibody fragment that targets TNF, such as orizizumab, V565, or PF-05230905.

[0339] In some embodiments, X is a fragment of an antibody that targets IL-6R, such as vobalizumab.

[0340] In some embodiments, X is an antibody fragment portion targeting BCMA, such as LCAR-B38M.

[0341] In some embodiments, X is an antibody fragment portion targeting ADAMTS5, such as M6495.

[0342] In some embodiments, X is an antibody fragment portion targeting CX3CR1, such as BI 655088.

[0343] In some embodiments, X is an antibody fragment portion targeting CXCR4, such as AD-214 or ALX-0651.

[0344] In some embodiments, X is an antibody fragment portion targeting TfR1, such as TXB4.

[0345] In some embodiments, X is an antibody fragment portion targeting VEGFR, such as CDP791.

[0346] In some embodiments, X is an antibody fragment portion that targets PSMA, such as GY1.

[0347] 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.

[0348] In some embodiments, X further comprises an imaging developer. In some embodiments, the imaging developer is a protein.

[0349] Connector section In some embodiments, L is bonded to X via a cystine or lysine residue on X.

[0350] In some embodiments, L is a non-breakable connector.

[0351] In some embodiments, L is a detachable connector.

[0352] In some embodiments, L comprises one or more amino acids.

[0353] In some embodiments, L comprises a polypeptide.

[0354] In some embodiments, L 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.

[0355] In some embodiments, L 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.

[0356] In some embodiments, L comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkyneene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkyl moieties; wherein each alkylene, alkenylene, alkyneene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkyl moieties may be independently and optionally substituted by one to five substituents independently selected from: oxo, halogenated, C-substituted. 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups.

[0357] In some embodiments, L is an alkylene linker that optionally includes one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thiosuccinimide, or ketone functional groups.

[0358] In some embodiments, the connector L 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 independently a 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 alkylene, alkenylene, ynylene, arylene, or heteroarylene is independently and optionally substituted by one to five independent substituents selected from: 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.

[0359] In some embodiments, L 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 independently a key, -NR 110 -、-O-、-S(O) 0-2 -、-NR 110 C(O)-、-C(O)NR 110 -、-NR110 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 alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkylene is independently and optionally substituted by one to five independent substituents selected from: 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.

[0360] In some embodiments, each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, 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 connector is not a bond.

[0361] The connector L may contain 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.

[0362] In some embodiments, the connector includes one or more of the following: , , , , , or .

[0363] In some embodiments, the connector includes one or more of the following: , , , , , , or .

[0364] In some embodiments, the connector includes one or more of the following: , , , , or .

[0365] In some embodiments, the connector comprises one or more .

[0366] In some embodiments, the connector comprises one or more .

[0367] In some embodiments, the connector is or includes one or more of the following: , , , , , , , , , , , , , , , or .

[0368] In some embodiments, the connector is or includes one or more of the following: , , , , or .

[0369] In some embodiments, the linker 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 constituting a protein or peptide sequence. The linker may also contain a self-sacrificing spacer that separates the drug from the protein peptide sequence.

[0370] In some embodiments, the connector may be a connector containing “A—Y—Z—X”. 2 —W” indicates the linker peptide, where “A” is a carboxyl acyl unit, “Y” and “Z” are each one or more natural or non-natural amino acids that together form the peptide sequence, and “X” is a linker 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, which separate the peptide from the payload, D, or bioorthogonal portion. In some embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0371] 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.

[0372] 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.

[0373] Exemplary YZ combinations include valine-citrulline; valine-alanine; and alanine-alanine.

[0374] In some embodiments, A is -OC(O)-.

[0375] In some embodiments, X 2 It is -OC(O)-.

[0376] In some embodiments, W is -OC(O)-. In some embodiments, X 2 It does not exist and W is -OC(O)-.

[0377] In some embodiments, part —X 2 —W includes In some embodiments, part —X 2 for .

[0378] In some embodiments, —X—W is .

[0379] In some embodiments, —X—W is .

[0380] 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.

[0381] 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.

[0382] 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.

[0383] In some embodiments, connector L is or includes one or more of the following: , , , , , , , , , , , , , , , , , , , , , , , , or .

[0384] In some embodiments, connector L includes one or more of the following: (For example, ), (For example, ), , , , , , , , , or .

[0385] In some embodiments, connector L includes one or more of the following: (For example, ), , , , , , , , , or .

[0386] The aforementioned linker can bond to the side chain of an amino acid present on X, such as lysine or cysteine ​​(e.g., , , , , , ).

[0387] In some embodiments, connector L 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–C1-4 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 independently selected substituents from the group consisting of: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, cyano, and nitro groups.

[0388] In some embodiments, the connector L comprises a carbonyl moiety conjugated to the connector or X. For example, the connector may comprise a polypeptide moiety (PPM) having lysine residues and lysine side chains, and the PPM may also have additional lysine or other amino acid side chains conjugated to the carbonyl moiety. In some embodiments, the connector L may include .

[0389] In some embodiments, connector L is or includes one or more of the following: , , , , , , , or .

[0390] In some embodiments, connector L is or includes one or more of the following: , , or .

[0391] In some embodiments, connector L is or includes one or more of the following: , , , , , or .

[0392] In some embodiments, connector L is or includes one or more of the following: , , or .

[0393] In some embodiments, connector L is or includes one or more of the following: , , , , , , , , , , or .

[0394] In some embodiments, connector L is or includes one or more of the following: .

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

[0396] In some embodiments, connector L is or includes one or more of the following: .

[0397] In some embodiments, connector L is or includes one or more of the following: .

[0398] In some embodiments, connector L is or includes one or more of the following: .

[0399] In some embodiments, connector L is or includes one or more of the following: .

[0400] In some embodiments, connector L is or includes one or more of the following: .

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

[0402] In some embodiments, connector L is or includes one or more of the following: .

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

[0404] In one embodiment, the targeting portion has Equation I: I in: p ranges from 1 to 16; 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 connecting atoms, which optionally contains one or more ethylene-oxy, amine, ester, amide, urethane, carbonate or ketone functional groups; 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; X represents an antibody fragment targeting the following: HER2, TROP2, cohesin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC or ECM, EPCAM, CEA or CEACAM5; and L independently selects a connector from the group consisting of the following items each time it appears: , , , , , , , , , , and .

[0405] In one embodiment, the targeting portion has Formula II: II in: X represents an antibody fragment; p ranges from 1 to 16; L is a connector independently 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 30Each 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; t is independently 0, 1, 2, 3 or 4 each time it appears; X represents an antibody fragment targeting the following: HER2, TROP2, cohesin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC or ECM, EPCAM, CEA or CEACAM5; and L independently selects a connector from the group consisting of the following items each time it appears: , , , , , , , , , , and .

[0406] In one embodiment, the targeting portion 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 an antibody fragment; p ranges from 1 to 16; L is a connector independently each time it appears; R 1Each time it appears, independently select 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 group is optionally defined by one to three Z-terminals. 1 replace; R 2 Each time it appears independently of halogen, cyano, nitro, hydroxy, 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 defined 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; t is independently 0, 1, 2, 3 or 4 each time it appears; X represents an antibody fragment targeting the following: HER2, TROP2, cohesin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC or ECM, EPCAM, CEA or CEACAM5; and L independently selects a connector from the group consisting of the following items each time it appears: , , , , , , , , , , and .

[0407] 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; wherein each may optionally be substituted.

[0408] In some embodiments, ring A is a phenyl group.

[0409] In some embodiments, X is an antibody fragment portion that targets HER2, TROP2, cohesin-4, FN1, FAP, TNC, or ECM.

[0410] In some embodiments, X is zobetuximab, clodiximab, adicaliximab, arnetumab, amatoximab, MMOT-0530A, L19, NJB2, F19, OMTX005, sibutruzumab, F16 or R6N, trastuzumab, enflutuzumab or sacitrullumab, datoporta, or an antibody fragment derived therefrom.

[0411] In some embodiments, X is an antibody fragment derived from L19, NJB2, F19, OMTX005, sibutrazole, F16 or R6N, trastuzumab, entferutuzumab or sacitrus.

[0412] In some embodiments, p is 1 to 12. In some embodiments, p is 1 to 6 or 5 to 6.

[0413] In some embodiments, p is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10.

[0414] In some embodiments, p is 1 to 16, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2.

[0415] In some embodiments, p is 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, and X is 15. Antibody fragments ranging from kDa to 55 kDa, or from 25 kDa to 55 kDa, or from 15 kDa to 50 kDa, or less than 25 kDa, or less than 35 kDa, or less than 45 kDa, or about 50 kDa, or less than 55 kDa.

[0416] In some embodiments, p depends on the size and / or number of available binding sites on X for forming a covalent bond with L. In some embodiments, p is 1 to 4 when X is an antibody fragment portion between 25 kDa and 55 kDa. In some embodiments, p is 1 to 4 when X is an antibody fragment portion between 45 kDa and 55 kDa. In some embodiments, p is 1 to 3, or 2 to 3, or 1 to 2, or about 1, about 2, or about 3 when X is an antibody fragment portion less than 45 kDa. In some embodiments, p is 1 to 3, or 2 to 3, or 1 to 2, or about 1, about 2, or about 3 when X is an antibody fragment portion less than 25 kDa.

[0417] C. Payload-TCO conjugate Trans-cyclooctene functionalized prodrugs (load-TCO conjugates) are known in the art, including prodrugs of anticancer agents such as those described in WO2018 / 187740, WO2014 / 205126, WO2015 / 139025 and WO2017 / 044983 (these contents are incorporated herein by reference) and / or chelating agents (with or without therapeutic or diagnostic radioligands). Further exemplary embodiments are described below.

[0418] In some embodiments, the payload-TCO conjugate is a conjugate consisting of a payload attached to one or more trans-cyclooctene moieties.

[0419] In some embodiments, the payload-TCO conjugate (or trans-cyclooctene functionalized prodrug) comprises an immunomodulatory payload, such as, for example, an immunomodulatory payload selected from the group consisting of cytokines, chemokines, chemokine antagonists, therapeutic monoclonal antibodies, and immune checkpoint inhibitor payloads; or a pharmaceutically acceptable salt thereof.

[0420] In some embodiments, the immunomodulatory payload is an inhibitor of the cytokine payload or a pharmaceutically acceptable salt thereof.

[0421] In some embodiments, the inhibitor of the cytokine payload is an inhibitor of TNF-α, infliximab, sertolizumab, TGF-β, galunisertib, non-hematoxylin and tadalafil, M7824, CSF-1, pidatinib, or carbillizumab.

[0422] In some embodiments, the payload-TCO conjugate comprises a monoclonal antibody or a pharmaceutically acceptable salt thereof.

[0423] In some embodiments, the payload-TCO conjugate comprises a therapeutic protein payload or a pharmaceutically acceptable salt thereof.

[0424] In some embodiments, the therapeutic protein payload is an antibody-based drug, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, a growth factor, a hormone, an interferon, an interleukin, or a thrombolytic agent.

[0425] In some embodiments, the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen.

[0426] In some embodiments, the therapeutic protein payload is the payload of erythropoietin (EPO, such as natural EPO or synthetic EPO (see, for example, US 2003 / 0191291), such as, but not limited to, PROCRIT®, EPREX®, or EPOGEN® (eportine-α), ARANESP® (dabepoetine-α), NEORECORMON®, EPOGIN® (eportine-β), etc.); growth hormone (e.g., growth hormones such as GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®, S... EROSTIM®, HUMATROPE®, etc.; therapeutic monoclonal antibodies (e.g., atezolizumab, avelumumab, bevacizumab, cimiprimab, cetuximab, daratumumab, dartuximab, duvalumab, erlotuximab, ipilimumab, esatuximab, moglizumab, nexituzumab, nivolumab, oxalitumumab, ofamumumab, olatumumab, panitumumab, pembrolizumab, pertuzumab, ramorumab, etc.). Rituximab, trastuzumab, etc.); Human growth hormone (hGH); Bovine growth hormone (bGH); Follicle-stimulating hormone (FSH); Interferons (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ, compound interferons, etc.); Insulin (e.g., NovoMed, Humulin, Humalog, Lantus, long-acting insulin, etc.); Insulin-like growth factor (e.g., IGF-I, IGF-II); Blood factors (e.g., Factor X, tissue plasminogen activator (TPA), such as but not limited to ACTIVASE® (alteplase) tissue plasminogen activator, NOVOSEVEN® (recombinant human factor VIIa), factor VIIa, factor VIII (e.g., KOGENATE®), factor IX, β-globin, hemoglobin, etc.); Colony-stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g., NEUPOGEN® (filgrastim)), macrophage-CSF). (M-CSF), granulocyte-macrophage-CSF (GM-CSF), nelamivir (pegylated filgrastim), granulocyte-monocyte colony-stimulating factor, megakaryocyte colony-stimulating factor, etc.), transforming growth factor (e.g., TGF-β, TGF-α); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®), IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IE-12, etc.);Growth factors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF, such as REGRANEX® (beclapermin)), fibroblast growth factor (FGF, such as aFGF, bFGF, such as FIB LAST® (trafermin)), glial cell line-derived growth factor (GDNF), nerve growth factor (NGF), stem cell factors (e.g., STEMGEN® (ancestim)), keratinocyte growth factor, hepatocyte growth factor, etc.); soluble receptors (e.g., TNF-α binding soluble receptors such as ENBREL® (etanercept), soluble VEGF receptors, soluble interleukin receptors, soluble γ / δ receptors). T-cell receptors, etc.; enzymes (e.g., α-glucosidase, CERAZYME® (imiglucosidase), β-glucocerebrosidase, CEREDASE® (aragidase)); enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10, Mig, Groα / IL-8, regulated and normal T-cell expression and secretion (RANTES), MIP-1α, MIP-1ρ, MCP-1, PF-4, etc.); angiogenic agents (e.g., vascular endothelial growth factor (VEGF)); anti-angiogenic agents (e.g., soluble VEGF receptor); protein vaccines; neuroactive peptides, such as bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, glycopeptide, growth hormone-releasing hormone, bufotenide, warfarin, dynorphin. Neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, etc.; other proteins, such as thrombolytic agents, atrial natriuretic peptide, bone morphogenetic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human α-1 antitrypsin, leukemia inhibitory factor, transforming growth factor, tissue factor, insulin-like growth factor, luteinizing hormone, follicle-stimulating hormone, macrophage activating factor, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, tissue inhibitors of metalloproteinases; vasoactive intestinal peptide, angiopoietin, opsonin, fibrin; hirudin; leukemia inhibitory factor; or IL-1 receptor antagonists (e.g., Kinetet®); etc.

[0427] In some embodiments, the payload-TCO conjugate has formula VIII or a pharmaceutically acceptable salt thereof: VIII in m is an integer from 1 to 150; G is independently, each time it appears, the optionally substituted trans-cyclooctene moiety; D represents the payload; L 1 Each time it appears, it is a separate connector.

[0428] In some embodiments of the payload-TCO conjugates described herein, each trans-cyclooctene moiety is independently: in: R 1A Choose C independently each time it appears. 1-4 Alkyl, C 1-4 Halogenated alkyl groups and C 1-4 The group consisting of alkoxy groups; 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 an optionally substituted heterocyclic group.

[0429] In some embodiments, the payload-TCO conjugate has formula VIII or a pharmaceutically acceptable salt thereof. VIII in G is the trans-cyclooctene moiety, and G is independently present each time it appears. ; L 1 Each time it appears, it is a connector independently; m is an integer from 1 to 150; D represents the payload; R 1A Choose C independently each time it appears. 1-4 Alkyl, C 1-4Halogenated alkyl groups and C 1-4 The group consisting of alkoxy groups; 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 an optionally substituted heterocyclic group.

[0430] In some embodiments, q1 is 1.

[0431] In some embodiments, the payload is an immunomodulatory payload.

[0432] In some embodiments, the payload is a therapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, or immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.

[0433] In some embodiments, the payload is selected from therapeutic agents used to treat cancer (e.g., doxorubicin, danomycin, PNU-159682, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, berry gibberellin III, gemcitabine, podophyllotoxin, carmustine, ixaprilone, partopilon (epotassium styrax), platinum-based drugs, ixotecan, reoxetine (saurus 10, MMAE, MMAD, MMAF), pyrrocin, pyrrolobenzodiazepine dimer, mitomycin C, bleomycin, carrichomycin, asteroidin, succinate). Antifungal agents (e.g., cyclosporine A, rapamycin, etc.), immunosuppressants (e.g., cyclosporine A, rapamycin, etc.), antifungal agents (e.g., amphotericin B, etc.), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, etc.), matrix metalloproteinase (MMP) inhibitors, L-DOPA, oseltamivir, cephalexin, 5-aminopyruvic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and c-AMP.

[0434] In some embodiments, the payload is selected from therapeutic agents used to treat cancer (e.g., paclitaxel, doxorubicin, danomycin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixaprilone, partopirone (epotassium-like drugs), platinum-based drugs, ixotecan, reoxetine (saurus 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, carrichomycin, astrospore, hexasporin, etc.), and immunosuppressants (e.g., cyclosporine A, rapamycin). Antifungal agents (e.g., amphotericin B), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, etc.), rubitidine, gademolide, matrix metalloproteinase (MMP) inhibitors, L-DOPA, oseltamivir, cephalexin, 5-aminopyruvic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and c-AMP.

[0435] Reference payload refers to one or more atoms (including hydrogen or non-hydrogen atoms) of the original unmodified payload that have been covalently replaced with one or more linkers. The payload is derived from a known nuclear payload and modified to be covalently bonded via linkers to at least one optionally substituted trans-cyclooctene. Even after modification to obtain the compounds described herein, the payload retains biological activity comparable to that observed in the original unmodified payload. In some embodiments, the payload exhibits binding activity or inhibition at at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the activity observed in the original unmodified payload.

[0436] In some embodiments, hydrogen atoms bonded to heteroatoms (e.g., N, O, or S) of the original unmodified payload are replaced by covalent bonds with the connector. In some embodiments, halogen atoms on the payload are replaced to attach to the remainder of the compound. In some embodiments, hydrogen atoms on the payload are replaced to attach to the remainder of the compound. In some embodiments, hydrogen atoms are located on heteroatoms. In some embodiments, hydrogen atoms are located on nitrogen. In some embodiments, hydrogen atoms are located on oxygen. In some embodiments, hydrogen atoms are located on carbon.

[0437] In some embodiments, G is independently defined for each occurrence. .

[0438] In some embodiments, G is independently defined for each occurrence. .

[0439] In some embodiments, the payload is a monoclonal antibody payload. The monoclonal antibody used as the payload herein can be a complete monoclonal antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In some embodiments, the antibody is an immune cell conjugate and thus induces or triggers an immune response. In some embodiments, the monoclonal antibody or fragment thereof targets one or more of the following: CD3 (NCBI gene ID 916), CD28 (NCBI gene ID 940), CD137 (4-1BB) (NCBI gene ID 3604), CD16 (NCBI gene ID 2214), NKG2D (NCBI gene ID 22914), CD64 (NCBI gene ID 2209), GITR / TNFRSF18 (NCBI gene ID 8487), CD25 (NCBI gene ID 3559), CD40 (NCBI gene ID 958), CD4 (NCBI gene ID 920), CXCR4 (NCBI gene ID 7852), G-CSFR (NCBI gene ID 1441), GM-CSFR (NCBI gene ID 1438), CD122 (NCBI gene ID 3560), PD1 (NCBI gene ID 5133), CTLA4 (NCBI gene ID 5133), and CD25 (NCBI gene ID 916). 1493), LAG3 (NCBI gene ID 3902), TIGIT (NCBI gene ID 201633), NCR1 (NCBI gene ID 9437), TIM3 (NCBI gene ID 84868), VISTA (NCBI gene ID 64115), CD134 (NCBI gene ID 7293), CD27 (NCBI gene ID 939), CD40L (NCBI gene ID 959), ICOS (NCBI gene ID 29851), BAFFR (NCBI gene ID 115650), LFA-1 (NCBI gene ID 3689), or BTLA (NCBI gene ID 151888).

[0440] In some embodiments, the payload is an antibody or antibody fragment targeting CD3, such as OKT3, SP34, UCHT1, terplezumab, octexizumab, vesilizumab, or frerumab, or an antibody fragment derived therefrom.

[0441] In some embodiments, the payload is an antibody or antibody fragment targeting CD28, such as cilalizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.

[0442] In some embodiments, the payload is an antibody or antibody fragment targeting CD137 (4-1BB), such as utolumab, urerucumab, LVGN6051 or AGEN2373, or antibody fragments derived therefrom.

[0443] In some embodiments, the payload is an antibody or antibody fragment targeting CD16, such as AFM13, or an antibody fragment derived therefrom.

[0444] In some embodiments, the payload is an antibody or antibody fragment targeting NKG2D, such as NNC0152-0002 or JNJ-64304500, or an antibody fragment derived therefrom.

[0445] In some embodiments, the payload is an antibody or antibody fragment targeting CD64, such as H22, or an antibody fragment derived therefrom.

[0446] In some embodiments, the payload is an antibody or antibody fragment targeting GITR / TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.

[0447] In some embodiments, the payload is an antibody or antibody fragment targeting CD25, such as daklizumab, RG6292, baliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0448] In some embodiments, the payload is an antibody or antibody fragment targeting CD40, such as icalimab, ABBV-323, brelelumab (ASKP-1240), BI-655064, FFP-104, BMS986090, dasastatumab, or rucastatumab, or antibody fragments derived therefrom.

[0449] In some embodiments, the payload is an antibody or antibody fragment targeting CD4, such as MAX.16H5, IT1208, zhamumab (HuMax-CD4), UB-421, or MTRX1011A, or an antibody fragment derived therefrom.

[0450] In some embodiments, the payload is an antibody or antibody fragment targeting CXCR4, such as F50067, or an antibody fragment derived therefrom.

[0451] In some embodiments, the payload is an antibody or antibody fragment targeting G-CSFR, such as CSL324, or an antibody fragment derived therefrom.

[0452] In some embodiments, the payload is an antibody or antibody fragment targeting GM-CSFR, such as macfulimumab, or an antibody fragment derived therefrom.

[0453] In some embodiments, the payload is an antibody or antibody fragment targeting CD122, such as Hu-Mik(β)1, or an antibody fragment derived therefrom.

[0454] In some embodiments, the payload is an antibody or antibody fragment targeting PD-1, such as CC-90006, cimiprimab, camrelizumab, or TSR-042, or an antibody fragment derived therefrom.

[0455] In some embodiments, the payload is an antibody or antibody fragment targeting CTLA4, such as trimemumab or ipilimumab, or an antibody fragment derived therefrom.

[0456] In some embodiments, the payload is an antibody or antibody fragment targeting LAG3, such as renalalimab (BMS-986016), GSK2831781, cimiprimab (REGN3767), finazelimab, eralalimab, or malazelimab, or an antibody fragment derived therefrom.

[0457] In some embodiments, the payload is an antibody or antibody fragment targeting TIGIT, such as BMS-986207, tireliumab, vimbrolizumab, atelimab, dovanalimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or antibody fragments derived therefrom.

[0458] In some embodiments, the payload is an antibody or antibody fragment targeting NCR1, such as hNKp46.02, or an antibody fragment derived therefrom.

[0459] In some embodiments, the payload is an antibody or antibody fragment targeting TIM3, such as cobberalumab, Sym023, LY3321367, BMS-986258, SHR-1702, sabatolimab, or INCAGN02390, or an antibody fragment derived therefrom.

[0460] In some embodiments, the payload is an antibody or antibody fragment targeting VISTA, such as SG7, K01401-020, CI-8993 or JNJ-61610588, or an antibody fragment derived therefrom.

[0461] In some embodiments, the payload is an antibody or antibody fragment targeting CD134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.

[0462] In some embodiments, the payload is an antibody or antibody fragment targeting CD27, such as varigramab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.

[0463] In some embodiments, the payload is an antibody or antibody fragment targeting CD40L, such as dapilizumab, or an antibody fragment derived therefrom.

[0464] In some embodiments, the payload is an antibody or antibody fragment targeting ICOS, such as MEDI-570, KY1044, JTX-2011 or GSK3359609, or an antibody fragment derived therefrom.

[0465] In some embodiments, the payload is an antibody or antibody fragment targeting BAFFR, such as inarubicin, or an antibody fragment derived therefrom.

[0466] In some embodiments, the payload is an antibody or antibody fragment targeting LFA-1, such as fazlinumab, or an antibody fragment derived therefrom.

[0467] In some embodiments, the payload is an antibody or antibody fragment targeting BTLA, such as icatolimab, or an antibody fragment derived therefrom.

[0468] In some embodiments, the payload is an anti-CD3 (αCD3) monoclonal antibody or a derivative or analogue thereof. In some embodiments, the anti-CD3 (αCD3) monoclonal antibody is SP34, UCHT1, or OKT3, or a derivative or analogue thereof.

[0469] In some embodiments, at least one payload is selected from inhibitors of poly(ADP-ribose) polymerase (PARP), pyroxine, pyrrolobenzodiazepine (PBD), sclerosporin, HTI-286, anti-CD3 (αCD3) monoclonal antibody, rubitidine, MSA-2, gadequinoline, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, eczemab, and MMAE, or derivatives or analogs thereof.

[0470] In some embodiments, D is a payload selected from the following: an inhibitor of poly(ADP-ribose) polymerase (PARP), pyroximide, pyrrolobenzodiazepine (PBD), hexazosporin, HTI-286 and anti-CD3 (αCD3) monoclonal antibody, or derivatives or analogs thereof.

[0471] In some embodiments, at least one payload is selected from rubitidine, MSA-2, gadequinoline, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, eczemab, seco-carcinomacin SA and MMAE, or derivatives or analogs thereof.

[0472] In some embodiments, the payload is an inhibitor of poly(ADP-ribose) polymerase (PARP), or a derivative or analog thereof. In some embodiments, the poly(ADP-ribose) polymerase inhibitor (PARP inhibitor) is niraparib, taprazolepanib, olaparib, pamipanib, rucaparib, veliparib, inipanib, 3-aminobenzamide, CEP-9722, E7016, or a derivative or analog thereof.

[0473] In some embodiments, the payload is: , , , , , , , , , , , or .

[0474] In some embodiments, the payload is pyruvicin, or a derivative or analogue thereof. In some embodiments, pyruvicin is pyruvicin A, pyruvicin B1, pyruvicin B2, pyruvicin C1, pyruvicin C2, pyruvicin D, pyruvicin SA, CC-1065, adolacin, calcecin, pyruvicin, or a derivative or analogue thereof.

[0475] In some embodiments, the payload is: , , , , , , , , , , , or .

[0476] In some embodiments, the payload is pyrrolobenzodiazepine (PBD), or a derivative or analogue thereof. In some embodiments, pyrrolobenzodiazepine (PBD) is [1,2]diazazo[3,4-e]indole, or a derivative or analogue thereof.

[0477] In some embodiments, the payload is: , , , or .

[0478] In some embodiments, the payload is an inhibitor of microtubule polymerization. In some embodiments, the payload is hexasporin, HTI-286, or a derivative or analogue thereof.

[0479] In some embodiments, the payload is derived from: , or .

[0480] In some embodiments, the payload is: , or .

[0481] In some embodiments, the payload includes a topoisomerase inhibitor. In some embodiments, the payload includes camptothecin, or a derivative or analogue thereof. In some embodiments, the payload includes topotecan, irinotecan, cilaranotecan, cotecan, eczetidine, letopotecan, gemmatotecan, belotetidine, or rubitecan.

[0482] In some embodiments, the payload includes: or .

[0483] In some embodiments, the payload includes: or .

[0484] In some embodiments, the payload includes or .

[0485] In some embodiments, the payload includes .

[0486] In some embodiments, the payload includes .

[0487] In some embodiments, the payload includes .

[0488] In some embodiments, the payload includes or .

[0489] In some embodiments, the payload comprises a polypeptide. In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues. In some embodiments, the connector L 1 It covalently binds to lysine, serine, threonine, or tyrosine residues on the payload. In some embodiments, the polypeptide comprises one or more lysine residues. In some embodiments, the connector L 1 It covalently binds to lysine residues present on the payload.

[0490] In some embodiments, the payload comprises an N-terminal amino acid, wherein the linker L 1 It is covalently bound to the N-terminal amino acid.

[0491] In some embodiments, m is 1 to 20.

[0492] In some embodiments, the payload is an immunomodulatory payload.

[0493] In some embodiments, the immunomodulator payload is an antibody payload.

[0494] In some embodiments, the immunomodulator payload is an immune checkpoint inhibitor payload. In some embodiments, the immune checkpoint inhibitor payload is the payload of the following: pildizumab, sintilimab, AMP-224, atezolizumab, duvalumab, BMS-936559, trimelimab, indomod, icardolosat, TIGIT inhibitors (e.g., LAG-3, such as anti-LAG-3 antibodies; TIM-3, such as anti-TIM-3 antibodies), B7 molecules, or BTLA pathway antagonists.

[0495] In some embodiments, the immune checkpoint inhibitor payload is the immune checkpoint inhibitor antibody payload. In some embodiments, the immune checkpoint inhibitor antibody payload is the PD-1 inhibitor payload. In some embodiments, the PD-1 inhibitor payload is nivolumab, pembrolizumab, pildizumab, sintilimab, or AMP-224 payload.

[0496] In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload. In some embodiments, the PD-L1 inhibitor payload is atezolizumab, avelumab, durvalumab, or BMS-936559.

[0497] In some embodiments, the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload. In some embodiments, the CTLA4 inhibitor payload is an ipilimumab or trimemumab payload.

[0498] In some embodiments, the immune checkpoint inhibitor payload is an indoleamine 2,3-dioxygenase (IDO) inhibitor payload. In some embodiments, the IDO inhibitor payload is indomod or icardolstat.

[0499] In some embodiments, the immunomodulator payload is a cytokine payload.

[0500] In some embodiments, the cytokine payload is an interferon, interleukin, tumor necrosis factor, erythropoietin, MIP3a, ICAM, macrophage colony-stimulating factor, erythropoietin (EPO), granulocyte colony-stimulating factor (GCSF), or granulocyte-macrophage colony-stimulating factor payload.

[0501] In some embodiments, the interleukin payload is selected from IL-1 to IL-40. In some embodiments, the interleukin payload is IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0502] In some embodiments, the immunomodulatory payload is a type 1 cytokine (IL-2, IL-12, TNF-β, IFN-γ).

[0503] In some embodiments, the cytokine payload is selected from the group consisting of: IFN-α, IFN-β, IFN-γ, PEGylated IFN-α and apolipoprotein AI fusion protein with IFN-α, interleukin, IL-2, IL-2 covalently bound to immunoglobulins (e.g., sertozumab-amyloidin, RO6874281), IL-2 covalently bound to PEG molecules (e.g., NKTR-214), IL-10, PEGylated IL-10 (e.g., pegilodecakin), IL-7, IL-12, IL-15, recombinant deglycosylated IL-15, fusion protein of IL-15 and the binding domain of IL-15Rα (e.g., RLI), a triple fusion protein containing human IL-15, the binding domain of IL-15Rα and apolipoprotein AI, ALT-803 (with IgG1), and IL-15Rα. Fc domain fusions of IL-15, IL-18, IL-21, tumor necrosis factor (TNF-α, TNF-β), erythropoietin (EPO), MIP3a, ICAM, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF and talimogenelaherparepvec.

[0504] In some embodiments, the immunomodulator payload is a chemokine payload.

[0505] In some embodiments, the chemokine payload is a CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CXCL14 payload.

[0506] In some embodiments, the immunomodulator payload is a chemokine antagonist payload. In some embodiments, the chemokine antagonist payload is a plerixafor payload.

[0507] In some embodiments, the immunomodulator is a monoclonal antibody that is specific to cytokines or cytokine receptors.

[0508] In some embodiments, the immunomodulator payload comprises a polypeptide.

[0509] In some embodiments, the polypeptide contains one or more lysine residues.

[0510] In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.

[0511] In some embodiments, trans-cyclooctene is linked to one or more lysine residues.

[0512] In some embodiments, trans-cyclooctene is independently linked to one or more lysine, serine, threonine, or tyrosine residues.

[0513] In some embodiments, the polypeptide comprises an N-terminal amino acid, wherein the presence of a bioorthogonal moiety is linked to the N-terminal amino acid.

[0514] In some embodiments, m is 1 to 20. In some embodiments, m is 1 to 10. In some embodiments, m is 1 to 5. In some embodiments, m is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, m is 1.

[0515] In some embodiments, the payload-TCO conjugate has formula IX: IX Or its pharmaceutically acceptable salt, wherein: R 1a Each time it appears, independently choose a group consisting of the following items: hydrogen, C 1-4 Alkyl and C1-4 Halogenated alkyl groups; R 1b Each time it appears, independently choose a group consisting of the following items: hydrogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C(O)OH, C(O)OC 1-4 Alkyl, C(O)N(R) 1c CHR 1e CO2H, C(O)N(R) 1c CHR 1e C(O)OC 1-4 Alkyl, C(O)N(R) 1c )–C 1-6 Alkylene –CO2H and C(O)N(R) 1c )–C 1-6 Alkylene–C(O)OC 1-4 alkyl; R 1c 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; D is the payload independently each time it appears; L 1 Each time it appears, it is a connector independently; p' is independently 0, 1, or 2 each time it appears; and p'' is independently 1, 2, or 3 each time it appears.

[0516] In some embodiments, D is independently selected from the group consisting of: anticancer agent payload, toll-like receptor (TLR) agonist payload, and interferon gene stimulator (STING) agonist payload each time it appears.

[0517] In some embodiments, R 1a It is hydrogen.

[0518] In some embodiments, R 1a C 1-4 alkyl.

[0519] In some embodiments, R 1a It is CH3.

[0520] In some embodiments, R 1b Choose from the following groups: -C(O)OH, -C(O)OC 1-4Alkyl, -C(O)N(R) 1c CHR 1e CO2H, -C(O)N(R) 1c CHR 1e C(O)OC 1-4 Alkyl group, -C(O)N(R) 1c )–C 1-6 Alkylene –CO2H and -C(O)N(R) 1c )–C 1-6 Alkylene–C(O)OC 1-4 alkyl.

[0521] In some embodiments, R 1b Choose from the following groups: C(O)OH, C(O)N(R) 1c CHR 1e CO2H and C(O)N(R) 1c )CH2CO2H.

[0522] In some embodiments, R 1b Choose from the following groups: –NR 1c –CH2CH2–N(CH3)3 + –N(R) 1c –CH2CH2–SO3H、–N(R) 1c )–(CH2CH2O)3–CH2CH2N((CH2CH2O)3–CH2CH2–CO2H)2 and –N(R 1c )–CH(CH2O–CH2CH2–CO2H)2.

[0523] In some embodiments, the trans-cyclooctene moiety (G) is: , , , , , or .

[0524] In some embodiments, the trans-cyclooctene portion is: , or .

[0525] In some embodiments, the trans-cyclooctene portion is In some embodiments, the trans-cyclooctene moiety is .

[0526] In some embodiments, the trans-cyclooctene portion is .

[0527] In some embodiments, the trans-cyclooctene portion is .

[0528] In some embodiments, the trans-cyclooctene portion is .

[0529] In some embodiments, the trans-cyclooctene portion is .

[0530] In some embodiments, the trans-cyclooctene portion is .

[0531] In some embodiments, the trans-cyclooctene portion is .

[0532] In some embodiments, the trans-cyclooctene portion is And 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 substituted with polyether.

[0533] In some embodiments, the trans-cyclooctene portion includes .

[0534] In some embodiments, the trans-cyclooctene portion includes .

[0535] In some embodiments, the trans-cyclooctene portion includes .

[0536] In some embodiments, the trans-cyclooctene portion includes .

[0537] In some embodiments, R 1e It can be –CH2CO2H, –CH2CH2CO2H, –CH2CONH2, –CH2CH2CONH2, –CH2OH or –CH(CH3)OH.

[0538] In some embodiments, R 1e For –C 1-4 Alkylene – CO2H.

[0539] In some embodiments, R 1e It is –CH2CO2H.

[0540] In some embodiments, R 1b -C(O)N(R) 1c )–C 1-6 Alkylene – CO2H.

[0541] In some embodiments, R 1b -C(O)N(R) 1c )CH2CO2H.

[0542] In some embodiments, R 1c It is hydrogen.

[0543] In some embodiments, R 1b It is hydrogen.

[0544] In some embodiments, R 1b It is C(O)OH.

[0545] In some embodiments, these methods include targeted therapy. In some embodiments, this can be achieved by utilizing a TCO targeting portion and one or more pharmaceutically active agents (i.e., drugs or radioactive isotopes used in radiotherapy or radioligand therapy). Suitable drugs for use in the context of targeted drug delivery are known in the art. Optionally, the therapeutic probe may also contain detectable markers, such as one or more imaging agents. The “radionoid” (used interchangeably with “radioligand” herein) used for the trans-cyclooctene portion comprises a chelating agent and an isotope; such as isotopes selected from the group consisting of: 24 Na、 32 P, 33 P, 47 Sc、 59 Fe、 67 Cu、 76 As、 77 As、 80 Br、 82 Br、 89 Sr、 90 Nb, 90 Y、 103 Ru、 105 Rh、 109 Pd, 111 Ag、 111 In、 121 Sn、 127 Te、 131 I, 140 La、 141 Ce、 142 Pr, 143 Pr, 144 Pr, 149 Pm, 149 Tb, 151 Pm, 153 Sm、 159 Gd, 161 Tb, 165 Dy、 166 Ho、169 Er、 172 Tm、 175 Yb、 177 Lu、 186 Re、 188 Re、 198 Au、 199 Au、 211 At、 211 Bi、 212 Bi、 212 Pb, 213 Bi、 214 Bi、 223 Ra and 225 Ac. Radionuclides can be delivered via direct conjugation or chelation with a chelating agent. Exemplary radionuclides, chelating agents, and connectors for potential TCO conjugate payloads are described below.

[0546] Bifunctional chelating agents (BFC): Therapeutic radionuclides: In some embodiments, the connector includes a connector as shown below, where BFC refers to a bifunctional chelating agent (e.g., as disclosed herein) and the wavy line indicates the attachment point with the TCO.

[0547] In some embodiments, the payload-TCO conjugate has formula IX: IX Or its pharmaceutically acceptable salt, wherein R 1a Each time it appears, independently choose a group consisting of the following items: hydrogen, C 1-4 Alkyl and C 1-4 Halogenated alkyl groups; R 1b Each time it appears, independently choose a group consisting of the following items: hydrogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C(O)OH, C(O)OC 1-4 Alkyl, C(O)N(R) 1c CHR 1e CO2H, C(O)N(R) 1c CHR 1e C(O)OC 1-4 Alkyl, C(O)N(R)1c )–C 1-6 Alkylene –CO2H and C(O)N(R) 1c )–C 1-6 Alkylene–C(O)OC 1-4 alkyl; R 1c 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; D is the payload independently each time it appears; L 1 Each time it appears, it is a connector independently; p' is independently 0, 1, or 2 each time it appears; and p'' is independently 1, 2, or 3 each time it appears.

[0548] In some embodiments, D is a chelating agent suitable for use in radioligand therapy each time it appears.

[0549] In some embodiments, D is independently selected from the following each time it appears: DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAGA: 1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid, DTPA: diethylenetriaminepentaacetic acid, NTA: hypozoxytriacetic acid, EDTA: ethylenediaminetetraacetic acid, DO3A: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, NOOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid, NODAGA: 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononanetriazole heterocyclic butane, tetraoxane, macrocyclic maleimide, or mixtures thereof.

[0550] In some embodiments, the payload-TCO conjugate has formula IXA: IXA Or its pharmaceutically acceptable salt, wherein R 1a Choose the group composed of the following: hydrogen, C 1-4 Alkyl and C 1-4 Halogenated alkyl groups; D represents the payload; and L 1 For connectors.

[0551] In some embodiments, D is a chelating agent suitable for use in radioligand therapy. Exemplary, non-limiting chelating agents include DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAGA: 1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid, DTPA: diethylenetriaminepentaacetic acid, NTA: hypozoxytriacetic acid, EDTA: ethylenediaminetetraacetic acid, DO3A: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, NOOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid, NODAGA: 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononanetriazolehexacyclobutane, tetraoxane, macrocyclic maleimide, or mixtures thereof.

[0552] In some embodiments, the chelating agent complexes with a radioligand or a radionuclide.

[0553] In some embodiments, connector L 1 It may have 1 to 100 connecting atoms and may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, the connector may have 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.

[0554] In some embodiments, connector L 1 It may contain one or more (e.g., 1-10 or 1-5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1-10 or 1-5) alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic moieties; wherein each alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic moieties may be independently and optionally substituted by one to five substituents independently selected from: oxo, halogenated, C-substituted. 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups.

[0555] In some embodiments, connector L 1 It can have 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 alkylene, alkenylene, ynylene, arylene, heteroarylene, cycloalkylene, or heterocyclic alkylene is independently and optionally substituted by one to five independent substituents selected from: 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.

[0556] In some embodiments, the connector is a key.

[0557] In some embodiments, the connector is not a key. In some embodiments, each R 110 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclic; and each R 120 Independently hydrogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, aryl, heteroaryl, cycloalkyl or heterocyclic.

[0558] Representative connectors include, but are not limited to, those shown below: .

[0559] Representative connectors include, but are not limited to, those shown below: .

[0560] In some embodiments, connector L 1 It may contain 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(methylcarbamate), aryl (e.g., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. In some embodiments, connector L 1 It can include .

[0561] In some embodiments, connector L 1 It may include one or more natural or non-natural amino acids, which may be referred to as peptide linkers. When the drug (D) contains an amino moiety, the linker may be a peptide linker composed of a carboxyl unit and one or more amino acids constituting a protein or peptide sequence, which is then bonded to it. In some embodiments, the linker L 1 It may also contain self-destructing spacers that separate the drug from the protein peptide sequence.

[0562] In some embodiments, connector L 1 The peptide linker can be represented by "A—Y—Z—X—W", where "A" is a carboxyl unit, "Y" and "Z" are each one or more natural or non-natural amino acids that together form the peptide sequence, and "X" and "W" are optional additional linkers having 1 to 50 linking atoms, or 5 to 10 linking atoms, or 1 to 10 linking atoms, which separate the peptide from the drug, D, or bioorthogonal portion. In some embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0563] 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.

[0564] 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.

[0565] In some embodiments, the exemplary YZ combination includes valine-citrulline; valine-alanine; and alanine-alanine.

[0566] In some embodiments, A is -OC(O)-.

[0567] In some embodiments, X is -OC(O)-.

[0568] In some embodiments, W is -OC(O)-. In some embodiments, X does not exist and W is -OC(O)-.

[0569] In some embodiments, -XW is .

[0570] In some embodiments, -XW is .

[0571] In some embodiments, the peptide linker is specially tailored so that it will be selectively cleaved (e.g., enzymatically cleaved) to release drugs, such as via one or more tumor-associated proteases.

[0572] In some embodiments, the peptide linker has a chain length of two to four amino acid residues (i.e., 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.

[0573] In some embodiments, the peptide linkers are Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly [SEQ ID NO: ], Ala-Leu-Ala-Leu [SEQ ID NO: ], Phe-N 9 -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.

[0574] In some embodiments, connector L 1 for: , , (For example, ), , or .

[0575] The aforementioned linker can attach an amino acid side chain of D, such as lysine or cysteine, to the right side (e.g., , , , , , ).

[0576] In some embodiments, the payload is covalently bonded to the connector via an amide bond; for example, the payload may be an amine-containing payload for attaching the payload to a carbonyl group of the connector, or in other cases, the payload may be a carboxyl-containing payload for attaching the payload to an amine group of the connector. In some cases, the payload and the connector together form a urethane group; for example, the payload may be an amine-containing payload for attaching the payload to an acyloxy group of the connector. In some cases, the payload and the connector together form a carbonate group; for example, the payload may be a hydroxyl-containing payload for attaching the payload to an acyloxy group of the connector.

[0577] In some embodiments, L 1 for Or –O–; L 3a For key or C 1-6 Alkylene; L 4a For the bond, –NHN:, –N(R) 10 )–C 2-6 Alkylene–N(R) 11 )–、–N(R 12 )–C 2-3 Alkylene–N(R) 13 )C(O)–、–N(R 10 )–C 1-6 Alkylene –C(O)NHN:、 –NHNHC(O)C1-6 Alkylene –C(O)NHN:、 –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 10 R 11 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; R 17 Each time it appears, it is independently hydrogen or –CH2OC(O)–; and G x A phenyl group optionally substituted with 1 to 5 independently selected substituents from the group consisting of: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 Alkoxy, cyano, and nitro groups.

[0578] In some embodiments, connector L 1 It is -OC(O)-.

[0579] In some embodiments, L 1 for ; L 3a For key; L 4a for or ;as well as R 12 and R 13 Each is independently hydrogen or C 1-4alkyl.

[0580] In some embodiments, p'' is 1.

[0581] In some embodiments, for , , , , , , , , , , , , , , , , , , , , , , , or ; R 18 It is independently hydrogen or –CH2OC(O)NHD' each time it appears; R D The hydrogen or carbon atom on the nitrogen atom of the payload 1-4 Alkyl groups; and D and D' are independently the payload portions.

[0582] In some embodiments, D or D' is a cyclic dinucleotide payload portion, an imidazo[4,5-c]quinoline-4-amine payload portion, a TLR agonist payload portion, a STING agonist payload portion, or an anticancer agent payload portion.

[0583] In some embodiments, for or ; R 12 and R 13 Each is independently hydrogen or C 1-4 Alkyl groups; and D and D' are independently payload portions (e.g., anticancer agent payload portions).

[0584] In some embodiments, p' is 0.

[0585] In some embodiments, p'' is 2 or 3.

[0586] In some embodiments, p is 2 and for .

[0587] Those skilled in the art will recognize that the payload (D or D') bonded to the linker does not refer to the payload molecule itself, but rather to a portion of the payload molecule bonded to the linker. The payload (D or D') is released from the prodrug.

[0588] The payload (D or D') can be the anticancer agent payload of any anticancer agent described herein.

[0589] In some embodiments, the payload includes a TLR7 / 8 agonist.

[0590] In some embodiments, the payload includes a TLR7 / 8 agonist, and X is an antibody or antibody fragment targeting HER2, TROP2, cohesin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM). In some embodiments, the payload includes calcimodilamide, and X is an antibody or antibody fragment targeting HER2, TROP2, cohesin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM).

[0591] In some embodiments, the payload includes camptothecin or a derivative thereof. In some embodiments, the payload includes eczema. In some embodiments, the payload includes camptothecin or a derivative thereof, and X is an antibody or antibody fragment portion targeting HER2, TROP2, cohesin 4, or the extracellular matrix (ECM). In some embodiments, the payload includes eczema, and X is an antibody or antibody fragment portion targeting HER2, TROP2, cohesin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM).

[0592] In some embodiments, the payload includes MMAE. In some embodiments, the payload includes MMAE or a derivative thereof, and X is an antibody or antibody fragment portion targeting HER2, TROP2, cohesin 4, CEACAM5, fibronectin, or extracellular matrix (ECM).

[0593] In some embodiments, the payload includes paclitaxel. In some embodiments, the payload includes paclitaxel or a derivative thereof, and X is an antibody or antibody fragment targeting HER2, TROP2, cohesin 4, CEACAM5, fibronectin, or extracellular matrix (ECM).

[0594] In some embodiments, the payload includes docetaxel or a derivative thereof. In some embodiments, the payload includes docetaxel or a derivative thereof, and X is an antibody or antibody fragment targeting HER2, TROP2, cohesin 4, CEACAM5, fibronectin, or extracellular matrix (ECM).

[0595] In some embodiments, the payload-TCO conjugate has the following structure: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0596] In some embodiments, the payload-TCO conjugate is selected from: , , , , , , , , , , , , , and .

[0597] In some embodiments, the payload-TCO conjugate is selected from: , as well as .

[0598] D. Combination therapy In one aspect, 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 target fraction of this disclosure or a pharmaceutically acceptable salt or composition thereof; and a prodrug, such as those described herein; and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of anticancer agents or immunomodulators.

[0599] This disclosure also provides pharmaceutical combinations comprising the payload-TCO conjugates described herein or pharmaceutically acceptable salts or compositions thereof; and additional therapeutic agents, such as anticancer agents or immunomodulators, for treating or preventing cancer or for enhancing or inducing an immune response.

[0600] 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.

[0601] Methods and uses for treating cancer include applying / targeting specific portions at the tumor site.

[0602] Additional therapeutic agents may be administered simultaneously or sequentially with the payload-TCO conjugate and composition. Sequential administration includes administration before or after the payload-TCO conjugate and composition. Additional therapeutic agents may be administered before the payload-TCO conjugate and composition. Additional therapeutic agents may be administered after the payload-TCO conjugate and composition. Additional therapeutic agents may be administered simultaneously with the payload-TCO conjugate and composition. In some embodiments, one or more additional therapeutic agents may be administered in the same composition as the payload-TCO conjugate. In other embodiments, a time interval may exist between the administration of the additional therapeutic agent and the payload-TCO conjugate or composition. In some embodiments, administering additional therapeutic agents together with the payload-TCO conjugate or composition may allow for lower doses and / or lower frequency intervals of administration of the other therapeutic agents. When used in combination with one or more other active ingredients, the payload-TCO conjugate or composition of this disclosure, as well as the other active ingredients, may be used at lower doses than when used individually. Therefore, pharmaceutical compositions of this disclosure include pharmaceutical compositions containing one or more other active ingredients in addition to the payload-TCO conjugate of this disclosure.

[0603] anticancer agents Exemplary anticancer agents include, but are not limited to, abiraterone acetate, abiraterone (methotrexate), abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab (metasone), doxorubicin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib maleate, Afinitor (everolimus), Aldara (imiquimod), interleukin-aldextrin, alemtuzumab, Alimta (pemetrexed disodium), and Aloxi (palonosetron hydrochloride). Amboclin (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidronate disodium), Reninide (anastrozole), Aromasin (exemestane), Arranon (nerapine), arsenic trioxide, Arzerra (olfamumab), chrysanthemum asparaginase, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotine, Bexxar (tosimomab and I131 iodotosimomab), bicalutamide, bleomycin, bortezomib, bosutinib (bosutinib), bosutinib, Brentuximab vedotin, Busulex, Cabazitaxel, Cabozantinib S-malate, CAF, Camppath (alemumab), Camptosar (irinotecan hydrochloride), Capecitabine, CAPOX, Carboplatin, Carboplatin-paclitaxel, Carfilzomib, Casodex (bicalutamide), CeeNU (lomustine), Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant bivalent HPV vaccine), Cetuximab, Chlorambucil, Chlorambucil-prednisone, CHOP, Cisplatin, Clafen (cyclophosphamide), Clofarabine Clofarex (clofarabine), Clolar (clofarabine), CMF, Cometriq (cazobantinib-S-malic acid), COPP, COPP-ABV, Cosmegen (actinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Cytarabine, Cytarabine liposomes, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (decitabine), Actinomycin, Dasatinib, Dazorubicin Hydrochloride, Decitabine, Degarelix, DenileukinDiftitox, Denosumab, DepoCyt (cytarabine liposome), DepoFoam (cytarabine liposome), Dexazosin hydrochloride, Docetaxel, Doxil (doxorubicin hydrochloride liposome), Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Efudex (fluorouracil), Elitek (raburicase), Ellence (epirubicin hydrochloride), Eloxatin (oxaliplatin), Eltrombopagamine, Emend (aprepitant), Enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab). Anti), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia), Etoposide (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Faslodex (Flavivestram), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluoruracil), Fluoruracil, Foles (Methotrexate), Foles PFS (Methotrexate), Folfiri, Folfiri-Bevacizumab, Folfiri-Cetuximab, Folfirinox, Folfox (Leucovorin, Fluorouracil, Oxaliplatin), Folotyn (Platrexate), FU-LV, Fulvestrant, Gardasil (Recombinant Quadrivalent HPV Vaccine), Gazyva (Oxytocin), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine Oxaliplatin, Gemcitabine, Gemzar, Afatinib Maleate, Imatinib Mesylate, Carboxypeptidase, Goserelin Acetate, Eribulin Mesylate, Trastuzumab, Recombinant Bivalent HPV Vaccine, Recombinant Quadrivalent HPV Vaccine, Hycamtin, Hyper-CVAD, Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig, Ifex, Ifosfamidum, Imatinib Mesylate, Imbruvica, Imiquimod, Inlyta, IntronA (recombinant interferon Alfa-2b), Iodine131 (tosimomumab and tosimomumab), ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidesin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (ruxotinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab mettansine), Keoxifene ( Raloxifene hydrochloride, Kepivance (palivmin), Kyprolis (carfilzomib), lapatinib besylate, lenalidomide, letrozole, calcium leucovorin, Leukeran (chlorambucil), leuprolide acetate, Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), liposomal cytarabine, lomustine, Lupron (leuprolide acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Nitrogen Mustard Hydrochloride, Megace (Megestrol Acetate), Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex Mesna, Methazolastone (temozolomide), Methotrexate, Methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), Mitomycin C, Mitozytrex (mitomycin C), MOPP, Mozobil (prexafo), Mustargen (nitrogen mustard hydrochloride), Promycin (mitomycin C), Myleran (busulin), Mylosar (azacitidine), Mylotarg (geutzumab ozomicin) , paclitaxel nanoparticles (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), Nelarabine, Neosar (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romistastatin), oxotuzumab, oxfatumumab, omaxitase mesosuccinate, Oncaspar (polyasparaginase), Ontak (Denileukin)Diftitox, OEPA, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, Palifermin, Palonosetron hydrochloride, Pamidronate disodium, Panitumumab, Paraplatin (carboplatin), Paraplatin (carboplatin), Pazopanib hydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pemetrexed disodium, Perjeta (pertuzumab), Pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Prednisolone, Pomalidomide, Pomalyst (pomalidomide), Panatinib hydrochloride, Pralatrexate, Prednisone, Procarbazine hydrochloride, Proleukin Aldesleukin, Prolia (denomab), Promacta (iridropa ethanolamine), Provenge (Cyproxetine-T), Mercaptopurine, Radium-223 dichloride, Raloxifene hydrochloride, Raburicase, R-CHOP, R-CVP, Recombinant Bivalent HPV Vaccine, Recombinant Quadrivalent HPV Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Lenalidomide, Rheumatoid Arthritis (Methotrexate), Rituximab (Rituximab), Rituximab, Romidisin, Romilastine, Erythromycin (Daunorubicin Hydrochloride), Ruxotetinib Phosphate, Sclerosing Agent Intrapleural Aerosol (Talc), Ciproxetine-T, Sorafenib Tosylate, Sprycel (Dasatinib), Stanford V. Sterile talc, Steritalc, Stivarga (regorafenib), Sunitinib malate, Sunitinib malate, Sylatron (pegylated interferon Alfa-2b), Synovir (thalidomide), Synribo (Omacetaxine Mepesuccinate), Tafinlar (dabrafenib), Tamoxifen citrate, TarabinePFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotin), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temoda (temozolomide), Temozolomide, Tesirolimus, Thalidomide, Thalidomide (thalidomide), Topoxadoxane (etoposide), Topotecan hydrochloride, Toremifene, Torisel (tesirolimus), Tosimomab and 1131I-tosimomab, Totect (dexrazoxen hydrochloride), Trametinib Trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib dimethylbenzenesulfonate), vandetanib, VAMP, Vectibix (panitumumab), VelP, Velban (vincrine sulfate), Velcade (bortezomib), Velsar (vincrine sulfate), Vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomes, vinorelbine tartrate, Vismodegib, Voraxaze (glucosacciase), Voronostat, 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), Zinecard (dexrazoxen hydrochloride), Ziv-Aflibercept, Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), and Zytiga (abiraterone acetate).

[0604] Anticancer agents may include PBD dimer, calcitrazine, seminosporin, microtubule lysin B, rhizomycin, dolalastatin, diphenhydramine B, camptothecin, CBI, tesiromoxim, actinomycin D, epothilone B, paclitaxel, cryptophytin, SN38, velcade, buspirone, DAVLBH, DM1, chlorophyll, alimta, T2 toxin, MMC, vastalani, vinorelbine, brefidobacterium, sunitinib, daunorubicin, semasanib, erlotinib, gefitinib, irinotecan, LY-541503, gerdemycin, gemcitabine, methotrexate, glimepiride, topotecan, bleomycin, doxorubicin, cisplatin, N-muscarine, etoposide, or 5-FU.

[0605] 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 monomethylolpropionate E.

[0606] Compound Synthesis The targeted moieties can be prepared using the methods disclosed herein, as well as their conventional modifications, which will be apparent given 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.

[0607] It should be understood that when typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given, other process conditions may also be used unless otherwise specified. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through conventional optimization procedures.

[0608] Additionally, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. The appropriate protecting groups for various functional groups, and suitable conditions for protecting specific functional groups and deprotecting them, are well known in the art. For example, numerous protecting groups are described in Wuts, PGM, Greene, TW, and Greene, TW (2006). Greene's protective groups in organic synthesis. Hoboken, NJ, Wiley-Interscience, and the references cited therein. In some embodiments, the term "leaving group" refers to an electron-withdrawing atom (or group of atoms) that can be replaced by a stable substance, carrying bonding electrons with it. Examples of suitable leaving groups include halides (e.g., Br, Cl, I), sulfonates (e.g., trifluoromethanesulfonates, methanesulfonates, toluenesulfonates, and p-bromobenzenesulfonates), and nitrophenols.

[0609] As shown in Scheme I, compound V (where each dashed line, R) 1 R 2 R 3 R 4 The rings A, t, L, p, and X are defined independently in this paper, and R 50 For the synthetic stem used to bond with L, a leaving group (e.g., halogen) or a portion of L capable of connecting to X or another part of L (e.g., hydroxyl, amino, methylamino, etc.) can be prepared by coupling a compound of formula I-2 with a suitably functionalized antibody fragment portion X. Suitable methods can be adapted according to the literature (see, for example, WO2020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025 and WO2014 / 205126). The compound of formula I-2 can be prepared by reacting compound I-1 with a precursor of L under suitable coupling reaction conditions. In some embodiments, X is an antibody fragment. Suitable coupling methods include, but are not limited to, using a succinimide functional group capable of forming an amide bond with a major amine on the antibody fragment, or functionalizing L with a group capable of forming a covalent bond with a cysteine ​​residue on the antibody fragment portion (such as pyrrole-2,5-dione).

[0610] Option I Compound I-1 can be prepared according to Scheme II, wherein each of the dashed lines, R 1 R 2 R 3 R 4Rings A, t, and L are defined independently in this paper, and R 50 For the synthetic stem used to bond with L, such as a leaving group (e.g., halogen or thioether), or a portion of L that can be connected to another part of X or L (e.g., hydroxyl, amino, methylamino, etc.), and each LG is independently a leaving group (e.g., halogen).

[0611] Option II As shown in Scheme II, compound II-3 is provided by coupling compound II-1 with compound II-2 in the presence of N2H4. Compound II-5 and / or compound II-7 are provided by further modifying compound II-3 with compound II-4 and / or compound II-6 under standard coupling conditions. Alternatively, compound II-3 can be provided by coupling compound II-8 with compound II-9 in the presence of N2H4. Compound II-10 can be provided by contacting compound II-3 with a suitable oxidant (e.g., NaNO2). Alternatively, compound II-3 can be provided by contacting compound II-10 with thiourea dioxide.

[0612] After each reaction is completed, each of the intermediates or the final compound can be recovered and optionally purified using conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration, etc.

[0613] It should be understood that any of the compounds or intermediates shown in Scheme I or II can be prepared using conventional methods or purchased from commercial sources. Furthermore, any of the intermediates or products obtained by the methods outlined in Scheme I or II can be derivatized at any step to provide compounds of various formulas V.

[0614] Exemplary payloads and their conjugates can be prepared according to methods adapted from the literature (see, for example, WO2022 / 032191, WO2021 / 007160, WO2020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025 and WO2014 / 205126, the methods of which are incorporated herein by way of their entirety). The examples below illustrate exemplary procedures for certain payloads, which can be applied to the preparation of other payloads, such as those disclosed herein.

[0615] Example The following examples illustrate specific embodiments of this disclosure. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques that work well in the practice of this disclosure and can therefore be considered to constitute a specific mode of practice therein. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the disclosed specific embodiments without departing from the spirit and scope of this disclosure and still obtaining the same or similar results.

[0616] LCMS analysis method: According to the manufacturer's protocol, the test item is subjected to PNG enzyme F (IgG) and DTT or DTT (Fab) alone in RapiGest. The sample is diluted with water to 100 µg / mL and centrifuged at 16.1k RCF for 10 min at 4 °C. The sample is then analyzed by LCMS (LC-Q-TOF) and the mass spectrum is reconstructed based on the charge gradient.

[0617] Example 1: Synthesis of the targeting moiety of tetrazine-trastuzumab Trastuzumab (22.1 mg / mL, 1.1 mL) in 0.01 M PBS was mixed with 20 equivalents of methyltetrazine-PEG4-NHS (Clickchemtools #1069-10).

[0618] Methyltetraazine-PEG4-NHS The reactants were thoroughly mixed and aged at room temperature for 1 hour, during which the reaction was quenched by adding 1 volume of 0.1 M Tris buffer. The resulting solution was buffer-exchanged to 0.01 M PBS to remove excess reagents and buffer salts. The resulting targeted moiety solution (6.3 mg / mL, 1.6 mL) was analyzed by SDS-Page and LCMS (data not shown) to confirm the formation of the targeted moiety and thus for use in subsequent studies. Based on the analysis, it is hypothesized that up to 12 methyltetraazine-PEG4 units are covalently bonded to the antibody.

[0619] Example 2: Tetraazine-Fab Targeted Component Fab was prepared from trastuzumab using a commercial kit (Pierce™ Fab Preparation Kit #44985) according to the manufacturer's protocol and purified using protein G resin (BioVision #6511-25). The purified Fab in 0.01 M PBS was mixed with 20 equivalents of methyltetrazine-PEG4-NHS (Clickchemtools #1069-10). The reaction mixture was thoroughly mixed and aged at room temperature for 1 hour, at which point the reaction was quenched by adding 1 volume of 0.1 M Tris buffer. The resulting solution was buffer-exchanged to 0.01 M PBS to remove excess reagents and buffer salts. The resulting target moiety solution (1.0 mg / mL, 10.3 mL) was analyzed by SDS-Page and LCMS (data not shown) to confirm the formation of the target moiety and thus for subsequent studies. Based on the analysis, it is hypothesized that up to approximately six methyltetrazine-PEG4 units are covalently bonded to Fab.

[0620] Example 3: Tetraazine-Fab Targeted Component Fab was prepared from entaftoxin using the following method. 0.1 mg of papain was pretreated with 1 mM DTT and 2 mM EDTA at a concentration of 0.5 mg / mL and incubated at 37°C for 30 min. The antibody was prepared in PBS buffer (10 mg, 0.5 mg / mL) at pH 7.4. The pretreated papain was mixed with the antibody at a (1:100) molar ratio and incubated at 37°C for 2 h. The digestion mixture was loaded onto an anti-CH1 affinity column, washed with 25 mM Tris, 150 mM NaCl, pH 8.0, and eluted with 50 mM sodium citrate, 150 mM NaCl, pH 3.0. The filtrate containing the Fab product was dialyzed into PBS. The purified Fab was concentrated to 0.2 mg / mL in PBS. Fab was mixed with Me-Tet-PEG9-NHS (prepared in 10 mM DMSO) at a 3:1 molar ratio and incubated at 37°C for 2 h. The conjugate was analyzed by LCMS and the DAR was calculated to be 2.66.

[0621] Example 4: Tetraazine-Fab Targeted Component Fab was prepared from brentuximab using the following method. 0.1 mg of papain was pretreated with 1 mM DTT and 2 mM EDTA at a concentration of 0.5 mg / mL and incubated at 37°C for 30 min. The antibody was prepared in PBS buffer (10 mg, 0.5 mg / mL) at pH 7.4. The pretreated papain was mixed with the antibody at a (1:100) molar ratio and incubated at 37°C for 2 h. The digestion mixture was loaded onto an anti-CH1 affinity column, washed with 25 mM Tris, 150 mM NaCl, pH 8.0, and eluted with 50 mM sodium citrate, 150 mM NaCl, pH 3.0. The filtrate containing the Fab product was dialyzed into PBS. The purified Fab was concentrated in PBS to a concentration of 0.2 mg / mL. Fab was mixed with Me-Tet-PEG9-NHS (prepared in 10 mM DMSO) at a molar ratio of 3:1 and incubated at 37°C for 2 hours. The resulting conjugate was analyzed by LCMS and the calculated DAR was 4.57.

[0622] Example 5A: Tetraazine-Fab Target Component Fab was prepared from sacitrus bacillus using a commercial kit (Pierce™ Fab Preparation Kit #44985) according to the manufacturer's protocol, and purified using protein G resin (BioVision #6511-25). Purified Fab 10 mM Me-Tet-PEG9-NHS was prepared in DMSO. The two components were reacted at 25°C for 2 hours at a drug-to-protein molar ratio of 3:1, followed by dialyzing with PBS (pH 7.4) to remove excess Me-Tet-PEG9-NHS compound from the protein fraction. The resulting targeted moiety solution was analyzed by SDS-Page and LCMS to confirm the formation of the targeted moiety. It was assumed that approximately two methyltetraazines were covalently bonded to each Fab, as confirmed by LCMS.

[0623] Example 5B: Tetraazine-Fab Targeting Component (Compound TM-2) The preparation and characterization of compound TM-2 are as follows.

[0624] Sacitocilizumab Fab amino acid sequence: Heavy chain: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQG SLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHT (SEQ ID NO. 9).

[0625] Light chain: DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTKSFNRGEC (SEQ ID NO. 10).

[0626] Formation of sacitrulline Fab-tetraazine conjugate The coding sequences for the heavy and light chain variable regions of sacitrus (human anti-TROP2) antibody were used to generate a construct expressing TROP2 Fab. The coding sequences were synthesized and subcloned into the PCDN3.4 expression vector. The constructed plasmid was transformed into... E. coliThe plasmids were proliferated and amplified. The purified plasmids were confirmed by sequencing. The constructs containing the heavy and light chains of TROP2 Fab were transfected into HEK293 cells (suspension) using a polymeric polyethyleneimine (PEI) reagent. The culture medium was harvested 6–7 days post-transfection. The TROP2 Fab-containing medium was centrifuged, filtered, and then loaded onto a KappaSelect affinity column (Mabselect Prism). The loading buffer was 25 mM Tris containing 150 mM NaCl (pH 8.0), and eluted with 100 mM sodium citrate buffer containing 150 mM NaCl (pH 2.5). The collected solution was neutralized with 1 M arginine and 400 mM succinate buffer (pH 9.0). The affinity-purified proteins were further purified by gel filtration (using a Superdex S-200 5 / 150GL column chromatography). The samples were eluted with 2X PBS (pH 7.4) as the mobile phase. The purified TROP2 Fab was analyzed by SDS-PAGE and SEC-HPLC.

[0627] Tetraazine conjugation of TROP2 Fab: The TROP2 Fab buffer was replaced with PBS (pH 7.4) overnight. Methyltetraazine-PEG9-NHS (SiChem #SC-8808) was dissolved in DMSO to prepare a 10 mM stock solution. For conjugation, the two components were reacted at 25°C for 2 hours at a molar ratio of 3:1 (methyltetraazine-PEG9-NHS to TROP2 Fab). The amount of Fab used for tetraazine conjugation varied between 30 mg and 100 mg, but the 3:1 ratio was maintained throughout the conjugation procedure. The solution buffer was then replaced with PBS (pH 7.4) to remove excess methyltetraazine-PEG9-NHS and buffer salts.

[0628] Characterization of sacitrulline Fab-Tz conjugate (compound TM-2):The prepared Fab-Tz conjugate samples were analyzed by SDS-PAGE and Q Exactive HF-X LC-MS (Thermo Scientific) equipped with an AcquityUPLC protein BEH C4 column (300A, 1.7 µm, 2.1 x 50 mm) to confirm conjugate formation and determine the tetrazine to antibody ratio. HPLC-SEC analysis was performed using an UltiMate 3000 HPLC (Thermo Scientific) equipped with an Xbridge BEH 200A SEC column (7.8 x 300 mm, Waters Corp). Proteins were eluted with PBS containing 15% isopropanol (pH 7.4). Size exclusion chromatograms showed that the Fab-tetrazine conjugates were 99% monomeric with minimal aggregation (1.0%). The tetrazine to antibody ratio was calculated to be 2.3.

[0629] Example 6: HER2 Fab-Tetraazine Targeting Component (Compound TM-1) The preparation and characterization of compound TM-1 and its isotype Fab-Tz are as follows.

[0630] Trastuzumab Fab amino acid sequence: Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGF YAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKV (SEQ ID NO. 3).

[0631] Light chain: DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 4).

[0632] Isotype Fab-Tz amino acid sequence: Heavy chain: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHT (SEQ ID NO. 5).

[0633] Light chain: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTKSFNRGEC (SEQ ID NO. 6).

[0634] Generation of HER2 Fab The coding sequences for the heavy and light chain variable regions of trastuzumab (human anti-HER2) antibody were used to generate a construct expressing HER2 Fab. The coding sequences were synthesized and subcloned into the PCDN3.4 expression vector. The constructed plasmid was transformed into... E. coliThe plasmids were proliferated and amplified. The purified plasmids were confirmed by sequencing. The heavy and light chain constructs containing HER2 Fab were transfected into HEK293 cells (suspension) using a polymeric polyethyleneimine (PEI) reagent. Six to seven days post-transfection, 6 liters of culture medium were harvested. The HER2 Fab-containing medium was centrifuged, filtered, and then loaded onto a KappaSelect affinity column (Mabselect Prism). The loading buffer was 25 mM Tris containing 150 mM NaCl (pH 8.0), and eluted with 100 mM sodium citrate buffer containing 150 mM NaCl (pH 2.5). The collected solution was neutralized with 1 M arginine and 400 mM succinate buffer (pH 9.0). The affinity-purified proteins were further purified by gel filtration (using a Superdex S-200 5 / 150GL column chromatography). The sample injection volume was 20 mL, the flow rate was 0.3 mL / min, and the mobile phase was 2X PBS (pH 7.4). The purified HER2 Fab was analyzed by SDS-PAGE and SEC-HPLC. The final yield was 742 mg, and the purified product was stored at -80°C for long-term storage.

[0635] Generation of the same type of Fab: The coding sequence was synthesized and subcloned into the pTT5 expression vector. The constructed plasmid was then transformed into... E. coli The plasmids were proliferated and amplified. The purified plasmids were confirmed by sequencing. The constructs containing the heavy and light chains of the isotype Fab were transfected into HEK293 cells (suspension) using a polymeric polyethyleneimine (PEI) reagent. Two liters of culture medium were harvested 7 days post-transfection. The culture medium containing the isotype Fab was centrifuged, filtered, and then loaded onto a KappaSelect affinity column (Mabselect Prism). The loading buffer was 25 mM Tris containing 150 mM NaCl (pH 8.0), and eluted with 50 mM sodium citrate buffer containing 150 mM NaCl (pH 3). The collected solution was neutralized with 1 M arginine and 400 mM succinate buffer (pH 9.0). The affinity-purified proteins were further purified by gel filtration (using a Superdex S-200 5 / 150GL column chromatography). The sample injection volume was 3 mL, the flow rate was 0.3 mL / min, and the mobile phase was 2X PBS (pH 7.4). The purified isoform Fab was analyzed by SDS-PAGE and SEC-HPLC. The final yield was 200 mg and the purified product was stored at -80°C for long-term storage.

[0636] Tetraazine conjugation of HER2 Fab and homologous Fab:The HER2 Fab and isotype Fab buffer were replaced with PBS (pH 7.4) overnight. Methyltetraazine-PEG9-NHS (SiChem #SC-8808) was dissolved in DMSO to prepare a 10 mM stock solution. For conjugation, the two components were reacted at 25°C for 2 hours at a 3:1 molar ratio (methyltetraazine-PEG9-NHS to HER2 Fab). The amount of Fab used for tetraazine conjugation varied between 30 mg and 100 mg, but the 3:1 ratio was maintained throughout the conjugation procedure. The solution buffer was then replaced with PBS (pH 7.4) to remove excess methyltetraazine-PEG9-NHS and buffer salts.

[0637] Methyltetraazine-PEG9-NHS Characterization of compound TM-1 and its isotype Fab-Tz: Samples of the prepared compound TM-1 or isoform Fab-Tz conjugates were analyzed by SDS-PAGE and Q Exactive HF-X LC-MS (Thermo Scientific) equipped with an Acquity UPLC protein BEHC4 column (300A, 1.7 µm, 2.1 x 50 mm) to confirm the formation of compound TM-1 and determine the tetrazine to antibody ratio. HPLC-SEC analysis was performed using an UltiMate 3000 HPLC (Thermo Scientific) equipped with an xBridge BEH 200A SEC column (7.8 x 300 mm, Waters Corp). Proteins were eluted with PBS containing 15% isopropanol (pH 7.4). Size exclusion chromatograms of compounds TM-1 and isoform Fab-Tz showed that >97% of the Fab-tetrazine conjugates were monomeric and exhibited minimal aggregation (≤3.0%). The ratio of tetrazine to antibody in compound TM-1 was calculated to be 2.2, and the ratio of tetrazine to antibody in isotype Fab-Tz was calculated to be 1.8.

[0638] Flow cytometry analysis of compound TM-1 and isotype Fab-Tz:Cell binding assays using NCI-N87 (HER2-positive) cells were performed by flow cytometry, with unconjugated HER2 Fab, compound TM-1, or isotype Fab-Tz controls as controls. NCI-N87 (HER2-positive) human gastric cancer cells were collected by centrifugation and resuspended in FACS buffer (PBS containing 2% FBS, pH 7.4). Cells were seeded into 96-well plates (200,000 cells per well) and centrifuged at 400 xg for 5 min. The supernatant was removed, and cells were incubated at 4°C for 1 h with a 10-fold titration starting at 400 nM of compound TM-1, unconjugated HER2 Fab, or isotype control (IgG). For isotype Fab-Tz cell binding assays, a 3-fold titration starting at 300 nM was performed. Centrifuge the plate, wash three times with FACS buffer, and resuspend in 100 mL of secondary antibody containing goat anti-human IgG Alexa 488 (catalog number #A-11013, Thermo Fisher Scientific, 1:500 dilution) or goat anti-human (Fab')2 fragment specific (109-116-097, Jackson Immunoresearch, PA, USA, 1:200 dilution) and incubate in the dark at 4°C for 1 hour. Remove the supernatant, wash the cells twice with PBS, and analyze using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) or Attune NxT (Thermo Scientific, MA, USA). Calculate the mean fluorescence intensity (MFI) value and plot it against antibody concentration. The binding of compound TM-1 to HER2-positive cells was observed to be comparable to that of unconjugated HER2 Fab, and no binding to isotype Fab-Tz was detected.

[0639] Example 7: A general procedure for preparing compound A A solution of compound 2 (800 mg, 1.79 mmol) in DMF (4.00 mL) was added to a solution of MMAE (1.40 g, 1.95 mmol) and DIEA (690 mg, 5.34 mmol) in DMF (4.00 mL) at 0 °C, and the mixture was stirred at 25 °C for 16 h. Then, a solution of HOBt (480 mg, 3.56 mmol) in DMF (0.50 mL) was added to the above reaction mixture at 0 °C, and the reaction mixture was stirred at 25 °C for 1.0 h. The reaction mixture was then cooled to 0 °C, and TBAF (1 M in THF, 4.45 mL) was added. After stirring the mixture at 25 °C for 2.0 h, another batch of TBAF (1 M in THF, 4.45 mL) was added at 0 °C, and the reaction mixture was stirred at 25 °C for another 12.0 h. LC-MS showed a single main peak with the desired mass. The resulting reaction mixture was purified by preparative HPLC (column: Welch XB-C18 7 μm 110A 250). 50 mm; mobile phase: [water (0.1% TFA) IN]; B%: 50%-70%-40 min. Injections: 2, retention time: 37 min, flow rate: 60 mL / min) to obtain compound A (450 mg, 99.0% purity; 64.6 mg, 99.2%, 31.2% yield). LCMS (m / z): 928.6 [M+H] + . 1 HNMR: (400 MHz, DMSO-d6): δ 8–46 - 8.28 (m, 1H),8–03 - 7.84 (m, 1H), 7.64 (d, J= 8.8 Hz, 1H), 7–35 - 7.23 (m, 4H), 7–21 - 7.13(m, 1H), 6–05 - 5.57 (m, 2H), 5.10 (s, 1H), 4–81 - 4.39 (m, 3H), 4–35 - 4.19(m, 1H), 4–05 - 3.92 (m, 2H), 3–40 - 3.09 (m, 11H), 3–08 - 2.83 (m, 5H), 2–48 - 2.37 (m, 2H), 2–31 - 2.09 (m, 5H), 2–07 - 1.89 (m, 3H), 1–88 - 1.64 (m,6H), 1–63 - 1.33 (m, 4H), 1–32 - 1.16 (m, 1H), 1–08 - 0.97 (m, 9H), 0–90 -0.68 (m, 18H).

[0640] Example 8: A general procedure for preparing compound B General procedure for preparing compound 4 Add DIEA (2.10 g, 16.3 mmol), EDCI (2.08 g, 10.9 mmol), and DMAP (1.33 g, 10.9 mmol) and compound 3 (1.61 g, 8.14 mmol) to a solution of compound 2 (1.00 g, 5.43 mmol) in DCM (10 mL). Stir the mixture at 25 °C for 16 hours. TLC indicated complete consumption of compound 2 and the formation of a new spot. Partition the reaction mixture between DCM (20 mL) and H₂O (10 mL). Separate the organic phase using... saturation The sample was washed with an aqueous solution of citric acid (3 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to give compound 4 (700 mg, 39.4% yield). 1 HNMR (400MHz, CDCl3): δ ppm 1.12 (s, 3 H), 1.60 (dd, J =15.45, 6.19 Hz, 1H), 1–79 - 1.87 (m, 2 H), 1.92 (br d, J= 5.88 Hz, 1 H), 1.95 (s, 1 H), 1–98 -2.00 (m, 1 H), 2.02 (br d, J = 4.13 Hz, 1 H), 2.26 (dd, J = 11.63, 3.88 Hz, 1 H),2–30 - 2.36 (m, 1 H), 2–77 - 2.89 (m, 1 H), 2–88 - 2.88 (m, 1 H), 3.00 (dd, J =16.95, 4.57 Hz, 1 H), 3.70 (s, 4 H), 3.75 (s, 3 H), 4.80 (dt, J = 8.00, 4.50Hz, 1H), 5.66 (dd, J = 16.63, 2.38 Hz, 1 H), 6–02 - 6.12 (m, 1 H), 6.54 (br d, J = 7.88 Hz, 2 H).

[0641] General procedure for preparing compound 6 Add Py (846 mg, 10.7 mmol) and compound 5 (1.72 g, 8.55 mmol) in DCM (5 mL) to a solution of compound 4 (700 mg, 2.14 mmol) in DCM (5 mL). Stir the mixture at 25 °C for 1 hour. TLC indicated that compound 4 was completely consumed and a new spot was formed. Partition the reaction mixture between DCM (20 mL) and H2O (10 mL). Separate the organic phase using... saturation The sample was washed with citric acid aqueous solution (3 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to give compound 6 (490 mg, 46.5% yield). 1 HNMR (400MHz, CDCl3): δ ppm 1.17 (s, 3H), 1–55 - 1.61 (m, 1 H), 1.58 (br s, 1 H), 1.76 (dd, J= 14.76, 6.25 Hz, 1 H),1–87 - 2.03 (m, 3 H), 2–06 - 2.15 (m, 1 H), 2–19 - 2.41 (m, 3 H), 2.82 (dd, J =17.13, 4.50 Hz, 1 H), 3.03 (dd, J = 17.07, 4.44 Hz, 1 H), 3.72 (s, 3 H), 3.77(s, 3 H), 4–78 - 4.86 (m, 1 H), 5.67 (dd, J = 16.70, 2.44 Hz, 1 H), 6–03 - 6.14(m, 1 H), 6.58 (br d, J = 7.88 Hz, 1 H), 7–40 - 7.45 (m, 2 H), 8–27 - 8.33 (m, 2 H).

[0642] General procedure for preparing compound 7 DIEA (64.3 mg, 497 µmol) and HOBt (202 mg, 1.49 mmol) were added to a solution of compound 6 (490 mg, 995 µmol) and MMAE (714 mg, 995 µmol) in DMF (4 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed that compound 6 was completely consumed and a main peak with the desired mass was detected. The residue was purified by preparative HPLC (0.1% TFA conditions) to give compound 7 (500 mg, 46.9% yield). 1 HNMR (400MHz, CDCl3): δ ppm 0.84 (br d, J = 6.75 Hz, 4 H), 0.89 (br d, J = 4.50 Hz, 5 H), 0.92(br d, J = 6.63 Hz, 4 H), 0.98 (br d, J = 6.25 Hz, 3 H), 1.04 (br d, J = 6.88 Hz, 3H), 1.16 (s, 3 H), 1–25 - 1.27 (m, 3 H), 1–59 - 1.74 (m, 3 H), 1.88 (br d, J=9.38 Hz, 4 H), 2.07 (br d, J = 8.38 Hz, 5 H), 2.27 (br s, 4 H), 2–36 - 2.43 (m,2 H), 2–45 - 2.53 (m, 1 H), 2.89 (br s, 6 H), 2–95 - 3.01 (m, 4 H), 3.04 (brs, 2 H), 3–29 - 3.34 (m, 3 H), 3–36 - 3.47 (m, 5 H), 3–67 - 3.73 (m, 4 H), 3.76 (s, 3 H), 3–82 - 3.89 (m, 1 H), 4–05 - 4.19 (m, 3 H), 4.28 (br s, 1 H),4–63 - 4.86 (m, 3 H), 4.96 (d, J = 2.50 Hz, 1 H), 5.24 (br s, 1 H), 5.63 (br d, J = 18.14 Hz, 1 H), 5.82 (br s, 1 H), 6–53 - 6.74 (m, 3 H), 7–30 - 7.41 (m, 5H).

[0643] General procedure for preparing compound B To a solution of compound 7 (500 mg, 467 µmol) in MeOH (5 mL), LiOH·H₂O (196 mg, 4.67 mmol) in H₂O (2 mL) was added. The mixture was stirred at 25 °C for 16 hours. LC-MS showed that compound 7 was completely consumed and a main peak with the desired mass was detected. The residue was then... saturation The pH was adjusted to approximately 2 by an aqueous citric acid solution, and then purified by preparative HPLC (0.1% TFA conditions) to give compound B (265 mg, 53.4% ​​yield). 1 HNMR(400MHz, CDCl3): δ ppm 0–80 - 1.04 (m, 25 H), 1.09 (s, 3 H), 1.24 (d, J = 6.88Hz, 3 H), 1–69 - 1.82 (m, 2 H), 1–88 - 1.94 (m, 3 H), 2–02 - 2.11 (m, 4 H), 2.16 (br d, J= 18.64 Hz, 1 H), 2–11 - 2.24 (m, 2 H), 2.32 (br d, J = 5.25 Hz, 2H), 2–40 - 2.45 (m, 1 H), 2.52 (br d, J = 5.50 Hz, 2 H), 2.81 (br dd, J = 14.01,4.88 Hz, 1 H), 2–94 - 3.04 (m, 2 H), 3.08 (s, 2 H), 3–14 - 3.27 (m, 6 H), 3.33 (s, 1 H), 3.39 (s, 3 H), 3–48 - 3.57 (m, 2 H), 3.94 (br d, J = 1.25 Hz, 1H), 4–05 - 4.18 (m, 4 H), 4.30 (br dd, J = 6.19, 4.82 Hz, 2 H), 4–54 - 4.67 (m,4 H), 4.91 (br d, J = 2.00 Hz, 2 H), 5.30 (br s, 1 H), 5–64 - 5.73 (m, 1 H), 5–79 - 5.89 (m, 1 H), 6.61 (br d, J = 7.38 Hz, 1 H), 7–30 - 7.42 (m, 5 H), 7–55 -7.64 (m, 1 H).

[0644] Example 9: Alternative pathways for compound B and synthesis of compound C.

[0645] ` General procedure for preparing compound 2 A solution of compound 1 (20.0 g, 83.2 mmol) in MeOH (80 mL) was added to a solution of compound 1 in H₂O (80 mL) containing KOH (8.19 g, 124 mmol). The mixture was stirred at 25 °C for 24 hours. The reaction was monitored by TLC (compound 1, PE / EtOAc = 5 / 1, R...). f=0.5). The reaction mixture was extracted with MTBE (3 × 400 mL). The combined organic layers were washed with water (100 mL), dried over Na2SO4, filtered, and concentrated under vacuum to provide the undesirable ester. The aqueous layer was acidified with 1 M HCl until pH = 4 while cooling in an ice-water bath (T < 7 °C). The aqueous layer was extracted with MTBE (3 × 400 mL). The combined MTBE layers were dried over Na2SO4, filtered, and concentrated under vacuum to provide compound 2 (5.50 g, 35.9% yield). The crude product was ready for the next step without further purification. 1 H NMR: (400 MHz, DMSO- d6 ): δ ppm 11.9 (brs, 1 H), 5–81 - 5.94 (m, 1 H), 5.58 (dd, J = 16.45, 2.31 Hz, 1 H), 4.65 (br s,1 H), 4.24 (br s, 1 H), 2–04 - 2.24 (m, 2 H), 1–87 - 2.03 (m, 1 H), 1–61 -1.86 (m, 4 H), 1–36 - 1.46 (m, 1 H), 0.97 (s, 3 H).

[0646] General procedure for preparing compound 3 DIEA (39.3 g, 304 mmol) and DSC (47.8 g, 186.4 mmol) were added to a solution of compound 2 (8.00 g, 43.4 mmol) in MeCN (160 mL). The mixture was stirred at 40 °C for 14 hours. The reaction was confirmed to be complete by TLC (compound 2, DCM / MeOH = 10 / 1, R...). f =0.5). The reaction mixture was poured into water (400 mL), and the temperature was raised from 20 °C to 27 °C. After 15 min, the mixture was cooled to 17 °C in an ice-water bath and stirred for 15 min. The solid was filtered, washed with water (3 × 20 mL), and dried under vacuum at 35 °C for 4 h to give crude compound 3 (9.60 g). Acetonitrile (20 mL) was added to the crude product, and the mixture was heated at 40 °C for 1 h with mechanical stirring. Heating was stopped, and the mixture was cooled to 8 °C in an ice-water bath for 15 min. The solid was filtered, washed with acetonitrile (2 × 10 mL), and dried under vacuum at 35 °C for 3 h to give compound 3 (6.65 g, 36.3% yield). 1H NMR: (400 MHz, CDCl3): δ 6–03 - 6.14 (m, 1 H), 5–60 - 5.67 (m, 1 H), 5.29 (br s, 1 H), 2–80- 2.88 (m, 8 H), 2–25 - 2.47 (m, 4 H), 1–94 - 2.18 (m, 4H), 1.29 (s, 3H).

[0647] General procedure for preparing compound 4 MMAE (136 mg, 0.19 mmol) and DIEA (61.2 mg, 0.47 mmol) were added to a solution of compound 3 (100 mg, 0.24 mmol) in DMF (20 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed a main peak with the desired mass. The residue was purified by preparative HPLC (water (0.1% I-ACN)) to give compound 4 (41.0 mg, 16.9% yield). LCMS (m / z): 1025.6 (M+H) + .

[0648] General procedure for preparing compound B DIEA (378 mg, 2.93 mmol) and DMAP (119 mg, 0.97 mmol) were added to a solution of compound 4 (500 mg, 0.49 mmol) and compound 4-1 (519 mg, 3.90 mmol) in DMF (10 mL). The mixture was stirred at 25 °C for 12 h. LC-MS showed a main peak with the desired mass. The residue was purified by preparative HPLC (water (0.1 IFA)-ACN) to give compound B (161 mg, 31.6% yield). 1H NMR: (400 MHz, MeOD–: δ7.73 - 8.00 (m, 1–H), 7.20 - 7.39 (m, 4–H), 5.78 - 5.96 (m, 1 H), 5.73 (br s,1–H), 5.20 - 5.28 (m, 1–H), 5.13 - 3.31 - 3.50 (m, 9–H), 3.28 - 3.30 (m, 3–H), 2.77 - 3.12 (m, 6–H), 2.44 - 2.58 (m, 2–H), 1.78 - 2.36 (m, 13–H), 1.56 - 1.72 (m, 2–H), 1.22 - 1.48 (m,3–H), 1.08 - 1.23 (m, 9-H), 0.80 - 1.07 (m, 18 H). LCMS (m / z):1043.62 (M+H) + 1065.61 (M+Na) + .

[0649] General procedure for preparing compound C DIEA (242 mg, 1.87 mmol) and DMAP (76.3 mg, 0.62 mmol) were added to a solution of compound 4 (320 mg, 0.31 mmol) and compound 4-2 (187 mg, 2.50 mmol) in DMF (3.2 mL). The mixture was stirred at 25 °C for 12 h. LC-MS showed that compound 4 was completely consumed and a main peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound C (92.0 mg, 29.9% yield). 1H NMR (400 MHz, MeOD–: δ 7.86 - 8.00 (m, 1–H), 7.15 - 7.45 (m, 5–H), 5.67 - 5.98 (m, 2 H), 5.17 (br s, 1–H), 4.50 - 4.74 (m, 2–H), 4.03 - 4.29 (m,3–H), 3.81 - 3.89 (m, 2–H), 3.51 - 3.77 (m, 2–H), 3.46 - 3.50 (m, 1–H), 3.33- 3.45 (m, 5 H), 3.30 (br s, 4 H), 3.20 (dt, J = 11.57, 7.47 Hz, 1–H), 3.02 -3.15 (m, 3–H), 2.90 - 3.01 (m, 1–H), 2.41 - 2.57 (m, 2–H), 1.65 - 2.39 (m,15–H), 1.52 - 1.64 (m, 1–H), 1.26 - 1.51 (m, 2–H), 1.08 - 1.23 (m, 9–H), 0.82- 1.07 (m, 18 H). LCMS (m / z):985.6 (M+H) + .

[0650] Example 10: Synthesis of Compound D General procedure for preparing compound 6 Add (2,5-dioxopyrrolidone-1-yl)2,2,2-trichloroethyl carbonate (210 g, 723 mmol) in dioxane (1 L) to a solution of compound 5 (150 g, 689 mmol, HCl) in NaOH (1 M, 1.38 L) and NaHCO3 (1 M, 1.38 L). Stir the mixture at 25 °C for 2 hours. Concentrate the reaction mixture under reduced pressure to remove dioxane. Extract the residue with MTBE (5 L), then use the aqueous phase… saturationThe pH was adjusted to approximately 4 with an aqueous solution of KHSO4 and extracted with EtOAc (5 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. SOCl2 (90.2 g, 758 mmol) was added to a MeOH (2 L) solution of the crude product, and the mixture was stirred at 25 °C for 2 hours. LC-MS showed that the reaction was complete, and a main peak with the desired mass was detected. The reaction mixture was then... saturation The pH was adjusted to approximately 9 to 10 with an aqueous NaHCO3 solution, followed by extraction with EtOAc (5 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was precipitated through PE (10 volumes) to give compound 6 (190 g, 74.4% yield). 1 H NMR: (400 MHz, CDCl3): δ 3.25 (br s, 1 H) 3.85 (s, – H) 4.64 - 4.83 (m,2 H) 5.30 (dd, J = 9.51, 1.13 Hz, 1 H) 5.92 (br d, J = 9.38 Hz, - H) 7.30 - 7.45(m, 5 H). LCMS (m / z):391.9 / 393.9 (M+H) + .

[0651] General procedure for preparing compound 7 To a solution of compound 6 (185 g, 499 mmol) in toluene (1.9 L), pyridine 4-methylbenzenesulfonic acid (3.90 g, 15.4 mmol) and 4-methoxybenzaldehyde dimethyl acetal (121 g, 666 mmol) were added. The mixture was stirred at 110 °C for 4 h. LC-MS showed a main peak with the desired mass. The reaction mixture was then cooled to 25 °C and concentrated under reduced pressure to remove toluene. The residue was diluted with H₂O (500 mL) and then extracted with EtOAc (500 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give compound 7 (285 g, crude), which was used as is.

[0652] General procedure for preparing compound 8 To a solution of compound 7 (285 g, crude) in MeOH (2000 mL), KOH (42.5 g, 758 mmol) in H₂O (1000 mL) was added. The mixture was stirred at 25 °C for 1 hour. LC-MS showed that compound 7 was completely consumed and a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was extracted with MTBE (5 L). The aqueous phase was then analyzed using... saturation The mixture was diluted with 1 L of KHSO4 aqueous solution and extracted with 5 L of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was precipitated by passing it through PE (10 volumes) to give compound 8 (95.0 g, 34.3% yield). 1 H NMR (400 MHz, MeOD): δ 3.82 (s, – H) 4.41- 4.47 (m, – H) 4.50 – 4.56 (m, 1 H) 4.60 (d, J = 4.88 Hz, 1 H) 5.47 (d, J = 4.75Hz, 1 H) 6.46 (s, - H) 6.86 - 6.94 (m, - H) 7.34 - 7.46 (m, 7 H). LCMS (m / z):495.9 (M+Na) + .

[0653] General procedure for preparing 7-Troc-berry gibberellin III DMAP (625 mg, 5.11 mmol), pyridine (14.2 g, 179 mmol), and 2,2,2-trichloroethyl chloroformate (15.2 g, 71.6 mmol) were added to a solution of 7-Troc-7-gibberellin III (30.0 g, 51.1 mmol) in DCM (300 mL). The mixture was stirred at 25 °C for 0.5 h. LC-MS showed complete consumption of 7-Troc-7-gibberellin III and a main peak of the desired mass was detected. The residue was diluted with water (300 mL) and extracted with DCM (300 mL), washed with water (200 mL) and brine (200 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give 7-Troc-7-gibberellin III (45.0 g, 34.3% yield). LCMS (m / z): 761.5 / 763.5 (M+Na) + .

[0654] General procedure for preparing compound 9 DMAP (4.20 g, 34.1 mmol) and DCC (21.1 g, 102 mmol) were added to a solution of 7-Troc-berry gibberellin III (26.0 g, 34.1 mmol) and compound 8 (32.4 g, 68.2 mmol) in DCM (1000 mL). The mixture was stirred at 0 °C for 1 hour. LC-MS showed that compound 8 was completely consumed and a main peak with the desired mass was detected. The reaction mixture was filtered. The crude product was passed through... saturation The sample was washed with 100 mL of aqueous NH4Cl solution and 1000 mL of water, dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 9 (35.0 g, crude). LCMS (m / z): 1240.0 / 1242.0 (M+Na) + .

[0655] General procedure for preparing compound 10 4-Methylbenzenesulfonic acid; hydrate (24.9 g, 131 mmol) was added to a solution of compound 9 (80.0 g, 65.6 mmol) in MeOH (350 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed approximately 50% of compound 9 remaining, and a main peak with the desired mass was detected. The reaction mixture was filtered, concentrated, and the residue was purified by preparative HPLC (water (0.1% TFA)-ACN). The eluent was concentrated under reduced pressure to remove the solvent, and then extracted with EtOAc (500 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give compound 10 (13.0 g, 17.9% yield). LCMS (m / z): 1120.2 (M+Na) + .

[0656] General procedure for preparing compound 11 Compound 10 (13.0 g, 11.8 mmol), DMAP (722 mg, 5.90 mmol), EDCI (2.70 g, 14.2 mmol), and benzoic acid (1.70 g, 14.2 mmol) were added to a solution of DCM (260 mL). The mixture was stirred at 25 °C for 1 hour. LC-MS showed that compound 10 was completely consumed and a main peak with the desired mass was detected. The reaction mixture was washed with saturated aqueous citric acid solution (100 mL), saturated aqueous NaHCO3 solution (100 mL), and water (200 mL), dried over NaSO4, filtered, and concentrated under reduced pressure to give compound 11 (11.0 g, 77.3% yield). LCMS (m / z): 1204.1 (M+H) + .

[0657] General procedure for preparing compound 12 Zn powder (21.6 g, 331 mmol) was added to a solution of compound 11 (20.0 g, 16.6 mmol) in MeOH (200 mL) and AcOH (200 mL). The mixture was stirred at 25 °C for 1 hour. LC-MS showed that compound 11 was completely consumed and a main peak with the desired mass was detected. The reaction mixture was filtered and diluted with H2O (500 mL), then with EtOAc (100 mL). 3) Extraction. The combined organic layers are then extracted using... saturation The sample was washed with 200 mL of NaHCO3 aqueous solution and 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound 12 (5.0 g, 21% yield). LCMS (m / z): 854.3 (M+H) + .

[0658] General procedure for preparing compound 13 Compound 12 (5.00 g, 5.90 mmol), DIEA (1.50 g, 11.7 mmol), and compound 3 (3.90 g, 8.80 mmol) were added to a solution of DMF (50 mL). The mixture was stirred at 25 °C for 16 hours. LC-MS showed approximately 50% of compound 12 remaining, and a main peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound 13 (505 mg, 7.4% yield). LCMS (m / z): 1161.4 (M+H) + .

[0659] General procedure for preparing compound D DMAP (94.7 mg, 0.78 mmol), compound 13-1 (66.7 mg, 0.65 mmol), and DIEA (100 mg, 0.78 mmol) were added to a solution of compound 13 (150 mg, 0.13 mmol) in DMF (1.50 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed that compound 13 was completely consumed and a main peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound D (75.0 mg, 50.5% yield). LCMS (m / z): 1148.5 (M) + .

[0660] Example 11: A general procedure for preparing compound E Compound 13 (350 mg, 0.30 µmol), DMAP (221 mg, 1.81 mmol), and compound 14 (249 mg, 0.39 mmol, HCl) were added to a solution of DMF (0.3 mL). The mixture was stirred at 25 °C for 16 h. LC-MS showed that compound 13 was completely consumed and a main peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound E (205 mg, 41.3% yield). LCMS (m / z): 1646.5 (M+H) + .

[0661] Example 12: Synthesis of ixenostatin prodrug compound F Procedure for preparing compound 2 NaOMe (5.40 M, 105 mL, 5.5 equivalents) in H2O (125 mL) was added to a solution of methyl compound 1 (25.0 g, 104 mmol, 1.0 equivalent) in MeOH (125 mL). The mixture was stirred at 25 °C for 24 h. The reaction was monitored by TLC (PE:EA = 2:1, product R...). f =0.20) indicator reactant (R f =0.60) was consumed. The reaction mixture was diluted with H2O (50 mL) and extracted with MTBE (4 × 500 mL). The aqueous layer was acidified with 1 M HCl until pH = 4 while cooling in an ice-water bath (T < 7 °C). The aqueous layer was extracted with MTBE (5 × 500 mL). The combined MTBE layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was then evaporated three times with MeCN to give compound 2 (9.1 g, 49.3 mmol, 47.4% yield). 1 H NMR: 400 MHz, CDCl3 δ ppm 1.11 (s, 3 H), 1.65 (brdd, J = 15.82, 6.19 Hz, 1–H), 1.78 - 2.02 (m, 4–H), 2.05 - 2.42 (m, 4 H), 4.49(br s, 1 H), 5.64 (dd, J = 16.63, 2.00 Hz, 1–H), 6.00 - 6.14 (m, 1 H).

[0662] Procedure for preparing compound 3 DIEA (32.4 g, 250 mmol, 43.6 mL, 7.0 equivalents) and DSC (39.4 g, 154 mmol, 4.3 equivalents) were added to a solution of compound 2 (6.6 g, 35.8 mmol, 1.0 equivalents) in MeCN (130 mL). The mixture was stirred at 25 °C for 16 hours. The reaction was monitored by TLC (DCM:MeOH = 10:1, product R...). f =0.60) indicator reactant (R f=0.20) was consumed. The reaction mixture was purified at 25 °C by recrystallization from H2O (350 mL), filtered, and concentrated under reduced pressure to give the residue. The crude product was evaporated three times with MeCN to give compound 3 (10.0 g, 21.6 mmol, 60.4% yield, 91.4% purity). HPLC: R t =2.14 min, purity: 91.4%. 1 H NMR: 400 MHz, CDCl3 δ ppm1.28 (s, 3–H), 1.97 - 2.17 (m, 4–H), 2.26 - 2.49 (m, 4–H), 2.82 - 2.86 (m, 8H), 5.29 (br s, 1 H), 5.63 (dd, J = 16.70, 2.19 Hz, 1–H), 6.02 - 6.15 (m, 1 H).

[0663] Procedure for preparing compound 5 DIEA (4.04 g, 31.2 mmol, 5.44 mL, 2.0 equivalent) and eczemab (6.23 g, 11.7 mmol, 0.75 equivalent) were added to a solution of compound 3 (6.6 g, 15.6 mmol, 1.0 equivalent) in DMF (70 mL). The mixture was stirred at 25 °C for 1 h. LC-MS (EC17183-7-P1A1) showed a main peak with the desired mass (RT = 0.44 min). The reactants were filtered and subjected to preparative HPLC (column: Phenomenex luna C18 (250 μL)). 70 mm, 10 μm); mobile phase: [wIr(FA)-ACN]; gradient: 40% to 70% B) purification to give compound 5 (6.0 g, 7.76 mmol, 49.6% yield, 96.1% purity).

[0664] LCMS (Monitoring System): R t =0.44 min, MS calculated value: 742.2, MS observed value: [M+H] + =743.1.

[0665] LCMS: R t =0.44 min, MS calculated value: 742.2, MS observed value: [M+H] + =743.3.

[0666] HPLC: R t =3.04 min, purity: 96.1%.

[0667] Procedure for preparing compound F DIEA (2.85 g, 22.0 mmol, 3.85 mL, 2.0 equivalent) and DMAP (2.70 g, 22.08 mmol, 2.0 equivalent) and compound 5 (8.2 g, 11.04 mmol, 1 equivalent) were added to a solution of compound 5-1 (9.95 g, 16.5 mmol, 1.5 equivalent) in DMSO (40 mL). The mixture was stirred at 25 °C for 16 h. LC-MS (EC17183-21-P1A11) showed a main peak (R0) with the desired mass. t =0.39 min). The reaction mixture was filtered, purified by preparative HPLC (FA conditions) and exchanged for AcOH salt to give compound F (7.5 g, 5.89 mmol, 53.3% yield, 96.5% purity).

[0668] LCMS (Monitoring System): R t =0.39 min, MS calculated value: 1227.5, MS observed value: [M+H] + =1228.8.

[0669] LCMS: R t =0.38 min, MS calculated value: 1227.5, MS observed value: [M+H] + =1228.7.

[0670] HPLC: R t =2.28 min, purity: 96.5%.

[0671] Example 13: Synthesis of MMAE-TCO compound G DMAP (95.3 mg, 780 μmol), compounds 1-3 (175 mg, 292 μmol), and DIEA (129 μL, 780 μmol) were added to a solution of compounds 1-2 (100 mg, 97.5 μmol) in DMF (1.00 mL). The mixture was stirred at 25 °C for 24 h. LC-MS showed a main peak with the expected mass (MS calculated value: 1510.91, MS observed value: [M+H)). +=1512.1). The mixture was filtered and purified by preparative HPLC (AcOH conditions) to give compound G as a white solid (44 mg, 29.8% yield).

[0672] LCMS: MS calculated value: 1510.91, MS observed value: [M+H] + =1511.1.

[0673] HPLC: R t =6.981 min, purity: 95.3% Example 14: Synthesis of MMAE-TCO compound H DMAP (95.3 mg, 780 μmol), compound 2-1 (97.6 mg, 780 μmol), and DIEA (129 μL, 780 μmol) were added to a solution of compounds 1-2 (100 mg, 97.5 μmol) in DMF (1.00 mL). The mixture was stirred at 25 °C for 16 hours. LC-MS showed a main peak with the expected mass (MS calculated value: 1035.3, MS observed value: [M+H)). + =1036.6). The mixture was filtered and purified by preparative HPLC to give compound H as a white solid (20 mg, 19.8% yield).

[0674] LCMS: MS calculated value: 1034.6, MS observed value: [M+H] + =1035.8.

[0675] HPLC: R t =4.566 min, purity: 95.06% Example 15: Synthesis of MMAE-TCO compound I DMAP (47.7 mg, 390 μmol), compound 3-1 (48.4 mg, 390 μmol), and DIEA (129 μL, 390 μmol) were added to a solution of compounds 1-2 (50.0 mg, 48.7 μmol) in DMF (0.50 mL). The mixture was stirred at 25 °C for 24 h. LC-MS showed a main peak with the expected mass (MS calculated value: 1033.6, MS observed value: [M+H)). + =1035.1). The mixture was filtered and purified by preparative HPLC to give compound I as a white solid (19 mg, 37.7% yield).

[0676] LCMS: MS calculated value: 1033.6, MS observed value: [M+H] + =1034.8.

[0677] HPLC: R t =5.863 min, purity: 96.39% Example 16: Synthesis of MMAE-TCO compound J DMAP (47.7 mg, 390 μmol), compound 4-1 (48.8 mg, 390 μmol), and DIEA (129 μL, 390 μmol) were added to a solution of compounds 1-2 (50.0 mg, 48.7 μmol) in DMF (0.50 mL). The mixture was stirred at 25 °C for 24 h. LC-MS showed a main peak with the expected mass (MS calculated value: 1034.6, MS observed value: [M+H)). + =1036.2). The mixture was filtered and purified by preparative HPLC to give compound J as a white solid (37 mg, 73.3% yield).

[0678] LCMS: MS calculated value: 1034.6, MS observed value: [M+H] + =1035.8.

[0679] HPLC: R t =7.609 min, purity: 95.48% Example 17: A general procedure for preparing intermediate 5-1B Triphosgene (405 mg, 1.36 mmol) was added fractionally over 5 minutes to a solution of tert-butyl 3-hydroxypropionate (0.50 g, 3.42 mmol) and pyridine (552 μL, 6.84 mmol) in THF (10.0 mL). The mixture was stirred at 0 °C for 1 hour. TLC indicated that reactant 1 was completely consumed and a new spot was formed. Aliquots of the reaction mixture were quenched with benzylamine and the desired mass was determined by LCMS (MS calculated value: 279.15, MS observed value: [M+H-56]). +=224.1). The reaction mixture was diluted with DCM (20 mL), washed with 0.5 M HCl (20 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. Intermediate 5-1B (520 mg, 2.49 mmol, 72.8% yield) was given as a colorless oil. The residue was used directly in the next step without purification.

[0680] Example 18: A general procedure for preparing intermediate 5-3B At 0 °C, DMAP (304 mg, 2.49 mmol) and 5-2 C (520 mg, 2.49 mmol) were added to a solution of 5-1B (321 mg, 1.24 mmol) in THF (10 mL). The mixture was stirred at 25 °C for 2 hours. LC-MS showed that 5-2 C was completely consumed and a main peak with the expected mass was detected (MS calculated value: 430.1, MS observed value: [M+Na)). + =453.1). The reaction mixture was filtered and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel 12 g, PE / EA = 0% to 100%) to give the product. Intermediate 5-3B was given as a colorless oil (502 mg, 1.16 mmol, 93.5% yield). LCMS: MS calculated value: 430.1, MS observed value: [M+Na] + =453.1. 1 H NMR: (400 MHz, CDC–3) δ: 7.35 - 7.22 (m, 5H), 5.38 (br d, J = 6.3 Hz, 1H), 5.04 (s, 2H), 4.35 (t, J = 6.1 H–, 2H), 3.71 - 3.60 (–, 2H), 3.59 - 3.51 (m, 2H), 2.52 (t, J =6.1 Hz, 2H), 1.38 (s, 9H).

[0681] Example 19: A general procedure for preparing intermediate 5-3C At 0 °C, HCl / dioxane (5 mL, 2 M) was added to a mixture of 5-3B (500 mg, 1.16 mmol), and the mixture was stirred at 25 °C for 48 hours. LC-MS showed that 5-3B was completely consumed, and a main peak with the expected mass was detected (MS calculated value: 374.1, MS observed value: [M+Na)). + =397.0). The reaction mixture was filtered and concentrated under reduced pressure to give a colorless oil. Intermediate ...

Claims

1. A method for forming in subjects who require this. in vivo A method for antibody-load conjugates, the method comprising: The subject is given an effective amount of the targeting portion, wherein the targeting portion comprises at least one antibody or a fragment thereof, the at least one antibody or a fragment thereof having at least one tetrazine portion covalently linked thereto; The subject is given a single dose of a therapeutically effective amount of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently linked thereto. The antibody or fragment thereof has binding affinity to a receptor on a tumor, and further wherein the ratio of the antibody-load conjugate at the tumor site to the antibody-load conjugate in plasma is greater than about 2:

1.

2. The method of claim 1, wherein the ratio of the antibody-load conjugate at the tumor site to the antibody-load conjugate in plasma is greater than about 5:1, about 8:1, or about 10:

1.

3. The method according to claim 1 or 2, wherein the application is sequential.

4. The method according to any of the preceding claims, wherein a single dose of the payload-TCO conjugate is administered to the subject between about 2 hours and about 48 hours, or about 3 hours and about 48 hours, or about 4 hours and about 48 hours after administration of the targeted portion to the subject.

5. The method according to any of the preceding claims, wherein the targeting portion is applied at least about 8 hours to about 24 hours before the application of the payload-TCO conjugate.

6. A method for administering a payload to a subject, the method comprising: a) Administering an effective amount of the target portion to the subject, wherein the target portion comprises Fab, the Fab having at least one tetrazine portion covalently linked thereto; as well as b) Administering a single therapeutically effective amount of the payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto; The single dose of the payload-TCO conjugate is administered to the subject between approximately 2 hours and approximately 48 hours, or approximately 3 hours and approximately 48 hours, or approximately 4 hours and approximately 48 hours after the administration of the targeted portion.

7. A method for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the target portion to the subject, wherein the target portion comprises Fab, the Fab having at least one tetrazine portion covalently linked thereto; as well as b) Administering a single therapeutically effective amount of the payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently linked thereto; The single dose of the payload-TCO conjugate is administered to the subject between approximately 2 and 48 hours, or approximately 3 and 48 hours, or approximately 4 and 48 hours after the administration of the targeted portion.

8. The method of claim 7 or 8, wherein a single dose of the payload-TCO conjugate is administered to the subject between approximately 8 hours and approximately 24 hours after administration of the targeted portion.

9. The method of claim 7 or 8, wherein a single dose of the payload-TCO conjugate is administered to the subject between approximately 8 and approximately 22 hours, between approximately 8 and approximately 16 hours, between approximately 8 and approximately 22 hours, between approximately 12 and approximately 16 hours, or between approximately 16 and approximately 20 hours after administration of the targeted portion to the subject.

10. The method according to any preceding claim, wherein the targeting portion has Formula I, Formula II, or Formula 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 an antibody fragment; p ranges from 1 to 20; L is a connector independently each time it appears; R 1 Each time it appears, independently select from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, ynyl, 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 defined by one to three Z 1 replace; R 2 Each time it appears independently of halogen, cyano, nitro, hydroxy, 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 defined 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 20 Each time it appears, it is independently selected from the group consisting of the following: hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, -CF3, -CF2R', -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 occurrence is an independent linker of 1 to 100 connecting atoms, the linker optionally comprising 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 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 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.

11. The method according to any preceding claim, wherein the targeting portion has formula I or II: in: X represents an antibody fragment; p ranges from 1 to 16; L is a connector independently 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 occurrence is an independent linker of 1 to 100 connecting atoms, the linker optionally comprising 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; 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 t is independently 0, 1, 2, 3 or 4 each time it appears.

12. The method according to any preceding claim, wherein the targeting portion has formula IIA: IIA。 13. The method according to any one of claims 10 to 12, wherein each R 20 It can be hydrogen or alkyl independently.

14. The method of claim 10, wherein the targeting portion has formula VII: VII。 15. The method according to claim 10 or 11, wherein 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.

16. The method according to claim 10 or 11, wherein ring A is phenyl.

17. The method according to any one of claims 10 to 16, wherein p is 1 to 16, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2.

18. The method according to any one of claims 10 or 14 to 17, wherein each R 1 It can be hydrogen or alkyl independently.

19. The method according to any one of claims 14 to 18, wherein each R 2 It is independently a halo, alkyl, or haloalkyl group.

20. The method according to any one of claims 14 to 18, wherein t is 0 each time it occurs.

21. The method according to any one of claims 5 to 16, wherein X is an antibody fragment targeting the following: TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, cohesin 4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

22. The method according to any one of claims 10 to 21, wherein X is an antibody fragment derived from: daklizumab, RG6292, baliximab, HuMax-TAC, labezizumab, 15-1-32, PR1A3, cT84.66, tasicitumab, CC4, PAN-622, cetuximab, nixituzumab, nimotuzumab, mateuzumab, AMG595, deperazine. Cialis, Daperazolizumab, Dugoutuzumab, Votoxizumab, GC1118, Imtracum, Panitumumab, Alutuzumab, Toltuzumab, Latoxizumab, Moozumab, Sitaturuzumab, Tocozizumab, Caputoxumab, Ezekielumab, Ademumab, Ramoximumab, Ramorumab, Vulinaxizumab, Olatoxizumab, Ramorumab, Saxitoxizumab, Pr1E11, Envertuzumab J591, MLN591, Belantuzumab, Mosetuzumab, Intocilizumab, Iprazumab, Pinatuzumab, Utoximab, Ofamumab, Rituximab, Obinutuzumab, Tosimomab, Tiimumab, Rontoximab, XMAB-5574, MOR208, Cortuzumab, Diphentoximab, Taritumab, MDX-1342, Polazumab, Isaltuzumab, Daremucil Monoclonal antibodies, MOR202, TAK-079, I-131-BC8, Iomab-B, caputuximab, bematuximab, apratuximab, rupatuximab, zotocinimab, clodioxanum, anddaliximab, mituximab, faletocinimab, MORAb-202, MORAb-003, SP8166, lovatocinimab, indextuximab, lovotocinimab, propimizumab, 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, Lintocizumab, KHK2823, Tacrolimus, G4723A, Gabatocizumab, Teritolus, Onatuzumab, SAIT301, Tesxotuzumab, Lifatocizumab, Indojutumab, Vantocizumab, Sofostocizumab, Voseltuzumab, Bivaliruzumab, Karacizumab, Orizizumab, V565, PF-05230905, Wabalizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, Sirolizumab, F16, R6N, Dapoxetine, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, 74HZ.

23. The method according to any one of claims 1 to 22, wherein the targeting portion further comprises an imaging contrast agent.

24. The method of claim 23, wherein the imaging contrast agent is a protein.

25. The method according to any one of claims 10 to 24, wherein L is independently bonded to X via a cystine or lysine residue on X each time it appears.

26. The method according to any one of claims 10 to 25, wherein each L comprises one or more amino acids.

27. The method according to any one of claims 10 to 26, wherein each L comprises a polypeptide.

28. The method according to any one of claims 10 to 27, wherein each L independently 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.

29. The method according to any one of claims 10 to 28, wherein each L independently comprises 5 to 50 linking atoms; comprises one or more chain heteroatoms and one or more alkylene, alkenylene, ynylene, arylene, or heteroaryl groups; wherein each alkylene, alkenylene, ynylene, arylene, or heteroaryl group may be independently and optionally substituted by one to five substituents independently selected from: oxo, halogenated, C-substituted ... 1-4 Alkyl, C 1-4 Alkoxy and C 1-4 Halogenated alkyl groups.

30. The method according to any one of claims 10 to 29, wherein X is an antibody fragment targeting the following: HER2, TROP2, cohesin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC or ECM, EPCAM, CEA or CEACAM5; and each L is independently selected from the group consisting of: , , , , and .

31. The method according to any one of claims 10 to 14, wherein X is an antibody fragment targeting HER2; p is 1 to 5; and each L is independently selected from the group consisting of: , , , , and .

32. The method according to any preceding claim, wherein the targeting portion has the formula IIF: IIF。 33. The method of claim 32, wherein X is an antibody fragment targeting HER2; and p is 1 to 5.

34. The method according to any preceding claim, wherein the payload-TCO conjugate has formula VIII or a pharmaceutically acceptable salt thereof: VIII in: G is independent each time it appears. ; L 1 Each time it appears, it is a connector independently; m is an integer from 1 to 150; D represents the payload; R 1A Each time it appears, independently select the group consisting of the following items: C 1-4 Alkyl, C 1-4 Halogenated alkyl groups 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 the 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) 1c CHR 1e C(O)OC 1-6 Alkyl, -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) 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, -N(R) 1c )-C 1-6 Alkylene-C(O)OC 1-6 Alkyl 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 an optionally substituted heterocyclic group.

35. The method according to any of the preceding claims, wherein the effective payload is paclitaxel, doxorubicin, doxorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixaprilone, partoprilone, platinum-based drugs, eczemaconazole, delutec, serotonin 10, MMAE, MMAD, MMAF, mitomycin C, bleomycin, carrichomycin, astrospore, hexasporin, seco-DUBA, pyroxine, cyclosporine A, or rapamycin.

36. The method according to any preceding claim, wherein the payload-TCO conjugate is selected from: , , , , , , , , , , , , , and .

37. A method of administering a therapeutically effective amount of monomethylolpropamine E (MMAE) to a subject suffering from cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion has the formula IIF: IIF Where X is a trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); or X is a sacitrullab Fab containing (SEQ ID NO.9) and (SEQ ID NO.10); and p is 1 to 5; as well as b) Administer a single therapeutically effective dose of the MMAE-TCO conjugate having the following structure to the subject: ； The single dose of the MMAE-TCO conjugate is administered to the subject between approximately 2 hours and approximately 48 hours or between approximately 8 hours and approximately 24 hours after the administration of the targeted portion.

38. A method for treating cancer or enhancing or inducing an immune response in a subject with cancer, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion has the formula IIF: IIF Where X is a trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); or X is a sacitrullab Fab containing (SEQ ID NO.9) and (SEQ ID NO.10); and p is 1 to 5; as well as b) Administer a single therapeutically effective dose of the MMAE-TCO conjugate having the following structure to the subject: ； The single dose of the MMAE-TCO conjugate is administered to the subject between approximately 2 hours and approximately 48 hours or between approximately 8 hours and approximately 24 hours after the administration of the targeted portion.

39. A method for reducing the tumor volume of a subject with a tumor, the method comprising: a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion has the formula IIF: IIF Where X is a trastuzumab Fab containing (SEQ ID NO.3) and (SEQ ID NO.4); or X is a sacitrullab Fab containing (SEQ ID NO.9) and (SEQ ID NO.10); and p is 1 to 5; as well as b) Administer a single therapeutically effective dose of the MMAE-TCO conjugate having the following structure to the subject: ； The single dose of the MMAE-TCO conjugate is administered to the subject between approximately 2 hours and approximately 48 hours or between approximately 8 hours and approximately 24 hours after the administration of the targeted portion.

40. The method according to any one of claims 37 to 39, wherein a single dose of the MMAE-TCO conjugate is administered to the subject between approximately 8 hours and approximately 24 hours after administration of the targeted portion to the subject.

41. The method according to any one of claims 37 to 39, wherein a single dose of the MMAE-TCO conjugate is administered to the subject between about 8 hours and about 12 hours, between about 8 hours and about 16 hours, between about 8 hours and about 22 hours, between about 12 hours and about 16 hours, or between about 16 hours and about 20 hours after the administration of the targeted portion to the subject.

42. The method according to any preceding claim, wherein the subject has cancer, and the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal cancer / 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.

43. The method of claim 42, wherein the cancer is a solid tumor.

44. The method of claim 42, wherein the cancer is breast cancer, lung cancer, or stomach cancer.

45. The method according to claim 42, wherein the cancer is a hematologic malignancy, such as myelodysplastic syndrome, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal globulinosis, 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 Hodgkin lymphoma predominantly composed of nodular lymphocytes.

46. ​​The method according to any of the preceding claims, wherein the method further comprises administering a therapeutically effective amount of an additional therapeutic agent.

47. The method of claim 46, wherein the additional therapeutic agent is selected from the group consisting of anticancer agents or immunomodulators.

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