Method for targeted delivery of payload in vivo

Abstract

This disclosure generally relates to methods utilizing antibody-tetrazine conjugates for bioorthogonal delivery of payloads to target sites in a subject, the conjugates having applications, for example, in cancer treatment, tumor growth, and immunotherapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application asserts interests under Section 119(e) of the United States Patent Act in relation to U.S. Provisional Patent Application No. 63 / 583,189 filed on 15 September 2023, No. 63 / 660,911 filed on 17 June 2024, and No. 63 / 681,755 filed on 9 August 2024, all of which are incorporated in their entirety by reference.

[0002] This disclosure relates to a method of utilizing an antibody-tetrazine conjugate for bioorthogonal delivery of a payload to a target site in a subject, the conjugate having applications, for example, in cancer treatment, tumor growth, and immunotherapy. [Background technology]

[0003] Bioorthogonal conjugations, or click reactions, are selective and orthogonal (non-interacting) functions found in biological systems that have found use in a variety of applications in the fields of chemistry, chemical biology, molecular diagnostics, and medicine. They can be used to facilitate the selective manipulation of molecules, cells, particles, and surfaces, as well as the tagging and tracking of biomolecules in vitro and in vivo. These reactions include Staudinger ligation, azido-cyclooctin ring addition, and reverse electron-demanded Diels-Alder reactions. [Overview of the project]

[0004] This disclosure relates to a method for delivering a payload to a target location in an object. In one embodiment, a method is provided for forming an antibody-payload conjugate in vivo in an object requiring an antibody-payload conjugate, the method being: Administering an effective amount of a targeted portion, wherein the targeted portion comprises at least one antibody or fragment thereof having at least one tetrazine portion covalently bound thereto. The administration of a single-dose therapeutically effective payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently bound thereto. The antibody or its fragments have binding affinity to receptors on the tumor, and furthermore, the amount of antibody-payload conjugate formed in vivo is greater at the tumor site compared to the plasma.

[0005] In one embodiment, a method is provided for administering a therapeutically effective payload, and this method is: a) Administering an effective amount of the targeted portion, wherein the targeted portion comprises a Fab having at least one tetrazine portion covalently bound thereto. b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1-3, 1-2, or 1) trans-cyclooctene moiety covalently bound thereto, The single-dose payload-TCO conjugate, which includes the target portion, is administered to the subject within a time frame of approximately 2 to 48 hours, 3 to 48 hours, or 4 to 48 hours after the target portion is administered to the subject.

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

[0007] The targeted portion for use in the methods disclosed herein is designed to localize to a target site within the subject upon administration. The targeted portion can be administered topically or systemically. Upon administration, a prodrug comprising a payload and one or more complementary bioorthogonal components (i.e., trans-cyclooctene moieties) is administered, which, upon contact with the targeted portion in vivo, enables targeted delivery of the payload or therapeutic agent.

[0008] In one embodiment, a method is provided for treating cancer or enhancing or inducing an immune response in a subject having cancer, and this method is: a) Administering an effective amount of a targeted portion, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine portions covalently bound thereto, b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1-3, 1-2, or 1) trans-cyclooctene moiety covalently bound thereto, The single-dose payload-TCO conjugate, which includes the targeting portion, is administered to the subject more than 1 hour but less than 48 hours after the targeting portion has been administered to the subject.

[0009] In one embodiment, a method is provided for treating cancer or enhancing or inducing an immune response in a subject having cancer, and this method is: a) Administering an effective amount of a targeted portion, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine portions covalently bound thereto, b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1-3, 1-2, or 1) trans-cyclooctene moiety covalently bound thereto, The single-dose payload-TCO conjugate, which includes the targeting portion, is administered to the subject more than 4 hours but less than 48 hours after the targeting portion has been administered to the subject.

[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, 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 lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

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

[0012] In some embodiments, a targeting portion of formula IIF is provided, [ka] In the formula, p is 1 to 10, and X is the antibody fragment portion containing SEQ ID NOs: 9 and SEQ ID NOs: 10. In some embodiments, p is 1 to 5.

[0013] In one embodiment, a method is provided for reducing the tumor volume in a subject having a tumor, and this method is a) Administering an effective amount of a targeted portion, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine portions covalently bound thereto, b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1-3, 1-2, or 1) trans-cyclooctene moiety covalently bound thereto, The single-dose payload-TCO conjugate, which includes the targeting portion, is administered to the subject more than 1 hour but less than 48 hours after the targeting portion has been administered to the subject.

[0014] In one embodiment, a method is provided for reducing the tumor volume in a subject having a tumor, and this method is a) Administering an effective amount of a targeted portion, wherein the targeted portion comprises a Fab having at least one (e.g., 1 to 5, 1 to 4, or 1 to 3) tetrazine portions covalently bound thereto, b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one (e.g., 1-3, 1-2, or 1) trans-cyclooctene moiety covalently bound thereto, The single-dose payload-TCO conjugate, which includes the targeting portion, is administered to the subject more than 4 hours but less than 48 hours after the targeting portion has been administered to the subject. [Brief explanation of the drawing]

[0015] [Figure 1A] The tumor volume of NCI-N87 tumors in SCID CB17 mice after treatment with the vehicle, compound TM-1, and compound B, as well as with the isotype Fab-Tz and compound B. Compound B was administered 4 hours after administration of compound TM-1. N=5 mice per group. [Figure 1B] The tumor volume of NCI-N87 tumors in SCID CB17 mice after treatment with the vehicle, compound TM-1, and compound B, as well as with the isotype Fab-Tz and compound B. Compound B was administered 8 hours after administration of compound TM-1. N=5 mice per group. [Figure 1C] The tumor volume of NCI-N87 tumors in SCID CB17 mice after treatment with the vehicle, compound TM-1, and compound B, as well as with the isotype Fab-Tz and compound B. Compound B was administered 24 hours after administration of compound TM-1. N=5 mice per group. [Figure 1D] The tumor volume of NCI-N87 tumors in SCID CB17 mice after treatment with the vehicle, compound TM-1, and compound B, as well as with the isotype Fab-Tz and compound B. Compound B was administered 48 hours after administration of compound TM-1. N=5 mice per group. [Figure 2A] This shows the schedule for medication administration and tumor collection. [Figure 2B] This study demonstrates that compound TM-1 activates compound B, leading to the release of MMAE within the tumor 15 minutes after administration. [Figure 3A] This shows the quantification of total Fab in HER2-positive tumors. [Figure 3B] This shows that compound TM-1 is localized to HER2-positive tumors. [Figure 4A] This shows the total Fab detected at the tumor site relative to the plasma. [Figure 4B] This shows the total tetrazine detected at the tumor site relative to the plasma. [Figure 5] This indicates that Fab exposure was prolonged in both plasma and tumors (Tz levels decreased rapidly in plasma but were prolonged in tumors). [Figure 6] This shows the total tetrazine detected at the tumor site at a specific time interval. [Figure 7] This shows the total Fab detected at the tumor site over a specific time interval. [Figure 8] This shows the ratio of tetrazine (Fab-biotin) in the tumor site to the surrounding tissue. [Figure 9] This shows the total Fab ratio in the tumor site relative to the tissue. [Figure 10] This shows preferential accumulation of compound TM-1 in tumors compared to plasma. [Figure 11] This shows preferential accumulation of compound TM-1 in tumors compared to plasma. [Modes for carrying out the invention]

[0016] The following description illustrates exemplary embodiments of the Technology. However, such descriptions should be understood not as limiting the scope of the Disclosure, but rather as being provided solely for illustrative purposes.

[0017] definition For clarity, it is understood that certain features of the Disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure described in the context of a single embodiment may also be provided separately or in any preferred partial combination. All combinations of embodiments belonging to the Disclosure are specifically encompassed by the Disclosure and are disclosed herein as if each and all combinations were individually and expressly disclosed to the extent that such combinations encompass a subject, for example, a stable compound (i.e., a compound that can be prepared, isolated, characterized, and tested for biological activity). Furthermore, all partial combinations of various embodiments and their elements (e.g., elements of chemical groups enumerated in embodiments describing such variables) are also specifically encompassed by the Disclosure and are disclosed herein as if each and all such partial combinations were individually and expressly disclosed herein.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, this specification, including its definitions, shall prevail. Exemplary methods and materials are described below, but similar or equivalent methods and materials may be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0019] The terms “comprise(s),” “include(s),” “have,” “possess,” “can have,” and “contain,” and their variations, when used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an,” and “the” include multiple references unless the context otherwise explicitly indicates. This disclosure also contemplates other embodiments that “include,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly described or not.

[0020] The modifier "approximately" used in relation to quantity includes the stated value and has a meaning determined by the context (for example, at least the degree of error associated with measuring a particular quantity). The modifier "approximately" should also be considered to disclose a range defined by the absolute values ​​of two endpoints. For example, the expression "approximately 2 to approximately 4" also discloses a range of "2 to 4". The term "approximately" can refer to plus or minus 10% of the stated number. For example, "approximately 10%" may indicate a range of 9% to 11%, and "approximately 1" may mean 0.9 to 1.1. Other meanings of "approximately" may become apparent from contexts such as rounding, so for example, "approximately 1" may also mean 0.5 to 1.4.

[0021] The conjunction “or” includes any and all combinations of one or more enumerated elements associated with the conjunction. For example, the phrase “a device containing A or B” could mean a device containing A without B, a device containing B without A, or a device containing both A and B. The phrase “at least one of A, B, ... and N” or “at least one of A, B, ... N, or any combination thereof” is defined in its broadest sense to mean one or more elements selected from the group containing A, B, ... and N, i.e., any combination of one or more elements A, B, ... or N that contains any one element alone or in combination with one or more of the other elements, and may also include additional elements not enumerated.

[0022] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are defined as follows: Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Identified according to the ed. and inside cover, specific functional groups are generally defined as described therein. Additionally, for general principles of organic chemistry, as well as specific functional parts and reactivity, see *Organic Chemistry*, Thomas Sorrell, University Science Books, Sausalito, 1999, and *Smith and March March's Advanced Organic Chemistry*, 5 th 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, 3 rdThis information is contained in Edition, Cambridge University Press, Cambridge, 1987, and its entirety is incorporated herein by reference.

[0023] As used herein, the term "alkyl" means a straight or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. 1- The term "C6-alkyl" refers to a linear or branched hydrocarbon containing 1 to 6 carbon atoms. The term "C1-C3-alkyl" refers to a linear or branched hydrocarbon containing 1 to 3 carbon atoms. Typical examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

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

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

[0026] As used herein, the term "alkynyl" refers to a linear or branched monovalent hydrocarbyl group having 2 to 30 carbon atoms, e.g., 2 to 20 or 2 to 10 carbon atoms, and having at least one triple-unsaturated site. The term "alkyne" also includes non-aromatic cycloalkyl groups having 5 to 20 carbon atoms, e.g., 5 to 10 carbon atoms, and having one or more rings and at least one triple bond. Examples of such alkynyl groups include, but are not limited to, acetylenyl (-C≡CH) and propargyl (-CH2C≡CH), and cycloalkynyl moieties such as substituted or unsubstituted cyclooctin moieties.

[0027] When used herein, the term "alkoxyalkyl" refers to an alkoxy group as defined herein, attached to the parent molecule via an alkyl group as defined herein.

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

[0029] The term "amino acid" refers to both natural and unnatural amino acids, protected natural and unnatural amino acids, and amino acid analogs and mimics that function in a similar manner 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. Unnatural amino acids refer to amino acid analogs that have the same basic chemical structure as naturally occurring amino acids, i.e., α-carbons bonded to hydrogen, carboxyl groups, amino groups, and R groups, for example only. Such analogs may have modified R groups (e.g., norleucine, for example) or may retain a modified peptide skeleton while retaining the same basic chemical structure as natural amino acids. Non-exclusive examples of unnatural amino acids or amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, homophenylalanine, ornithine, formylglycine, phenylglycine, para-azidophenylglycine, para-azidophenylalanine, para-acetophenylalanine, 4-(3-methyl-(1,2,4,5-tetrazine))-phenylglycine, and 4-(3-methyl-(1,2,4,5-tetrazine))-phenylalanine.

[0030] The term "aryl," as used herein, refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or multiple rings including a condensed system (e.g., bicyclic or tricyclic). Typical examples of aryls include, but are not limited to, phenyl, naphthyl, and anthracenyl. Monocyclic, bicyclic, and tricyclic aryls are connected to the parent molecule via any carbon atoms contained within the ring and can be unsubstituted or substituted. Aromatic bicyclic or aromatic tricyclic ring systems do not contain a non-aromatic ring. Therefore, if a bicyclic or tricyclic ring system contains a non-aromatic ring, the ring system is cycloalkyl or heterocyclyl, depending on whether the heteroatom is present on the non-aromatic ring, regardless of the bonding site to the rest of the molecule.

[0031] In some embodiments, the term “aryl” as used herein refers to a phenyl group, or a bicyclic or tricyclic aryl fused ring system. A bicyclic fused ring system is exemplified by a phenyl group attached to the parent molecule and fused to a phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to the parent molecule and fused to two other phenyl groups. Typical examples of bicyclic aryls include, but are not limited to, naphthyl. Typical examples of tricyclic aryls include, but are not limited to, anthracenyl. Monocyclic, bicyclic, and tricyclic aryls can be attached to the parent molecule via any carbon atoms contained within the ring and can be unsubstituted or substituted.

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

[0033] As used herein, the term "cycloalkyl" refers to a non-aromatic carbocyclic ring system containing 3 to 10, or 3 to 8, or 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; therefore, the term cycloalkyl includes cycloalkenyl ring systems. Typical examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl. "Cycloalkyl" also includes carbocyclic ring systems in which the cycloalkyl group is condensed to an aryl or heteroaryl as defined herein, regardless of its bonding site to the rest of the molecule.

[0034] In some embodiments, the term “cycloalkyl” refers, as used herein, to a carbocyclic ring system containing 3 to 10 carbon atoms, zero heteroatoms, and zero double bonds. Typical examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. “Cycloalkyl” also includes carbocyclic ring systems in which a cycloalkyl group is added to a parent molecule and condensed into an aryl group, a heteroaryl group, or a heterocycle as defined herein.

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

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

[0037] As used herein, the term "fluoroalkyl" means an alkyl group as defined herein, in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by fluorine. Typical examples of fluoroalkyls include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl, for example, 3,3,3-trifluoropropyl.

[0038] When used herein, the term "alkoxyfluoroalkyl" refers to an alkoxy group as defined herein, which is attached to the parent molecule via a fluoroalkyl group, as defined herein.

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

[0040] The terms "halogen" or "halo" as used herein mean Cl, Br, I, or F.

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

[0042] When used herein, the term "haloalkoxy" means at least one haloalkyl group as defined herein, attached to the parent molecule via an oxygen atom.

[0043] As used herein, the term "heteroalkyl" means an alkyl group as defined herein, in which one or more carbon atoms are replaced by heteroatoms selected from S, Si, O, P, and N. Heteroatoms can be oxidized. Typical examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkylamines, and alkyl sulfides.

[0044] As used herein, the term "heteroaryl" refers to an aromatic group having a monocyclic, polycyclic, or multiple fused ring, where 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 ring, an aromatic bicyclic ring system, or an aromatic tricyclic ring system. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridine-2-yl, pyridine-3-yl, and pyridine-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl. Representative examples of bicyclic heteroaryls include, but are not limited to, clomenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranil, indolyl, quinolinyl, imidazopyridine, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranil and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryls are linked to the parent molecule via any carbon or nitrogen atom contained within the ring and can be unsubstituted or substituted. In some embodiments, aromatic bicyclic or aromatic tricyclic ring systems do not contain a non-aromatic ring. Therefore, if a bicyclic or tricyclic ring system contains a non-aromatic ring, the ring system is cycloalkyl or heterocyclyl, depending on whether the heteroatom is present in the non-aromatic ring, regardless of the bonding site to the rest of the molecule.

[0045] In some embodiments, a five-membered aromatic monocyclic ring has two double bonds, and a six-membered aromatic monocyclic ring has three double bonds. In some embodiments, exemplary bicyclic heteroaryl groups are exemplified by monocyclic heteroaryl rings fused to a parent molecule and a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic heterocycle as defined herein. In some embodiments, tricyclic heteroaryl groups are exemplified by monocyclic heteroaryl rings fused to two of a parent molecule and a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic heterocycle as defined herein.

[0046] As used herein, the terms “heterocyclyl,” “heterocycle,” or “heterocyclic” refer to a non-aromatic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10 carbon atoms, at least one heteroatom (e.g., 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1), and optionally one or more oxo and / or double bonds. The terms “heterocyclyl,” “heterocycle,” 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 3, 4, 5, 6, 7, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, the 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. In some embodiments, the 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the 7- and 8-membered rings contain zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S.Typical examples of monocyclic heterocycles include azetidinyl, azepanyl, azilidinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, and oxetanyl. Examples include, but are not limited to, piperazinil, piperidinil, pyranil, pyrazolinil, pyrazolidinil, pyrrolinil, pyrrolidinil, tetrahydrofuranil, tetrahydropyranil, tetrahydropyridinil, tetrahydrothienyl, thiadiazolinil, thiadiazolidinil, 1,2-thiadinil, 1,3-thiadinil, thiazolinil, thiazolidinil, thiomorpholinil, 1,1-dioxidethiomorpholinil (thiomorpholine sulfone), thiopyranil, and trithianil. A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl group, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiroheterocyclic group, or a bridged monocyclic heterocycle system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hepta-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl.Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are connected by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 decane), but are not limited thereto. Monocyclic, bicyclic, and tricyclic heterocycles can be connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring, and can be unsubstituted or substituted.

[0047] The term "hydroxyl" as used herein refers to a -OH group.

[0048] The term "hydroxyalkyl" as used herein refers to an alkyl group as defined herein in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by a hydroxyl group.

[0049] In some instances, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix "C x -C y -" or "C x-y " (where x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms in the substituent). Thus, for example, "C1-C3-alkyl" and "C 1-3 alkyl" refer to an alkyl substituent containing from 1 to 3 carbon atoms. The two conventional expressions "C x -C y -" and "Cx-y The terms "..." and "..." are used interchangeably and have the same meaning.

[0050] The term "substituted" refers to a group that may be further substituted with one or more nonhydrogen substituents. Substituent groups 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, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketones, amides, carbamates, and acyls.

[0051] The term "tetrazine" refers to a substituted or unsubstituted aromatic cyclic group having a single ring with three double bonds, comprising two carbon atoms and four nitrogen atoms. Examples of tetrazine groups include 1,2,3,4-tetrazine and 1,2,4,5-tetrazine. As used herein, 1,2,4,5-tetrazine is referred to as the "Tz" group.

[0052] The term “selectively deliver” refers to delivering a drug (e.g., 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 targeted portion described herein, or therapeutic targeted portion, does not have a therapeutic effect itself, but rather is designed to enable the selective or targeted delivery of a therapeutic agent. However, a targeted portion may have a therapeutic effect, and such constructs are therefore not excluded by this disclosure.

[0053] The term "payload" refers to a drug intended for delivery to a target site in an object. In some embodiments, the payload is a therapeutic agent. In some embodiments, the payload is a diagnostic agent.

[0054] The term "therapeutic agent" refers to a drug capable of treating and / or improving a condition or disease or one or more symptoms thereof. The therapeutic agents of this disclosure also include prodrug forms of therapeutic agents, and chelating agents having or not having radionuclides (e.g., for diagnostic or therapeutic purposes).

[0055] As used herein, therapeutic “radionuclide” or “radioligand” refers to a radioactive substance used to treat a medical condition, particularly cancer, and may also be called a radiopharmaceutical. The radionuclides used in the transcyclooctene portion described herein include chelating agents and isotopes, for example. 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, 169Er, 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 It contains isotopes selected from the group consisting of Ac. Radionuclides can be delivered via direct conjugation or chelation with a chelating agent. Exemplary radionuclides, chelating agents, and linkers for potential TCO conjugate payloads are described below.

[0056] The term “diagnostic agent” refers to a drug used to aid in the diagnosis of a condition or disease. Typical 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 magnetic field. Examples of paramagnetic agents include, but are not limited to, iron particles, including iron nanoparticles and iron microparticles. Optical probes are fluorescent compounds that can be detected by excitation with radiation of one wavelength and detection with radiation of a different second wavelength. Examples of optical probes in this disclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo detectable radioactive decay. Radionuclides useful in embodiments of this disclosure, such as diagnostic radionuclides, 3 H, 11 C, 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67Cu, 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, including but not limited thereto.

[0057] The term "targeting agent" refers to a chemical or biological agent that specifically binds to a target (e.g., a targeted organ or tissue), thereby forming a stable association between the targeting agent and the specific target. "Stably associated" or "stable association" means that a moiety binds to or otherwise associates with another moiety or structure under standard physiological conditions. The binding may include covalent bonds and non-covalent interactions including, but not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. Targeting agents include ligands that specifically bind (or bind substantially specifically) to a particular clinically relevant target receptor or cell surface target.

[0058] 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 encompassed within the antigen-binding portion of an antibody include, but are not limited to, Fab fragments. In some embodiments, the Fab fragment comprises at least V L and V H is a monovalent fragment containing In some embodiments, the Fab fragment is VL , V H , C L , and C H 1 domain. Fab fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. For example, antibody fragments can be produced by recombinant DNA technology, or by enzymatic or chemical cleavage of intact immunoglobulins. Additional examples are included in the various embodiments disclosed herein.

[0059] "Specifically binds" 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 moiety having one or more antigenic determinants) or a cell or tissue bearing the target molecule (e.g., a cell surface antigen, such as a receptor or ligand), and does not associate with cells or tissues lacking the target molecule. It is recognized that some degree of non-specific interaction may occur between a binding moiety and a non-target molecule (present alone or in combination with a cell or tissue). Nevertheless, specific binding can be identified as being mediated via the specific recognition of the target antigen. Specific binding results in a stronger association between a targeting moiety (e.g., a Fab) and, for example, a cell bearing a target molecule (e.g., an antigen) than between the binding moiety and, for example, a cell lacking the target molecule. Specific binding typically results in a greater than 2-fold, greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in the amount of binding moiety (per unit time) bound to, for example, a cell or tissue bearing the target molecule or marker, as compared to a cell or tissue lacking the target molecule or marker. In some embodiments, the binding moiety is, for example, 10 -5 less than M, 10 -7 less than M, 10 -8 less than M, 10 -9 less than M, 10 -10 less than M, 10 -11 less than M, 10 -12 less than M, 10 -13 less than M, 10 -14 less than M, or 10 -15It binds to target molecules or markers with a dissociation constant of less than M. Various assay formats, such as solid-phase ELISA immunoassay, are suitable for measuring binding.

[0060] The term "targeted organ or tissue" refers to an organ or tissue that is targeted for payload delivery. Typical organs and tissues 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.

[0061] The term "contact" or "to come into contact" refers to the process of bringing at least two different species into contact so that they can interact with each other, such as in non-covalent or covalent interactions or bonding reactions. However, it should be understood that the resulting complex or reaction product can be formed directly from interactions or reactions between added reagents, or from intermediates from one or more of the added reagents or parts that can be formed in the contact mixture.

[0062] The term “administering” refers to any preferred route of administration to the subject, including, but not limited to, oral administration, suppository administration, topical contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration, or subcutaneous administration, subarachnoid administration, or implantation of a sustained-release device to the subject, such as a mini osmotic pump.

[0063] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0064] The terms “pharmaceutical effective dose” and “therapeutic effective dose” refer to the amount of a compound sufficient to treat one or more of a particular disorder or disease or its symptoms, and / or to prevent or reduce the risk of the onset or recurrence of the disease or disorder or its symptoms. With respect to tumorigenic proliferative disorders, the pharmaceutical effective dose or therapeutic effective dose includes, among other things, an amount sufficient to shrink the tumor or slow its growth rate.

[0065] As used herein, the terms “subject,” “patient,” or “organism” include humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which the drugs of this disclosure may be administered may include mammals, in particular primates, and especially humans. For veterinary use, suitable subjects may include, for example, livestock such as cattle, sheep, goats, and pigs; poultry such as chickens, ducks, geese, and turkeys; and domesticated animals, in particular pets such as dogs and cats. For diagnostic or research use, suitable subjects may include mammals such as rodents (e.g., mice, rats, and hamsters), rabbits, primates, and pigs such as inbred pigs.

[0066] When used herein, “to treat” or “treatment” means treating or treating a disease or medical condition or symptom(s) in a patient, such as a mammal (e.g., human), which includes (a) improving the disease or medical condition or symptom(s) in a patient, for example, eliminating or causing regression of the disease or medical condition or symptom(s) in a patient; (b) suppressing the disease or medical condition or symptom(s) in a patient, for example, by delaying or stopping the progression of the disease or medical condition or symptom(s) in a patient; or (c) reducing the symptoms of the disease or medical condition or symptom(s) in a patient.

[0067] The term "physiological conditions" is intended to encompass conditions that are compatible with living cells, such as temperature, pH, salinity, and other primarily aqueous conditions that are compatible with living cells.

[0068] For the compounds described herein, the groups and substituents may be selected according to the allowable valencies of the atoms and substituents, and the selection and substitution result in stable compounds that do not undergo spontaneous transformations such as rearrangement, cyclization, or elimination.

[0069] Where a numerical range is indicated, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of that range (up to one-tenth of the lower limit unit), as well as any other values ​​or intermediate values ​​indicated within that range, should be understood to be included in this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also included in this disclosure according to any specifically excluded limits within the indicated range. If one or both limits are included in the indicated range, the range excluding one or both of the included limits is also included in this disclosure.

[0070] For the purposes of enumerating numerical ranges as defined herein, each number intervening between them is explicitly intended with the same degree of precision. For example, for the range 6–9, the digits 7 and 8 are intended in addition to 6 and 9, and for the range 6.0–7.0, the digits 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 intended.

[0071] Compounds may exist as stereoisomers containing an asymmetric or chiral center. The stereoisomers are "R" or "S" depending on the arrangement of substituents around the chiral carbon atom. The terms "R" and "S" as used herein refer to the arrangements described in the IUPAC 1974 Recommendations in Section E, Fundamental Stereochemistry, Pure Appl. Chem., 1976, 45:13-30. This disclosure envisions various stereoisomers and mixtures thereof, which are specifically included within the scope of this disclosure. Examples of 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 the preparation of racemic mixtures followed by separation methods well known to those skilled in the art. These separation methods include (1) attachment of the enantiomer mixture to a chiral auxiliary, separation of the diastereomer mixture obtained by recrystallization or chromatography, and optional release of optically pure products from the auxiliary (Furniss, Hannaford, Smith, and Tatchell, “Vogel’s Textbook of Practical Organic Chemistry,” 5 th As exemplified by (1989 edition, Longman Scientific & Technical, Essex CM20 2JE, England), or (2) direct separation of a mixture of optical enantiomers on a chiral chromatography column, or (3) fractional recrystallization methods.

[0072] It should be understood that the compound may have tautomers and geometric isomers, and these also constitute aspects of this disclosure.

[0073] This disclosure also includes isotope-labeled compounds that are identical to those enumerated herein, except that one or more atoms are replaced by atoms having atomic masses or mass numbers different from those commonly found in nature. Examples of suitable isotopes to be included in the compounds of this disclosure are, but are not limited to, those listed above. 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 These include hydrogen (such as Cl), carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Deuterium, that is, 2 Substitution with heavier isotopes such as 1H can offer certain therapeutic benefits resulting from higher metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and may therefore be preferable in some situations. The compound may also incorporate positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated are: 11 C, 13 N, 15 O, and 18 F is the isotope-labeled compound disclosed herein can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the appended examples, using appropriate isotope-labeled reagents instead of non-isotope-labeled reagents.

[0074] Treatment methods This disclosure relates to a method for delivering a payload to a target location within an object.

[0075] In one embodiment, a method is provided for forming an antibody-payload conjugate in vivo in a subject requiring an antibody-payload conjugate, and this method is... Administering an effective amount of a targeted portion, wherein the targeted portion comprises at least one antibody or fragment thereof having at least one tetrazine portion covalently bound thereto. The administration of a single-dose therapeutically effective payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently bound thereto. The antibody or its fragments have binding affinity to receptors on the tumor, and furthermore, the amount of antibody-payload conjugate formed in vivo is greater at the tumor site compared to the plasma.

[0076] In one embodiment, a method is provided for forming an antibody-payload conjugate in vivo in a subject requiring an antibody-payload conjugate, and this method is... Administering an effective amount of a targeted portion, wherein the targeted portion comprises at least one antibody or fragment thereof having at least one tetrazine portion covalently bound thereto. The administration of a single-dose therapeutically effective payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently bound thereto. The antibody or its fragments contain a binding affinity to receptors on the tumor, and furthermore, the ratio of the antibody-payload conjugate at the tumor site to the plasma is greater than approximately 2:1.

[0077] In some embodiments, the ratio of antibody-payload conjugate in the tumor site to 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.

[0078] In some embodiments, the ratio of antibody-payload conjugate in the tumor site to plasma is greater than approximately 2:1, or greater than approximately 3:1, or greater than approximately 4:1, or greater than approximately 5:1, or greater than approximately 6:1, or greater than approximately 7:1, or greater than approximately 8:1, or greater than approximately 9:1, or greater than approximately 10:1.

[0079] In some embodiments, administration is simultaneous.

[0080] In some embodiments, administration is sequential. In some embodiments, the targeted portion is administered before the payload-TCO conjugate.

[0081] In some embodiments, the first dose of the payload-TCO conjugate is administered to the subject less than 48 hours after the targeted portion is administered to the subject, or more than 4 hours but less than 48 hours later.

[0082] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject less than 48 hours after the targeted portion is administered to the subject, or more than 4 hours but less than 48 hours later.

[0083] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject less than 48 hours after the targeted portion has been administered to the subject.

[0084] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject more than 4 hours but less than 48 hours after the targeted portion has been administered to the subject.

[0085] In some embodiments, the targeted portion is administered at least 8 to about 24 hours before the payload-TCO conjugate is administered.

[0086] In some embodiments, the targeted portion is administered within a serum half-life of 0–8. The timing of payload-TCO conjugate administration is intended to be calculated based on the properties of the tumor, which can be determined by the biology of the tumor and the antigen in the targeted portion. For example, in some embodiments, if the targeted portion includes a targeting format as shown in the table below, the payload-TCO conjugate is administered within the serum half-life range shown therein (see also Berland et al., Biomolecules 2021, 11(5), 637). [Table 1]

[0087] In one embodiment, a method is provided for administering a payload to a target, and this method is: a) Administering an effective amount of the targeted portion, wherein the targeted portion comprises a Fab having at least one tetrazine portion covalently bound thereto. b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently bound thereto. The single-dose payload-TCO conjugate, which includes [specific component], is administered to the subject at least approximately 2 hours, or at least approximately 3 hours, or at least approximately 4 hours, less than approximately 48 hours, or approximately 4 to 48 hours after the targeted portion has been administered to the subject.

[0088] In one embodiment, a method is provided for administering a payload to a target, and this method is: a) Administering an effective amount of the targeted portion, wherein the targeted portion comprises a Fab having at least one tetrazine portion covalently bound thereto. b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently bound thereto. The single-dose payload-TCO conjugate, which includes the targeting portion, is administered to the subject more than 4 hours but less than 48 hours after the targeting portion has been administered to the subject.

[0089] Selective binding occurs between bio-orthogonal binding partners (for example, between the tetradine in the targeting portion and its complementary trans-cyclooctene in the payload-TCO conjugate). As described above, for local administration of the targeting portion to a desired location in the target, the selective binding between the trans-cyclooctene in the payload-TCO conjugate and the tetradine in the targeting portion localizes the payload to the desired target location.

[0090] The methods disclosed herein enable selective and safe delivery of payloads, thereby reducing side effects or toxicity associated with off-target delivery. Furthermore, by administering the two agents described herein, only a single-dose payload-TCO conjugate is required for therapeutic efficacy.

[0091] As described herein, selective delivery of the payload can be achieved by administering the payload-TCO conjugate within a certain therapeutic window after administration of the targeting agent. A relatively inactive, systemically administered payload-TCO conjugate is activated by the targeting agent at a target site, e.g., a tumor, via a covalent click chemical reaction, and subsequently releases the active payload by chemical rearrangement.

[0092] In some embodiments, the subject has cancer. In one embodiment, a method is provided for treating cancer or enhancing or inducing an immune response in a subject having cancer, and this method is a) Administering an effective amount of the targeted portion, wherein the targeted portion comprises a Fab having at least one tetrazine portion covalently bound thereto. b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently bound thereto. The single-dose payload-TCO conjugate, which includes [specific component], is administered to the subject at least approximately 2 hours, or at least approximately 3 hours, or at least approximately 4 hours, less than approximately 48 hours, or approximately 4 to 48 hours after the targeted portion has been administered to the subject.

[0093] In one embodiment, a method is provided for treating cancer or enhancing or inducing an immune response in a subject having cancer, and this method is: a) Administering an effective amount of the targeted portion, wherein the targeted portion comprises a Fab having at least one tetrazine portion covalently bound thereto. b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently bound thereto. The single-dose payload-TCO conjugate, which includes the targeting portion, is administered to the subject more than 4 hours but less than 48 hours after the targeting portion has been administered to the subject.

[0094] In some embodiments, a single-dose payload-TCO conjugate is administered 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or less than 24 hours, or approximately 24 hours, after the targeting portion has been administered to the subject.

[0095] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject approximately 5 to 24 hours, or approximately 6 to 24 hours, or approximately 7 to 24 hours, or approximately 8 to 24 hours, or approximately 9 to 24 hours, or approximately 10 to 24 hours, or approximately 11 to 24 hours, or approximately 12 to 24 hours, or approximately 13 to 24 hours, or approximately 14 to 24 hours, or approximately 15 to 24 hours, or approximately 16 to 24 hours, or approximately 17 to 24 hours, or approximately 18 to 24 hours, or approximately 19 to 24 hours, or approximately 20 to 24 hours, or approximately 21 to 24 hours, or approximately 22 to 24 hours, or approximately 23 to 24 hours after the targeting portion has been administered to the subject.

[0096] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject approximately 5 to 22 hours, or approximately 6 to 22 hours, or approximately 7 to 22 hours, or approximately 8 to 22 hours, or approximately 9 to 22 hours, or approximately 10 to 22 hours, or approximately 11 to 22 hours, or approximately 12 to 22 hours, or approximately 13 to 22 hours, or approximately 14 to 22 hours, or approximately 15 to 22 hours, or approximately 16 to 22 hours, or approximately 17 to 22 hours, or approximately 18 to 22 hours, or approximately 19 to 22 hours, or approximately 20 to 22 hours, or approximately 21 to 22 hours after the targeting portion has been administered to the subject.

[0097] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject approximately 5 to 20 hours, or approximately 6 to 20 hours, or approximately 7 to 20 hours, or approximately 8 to 20 hours, or approximately 9 to 20 hours, or approximately 10 to 20 hours, or approximately 11 to 20 hours, or approximately 12 to 20 hours, or approximately 13 to 20 hours, or approximately 14 to 20 hours, or approximately 15 to 20 hours, or approximately 16 to 20 hours, or approximately 17 to 20 hours, or approximately 18 to 20 hours, or approximately 19 to 20 hours after the targeting portion has been administered to the subject.

[0098] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject approximately 8 to 12 hours, 8 to 16 hours, 8 to 22 hours, 12 to 16 hours, or 16 to 20 hours after the targeting portion has been administered to the subject.

[0099] 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 approximately 8 to 12 hours, 8 to 16 hours, 8 to 22 hours, 12 to 16 hours, or 16 to 20 hours after the targeting portion has been administered to the subject.

[0100] In some embodiments, a single-dose payload-TCO conjugate is administered to the subject approximately 16 to 20 hours after the targeting portion has been administered to the subject.

[0101] In one embodiment, a method is provided for selectively administering a payload to a tumor site in a subject requiring it, and this method is, a) Administering an effective amount of the targeted portion, wherein the targeted portion comprises a Fab having at least one tetrazine portion covalently bound thereto. b) Administering a single therapeutic dose of a payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently bound thereto. The single-dose payload-TCO conjugate, which includes the targeting portion, is administered to the subject approximately 8 to 18 hours after the targeting portion has been administered.

[0102] In some embodiments, the accumulation at the tumor site is at least about 10 times greater, or up to about 200 times greater, than in kidney, liver, or spleen tissue. In some embodiments, the targeted portion is Formula I, Formula II, or Formula V. [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 R represents an additional bond that forms dihydroceramide when both are present, however, when ring A is aryl. 3 and R 4 Assuming that both exist, X is the antibody fragment portion, p is between 1 and 20. In each appearance, L is independently a linker. R in each appearance 1These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -OR', -SR', -C(=O)R', -C(=S)R', -OC(=O)R''', -SC(=O)R''', -OC(=S)R''', -SC(=S)R''', -S(=O)R', -S(=O)2R''', -S(=O)2NR'R'', -C(=O)O-R', -C(=O)S-R', -C(=S)OR', -C(=S)SR', -C(=O)NR'R'', -C(=S)NR'R'', -NR'R Selected from the group consisting of '', -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'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl, optionally with 1 to 3 Z 1 It has been replaced with, R in each appearance 2 Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally a Z 1 It has been replaced with, R 3 and R 4If both are absent, or R 3 and R 4 Each of these is an independent group that can be removed after hydrogen or a trigger event. R in each appearance 20 These independently include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, 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( Selected from the group consisting of =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 in each appearance 22 Independently and optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups, is a linker of 1 to 100 linked atoms. R in each appearance 30 These are independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. each Z 1These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -OR', -SR', -C(=O)R', -C(=S)R', -OC(=O)R''', -SC(=O)R''', -OC(=S)R''', -SC(=S)R''', -S(=O)R', -S(=O)2R''', -S(=O)2NR'R'', -C(=O)O-R', -C(=O)S-R', Selected from -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'', In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0103] In some embodiments, the targeting portion is formula I or II. [ka] During the ceremony, X is the antibody fragment portion, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20These independently include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C( Selected from the group consisting of =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 in each appearance 22 Independently and optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups, is a linker of 1 to 100 linked atoms. R in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl, and in each occurrence, R''' is independently selected from aryl and alkyl. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0104] In some embodiments, the targeting portion is formula IIA, [ka] IIA, where X, p, L, and R 20 Each of these is independently defined as described herein.

[0105] Several embodiments, each R 20 These are independently hydrogen or alkyl.

[0106] In some embodiments, the targeting portion is Equation VII, [ka] VII, where X, p, L, ring A, R 1 , R 2 , and t are each independently as defined herein.

[0107] In some embodiments, ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl.

[0108] In some embodiments, ring A is phenyl.

[0109] In some embodiments, p is 1-16, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2.

[0110] Several embodiments, each R 1 These are independently hydrogen or alkyl.

[0111] Several embodiments, each R 2 These are independently halo, alkyl, or haloalkyl.

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

[0113] In some embodiments, X is an antibody fragment that targets TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin 4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

[0114] In some embodiments, X is an antibody fragment portion "derived" from the antibody. In some embodiments, the term "derived" is a portion of the antibody. H and V L This refers to an antibody fragment of an antibody containing [the specified substance], and therefore the antibody fragment portion, or Fab, specifically binds to the antigen.

[0115] In some embodiments, X is daclizumab, RG6292, basiliximab, HuMax-TAC, rabetsuzumab, 15-1-32, PR1A3, cT84.66, tusamithiamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matsuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, GC111 8. Imugatuzumab, Panitumumab, Artumumab, Tomzotuximab, Laprituximab, Oportuzumab, Sitatuzumab, Tucotsuzumab, Katsumakisomab, Edrecolomab, Adecatumumab, Ramucizumab, Ramucirumab, Valinacimab (vulinacimab), Oralatumab, Ramucirumab, Sacituzumab, Pr1E11, Enfortumab, J591, MLN591 , verantamab, moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ubrituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, ronkastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, tapritumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MOR 202, TAK-079, I-131-BC8, Iomab-B, Carotuximab, Bemarituzumab, Apulzumab, Rupalzumab, Zolbetuximab, Claudiximab, Andecaliximab, Milbetuximab, Farletuzumab, MORAb-202, MORAb-003, SP8166, Robalpituzumab, Indatuximab, Rorbotuzumab, Promiximab, BI 836826, Otreltuzumab, Naratuximab, Miratuzumab, Anetuzumab, Amatsuximab, MMOT-0530A, Sarilumab, Elotuzumab, Belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, Cribatuzumab, 8HuDS6, Gatipotuzumab, AR20.5. Cantuzumab, Codlituzumab, ECT204, MDX-1414, Pertuzumab, Trastuzumab, Margetuximab, Patrizumab, Cerivantuzumab, Lumuletuzumab, Elgemzumab, AV-203, CDX-3379, GSK284933, Brentuximab, Gemtuzumab, BI 835858, vadasutuximab, lintuzumab, KHK2823, tacrotuzumab, G4723A, grembatumumab, terisotuzumab, onarutuzumab, SAIT301, tisotuzumab, rifastuzumab, indusatuzumab, bundletuzumab, sofituzumab, borsetuzumab, vibatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, BI These are antibody fragments derived from 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, cibrotuzumab, F16, R6N, datopotamab, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, and 74Hz.

[0116] In some embodiments, the targeting portion further comprises an imaging contrast agent. In some embodiments, the imaging contrast agent is a protein.

[0117] In some embodiments, L in each occurrence is independently bound to X via a cysteine ​​or lysine residue on X.

[0118] In some embodiments, each L comprises one or more amino acids.

[0119] In some embodiments, each L contains a polypeptide.

[0120] In some embodiments, each L independently contains 1 to 100 linked atoms, 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0121] In some embodiments, each L independently comprises 5 to 50 linked atoms including one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, or heteroarylene moieties, and each alkylene, alkenylene, alkynylene, arylene, or heteroarylene moiety independently optionally comprises oxo, halo, C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

[0122] In some embodiments, X is an antibody fragment portion targeting HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, Mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5, and each L is independently [ka] It is selected from the group consisting of the following.

[0123] In some embodiments, X is an antibody fragment portion targeting HER2, p is 1-5, and each L is independently [ka] It is selected from the group consisting of the following.

[0124] In some embodiments, the targeting portion is formula IIF, [ka] In formula IIF, where X and p are defined independently as herein.

[0125] In some embodiments, X is an antibody fragment that targets HER2, and p is 1 to 5.

[0126] In some embodiments, the payload-TCO conjugate is formula VIII or a pharmaceutically acceptable salt thereof. [ka] During the ceremony, In each occurrence, G is independent, [ka] And, L in each appearance 1 It is independent and is a linker, m is an integer between 1 and 150. D is the payload, R in each appearance 1A C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Selected from the group consisting of alkoxys, q is 0, 1, or 2, q1 is 0 or 1, R in each appearance 1B G 1 -OH, -NR 1c -C 1-4 Alkilen-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 Alkilen) 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 Alkilen) 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 Selected from the group consisting of alkylene-CO2H)2, R in each appearance 1c and R 1d Independently, hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, R in each appearance 1f Hydrogen, C 1-6 Alkyl, or C 1-4 It is alkylene-CO2H. In each occurrence, n is independently 0, 1, 2, or 3. L in each appearance 2 Independently, -C(O)- and C 1-3 Selected from the group consisting of alkylenes, G in each appearance 1 These are heterocyclines that have been independently and arbitrarily substituted.

[0127] In some embodiments, the payload may include therapeutic agents for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepylone, patupylone (epoteron class), platinum-based drugs, exatecan, deruxtecan, orlistatin (drastatin 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, seco-DUBA, duocalmycin, etc.), immunosuppressants (e.g., cyclosporine A, rapamycin), and immunosuppressants (e.g., cyclosporine A, rapamycin). The following are selected from antifungal agents (e.g., amphotericin), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin beta, flucytosine, emtricitabine, gentamicin, colistin), lurubinectedin, gardiquimod, matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidyl (c-AMP).

[0128] In some embodiments, the payload is a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepyrone, patupyrone (epoteron class), platinum-based drugs, exatecan, deruxtecan, orlistatin (drastatin 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, seco-DUBA, duocalmycin, etc.) or an immunosuppressant (e.g., cyclosporine A, rapamycin, etc.).

[0129] In some embodiments, the payload is paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepyrone, patupyrone, platinum, exatecan, deruxtecan, dorastatin 10, MMAE, MMAD, MMAF, mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterin, seco-DUBA, duocalmycin, cyclosporine A, or rapamycin.

[0130] In some embodiments, the payload-TCO conjugate is [ka] [ka] Selected from.

[0131] In some embodiments, a method is provided for administering a therapeutically effective dose of monomethyl auristatin E (MMAE) to a subject with cancer, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is the trastuzumab Fab-tetradine targeted portion of formula IIF. [ka] In the formula, X is trastuzumab Fab including (SEQ ID NO: 3) and (SEQ ID NO: 4), and p is 1 to 5. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] The administration of a single dose of MMAE-TCO conjugate is performed on the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the target portion has been administered to the subject.

[0132] In some embodiments, methods are provided for treating cancer or enhancing or inducing an immune response in subjects having cancer, and these methods include a) Administering an effective amount of the targeted portion, wherein the targeted portion is the trastuzumab Fab-tetradine targeted portion of formula IIF. [ka] In the formula, X is trastuzumab Fab, including (SEQ ID NO: 3) and (SEQ ID NO: 4), and p is 1 to 5. The drug is administered to the target patient. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0133] In some embodiments, a method is provided for reducing tumor volume in a subject having a tumor, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is the trastuzumab Fab-tetradine targeted portion of formula IIF. [ka] In the formula, X is trastuzumab Fab including (SEQ ID NO: 3) and (SEQ ID NO: 4), and p is 1 to 5. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single dose of MMAE-TCO conjugate is administered to the target subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the target portion has been administered.

[0134] In some embodiments, a single dose of MMAE-TCO conjugate is administered to the subject approximately 8 to 24 hours after the targeting portion has been administered to the subject.

[0135] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject approximately 8 to 12 hours, 8 to 16 hours, 8 to 22 hours, 12 to 16 hours, or 16 to 20 hours after the targeting portion has been administered to the subject.

[0136] In some embodiments, a single dose of the MMAE-TCO conjugate is administered to the subject approximately 8 to 22 hours, or approximately 16 to 20 hours, after the targeting portion has been administered to the subject.

[0137] In some embodiments, a method is provided for administering a therapeutically effective dose of monomethyl auristatin E (MMAE) to a subject with cancer, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is formula IIF, [ka] In the formula, X is trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), or X is sacituzumab Fab containing (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5. The drug is administered to the subject. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0138] In some embodiments, methods are provided for treating cancer or enhancing or inducing an immune response in subjects having cancer, and these methods include a) Administering an effective amount of the targeted portion, wherein the targeted portion is formula IIF, [ka] In the formula, X is trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), or X is sacituzumab Fab containing (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5. The drug is administered to the subject. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0139] In some embodiments, a method is provided for reducing tumor volume in a subject having a tumor, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is formula IIF, [ka] In the formula, X is trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), or X is sacituzumab Fab containing (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5. The drug is administered to the subject. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0140] In some embodiments, a method is provided for administering a therapeutically effective dose of monomethyl auristatin E (MMAE) to a subject with cancer, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is the trastuzumab Fab-tetradine targeted portion of formula IIFa. [ka] In the formula, X is trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), and p is 2-3. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0141] In some embodiments, methods are provided for treating cancer or enhancing or inducing an immune response in subjects having cancer, and these methods include a) Administering an effective amount of the targeted portion, wherein the targeted portion is the trastuzumab Fab-tetradine targeted portion of formula IIFa. [ka] In the formula, X is trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), and p is 2-3. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0142] In some embodiments, a method is provided for reducing tumor volume in a subject having a tumor, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is the trastuzumab Fab-tetradine targeted portion of formula IIFa. [ka] In the formula, X is trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), and p is 2-3. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0143] In some embodiments, a method is provided for administering a therapeutically effective dose of monomethyl auristatin E (MMAE) to a subject with cancer, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is the sacituzumab Fab-tetradine targeted portion of formula IIF. [ka] In the formula, X is sacituzumab Fab, including (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0144] In some embodiments, methods are provided for treating cancer or enhancing or inducing an immune response in subjects having cancer, and these methods include a) Administering an effective amount of the targeted portion, wherein the targeted portion is the sacituzumab Fab-tetradine targeted portion of formula IIF. [ka] In the formula, X is sacituzumab Fab, including (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0145] In some embodiments, a method is provided for reducing tumor volume in a subject having a tumor, and this method is provided. a) Administering an effective amount of the targeted portion, wherein the targeted portion is the sacituzumab Fab-tetradine targeted portion of formula IIF. [ka] In the formula, X is sacituzumab Fab, including (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5. The drug is administered to the target population. b) Administering a single therapeutic dose of an effective MMAE-TCO conjugate having the following structure to the target: [ka] A single-dose MMAE-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

[0146] In some embodiments, a single dose of MMAE-TCO conjugate is administered to the subject approximately 8 to 24 hours after the targeting portion has been administered to the subject.

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

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

[0149] In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 10 mg / kg to approximately 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 10 mg / kg to approximately 50 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 20 mg / kg to approximately 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 20 mg / kg to approximately 50 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 30 mg / kg to approximately 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 30 mg / kg to approximately 50 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 40 mg / kg to approximately 60 mg / kg. In some embodiments, the targeted portion is administered as a single dose in an amount ranging from 40 mg / kg to approximately 50 mg / kg.

[0150] In some embodiments, the targeted portion is administered as a single dose in amounts of approximately 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, or 60 mg / kg.

[0151] In some embodiments, the payload-TCO conjugate is administered as a single dose in an amount ranging from 10 mg / kg to approximately 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 approximately 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 approximately 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 approximately 35 mg / kg.

[0152] In some embodiments, the payload-TCO conjugate is administered as a single dose in amounts of approximately 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg.

[0153] In some embodiments, the subject has cancer.

[0154] 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, 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 lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

[0155] 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.

[0156] In some embodiments, cancer is a solid tumor.

[0157] In some embodiments, the cancer is a soft tissue sarcoma.

[0158] In some embodiments, the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0159] In some embodiments, the method also includes enhancing or inducing an immune response. In some embodiments, the immune response is an increase in one or more of the following: leukocytes, lymphocytes, monocytes, and eosinophils.

[0160] In some embodiments, the method further comprises administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctenprodrugs thereof. Anticancer agents, immunomodulators, and trans-cyclooctenprodrugs thereof are known in the art.

[0161] The indications for this approach include both hematological and solid tumors. In certain embodiments, this approach can be used for the treatment and / or diagnosis of soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma, and all different subtypes of soft tissue sarcomas, as well as osteosarcoma. The composition may be for the treatment and / or diagnosis of pigmented villonodular synovitis.

[0162] In certain embodiments, the approach can be used to treat and / or diagnose hematological 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 gammaglobulinemia, plasmacytomyeloma, 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 others.

[0163] The compositions of this disclosure find use in the treatment and / or diagnosis of a condition or disease in a subject who may be treated or diagnosed by administration of a payload (e.g., a parent drug (i.e., a drug before conjugation into the composition)). "Treatment" means that at least improvement of the symptoms associated with the pathological condition afflicting the subject is achieved, where improvement is used in a broad sense to mean at least a reduction of a parameter associated with the condition being treated, e.g., the severity of the symptoms. Thus, treatment includes situations in which a pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated) so that the subject no longer suffers from that condition, or at least the symptoms that characterize that condition. Treatment may include inhibition, i.e., prevention of the onset or further onset of clinical symptoms, e.g., alleviation or complete inhibition of an active disease. Treatment may include alleviation, i.e., causing a regression of clinical symptoms. For example, in the context of cancer, the term “treating” includes any or all of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing overall tumor volume, extending survival time, and improving one or more cancer-related symptoms.

[0164] The subject to be treated may be any subject in need of therapy, and hereby the subject to be treated is any subject that can be treated with the parent drug. Thus, a variety of subjects may be treated using the compositions disclosed herein. Generally, such subjects are “mammals,” and humans are among the subjects. 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., animal models of diseases), and non-human primates (e.g., chimpanzees and monkeys).

[0165] In certain embodiments, additional therapeutic agents and methods, though not limited to these, can be used for the treatment, prevention, and / or diagnosis of solid tumors including, but not limited to, melanoma (e.g., unresectable metastatic melanoma), kidney cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer such as metastatic epithelial ovarian cancer), endometrial cancer, breast cancer (e.g., triple-negative breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma. The approach of this disclosure is suitable for use as an adjuvant / neoadjuvant system. For example, the particles disclosed herein can be placed during a biopsy, and upon return of the study results, the physician can deliver the appropriate cocktail to the desired site in the body. This would minimize tumor size, particularly in the case of surgically resectable tumors. Then, at the end of surgery, the surgeon can administer additional targeted portions to target surgical cavities and treat the patient with further doses of therapy (e.g., chemotherapy using the approach of this disclosure) to minimize the risk from cancer cells that may have been missed at the resection margin.

[0166] In certain embodiments, the targeted portions disclosed herein can be administered, allowing a physician to deliver the appropriate cocktail to a desired site in the body. This would minimize tumor size, particularly in the case of surgically resectable tumors. Then, at the end of surgery, the surgeon can administer additional targeted portions to target surgical cavities and treat the patient with further doses of therapy (e.g., chemotherapy according to the approach of this disclosure) to minimize the risk from cancer cells that may have been missed at the resection margin.

[0167] In certain embodiments, the method disclosed herein provides the ability to position particles as disclosed herein during a biopsy. Upon return of the results, the physician can deliver the immunomodulator to the biopsy site.

[0168] In certain embodiments, the methods of the present disclosure provide the ability for a physician to deliver immunomodulatory agents such as TLR agonists, STING agonists, chemokines (agents that attract cancer cells and / or immune cells), and adjuvants, as well as chemotherapeutic agents in combination with immunotherapeutic agents, to enhance the immune system with fewer side effects. This combination approach would be beneficial to the patient. The chemotherapeutic agent would treat a solid tumor or a specific site, while the enhanced response of the immunotherapy would help distant metastatic sites. For example, in certain embodiments, the compositions and methods of the present disclosure can enhance the efficacy of one or more immunomodulatory agents such as ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer) using anthracyclines, taxanes, gemcitabine, and other agents.

[0169] cancer The methods of this disclosure may be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that may include persistent growth signaling, evasion of growth inhibitors, resistance to cell death, activation of replication immortality, induction of angiogenesis, and activation of invasion and metastasis. The methods of this disclosure may 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.

[0170] Cancers that can be treated by the methods disclosed herein include, but are not limited to, astrocytoma, adrenocortical carcinoma, appendiceal cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain cancer, brainstem cancer, brainstem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse endogenous pontine glioma, ductal carcinoma, endometrial cancer, ependymoma, Ewing's sarcoma, esophageal cancer, eye cancer, fibrosarcoma, gallbladder cancer, and gastric cancer. Cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, macroglobulinemia, melanoma, mesothelioma, oral cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, solid tumor, squamous cell carcinoma, stomach cancer This includes cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thyroid cancer, gestational trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, non-small cell lung cancer (NSCLC), diffuse large B-cell lymphoma (DLBCL), or oral-lingual squamous cell carcinoma (OTSCC).

[0171] In some embodiments, the cancers that can be treated by the methods of the present disclosure are melanoma, kidney cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a soft tissue cancer. In some embodiments, the cancer is a fibrosarcoma. In some embodiments, the cancer is a diffuse endogenous pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0172] In some embodiments, cancers that can be treated by the methods of the present disclosure are hematological 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 gammaglobulinemia, plasmacytomyeloma, 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 others.

[0173] While not constrained by any particular theory, local release of certain anticancer agents using the compounds and methods of this disclosure may induce or contribute to 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 an effective antitumor immune response by activating dendritic cells (DCs) and, consequently, certain T cell responses. ICD is characterized by the secretion of damage-associated molecular patterns (DAMPs). Calreticulin (CRT), one of the DAMP molecules normally located in the lumen of the endoplasmic reticulum (ER), translocates to the surface of dying cells after inducing immunogenic apoptosis, where it functions as an "eat me" signal to professional phagocytic cells. 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 the cell surface, they have immunostimulatory effects based on their interaction with several antigen-presenting cell (APC) surface receptors, such as CD91 and CD40, and also promote the cross-presentation of antigens derived from tumor cells on MHC class I molecules, which then leads to a CD8+ T cell response. Other important DAMPs characteristic of ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is considered a late apoptosis marker, and its release into the extracellular space appears to be required 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, which are expressed on APCs. The most recently discovered DAMP released during immunogenic cell death is ATP, which, when secreted, acts as a "find-me" signal for monocytes, inducing their attraction to apoptotic sites. Kroemer et.al.Curr.Op.Immunol.2008(20),504-511.

[0174] Therefore, the local release of ICD-inducing substances using the compounds and methods of this disclosure can be beneficially combined with one or more immunomodulators.

[0175] In certain embodiments, the targeted portion can be used for the treatment, prevention, and / or diagnosis of solid tumors including, but are not limited to, melanoma (e.g., unresectable metastatic melanoma), kidney cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer such as metastatic epithelial ovarian cancer), breast cancer (e.g., triple-negative breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, and pancreatic cancer.

[0176] The approach of this disclosure is suitable for use as an adjuvant / neoadjuvant system. For example, the targeted portion disclosed herein can be placed during a biopsy, and upon return of the study results, the physician can administer an appropriate cocktail (compounds disclosed herein and any additional therapeutic agents) to deliver the treatment to the desired site in the body. The biopsy results may indicate the amount and type of treatment to be delivered to the tumor site. For example, chemokines (agents that attract cancer cells and / or immune cells) and adjuvants to enhance the immune system with fewer side effects, as well as chemotherapy agents, can be delivered and combined with immunotherapeutic agents.

[0177] The methods disclosed herein may be used to deliver a functionalized payload to these locations by systemic or topical administration. In some embodiments, the targeted portion is delivered systemically. In some embodiments, both the targeted portion and the payload-TCO conjugate are delivered systemically.

[0178] The compounds and compositions of this disclosure may be administered prior to surgical resection. The methods of this disclosure may minimize the size of the tumor prior to surgical resection. This will minimize the size of the tumor, particularly in the case of surgically resectable tumors. The conjugates, compounds, and compositions of this disclosure may be administered during surgical resection. The conjugates, compounds, and compositions of this disclosure may be administered after surgical resection. The targeted portion may be positioned around the surgical cavity at the end of the surgical resection, and the target may then be treated with additional doses to minimize the risk from cancer cells that may have been missed at the resection margin.

[0179] The functionalized payloads disclosed herein may function as adjuvants. This combination approach may be beneficial to patients. While chemotherapeutic agents may treat solid tumors or specific sites and enhance or induce immune responses, the enhanced response of immunotherapy with functionalized payloads and / or separate agents may aid distant metastatic sites. For example, in certain embodiments, the compositions and methods of the disclosed herein can enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer) using anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, camptothecin analogs, and other agents.

[0180] The methods described herein may be used to treat diffuse endogenous pontine glioma. Diffuse endogenous pontine glioma (DIPG) is a pediatric brainstem tumor that can be highly malignant and difficult to treat. There is no known curative treatment for DIPG, and survival rates have remained bleak for the past 40 years. Patients with DIPG have a median overall survival of only 11 months, and a 2-year survival rate of less than 10%. DIPG accounts for 75–80% of pediatric brainstem tumors, affecting an estimated 200–300 children each year in the United States. The rarity of this devastating disease and the lack of existing experimental model systems have hindered research, and survival rates have remained the same for the past 40 years. Diagnosis of DIPG may begin with clinical symptoms and may be confirmed by MRI. The disease may begin with several months of systemic symptoms, including behavioral changes and school difficulties, diplopia, abnormal or restricted eye movements, asymmetrical smiles, loss of balance, and weakness. Alternatively, severe neurological deterioration may occur more rapidly, with symptoms present for less than a month before diagnosis. Clinical examination may reveal polyneuropathy, cord signs such as hyperreflexia and clonus, and the triad of ataxia. Dilatation of the pontine portion of the brainstem can lead to obstructive hydrocephalus and increased intracranial pressure.

[0181] The nuclei essential for life-sustaining functions such as respiration and heart rate are located in the pons, and without treatment, DIPG can impair respiration and heart rate.

[0182] In certain embodiments, the methods disclosed herein involve administering both the targeted portion and the payload-TCO conjugate parenterally. As used herein, the term “parenteral” refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0183] For parenteral administration, the conjugates, compounds, or compositions disclosed herein may be dissolved or suspended in a physiologically acceptable diluent such as water, a buffer, oil with or without a solubilizer, a surfactant, a dispersant, or an emulsifier. Suitable oils include, for example, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil, and sesame oil. For parenteral administration, the conjugates, compounds, or compositions disclosed herein may be administered in the form of aqueous, lipid, oily, or other types of solutions or suspensions, or in the form of liposomes or nanosuspensions.

[0184] The amount of composition administered to the subject can be initially determined based on the dose guidance of the parent drug. Generally, the composition can provide targeted delivery and / or extension of the serum half-life of the conjugated drug, and thus can provide at least one of a dose reduction or a reduction in the number of administrations in the administration regimen. Thus, the composition can result in a dose reduction and / or a reduction in the number of administrations in the administration regimen compared to the parent drug before use in the method of this disclosure.

[0185] Pharmaceutical formulations may be provided in unit dosage forms. In such forms, the pharmaceutical formulation may be subdivided into unit doses containing an appropriate amount of the composition of the present disclosure. The unit dosage forms may be packaged formulations, and the packaging may contain discrete amounts of formulations such as packetized tablets, capsules, and powder in pouches, vials, or ampoules.

[0186] In some embodiments, a kit is provided comprising a targeted portion described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, and instructions for use thereof.

[0187] In some embodiments, the kit further includes a prodrug.

[0188] The compositions of this disclosure may be present in any preferred amount and may depend on a variety of factors, including but not limited to the subject's weight and age, disease state, etc. Preferred dosage ranges for the compositions of this disclosure include 0.1 mg to 10,000 mg, or 1 mg to 1,000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For example, preferred doses of the compositions of this disclosure include 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1,000 mg.

[0189] The compositions of this disclosure may be co-administered with other activators. Co-administration involves administering the compositions of this disclosure and the activators to each other within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours. Co-administration also involves administering the compositions of this disclosure and the activators simultaneously or nearly simultaneously (e.g., within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes) to each other, or sequentially in any order. In addition, the additional activators may be administered once daily, or two, three, or more times daily, to provide a desired daily dose level. Co-administration may be achieved by co-implantation or co-injection.

[0190] In some embodiments, co-administration can be achieved by co-formulation, for example, by preparing a single pharmaceutical formulation containing both the composition and the activator of the Disclosure. In other embodiments, the composition and the activator of the Disclosure can be formulated separately and co-administered to a subject.

[0191] The compositions and activators of this disclosure may be present in formulations in any suitable weight ratio, for example, 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 activators of this disclosure may 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 activators of this disclosure are preferred in formulations and methods described herein.

[0192] targeting part This specification provides a targeted moiety comprising an antibody fragment moiety covalently bound to one or more tetrazine moieties. The targeted moieties described herein are designed to localize to a target site within a subject upon administration. The targeted moiety can be administered topically or systemically. In some embodiments, the targeted moiety is a therapeutic targeted moiety. After administration, a prodrug containing a complementary bioorthogonal component (i.e., a trans-cyclooctene moiety) can be administered, which, upon contact with the targeted moiety in vivo, enables targeted drug delivery of a payload or therapeutic agent. In some embodiments, the targeted moiety described herein includes a diagnostic agent so that the targeted moiety can be used for the diagnosis of a condition or disease, whether or not a payload or therapeutic agent is administered.

[0193] In some embodiments, the antibody fragment portion is selected from the group consisting of single-stranded variable fragments (scFv), divalent (or bivalent) single-stranded variable fragments (di-scFvs, bi-scFvs), antigen-binding fragments (Fab), single-domain antibodies (sdAb), single-domain antibodies (sdAb), antigen-binding proteins, DotBody, afibody, DARPin, DART, TandAb, diabody, ribobody, centinrin, nottin, affinin, afimer, alphabody, antikalin, atrimer, avimer, finomer, Knitz domain, obody, pronectin, repebody, and bicyclic peptides or Humabody.

[0194] In some embodiments, the antibody fragment portion is selected from the group consisting of Fab2, Fab, scFV, minibody, diabody, VHH, V-NAR, or a fragment or polypeptide that targets a tumor by forming a targeting portion (e.g., peptide)-antigen complex.

[0195] In some embodiments, the antibody fragment portion is selected from the group consisting of single-strand variable fragments (scFv), divalent (or bivalent) single-strand variable fragments (di-scFvs, bi-scFvs), antigen-binding fragments (Fab), single-domain antibodies (sdAb), and single-domain antibodies (sdAb).

[0196] In some embodiments, the antibody fragment portion is an antigen-binding protein, DotBody, afibody, DARPin, DART, TandAb, diabody, ribobody, centinrin, nottin, affinrin, afimer, alphabody, antikalin, atrimer, avimer, finomer, Knitz domain, obody, pronectin, repebody, bicyclic peptide, or Humabody.

[0197] This specification provides a targeting portion of formula I, formula II, or formula V, [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 R represents an additional bond that forms dihydrotetrazine when both are present, however, when ring A is aryl. 3 and R 4 Assuming that both exist, X is the antibody fragment portion, p is between 1 and 20. In each appearance, L is independently a linker. R in each appearance 1 These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of 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'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally associated with 1 to 3 Z 1 It has been replaced with, R in each appearance 2Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally a Z 1 It has been replaced with, R 3 and R 4 If both are absent, or R 3 and R 4 Each of these is an independent group that can be removed after hydrogen or a trigger event. R in each appearance 20 These independently include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C( Selected from the group consisting of =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 in each appearance22 Independently and optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups, is a linker of 1 to 100 linked atoms. R in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. each Z 1 These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R Selected from ', 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'', In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0198] Compounds of formulas I, II, IIA, IIB, IIC, and III: In one embodiment, the targeting portion is given by formula I, [ka] During the ceremony, X is the antibody fragment portion, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C( Selected from the group consisting of =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 in each appearance 22 It is independently and optionally comprises a linker of 1 to 100 linked atoms containing one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups.

[0199] In one embodiment, the targeting portion is given by formula II, [ka] During the ceremony, X is the antibody fragment portion, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C( Selected from the group consisting of =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 in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0200] One embodiment: R in each occurrence 22 These are independently linkers of 1 to 100 linked atoms and may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups.

[0201] In one embodiment, the targeting portion is formula IIA, [ka] In the formula, L, p, X, and R 20 Each of these is independently defined as described herein.

[0202] In one embodiment, the targeting portion is formula IIB, [ka] In the formula, L, p, and X are each independently as defined herein.

[0203] In one embodiment, the targeting portion is formula IIC, [ka] In the formula, L, p, and X are each independently as defined herein.

[0204] In one embodiment, the targeting portion is given by formula III, [ka] During the ceremony, X is the antibody fragment portion, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C( Selected from the group consisting of =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 in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0205] In some embodiments, the targeting portion is formula IID, [ka] In the formula, X and R 20 Each of these is independently defined herein. In some embodiments, R 20X is methyl. In some embodiments, X is an antigen-binding protein. In some embodiments, X is an antigen-binding protein that targets HER2.

[0206] In some embodiments, the targeting portion is formula IIE, [ka] In the formula, p and X are each independently as defined herein.

[0207] In some embodiments, the targeting portion is formula IIF, [ka] In the formula, p and X are each independently as defined herein.

[0208] In some embodiments, the targeting portion is formula IIF, [ka] In the formula, p is between 1 and 10, and X is the antibody fragment portion containing SEQ ID NOs: 9 and SEQ ID NOs: 10.

[0209] In some embodiments, p is 1 to 5.

[0210] In some embodiments, a method is provided for administering a payload to a target payload, the method of which is a) an effective dose of formula IIF [ka] a) administering a targeted portion which is (wherein p is 1 to 10, or p is 1 to 5, and X is an antibody fragment portion which includes SEQ ID NO: 9 and SEQ ID NO: 10) to a target; and b) administering a therapeutically effective amount of a payload-TCO conjugate which comprises a payload having at least one trans-cyclooctene portion covalently bound thereto.

[0211] In some embodiments, a method is provided for forming an antibody-payload conjugate in vivo in a subject requiring an antibody-payload conjugate, and this method is... Effective quantity formula IIF [ka] The target portion is administered to the antibody fragment portion that is (wherein p is 1 to 10, or p is 1 to 5, and X is the antibody fragment portion containing SEQ ID NOs. 9 and SEQ ID NOs. 10). The administration of a single-dose therapeutically effective payload-TCO conjugate, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently bound thereto. The antibody or its fragments have binding affinity to receptors on the tumor, and furthermore, the amount of antibody-payload conjugate formed in vivo is greater at the tumor site compared to the plasma.

[0212] In some embodiments, the ratio of antibody-payload conjugate in the tumor site to 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. Larger than approximately 14:1, or larger than approximately 15:1, or larger than approximately 16:1, or larger than approximately 17:1, or larger than approximately 18:1, or larger than approximately 19:1, or larger than approximately 2:1, or larger than approximately 3:1, or larger than approximately 4:1, or larger than approximately 5:1, or larger than approximately 6:1, or larger than approximately 7:1, or larger than approximately 8:1, or larger than approximately 9:1, or larger than approximately 10:1.

[0213] In some embodiments, the targeting portion is formula IIG, [ka] In the formula, p and X are each independently as defined herein.

[0214] In some embodiments, compounds of formula IIG are provided, [ka] In the formula, p is 1 to 5, and X is the antibody fragment portion containing SEQ ID NOs: 9 and SEQ ID NOs: 10. In some embodiments, p is 2 to 3.

[0215] In some embodiments of formula IIA, [ka] [ka] That is the case.

[0216] In some embodiments of formula IIA, [ka] In the formula, R 20 This is defined herein.

[0217] In some embodiments of formula IIA, [ka] That is the case.

[0218] In some embodiments of formula IIA, [ka] In the formula, R 20 This is defined herein.

[0219] In some embodiments of formula IIA, [ka] That is the case.

[0220] 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 (e.g., Fab).

[0221] Compounds of formulas V, VI, and VII: In some embodiments, the targeting portion is given by formula V, [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 R represents an additional bond that forms dihydrotetrazine when both are present, however, when ring A is aryl. 3 and R 4 Assuming that both exist, X is the antibody fragment portion, p is between 1 and 20. In each appearance, L is independently a linker. R in each appearance 1These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -OR', -SR', -C(=O)R', -C(=S)R', -OC(=O)R''', -SC(=O)R''', -OC(=S)R''', -SC(=S)R''', -S(=O)R', -S(=O)2R''', -S(=O)2NR'R'', -C(=O)O-R', -C(=O)S-R', -C(=S)OR', -C(=S)SR', -C(=O)NR'R'', -C(=S)NR'R'', -NR'R Selected from the group consisting of '', -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'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl, optionally with 1 to 3 Z 1 It has been replaced with, R in each appearance 2 Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally a Z 1 It has been replaced with, R 3 and R 4If both are absent, or R 3 and R 4 Each of these is an independent group that can be removed after hydrogen or a trigger event. each Z 1 These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -OR', -SR', -C(=O)R', -C(=S)R', -OC(=O)R''', -SC(=O)R''', -OC(=S)R''', -SC(=S)R''', -S(=O)R', -S(=O)2R''', -S(=O)2NR'R'', -C(=O)O-R', -C(=O)S-R', Selected from -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'', In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0222] In this specification, the targeting portion of formula V is provided, [ka] During the ceremony, Ring A is a cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R4 This represents an additional bond that forms dihydrotetrazine when both are present. X is the antibody fragment portion, p is between 1 and 20. In each appearance, L is independently a linker. R in each appearance 1 These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of 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'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally associated with 1 to 3 Z 1 It has been replaced with, R in each appearance 2Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally a Z 1 It has been replaced with, R 3 and R 4 Both are either absent or R 3 It is a base that can be removed after the trigger event, R 4 is hydrogen or R 3 And, each Z 1 These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', and C(=O)S-R Selected from ', 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'', In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0223] In some embodiments, the targeting portion is formula VI, [ka] In the formula, R 1 , R 2 , R 3 , R 4 Each of the rings A, L, p, t, and X is independently defined as herein.

[0224] In some embodiments, R 4 It is hydrogen.

[0225] In the targeted portion described herein, R 3 is a base that can be removed after the trigger event. In some embodiments, the trigger event occurs in vivo. When the trigger event occurs, R 3 When removed, the dihydrotetrazine portion is oxidized to obtain tetrazine as shown in formula VII. [ka] In the formula, R 1 , R 2 Each of the rings A, L, p, t, and X is independently defined as herein.

[0226] The trigger event is initiated after administration to the targeted portion and can be initiated by any means, such as internal means (e.g., via enzymatic cleavage of functional groups, and optionally subsequent degradation) or external means (e.g., a photocleavable linker). In some embodiments, R 3 This includes a targeted portion such as an antibody fragment as described herein.

[0227] In some embodiments, R 3 It contains an amino acid sequence specific to cleavage by proteases or esterases.

[0228] In some embodiments, R 3 As shown in Table 1A, it contains an amino acid sequence specific to cleavage by proteases. [Table 2-1] [Table 2-2] [Table 2-3]

[0229] In some embodiments, R 3 This includes an amino acid sequence specific to cleavage by cathepsin, matrix metalloproteinase (MMP), or PSMA. For example, in some embodiments, R 3 This includes Val-Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly for cathepsin-mediated cleavage. In some embodiments, R 3 This comprises Ac-γE-PLG-S(Obn)YL or Ac-PLG-HofOrnL, where Hof is homophenylalanine and Orn is ornithine for cleavage by MMP. In some embodiments, R 3 It contains the amino acid sequence shown in Table 1B. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0230] Additional cleavable groups are described in Choi, et al., Theranostics. 2012;2(2):156-178, and Table 2 therein is incorporated herein by reference.

[0231] In some embodiments, R 3 It is photodegradable. In some embodiments, the photodegradable group is unstable or decomposes upon exposure to light of a wavelength that matches the absorbance profile of the photodegradable group.

[0232] In some embodiments, R 3 teeth, [ka] And, L 5 This is a direct link or linker, X 1 This is an optionally substituted peptide unit containing -NO2, an optionally substituted sugar moiety, or one or more natural or unnatural amino acids.

[0233] In some embodiments, at least one of the following parts: [ka] teeth, [ka] [ka] It is expressed by an expression selected from, where R 1 , R 2 , R 3 , and R 4 Each of them is independent as defined herein, and optionally, ring A portion may be one or more R 2 It can be replaced in parts.

[0234] In some embodiments, at least one of the following parts: [ka] teeth, [ka] It is expressed by an expression selected from, where X 2 is optionally an alkyl (e.g., methyl) substituted with PEG, amino acids, esters, amides, amines, -C(O)OH, -SO2, -SO3, -PO3, -PO4, or other solubility-enhancing substituents, and L, ring A, R 1 , R 2 Each of t, p, and X is independently defined as herein.

[0235] In some embodiments, ring A is a cycloalkyl group. In some embodiments, ring A is a heterocyclyl group. In some embodiments, ring A is a heteroaryl group. In some embodiments, ring A is an aryl group.

[0236] In some embodiments, ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl.

[0237] In some embodiments, ring A is phenyl.

[0238] In some embodiments, at least one of the following parts: [ka] teeth, [ka] It is expressed by an expression selected from, where R 1 and R 2 Each of these is independent and as defined herein.

[0239] Several embodiments, R in each occurrence 1 Each is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl, and each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with.

[0240] Several embodiments, R in each occurrence 1 Independently, hydrogen or of any choice, 1 to 3 Z 1 It is an alkyl group substituted with [a specific compound].

[0241] In some embodiments, Z in each occurrence 1 These are independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'.

[0242] Several embodiments, R in each occurrence 2 R is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl. In some embodiments, R in each appearance 2 R is independently a halo, alkyl, or haloalkyl. In some embodiments, R in each occurrence 2 These are independently halo or alkyl.

[0243] In some embodiments, t is 0 in each occurrence.

[0244] In some embodiments, the targeting portion is given by formula VA, [ka] In the formula, p and X are each independently as defined herein.

[0245] In some embodiments, the targeting portion is formula VB, [ka] In the formula, p and X are each independently as defined herein.

[0246] In some embodiments, ring A is something other than pyridyl. In some embodiments, ring A is something other than aryl. In some embodiments, ring A is something other than phenyl.

[0247] Antibody fragment part In some embodiments, X is 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), Nectin 4 (NCBI gene ID 81607), PSMA ( NCBI gene ID 2346), BCMA (NCBI gene ID 608), CD22 (NCBI gene ID 933), CD20 (NCBI gene ID 920), CD19 (NCBI gene ID 930), CD79b (NCBI gene ID 974), CD38 (NCBI gene ID 952), CD45 (NCBI gene ID 5788), Endoglin (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), folate receptor (NCBI gene ID 2348), DLL3 (NCBI gene ID 10683), CD138 (NCBI gene ID 6382), CD56 (NCBI gene ID 4684), CD37 (NCBI gene ID 951), CD74 (NCBI gene ID 972), mesothelin (NCBI gene ID 10232), IL-6R (NCBI gene ID 3570), SLAMF7 (NCBI gene ID 57823), BAFF (NCBI gene ID 10673), MUC1 (NCBI gene ID 4582), GPC3 (NCBI gene ID 2719), HER2 (NCBI gene ID 2064), HER3 (NCBI gene ID 2065), CD30 (NCBI gene ID 943), CD33 (NCBI gene ID 945), CD123 (NCBI gene ID 3563), GPNMB (NCBI gene ID 10457), cMET (NCBI Gene ID 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 ( This antibody fragment targets one or more of the following: 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).

[0248] In some embodiments, X is CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin 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), This is an antibody fragment that targets GPNMB (NCBI gene ID 10457), GPC1 (NCBI gene ID 2817), ITGB6 (NCBI gene ID 3694), SEZ6 (NCBI gene ID 124925), SLITRK6 (NCBI gene ID 84189), NaPi-2b (NCBI gene ID 20531), ZIP6 (NCBI gene ID 25800), ROR1 (NCBI gene ID 4919), or ROR2 (NCBI gene ID 4920).

[0249] In some embodiments, X is 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), Nectin 4 (NCBI gene ID 81607), PSMA ( NCBI gene ID 2346), BCMA (NCBI gene ID 608), CD22 (NCBI gene ID 933), CD20 (NCBI gene ID 920), CD19 (NCBI gene ID 930), CD79b (NCBI gene ID 974), CD38 (NCBI gene ID 952), CD45 (NCBI gene ID 5788), Endoglin (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), folate receptor (NCBI gene ID 2348), DLL3 (NCBI gene ID 10683), CD138 (NCBI gene ID 6382), CD56 (NCBI gene ID 4684), CD37 (NCBI gene ID 951), CD74 (NCBI gene ID 972), mesothelin (NCBI gene ID 10232), IL-6R (NCBI gene ID 3570), SLAMF7 (NCBI gene ID 57823), BAFF (NCBI gene ID 10673), MUC1 (NCBI gene ID 4582), GPC3 (NCBI gene ID 2719), HER2 (NCBI gene ID 2064), HER3 (NCBI gene ID 2065), CD30 (NCBI gene ID 943), CD33 (NCBI gene ID 945), CD123 (NCBI gene ID 3563), GPNMB (NCBI gene ID 10457), cMET (NCBI gene ID 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 35 70) This is an antibody fragment that targets one or more of the following: 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).

[0250] In some embodiments, X is an antibody fragment that targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin 4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

[0251] In some embodiments, X is an antibody fragment that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, Mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5.

[0252] In some embodiments, X is an antibody fragment that targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin 4, PSMA, BCMA, HER2, or CD25.

[0253] In certain embodiments, X is an antibody fragment that targets CD25, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0254] In certain embodiments, X is an antibody fragment that targets CEA, such as rabetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment derived therefrom.

[0255] In certain embodiments, X is an antibody fragment that targets CEACAM5, such as tusamitiamab or CC4, or an antibody fragment derived therefrom.

[0256] In certain embodiments, X is an antibody fragment portion that targets ASPH, for example, PAN-622, or an antibody fragment portion derived therefrom.

[0257] In certain embodiments, X is an antibody fragment portion that targets EGFR, such as cetuximab, necitumumab, nimotuzumab, matsuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, GC1118, imugatuzumab, panitumumab, artumumab, tomzotuximab, or laprituximab, or an antibody fragment portion derived therefrom.

[0258] In a particular embodiment, X is an antibody fragment portion that targets EPCAM, such as oportuzumab, sitatuzumab, tucotzumab, catumakisomab, edrecolomab, or adecatumumab, or an antibody fragment portion derived therefrom.

[0259] In certain embodiments, X is an antibody fragment that targets VEGFR, such as ramucizumab, ramucirumab, or vrinacimab, or an antibody fragment derived therefrom.

[0260] In a particular embodiment, X is an antibody fragment that targets PDGFR, for example, olaratumab or ramucirumab, or an antibody fragment derived therefrom.

[0261] In a particular embodiment, X is an antibody fragment that targets TROP2, such as sacituzumab, datopotamab, or Pr1E11, or an antibody fragment derived therefrom.

[0262] In a particular embodiment, X is an antibody fragment portion that targets nectin 4, such as enfortomab, 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.

[0263] In a particular embodiment, X is an antibody fragment portion that targets PSMA, for example, J591 or MLN591, or an antibody fragment portion derived therefrom.

[0264] In a particular embodiment, X is an antibody fragment portion that targets BCMA, for example, verantamab, or an antibody fragment portion derived therefrom.

[0265] In a particular embodiment, X is an antibody fragment that targets CD22, such as moxetumomab, inotuzumab, epratuzumab, or pinatuzumab, or an antibody fragment derived therefrom.

[0266] In certain embodiments, X is an antibody fragment that targets CD20, such as ubrituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, or ibritumomab, or an antibody fragment derived therefrom.

[0267] In certain embodiments, X is an antibody fragment that targets CD19, such as roncustuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, tapritumomab, or MDX-1342, or an antibody fragment derived therefrom.

[0268] In a particular embodiment, X is an antibody fragment that targets CD79b, for example, polatuzumab, or an antibody fragment derived therefrom.

[0269] In certain embodiments, X is an antibody fragment portion that targets CD38, such as isatuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment portion derived therefrom.

[0270] In a particular embodiment, X is an antibody fragment that targets CD45, for example, I-131-BC8 or Iomab-B, or an antibody fragment derived therefrom.

[0271] In a particular embodiment, X is an antibody fragment that targets endoglin, for example, carotuximab, or an antibody fragment derived therefrom.

[0272] In a particular embodiment, X is an antibody fragment that targets FGFR2, for example, bemarituzumab or appletuzumab, or an antibody fragment derived therefrom.

[0273] In a particular embodiment, X is an antibody fragment portion that targets C4.4A, for example, lupalzumab, or an antibody fragment portion derived therefrom.

[0274] In certain embodiments, X is an antibody fragment portion that targets claudin-18.2, for example, zolbetuximab or clodiximab, or an antibody fragment portion derived therefrom.

[0275] In a particular embodiment, X is an antibody fragment portion that targets MMP9, for example, andecaliximab, or an antibody fragment portion derived therefrom.

[0276] In certain embodiments, X is an antibody fragment that targets a folate receptor, such as milbetuximab, farletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment derived therefrom.

[0277] In a particular embodiment, X is an antibody fragment portion that targets DLL3, for example, lovalpituzumab, or an antibody fragment portion derived therefrom.

[0278] In a particular embodiment, X is an antibody fragment portion that targets CD138, for example, indatuximab, or an antibody fragment portion derived therefrom.

[0279] In certain embodiments, X is an antibody fragment portion that targets CD56, such as lorbotuzumab, promiximab, or an antibody fragment portion derived therefrom.

[0280] In a particular embodiment, X is an antibody fragment portion that targets CD37, for example, BI 836826, otreltuzumab, or naratuximab, or an antibody fragment portion derived therefrom.

[0281] In a particular embodiment, X is an antibody fragment portion that targets CD74, for example, milatuzumab, or an antibody fragment portion derived therefrom.

[0282] In a particular embodiment, X is an antibody fragment that targets mesothelin, such as anetumab, amatuximab, or MMOT-0530A, or an antibody fragment derived therefrom.

[0283] In certain embodiments, X is an antibody fragment that targets IL-6R, such as tocilizumab or sarilumab, or an antibody fragment derived therefrom.

[0284] In certain embodiments, X is an antibody fragment that targets SLAMF7, for example, elotuzumab, or an antibody fragment derived therefrom.

[0285] In a particular embodiment, X is an antibody fragment portion that targets BAFF, for example, belimumab, or an antibody fragment portion derived therefrom.

[0286] In a particular embodiment, X is an antibody fragment that targets MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, cribatuzumab, 8HuDS6, gatipotuzumab, AR20.5, or cantuzumab, or an antibody fragment derived therefrom.

[0287] In certain embodiments, X is an antibody fragment that targets GPC3, such as codolituzumab, ECT204, or MDX-1414, or an antibody fragment derived therefrom.

[0288] In certain embodiments, X is an antibody fragment that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment derived therefrom.

[0289] In certain embodiments, X is an antibody fragment that targets HER3, such as patritumab, cerivanthumab, lumuretuzumab, elgemtumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment derived therefrom.

[0290] In a particular embodiment, X is an antibody fragment portion that targets CD30, for example, brentuximab, or an antibody fragment portion derived therefrom.

[0291] In certain embodiments, X is an antibody fragment that targets CD33, such as gemtuzumab, BI 835858, vadasutuximab, or lintuzumab, or an antibody fragment derived therefrom.

[0292] In a particular embodiment, X is an antibody fragment that targets CD123, such as KHK2823, tacrotuzumab, or G4723A, or an antibody fragment derived therefrom.

[0293] In a particular embodiment, X is an antibody fragment portion that targets GPNMB, for example, glembatumumab, or an antibody fragment portion derived therefrom.

[0294] In a particular embodiment, X is an antibody fragment that targets cMET, such as terisotuzumab, onarutuzumab, or SAIT301, or an antibody fragment derived therefrom.

[0295] In a particular embodiment, X is an antibody fragment portion that targets CD142, for example, tisotumab, or an antibody fragment portion derived therefrom.

[0296] In a particular embodiment, X is an antibody fragment that targets NaPi2B, for example, rifastuzumab, or an antibody fragment derived therefrom.

[0297] In certain embodiments, X is an antibody fragment portion that targets GCC, for example, indusatumab, or an antibody fragment portion derived therefrom.

[0298] In a particular embodiment, X is an antibody fragment portion that targets STEAP1, for example, bundletuzumab, or an antibody fragment portion derived therefrom.

[0299] In a particular embodiment, X is an antibody fragment that targets MUC16, for example, sofituzumab, or an antibody fragment derived therefrom.

[0300] In a particular embodiment, X is an antibody fragment portion that targets CD70, for example, borsetuzumab, or an antibody fragment portion derived therefrom.

[0301] In a particular embodiment, X is an antibody fragment that targets CD44, for example, vivacuzumab, or an antibody fragment derived therefrom.

[0302] In a particular embodiment, X is an antibody fragment portion that targets vWF, for example, caplacizumab, or an antibody fragment portion derived therefrom.

[0303] In certain embodiments, X is an antibody fragment that targets TNF, such as ozoralizumab, V565, or PF-05230905, or an antibody fragment derived therefrom.

[0304] In certain embodiments, X is an antibody fragment that targets IL-6R, such as bovalilizumab, or an antibody fragment derived therefrom.

[0305] In certain embodiments, X is an antibody fragment that targets BCMA, for example, LCAR-B38M, or an antibody fragment derived therefrom.

[0306] In a particular embodiment, X is an antibody fragment portion that targets ADAMTS5, for example, M6495, or an antibody fragment portion derived therefrom.

[0307] In a particular embodiment, X is an antibody fragment portion that targets CX3CR1, for example, BI655088, or an antibody fragment portion derived therefrom.

[0308] In a particular embodiment, X is an antibody fragment portion that targets CXCR4, for example, AD-214 or ALX-0651, or an antibody fragment portion derived therefrom.

[0309] In a particular embodiment, X is an antibody fragment portion that targets TfR1, for example, TXB4, or an antibody fragment portion derived therefrom.

[0310] In a particular embodiment, X is an antibody fragment that targets VEGFR, for example, CDP791, or an antibody fragment derived therefrom.

[0311] In a particular embodiment, X is an antibody fragment portion that targets PSMA, for example, GY1, or an antibody fragment portion derived therefrom.

[0312] In a particular embodiment, X is an antibody fragment portion that targets FN1, for example, L19 or NJB2, or an antibody fragment portion derived therefrom.

[0313] In a particular embodiment, X is an antibody fragment portion that targets FAP, such as F19, OMTX005, or cibrotuzumab, or an antibody fragment portion derived therefrom.

[0314] In a particular embodiment, X is an antibody fragment portion that targets the TNC, for example, F16 or R6N, or an antibody fragment portion derived therefrom.

[0315] In some embodiments, the antibody fragment portion is daclizumab, RG6292, basiliximab, HuMax-TAC, rabetuzumab, 15-1-32, PR1A3, cT84.66, tusamithiamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matsuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, GC 1118, imugatuzumab, panitumumab, artumumab, tomzotuximab, laprituximab, oportuzumab, sitatuzumab, tucotsuzumab, catumakisomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, valinacimab (vulinacimab), oraratuzumab, ramucirumab, sacituzumab, Pr1E11, enfortumab, J591, MLN5 91, verantamab, moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ubrituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, ronkastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, tapritumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MO R202, TAK-079, I-131-BC8, Iomab-B, carotuximab, bemarituzumab, apulzumab, rupalzumab, zolbetuximab, clodiximab, andecaliximab, milbetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, lovalpituzumab, indatuximab, rorbotuzumab, promiximab, BI 836826, Otreltuzumab, Naratuximab, Miratuzumab, Anetuzumab, Amatsuximab, MMOT-0530A, Sarilumab, Elotuzumab, Belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, Cribatuzumab, 8HuDS6, Gatipotuzumab, AR20.5. Cantuzumab, Codlituzumab, ECT204, MDX-1414, Pertuzumab, Trastuzumab, Margetuximab, Patrizumab, Cerivantuzumab, Lumuletuzumab, Elgemzumab, AV-203, CDX-3379, GSK284933, Brentuximab, Gemtuzumab, BI 835858, vadasutuximab, lintuzumab, KHK2823, tacrotuzumab, G4723A, grembatumumab, terisotuzumab, onarutuzumab, SAIT301, tisotuzumab, rifastuzumab, indusatuzumab, bundletuzumab, sofituzumab, borsetuzumab, vibatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, cibrotuzumab, F16, R6N, datopotamab, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, derived from 74Hz (e.g., at least V). H and V L (This is a Fab that includes...)

[0316] In some embodiments, X is an antibody selected from atezolizumab, avelumab, bevacizumab, semiprimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, and trastuzumab.

[0317] In some embodiments, X or the antibody fragment portion is an antigen-binding fragment (Fab). The Fab is a region on the antibody that binds to the antigen and consists of one constant domain and one variable domain in both the heavy and light chains. In some embodiments, the Fab comprises four domains: VH, CH1, VL, and CL1. In some embodiments, the Fab contains 400–500 amino acids, or 440–480 amino acids. In some embodiments, the Fab has a molecular weight of approximately 50 kDa, or 40–55 kDa, or 45–50 kDa, or 45–55 kDa.

[0318] In some embodiments, Fab is trastuzumab, enfortumab, brentuximab, sacituzumab, L19 (gene ID 2335) that binds to FN-1, or F16 (gene ID 3371) that binds to TNC.

[0319] In some embodiments, the antibody fragment portion includes one or more PEG units that can improve circulating lifetime.

[0320] In some embodiments, the antibody fragment portion is an antigen-binding protein. The antigen-binding protein is a protein designed to be an antibody mimetic that exhibits high affinity and specificity to a given target. In some embodiments, the antigen-binding protein is a single-chain antigen-binding protein, which is a novel recombinant polypeptide consisting of an antibody variable light chain amino acid sequence (VL) linked to a variable heavy chain sequence (VH) by a peptide designed to link the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence.

[0321] In some embodiments, the antigen-binding protein is about 5–10 kDa or about 7 kDa. In some embodiments, the antigen-binding protein is about 50–80, or 60–70, or 66 amino acids long. 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.

[0322] In some embodiments, the antibody fragment portion is an antigen-binding protein targeting TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin 4, PSMA, BCMA, HER2, or CD25. In some embodiments, the antibody fragment portion is an antigen-binding protein targeting HER2. Antigen-binding proteins can be prepared and tested according to standard methods or purchased from commercial sources (e.g., Affilogic).

[0323] In some embodiments, the antibody fragment portion is daclizumab, RG6292, basiliximab, HuMax-TAC, rabetuzumab, 15-1-32, PR1A3, cT84.66, tusamithiamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matsuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, gc 1118, imugatuzumab, panitumumab, artumumab, tomzotuximab, laprituximab, oportuzumab, sitatuzumab, tucotsuzumab, catumakisomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, valinacimab (vulinacimab), oraratuzumab, ramucirumab, sacituzumab, Pr1E11, enfortumab, J591, MLN5 91, verantamab, moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ubrituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, ronkastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, tapritumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MO R202, TAK-079, I-131-BC8, Iomab-B, carotuximab, bemarituzumab, apulzumab, rupalzumab, zolbetuximab, clodiximab, andecaliximab, milbetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, lovalpituzumab, indatuximab, rorbotuzumab, promiximab, BI 836826, Otreltuzumab, Naratuximab, Miratuzumab, Anetuzumab, Amatsuximab, MMOT-0530A, Sarilumab, Elotuzumab, Belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, Cribatuzumab, 8HuDS6, Gatipotuzumab, AR20.5. Cantuzumab, Codlituzumab, ECT204, MDX-1414, Pertuzumab, Trastuzumab, Margetuximab, Patrizumab, Cerivantuzumab, Lumuletuzumab, Elgemzumab, AV-203, CDX-3379, GSK284933, Brentuximab, Gemtuzumab, BI 835858, vadasutuximab, lintuzumab, KHK2823, tacrotuzumab, G4723A, grembatumumab, terisotuzumab, onarutuzumab, sait301, tisotuzumab, rifastuzumab, indusatuzumab, bundletuzumab, sofituzumab, borsetuzumab, vibatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, BI Derived from 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, cibrotuzumab, F16, R6N, datopotamab, 15A7.5_H1L3, hNec.4.05, 14A5.2, 42D20-Hz3, 42D20-Hz10, HZD6.1C, HZD6.2C, and 74Hz.

[0324] In some embodiments, X is an antibody fragment portion derived from atezolizumab, avelumab, bevacizumab, semiprimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, or trastuzumab.

[0325] In a particular embodiment, X is an antibody fragment that targets vWF, for example, caplacizumab.

[0326] In certain embodiments, X is an antibody fragment that targets TNF, for example, ozoralizumab, V565, or PF-05230905.

[0327] In a particular embodiment, X is an antibody fragment that targets IL-6R, such as bovalilizumab.

[0328] In a particular embodiment, X is an antibody fragment that targets BCMA, for example, LCAR-B38M.

[0329] In a particular embodiment, X is an antibody fragment portion that targets ADAMTS5, for example, M6495.

[0330] In a particular embodiment, X is an antibody fragment that targets CX3CR1, for example, BI 655088.

[0331] In certain embodiments, X is an antibody fragment portion that targets CXCR4, for example, AD-214 or ALX-0651.

[0332] In a particular embodiment, X is an antibody fragment that targets TfR1, for example, TXB4.

[0333] In a particular embodiment, X is an antibody fragment that targets VEGFR, for example, CDP791.

[0334] In a particular embodiment, X is an antibody fragment that targets PSMA, for example, GY1.

[0335] In some embodiments, the antibody fragment portion is caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, M6495, BI 655088, AD-214, ALX-0651, TXB4, CDP791, or GY1.

[0336] In some embodiments, X further comprises an imaging contrast agent. In some embodiments, the imaging contrast agent is a protein.

[0337] Linker section In some embodiments, L is bound to X via a cystine or lysine residue on X.

[0338] In some embodiments, L is an inseparable linker.

[0339] In some embodiments, L is a severable linker.

[0340] In some embodiments, L comprises one or more amino acids.

[0341] In some embodiments, L includes a polypeptide.

[0342] In some embodiments, L comprises one or more of the following: hydrazone, hydrazide, disulfide, N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), 4-(4'-acetylphenoxy)butanoic acid (AcBut), one or more linear or branched, natural or unnatural amino acids, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety.

[0343] In some embodiments, L includes 1 to 100 linked atoms, 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0344] In some embodiments, L comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety independently and optionally oxo, halo, C 1-4 Alkyl, C 1-4Alkoxy, and C 1-4 It may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

[0345] In some embodiments, L is an alkylene linker comprising, optionally, one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.

[0346] In some embodiments, the linker L is given by the following formula: -Y 10 -(CHR 130 ) n’ -Y 20 -(CHR 140 ) n’’ -Y 30 -(CHR 150 ) m’’ -Y 40 - During the ceremony, Y 10 , Y 20 , Y 30 , and Y 40 Each of them is independent, combined, -NR 110 -, -O-, -S(O) 0-2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, -(CH2CH2O) 1-5 -, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, arylene, or heteroarylene can be independently and optionally oxo, halo, or C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R110 Hydrogen, C 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 130 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains, Each R 140 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains, Each R 150 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains, n', n'', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0347] In some embodiments, L is given by the following formula: -Y 10 -(CH2) n’ -Y 20 -(CH2) m’’ -Y 30 - During the ceremony, Y 10 , Y 20 , and Y 30 Each of them is independent, combined, -NR 110 -, -O-, -S(O) 0-2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110-, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, and each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene can be independently and optionally oxo, halo, or C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 120 Hydrogen, C 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. n' and m'' are independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0348] In a particular embodiment, each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 It is a haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. In certain embodiments, the linker is not a bond.

[0349] Linker L may contain one or more of the following: polyethylene glycol (e.g., PEG having an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diyrbis(methyl carbamate), arylene (e.g., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.

[0350] In some embodiments, the linker is [ka] Includes one or more of the following.

[0351] In some embodiments, the linker is [ka] Includes one or more of the following.

[0352] In some embodiments, the linker is [ka] It is either or includes it.

[0353] In some embodiments, the linker is one or more [ka] Includes.

[0354] In some embodiments, the linker is one or more [ka] Includes.

[0355] In some embodiments, the linker is one or more [ka] It is either or includes it.

[0356] In some embodiments, the linker is one or more [ka] It is either or includes it.

[0357] In some embodiments, the linker may comprise one or more natural or non-natural amino acids, which may be referred to as peptide linkers. The linker may also be a peptide linker composed of a carboxylacyl unit and one or more amino acids that constitute a protein or peptide sequence. The linker may also contain self-immolating spacers that space the drug and protein peptide sequences.

[0358] In some embodiments, the linker is "AYZX 2 It may also be a peptide-containing linker represented as "-W", where "A" is a carboxylate acyl unit, and "Y" and "Z" are each one or more natural or non-natural amino acids, together forming a peptide sequence, "X 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, that space the peptide and payload, D, or bioorthogonal portion. In certain embodiments, one or more amino acids in the peptide linker are N-methylated.

[0359] In some embodiments, Y may 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 may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0360] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, acetyl or formyl-protected lysine, arginine, tosyl or nitro-protected arginine, histidine, ornithine, acetyl or formyl-protected ornithine, and citrulline.

[0361] Examples of YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0362] In some embodiments, A is -OC(O)-.

[0363] In some embodiments, X 2 It is -OC(O)-.

[0364] In some embodiments, W is -OC(O)-. In some embodiments, X 2 It is absent, and W is -OC(O)-.

[0365] In some embodiments, part-X 2 -W is, [ka] Includes. In some embodiments, part-X 2 teeth, [ka] That is the case.

[0366] In some embodiments, -XW is [ka] That is the case.

[0367] In some embodiments, -XW is [ka] That is the case.

[0368] In some embodiments, the peptide linker is specifically modified so that it is selectively cleaved (e.g., enzymatically) by one or more tumor-associated proteases, thereby releasing the drug.

[0369] In some embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., dipeptide, tripeptide, or tetrapeptide). However, it will be understood that peptide linkers with up to 5, 6, 7, or 8 amino acid residues can also be suitably used.

[0370] 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 -Tosyl-Arg, or Phe-N 9 It is -nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0371] In some embodiments, the linker L is one or more of the following: [ka] [ka] [ka] [ka] It is one or more of the following, or includes one of them.

[0372] In some embodiments, the linker L is [ka] Includes one or more of the following.

[0373] In some embodiments, the linker L is [ka] Includes one or more of the following.

[0374] The aforementioned linker is an amino acid side chain present on X, such as lysine or cysteine. [ka] It can be combined with other things.

[0375] In some embodiments, the linker L is -C(O)L 4 -or -C(O)C 1-6 Alkilen C(O)L 4 -and, L 4 This is a bond, -N(R 12 )-C 2-3 Alkylene-N(R) 13 )C(O)-,-CH(NHC(O)R 14 )C 1-4 Alkylene-SSC 1-4 Alkylene-OC(O)-,-NHNHC(O)CH(NHC(O)R 15 )CH2C(O)-, -C 1-6 Alkylene-CH(G x )OC(O)-, [ka] And, R 12 , R 13 , R 14 , R 15 , and R 19 Each of them independently consists of hydrogen or C 1-4 It is alkyl, R 16 is hydrogen, C 1-4 Alkyl, -C 1-4 Alkylene-OH,-C 1-4 Alkylene-OC 1-4 Alkyl, -C 1-4 Alkylene-CO2H, or -C 1-4 It is alkylene-CONH2, G x The following are optional choices: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 This is a phenyl compound substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0376] In some embodiments, linker L includes a carbonyl moiety for conjugating the tetrazine moiety to the linker or X. For example, the linker may include a polypeptide moiety (PPM) having a lysine residue and a lysine side chain, and the PPM may also include additional lysine or other amino acid side chains conjugated to the carbonyl moiety. In some embodiments, linker L is [ka] It may include.

[0377] In some embodiments, the linker L is one or more of the following: [ka] That is the case.

[0378] In some embodiments, the linker L is one or more of the following: [ka]

[0379] In some embodiments, the linker L is one or more of the following: [ka] Includes one or more of the following.

[0380] In some embodiments, the linker L is one or more of the following: [ka] That is the case.

[0381] In some embodiments, the linker L is one or more of the following: [ka]

[0382] In some embodiments, the linker L is one or more of the following: [ka]

[0383] In some embodiments, the linker L is one or more of the following: [ka]

[0384] In some embodiments, the linker L is one or more of the following: [ka]

[0385] In some embodiments, the linker L is one or more of the following: [ka]

[0386] In some embodiments, the linker L is [ka] It is one or more of the following, or includes one of them.

[0387] In some embodiments, the linker L is [ka] It is one or more of the following, or includes one of them.

[0388] In some embodiments, the linker L is [ka] It is one or more of the following, or includes one of them.

[0389] In some embodiments, the linker L is [ka] It is one or more of the following, or includes one of them.

[0390] In some embodiments, the linker L is [ka] It is one or more of the following, or includes one of them.

[0391] In some embodiments, the linker L is [ka] It is one or more of the following, or includes one of them.

[0392] In one embodiment, the targeting portion is given by formula I, [ka] During the ceremony, p is between 1 and 16. R in each appearance 20 These independently include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C( Selected from the group consisting of =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 in each appearance 22 Independently and optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups, is a linker of 1 to 100 linked atoms. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. X is an antibody fragment that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, Mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5. L in each occurrence is independent, [ka] This linker is selected from the group consisting of the following:

[0393] In one embodiment, the targeting portion is given by formula II, [ka] During the ceremony, X is the antibody fragment portion, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20 These independently include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C( Selected from the group consisting of =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 in each appearance 30 These are independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a, and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4. X is an antibody fragment that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, Mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5. L in each occurrence is independent, [ka] This linker is selected from the group consisting of the following:

[0394] In one embodiment, the targeting portion is given by formula V, [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 R represents an additional bond that forms dihydrotetrazine when both are present, however, when ring A is aryl. 3 and R 4 Assuming that both exist, X is the antibody fragment portion, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 1These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of 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'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally associated with 1 to 3 Z 1 It has been replaced with, R in each appearance 2 Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally a Z 1 It has been replaced with, R 3 and R 4 If both are absent, or R 3and R 4 Each of these is an independent group that can be removed after hydrogen or a trigger event. each Z 1 These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', and C(=O)S-R Selected from ', 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'', In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4. X is an antibody fragment that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, Mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5. L in each occurrence is independent, [ka] This linker is selected from the group consisting of the following:

[0395] In some embodiments, ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl, each of which may be optionally substituted.

[0396] In some embodiments, ring A is phenyl.

[0397] In some embodiments, X is an antibody fragment that targets HER2, TROP2, Nectin-4, FN1, FAP, TNC, or ECM.

[0398] In some embodiments, X is zolbetuximab, clodiximab, anddecaliximab, anetumab, amatsuximab, MMOT-0530A, L19, NJB2, F19, OMTX005, cibrotuzumab, F16, or R6N, trastuzumab, enfortumab, or sacituzumab, datopotamab, or an antibody fragment portion derived therefrom.

[0399] In some embodiments, X is an antibody fragment portion derived from L19, NJB2, F19, OMTX005, cibrotuzumab, F16, or R6N, trastuzumab, enfortumab, or sacituzumab.

[0400] In some embodiments, p is 1 to 12. In some embodiments, p is 1 to 6, or 5 to 6.

[0401] In some embodiments, p is 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 2-10, or 2-9, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 2-3, or 3-10, or 3-9, or 3-8, or 3-7, or 3-6, or 3-5, or 3-4, or 4-10, or 4-9, or 4-8, or 4-7, or 4-6, or 4-5, or 5-10, or 5-9, or 5-8, or 5-7, or 5-6, or 6-10, or 6-9, or 6-8, or 6-7, or 7-10, or 7-9, or 7-8, or 8-10, or 8-9, or 9-10.

[0402] In some embodiments, p is 1-16, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2.

[0403] In a particular embodiment, p is 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 2-10, or 2-9, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 2-3, or 3-10, or 3-9, or 3-8, or 3-7, or 3-6, or 3-5, or 3-4, or 4-10, or 4-9, or 4-8, or 4-7, or 4-6, and X is 4-5, or 5-10, or 5-9, or 5-8, or 5-7, or 5-6, or 6-10, or 6-9, or 6-8, or 6-7, or 7-10, or 7-9, or 7-8, or 8-10, or 8-9, or 9-10, and X is an antibody fragment portion of 15kDa-55kDa, or 25-55kDa, or 15-50kDa, or less than 25kDa, or less than 35kDa, or less than 45kDa, or about 50kDa, or less than 55kDa.

[0404] In certain embodiments, p depends on the size and / or number of available binding sites on X for forming a covalent bond to L. In certain embodiments, if X is an antibody fragment portion of 25–55 kDa, p is 1–4. In certain embodiments, if X is an antibody fragment portion of 45–55 kDa, p is 1–4. In certain embodiments, if X is an antibody fragment portion of less than 45 kDa, p is 1–3, or 2–3, or 1–2, or about 1, about 2, or about 3. In certain embodiments, if X is an antibody fragment portion of less than 25 kDa, p is 1–3, or 2–3, or 1–2, or about 1, about 2, or about 3.

[0405] C. Payload-TCO Conjugate Trans-cyclooctene-functionalized prodrugs (payload-TCO conjugates), including prodrugs of anticancer agents such as those described in WO2018 / 187740, WO2014 / 205126, WO2015 / 139025, and WO2017 / 044983, which are incorporated herein by reference, and / or chelating agents with or without therapeutic or diagnostic radioligands, are known to those skilled in the art. Further exemplary embodiments are shown below.

[0406] In some embodiments, the payload-TCO conjugate is a conjugate consisting of a payload connected to one or more trans-cyclooctene portions.

[0407] In some embodiments, the payload-TCO conjugate (or trans-cyclooctene functionalized prodrug) includes an immunomodulatory payload, such as 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.

[0408] In some embodiments, the immunomodulatory payload is an inhibitor of the cytokine payload or a pharmaceutically acceptable salt thereof.

[0409] In some embodiments, the cytokine payload inhibitor is an inhibitor of TNF-α, infliximab, certolizumab, TGF-β, garnicertib, fresolimmab, M7824, CSF-1, pexidartinib, or kabilizumab.

[0410] In some embodiments, the payload-TCO conjugate comprises a monoclonal antibody or a pharmaceutically acceptable salt thereof.

[0411] In some embodiments, the payload-TCO conjugate comprises a therapeutic protein payload or a pharmaceutically acceptable salt thereof.

[0412] 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.

[0413] In some embodiments, the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen.

[0414] In some embodiments, the therapeutic protein payload may be, but is not limited to, erythropoietin (EPO, e.g., natural EPO or synthetic EPO (see, e.g., US2003 / 0191291)) such as PROCRIT®, EPREX®, or EPOGEN® (epoetin-α), ARANESP® (darbepoetin-α), NEORECORMON®, EPOGIN® (epoetin-β), etc.). Growth hormone (e.g., somatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®, SEROSTIM®, HUMATROPE®, etc.); Therapeutic monoclonal antibodies (e.g., atezolizumab, avelumab, bevacizumab, semiprimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab) Mab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, trastuzumab, etc.); human growth hormone (hGH); bovine growth hormone (bGH); follicle-stimulating hormone (FSH); interferon (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ, consensus interferon, etc.); insulin (e.g., Novolin, Humulin, Humalog, Lantus) (e.g., Ultralente), insulin-like growth factors (e.g., IGF-I, IGF-II); blood factors (but not limited to these, e.g., ACTIVASE® (alteplase) tissue plasminogen activator, NOVOSEVEN® (recombinant human factor VIIa), factor VIIa, factor VIII (e.g., KOGENATE®), factor IX, β-globin, hemoglobin, etc., e.g., factor X, tissue plasminogen activator (TPA), etc.);Colony-stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g., NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), Neurasta (pegfilgrastim), granulocyte-monocyte colony-stimulating factor, megakaryocyte colony-stimulating factor, etc.), transforming growth factors (e.g., TGF-beta, TGF-alpha); 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, e.g., REGRANEX® (beclapermin)), fibroblast growth factor (FGF, e.g., aFGF, bFGF, e.g., 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., ENBREL® (Etanercept), soluble VEGF receptor, soluble interleukin receptor, soluble γ / δ T-cell receptors, such as TNF-α-binding soluble receptors; enzymes (e.g., α-glucosidase, CERAZYME® (imiglucarase, β-glucocerebrosidase, CEREDASE® (algluculose)); enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10, Mig, Groα / IL-8, expressed and secreted regulated normal T cells (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;Bradykinin, cholecystokinin, gustin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, galanin, growth hormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, and other neuroactive peptides; thrombolytic agents, atrial natriuretic peptide, bone morphogenetic protein, etc. Other proteins such as rhombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human alpha-1 antitrypsin, leukemia inhibitors, transforming growth factors, tissue factor, insulin-like growth factor, luteinizing hormone, follicle-stimulating hormone, macrophage activator, tumor necrosis factor, neutrophil chemotactic factors, nerve growth factor, and other tissue inhibitors of metalloproteinases; vasoactive intestinal peptides, angiogenin, angiotropin, fibrin; hirudin; leukemia inhibitors; or payloads such as IL-1 receptor antagonists (e.g., Kineret® (Anakinra)).

[0415] In some embodiments, the payload-TCO conjugate is formula VIII or a pharmaceutically acceptable salt thereof. [ka] During the ceremony, m is an integer between 1 and 150. In each occurrence, G is independently a trans-cyclooctene moiety that has been substituted by arbitrary choice. D is the payload, L in each appearance 1 It is an independent linker.

[0416] In some embodiments of the payload-TCO conjugate described herein, each trans-cyclooctene portion is independently: [ka] During the ceremony, R in each appearance 1A C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Selected from the group consisting of alkoxys, q is 0, 1, or 2, q1 is 0 or 1, R in each appearance 1B G 1 -OH, -NR 1c -C 1-4 Alkilen-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 Alkilen) 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 Alkilen) 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 Selected from the group consisting of alkylene-CO2H)2, R in each appearance 1c and R 1d Independently, hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, R in each appearance 1f Hydrogen, C 1-6 Alkyl, or C 1-4 It is alkylene-CO2H. In each occurrence, n is independently 0, 1, 2, or 3. L in each appearance 2 Independently, -C(O)- and C 1-3 Selected from the group consisting of alkylenes, G in each appearance 1 These are heterocyclines that have been independently and arbitrarily substituted.

[0417] In some embodiments, the payload-TCO conjugate is formula VIII or a pharmaceutically acceptable salt thereof. [ka] During the ceremony, G is the trans-cyclooctene moiety, and in each occurrence, G is independently defined as follows: [ka] L in each appearance 1 It is independent and is a linker, m is an integer between 1 and 150. D is the payload, R in each appearance 1A C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Selected from the group consisting of alkoxys, q is 0, 1, or 2. q1 is 0 or 1, R in each appearance 1B G 1 , OH, -NR 1c -C 1-4 Alkilen-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 Alkilen) 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 Alkilen) 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 Selected from the group consisting of alkylene-CO2H)2, R in each appearance 1c and R 1d Independently, hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, R in each appearance 1f Hydrogen, C 1-6 Alkyl, or C 1-4 It is alkylene-CO2H. In each occurrence, n is independently 0, 1, 2, or 3. L in each appearance 2 Independently, -C(O)- and C 1-3 Selected from the group consisting of alkylenes, G in each appearance 1 These are heterocyclines that have been independently and arbitrarily substituted.

[0418] In some embodiments, q1 is 1.

[0419] In some embodiments, the payload is an immunomodulatory agent payload.

[0420] In some embodiments, the payload is a therapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, or immune checkpoint inhibitor, or a pharmaceutically acceptable salt thereof.

[0421] In some embodiments, the payload is a therapeutic agent for treating cancer (e.g., doxorubicin, daunorubicin, PNU-159682, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, baccatin III, gemcitabine, podophyllotoxin, carmustine, ixabépirone, patupirone (epoterone class), platinum-based drugs, exatecan, orlistin (drastatin 10, MMAE, MMAD, MMAF), duocalmycin, pyrrolobenzodiazapene dimer, mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin), immunosuppressants. The following are selected from disease control agents (e.g., cyclosporine A, rapamycin, etc.), antifungal agents (e.g., amphotericin, etc.), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, etc.), matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidyl (c-AMP).

[0422] In some embodiments, the payload may include therapeutic agents for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepylone, patupylone (epoteron class), platinum-based drugs, exatecan, orlistin (drastatin 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, etc.), immunosuppressants (e.g., cyclosporine A, rapamycin, etc.), and antifungal agents (e.g., A The following are selected from (e.g., muphotericin), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin beta, flucytosine, emtricitabine, gentamicin, colistin, etc.), lurubinectedin, gardiquimod, matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidyl (c-AMP).

[0423] Reference to a payload means that one or more atoms, including hydrogen or non-hydrogen atoms, of the original unmodified payload are replaced by covalent bonds to one or more linkers. The payload is modified to originate from a known nuclear payload and to covalently bond to trans-cyclooctene, which is substituted by at least one optional linker. Even after being modified to reach the compounds described herein, the payload maintains equivalent biological activity to that observed in the original unmodified payload. In certain embodiments, the payload exhibits binding activity or inhibition that is at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of that observed in the original unmodified payload.

[0424] In certain embodiments, hydrogen atoms bonded to heteroatoms (e.g., N, O, or S) in the original unmodified payload are replaced by covalent bonds to the linker. In certain embodiments, halogen atoms on the payload are replaced for bonding to the rest of the compound. In certain embodiments, hydrogen atoms on the payload are replaced for bonding to the rest of the compound. In certain embodiments, the hydrogen atom is on a heteroatom. In certain embodiments, the hydrogen atom is on nitrogen. In certain embodiments, the hydrogen atom is on oxygen. In certain embodiments, the hydrogen atom is on carbon.

[0425] In some embodiments, G in each occurrence is independent, [ka] That is the case.

[0426] In some embodiments, G in each occurrence is independent, [ka] That is the case.

[0427] In some embodiments, the payload is a monoclonal antibody payload. The monoclonal antibody used herein as the payload may be the entire monoclonal antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In some embodiments, the antibody is an immune cell engager and will therefore induce or trigger an immune response. In some embodiments, the monoclonal antibody or fragment thereof is 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 920). The target is one or more of the following genes: gene ID 3560, PD1 (NCBI gene ID 5133), CTLA4 (NCBI gene ID 1493), LAG3 (NCBI gene ID 3902), TIGIT (NCBI gene ID 201633), NCR1 (NCBI gene ID 9437), TIM3 (NCBI gene ID 84868), VISTA (NCBI gene ID 64115), CD134 (NCBI gene ID 7293), CD27 (NCBI gene ID 939), CD40L (NCBI gene ID 959), ICOS (NCBI gene ID 29851), BAFFR (NCBI gene ID 115650), LFA-1 (NCBI gene ID 3689), or BTLA (NCBI gene ID 151888).

[0428] In certain embodiments, the payload is a CD3-targeting antibody or antibody fragment, or an antibody fragment derived therefrom, such as OKT3, SP34, UCHT1, teplizumab, otelixizumab, bicilizumab, or foralumab.

[0429] In certain embodiments, the payload is a CD28-targeting antibody or antibody fragment, such as teralizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.

[0430] In certain embodiments, the payload is an antibody or antibody fragment that targets CD137(4-1BB), such as utomirumab, urerumab, LVGN6051, or AGEN2373, or an antibody fragment derived therefrom.

[0431] In a particular embodiment, the payload is an antibody or antibody fragment that targets CD16, such as AFM13, or an antibody fragment derived therefrom.

[0432] In a particular embodiment, the payload is an antibody or antibody fragment that targets NKG2D, such as NNC0152-0002 or JNJ-64304500, or an antibody fragment derived therefrom.

[0433] In a particular embodiment, the payload is an antibody or antibody fragment that targets CD64, such as H22, or an antibody fragment derived therefrom.

[0434] In certain embodiments, the payload is an antibody or antibody fragment that targets GITR / TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.

[0435] In certain embodiments, the payload is a CD25-targeting antibody or antibody fragment, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0436] In certain embodiments, the payload is a CD40-targeting antibody or antibody fragment, or an antibody fragment derived therefrom, such as iscarimab, ABBV-323, preserumab (ASKP-1240), BI-655064, FFP-104, BMS986090, dacetuzumab, or lucatumumab.

[0437] In certain embodiments, the payload is a CD4-targeting antibody or antibody fragment, or an antibody fragment derived therefrom, such as MAX.16H5, IT1208, zanorimumab (HuMax-CD4), UB-421, or MTRX1011A.

[0438] In a particular embodiment, the payload is an antibody or antibody fragment that targets CXCR4, such as F50067, or an antibody fragment derived therefrom.

[0439] In a particular embodiment, the payload is an antibody or antibody fragment that targets a G-CSFR such as CSL324, or an antibody fragment derived therefrom.

[0440] In a particular embodiment, the payload is a GM-CSFR-targeting antibody or antibody fragment, such as maprilimumab, or an antibody fragment derived therefrom.

[0441] In a particular embodiment, the payload is an antibody or antibody fragment that targets CD122, such as Hu-Mik(beta)1, or an antibody fragment derived therefrom.

[0442] In certain embodiments, the payload is a PD-1-targeting antibody or antibody fragment, such as CC-90006, semiprimab, camrelizumab, or TSR-042, or an antibody fragment derived therefrom.

[0443] In certain embodiments, the payload is an antibody or antibody fragment that targets CTLA4, such as tremelimumab or ipilimumab, or an antibody fragment derived therefrom.

[0444] In certain embodiments, the payload is an antibody or antibody fragment that targets LAG3, such as relatrimab (BMS-986016), GSK2831781, semiprimab (REGN3767), fabezerimab, ieramilimab, or mavezelimab, or an antibody fragment derived therefrom.

[0445] In certain embodiments, the payload is an antibody or antibody fragment that targets TIGIT, such as BMS-986207, tilagolumab, vivostrimab, etigirimab, dombanarimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.

[0446] In a particular embodiment, the payload is an antibody or antibody fragment that targets NCR1, such as hNKp46.02, or an antibody fragment derived therefrom.

[0447] In certain embodiments, the payload is an antibody or antibody fragment that targets TIM3, such as covolimab, Sym023, LY3321367, BMS-986258, SHR-1702, sabatrimab, or INCAGN02390, or an antibody fragment derived therefrom.

[0448] In certain embodiments, the payload is an antibody or antibody fragment that targets VISTA, such as SG7, K01401-020, CI-8993, or JNJ-61610588, or an antibody fragment derived therefrom.

[0449] In certain embodiments, the payload is an antibody or antibody fragment that targets CD134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.

[0450] In certain embodiments, the payload is an antibody or antibody fragment that targets CD27, such as varylumab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.

[0451] In a particular embodiment, the payload is a CD40L-targeting antibody or antibody fragment, such as dapyrolizumab, or an antibody fragment derived therefrom.

[0452] In certain embodiments, the payload is an antibody or antibody fragment that targets ICOS, such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.

[0453] In a particular embodiment, the payload is an antibody or antibody fragment that targets BAFFR, such as ianarumab, or an antibody fragment derived therefrom.

[0454] In a particular embodiment, the payload is an antibody or antibody fragment that targets LFA-1, such as efalizumab, or an antibody fragment derived therefrom.

[0455] In certain embodiments, the payload is an antibody or antibody fragment that targets BTLA, such as icatrimab, or an antibody fragment derived therefrom.

[0456] In some embodiments, the payload is an anti-CD3(αCD3) monoclonal antibody, or a derivative or analog thereof. In some embodiments, the anti-CD3(αCD3) monoclonal antibody is SP34, UCHT1, or OKT3, or a derivative or analog thereof.

[0457] In some embodiments, at least one payload is selected from poly(ADP-ribose) polymerase (PARP) inhibitors, duocalmycin, pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, anti-CD3 (αCD3) monoclonal antibodies, lurubinectedin, MSA-2, gardiquimod, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, exatecan, and MMAE, or derivatives or analogues thereof.

[0458] In some embodiments, D is a payload selected from poly(ADP-ribose) polymerase (PARP) inhibitors, duocalmycin, pyrrolobenzodiazepines (PBD), hemiasterlin, HTI-286, and anti-CD3 (αCD3) monoclonal antibodies, or derivatives or analogs thereof.

[0459] In some embodiments, at least one payload is selected from lurubinectedin, MSA-2, gardiquimod, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, exatecan, seco-duocalmycin SA, and MMAE, or derivatives or analogs thereof.

[0460] In some embodiments, the payload is a poly(ADP-ribose) polymerase (PARP) inhibitor, derivative thereof, or analogue. In some embodiments, the poly(ADP-ribose) polymerase inhibitor (PARP inhibitor) is niraparib, talazoparib, olaparib, pamiparib, lucaparib, veliparib, iniparib, 3-aminobenzamide, CEP-9722, E7016, or derivative thereof or analogue.

[0461] In some embodiments, the payload is [ka] It has a structure represented by an expression selected from.

[0462] In some embodiments, the payload is duocalmycin, or a derivative or analog thereof. In some embodiments, duocalmycin is duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, CC-1065, adzeresin, karzeresin, bizeresin, or a derivative or analog thereof.

[0463] In some embodiments, the payload is [ka] [ka] It has a structure represented by an expression selected from.

[0464] In some embodiments, the payload is pyrrolobenzodiazepine (PBD), or a derivative or analog thereof. In some embodiments, pyrrolobenzodiazepine (PBD) is [1,2]diazepino[3,4-e]indole, or a derivative or analog thereof.

[0465] In some embodiments, the payload is [ka] Includes.

[0466] In some embodiments, the payload is a tubulin polymerization inhibitor. In some embodiments, the payload is hemiasterlin, HTI-286, or a derivative or analog thereof.

[0467] In some embodiments, the payload is [ka] It originates from this.

[0468] In some embodiments, the payload is [ka] That is the case.

[0469] In some embodiments, the payload comprises a topoisomerase inhibitor. In some embodiments, the payload comprises camptothecin, or its derivatives or analogs. In some embodiments, the payload comprises topotecan, irinotecan, siratecan, cocitecan, exatecan, lurtotecan, gimatecan, berotecan, or rubitecan.

[0470] In some embodiments, the payload is [ka] Includes.

[0471] In some embodiments, the payload is [ka] Includes.

[0472] In some embodiments, the payload is [ka] Includes.

[0473] In some embodiments, the payload is [ka] Includes.

[0474] In some embodiments, the payload is [ka] Includes.

[0475] In some embodiments, the payload is [ka] Includes.

[0476] In some embodiments, the payload is [ka] Includes.

[0477] In some embodiments, the payload comprises a polypeptide. In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues. In some embodiments, the linker L 1 It is covalently bonded to a lysine, serine, threonine, or tyrosine residue present on the payload. In some embodiments, the polypeptide contains one or more lysine residues. In some embodiments, the linker L 1 It is covalently bonded to lysine residues present on the payload.

[0478] In some embodiments, the payload includes an N-terminal amino acid and a linker L 1 It is covalently bonded to the N-terminal amino acid.

[0479] In some embodiments, m is between 1 and 20.

[0480] In some embodiments, the payload is an immunomodulatory agent payload.

[0481] In some embodiments, the immunomodulatory agent payload is an antibody payload.

[0482] In some embodiments, the immunomodulatory agent payload is an immune checkpoint inhibitor payload. In some embodiments, the immune checkpoint inhibitor payload is a payload of pizilizumab, cintilimab, AMP-224, atezolizumab, durvalumab, BMS-936559, tremelimumab, indoximod, epacadostat, TIGIT inhibitors (e.g., LAG-3 such as an anti-LAG-3 antibody, TIM-3 such as an anti-TIM-3 antibody), B7 molecules, or BTLA pathway antagonists.

[0483] In some embodiments, the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload. In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload. In some embodiments, the PD-1 inhibitor payload is nivolumab, pembrolizumab, pizilizumab, cintilimab, or AMP-224 payload.

[0484] 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 payload.

[0485] 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 tremelimumab payload.

[0486] 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 indoximod or epacadostat payload.

[0487] In some embodiments, the immunomodulatory payload is a cytokine payload.

[0488] In some embodiments, the cytokine payload is 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.

[0489] 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.

[0490] In some embodiments, the immunomodulatory payload is type 1 cytokines (IL-2, IL-12, TNF-B, IFN-γ).

[0491] In some embodiments, the cytokine payload includes IFN-alpha, IFN-beta, IFN-gamma, PEGylated IFN-α, and apolipoprotein AI fusion proteins having IFN-α, interleukin, IL-2, IL-2 covalently bound to immunoglobulin (e.g., sergutuzumabu amnaleukin, RO6874281), IL-2 covalently bound to PEG molecules (e.g., NKTR-214), IL-10, PEGylated IL-10 (e.g., pegyrodecakin), IL-7, IL-12, IL-15, recombinant aglycosylated IL-15, IL-15 fusion proteins having an IL-15Rα binding domain (e.g., RLI), a triple fusion protein containing human IL-15, an IL-15Rα binding domain, and apolipoprotein AI, ALT-803 (IgG1 The group selected consists of IL-15 (fused to the Fc domain), IL-18, IL-21, tumor necrosis factor (TNF-alpha, TNF-beta), 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 talimogene laherparepvec.

[0492] In some embodiments, the immunomodulatory agent payload is a chemokine payload.

[0493] 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.

[0494] In some embodiments, the immunomodulatory payload is a chemokine antagonist payload. In some embodiments, the chemokine antagonist payload is a plerixafor payload.

[0495] In some embodiments, the immunomodulator is a monoclonal antibody specific to a cytokine or cytokine receptor.

[0496] In some embodiments, the immunomodulatory payload includes a polypeptide.

[0497] In some embodiments, the polypeptide comprises one or more lysine residues.

[0498] In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.

[0499] In some embodiments, trans-cyclooctene is ligated to one or more lysine residues.

[0500] In some embodiments, trans-cyclooctene is independently linked to one or more lysine, serine, threonine, or tyrosine residues.

[0501] In some embodiments, the polypeptide contains an N-terminal amino acid, and the appearance of the bioorthogonal moiety is linked to the N-terminal amino acid.

[0502] 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.

[0503] In some embodiments, the payload-TCO conjugate is expressed by formula IX [ka] A compound having, or a pharmaceutically acceptable salt thereof, wherein, R in each appearance 1a Hydrogen and C are independent of each other.1-4 Alkyl, and C 1-4 Selected from the group consisting of haloalkyls, R in each appearance 1b Hydrogen and C are independent of each other. 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 Selected from the group consisting of alkyl groups, R in each appearance 1c These are independently hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, In each occurrence, D is independently a payload. L in each appearance 1 It is independent and is a linker, In each occurrence, p' is independently 0, 1, or 2. In each occurrence, p'' is independently 1, 2, or 3.

[0504] In some embodiments, D in each occurrence is independently selected from the group consisting of anticancer drug payloads, Toll-like receptor (TLR) agonist payloads, and interferon gene stimulator (STING) agonist payloads.

[0505] In some embodiments, R 1a It is hydrogen.

[0506] In some embodiments, R 1a C 1-4 It is alkyl.

[0507] In some embodiments, R 1a This is CH3.

[0508] In some embodiments, R 1b -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 Selected from the group consisting of alkyl groups.

[0509] In some embodiments, R 1b C(O)OH, C(O)N(R) 1c )CHR 1e CO2H and C(O)N(R) 1c Selected from the group consisting of CH2CO2H.

[0510] In some embodiments, R 1b -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 It is selected from the group consisting of )-CH(CH2O-CH2CH2-CO2H)2.

[0511] In some embodiments, the trans-cyclooctene portion (G) is [ka] That is the case.

[0512] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0513] In some embodiments, the trans-cyclooctene portion is [ka] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0514] In some embodiments, the trans-cyclooctene portion is [ka] That is the case. In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0515] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0516] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0517] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0518] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0519] In some embodiments, the trans-cyclooctene portion is [ka] And R 2 R is -OH, 2-aminoethanesulfonic acid, N-linked natural or unnatural amino acids, or optionally substituted ethylenediamines, 2 This can optionally be further substituted with a polyether.

[0520] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0521] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0522] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0523] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0524] In some embodiments, R 1e These are -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH.

[0525] In some embodiments, R 1e is -C 1-4 It is alkylene-CO2H.

[0526] In some embodiments, R 1e It is -CH2CO2H.

[0527] In some embodiments, R 1b is -C(O)N(R 1c )-C 1-6 It is alkylene-CO2H.

[0528] In some embodiments, R 1b is -C(O)N(R 1c It is CH2CO2H.

[0529] In some embodiments, R 1c It is hydrogen.

[0530] In some embodiments, R 1b It is hydrogen.

[0531] In some embodiments, R 1b It is C(O)OH.

[0532] In some embodiments, the method includes targeted therapy. In some embodiments, this can be achieved by utilizing a TCO-targeting moiety and one or more pharmaceutically active agents (i.e., drugs or radioisotopes for radiotherapy or radioligand therapy). Suitable drugs for use in the context of targeted drug delivery are known to those skilled in the art. Optionally, the therapeutic probe may also include one or more detectable labels, such as imaging agents. The “radionuclide” (used herein interchangeably with “radiolid”) used in the trans-cyclooctene moiety described herein may be a chelating agent or isotope, for example. 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 It contains isotopes selected from the group consisting of Ac. Radionuclides can be delivered via direct conjugation or chelation with a chelating agent. Exemplary radionuclides, chelating agents, and linkers for potential TCO conjugate payloads are described below.

[0533] Bifunctional chelating agents (BFCs): [Table 4]

[0534] Radioactive nuclides for therapeutic use: [Table 5]

[0535] In some embodiments, the linker includes a linker as shown below, where BFC refers to a bifunctional chelating agent (e.g., as disclosed herein), and the dashed line indicates a binding point to TCO. [Table 6-1] [Table 6-2]

[0536] In some embodiments, the payload-TCO conjugate is expressed by formula IX [ka] or a pharmaceutically acceptable salt thereof, in the formula, R in each appearance 1a Hydrogen and C are independent of each other. 1-4 Alkyl, and C 1-4 Selected from the group consisting of haloalkyls, R in each appearance1b Hydrogen and C are independent of each other. 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 Selected from the group consisting of alkyl groups, R in each appearance 1c These are independently hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, In each occurrence, D is independently a payload. L in each appearance 1 It is independent and is a linker, In each occurrence, p' is independently 0, 1, or 2. In each occurrence, p'' is independently 1, 2, or 3.

[0537] In some embodiments, D in each appearance is a chelating agent suitable for use in radioligand therapy.

[0538] In some embodiments, D in each appearance is independently selected from 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: nitrilotriacetic acid, EDTA: ethylenediaminetetraacetic acid, DO3A: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid, NODAGA: 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononanetrizoxetane, tetraxetane, macrocyclic maleimide, or a mixture thereof.

[0539] In some embodiments, the payload-TCO conjugate is expressed by formula IXA [ka] or a pharmaceutically acceptable salt thereof, in the formula, R 1a However, hydrogen, C 1~4 Alkyl, and C 1~4 Selected from the group consisting of haloalkyls, D is the payload, L 1 But it's a linker.

[0540] In some embodiments, D is a chelating agent suitable for use in radioligand therapy. Exemplary, but not limited to, 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: nitrilotriacetic acid, EDTA: ethylenediaminetetraacetic acid, DO3A: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid, NODAGA: 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononanetrizoxetane, tetraxetane, macrocyclic maleimide, or mixtures thereof.

[0541] In some embodiments, the chelating agent is complexed with a radioactive ligand or radionuclide.

[0542] In some embodiments, linker L 1 It may have 1 to 100 linked atoms and may contain ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, a linker may have 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0543] In some embodiments, linker L 1It may contain one or more (e.g., 1 to 10 or 1 to 5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1 to 10 or 1 to 5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety independently and optionally oxo, halo, C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

[0544] In some embodiments, linker L 1 This may also be the following: -Y 10 -(CH2) n’ -Y 20 -(CH2) m’’ -Y 30 - During the ceremony, Y 10 , Y 20 , and Y 30 Each of them is independent, combined, -NR 110 -, -O-, -S(O) 0-2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, and each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene can be independently and optionally oxo, halo, or C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 120 Hydrogen, C 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. n' and m'' are independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0545] In a particular embodiment, the linker is a coupling.

[0546] In certain embodiments, the linker is not a link. In certain embodiments, each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.

[0547] Representative linkers, though not limited to these, include the following: [ka]

[0548] Representative linkers, though not limited to these, include the following: [ka]

[0549] In some embodiments, linker L1 This may include one or more of the following: polyethylene glycol (e.g., PEG having an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diyrbis(methyl carbamate), arylene (e.g., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. In some embodiments, linker L 1 teeth, [ka] It may include.

[0550] In some embodiments, linker L 1 This may contain one or more natural or non-natural amino acids and may be referred to as a peptide linker. If drug (D) contains an amino moiety, the linker may be linked to it using a peptide linker composed of a carboxyacyl unit and one or more amino acids that constitute a protein or peptide sequence. In some embodiments, linker L 1 It may also contain self-destructing spacers that space drug and protein peptide sequences.

[0551] In some embodiments, linker L 1 The linker may be a peptide linker represented as "AYZXW", where "A" is a carboxylacyl unit, "Y" and "Z" are one or more natural or non-natural amino acids, together forming a 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, that create a gap between the peptide and drug D or the bioorthogonal moiety. In certain embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0552] In some embodiments, Y may 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 may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0553] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, acetyl or formyl-protected lysine, arginine, tosyl or nitro-protected arginine, histidine, ornithine, acetyl or formyl-protected ornithine, and citrulline.

[0554] In some embodiments, exemplary YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0555] In a particular embodiment, A is -OC(O)-.

[0556] In a particular embodiment, X is -OC(O)-.

[0557] In a particular embodiment, W is -OC(O)-. In a particular embodiment, X does not exist, and W is -OC(O)-.

[0558] In a particular embodiment, -XW is, [ka] That is the case.

[0559] In a particular embodiment, -XW is, [ka] That is the case.

[0560] In certain embodiments, the peptide linker is specifically modified to be selectively cleaved (e.g., enzymatically cleaved) to release a drug, such as by one or more tumor-associated proteases.

[0561] In certain embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., dipeptide, tripeptide, or tetrapeptide). However, it will be understood that peptide linkers with up to 5, 6, 7, or 8 amino acid residues can also be suitably used.

[0562] In a particular embodiment, 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[sequence number], Ala-Leu-Ala-Leu[sequence number], Phe-N 9 -Tosyl-Arg, or Phe-N 9 It is -nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0563] In a particular embodiment, linker L 1 teeth, [ka] That is the case.

[0564] The aforementioned linker is the D amino acid side chain, such as lysine or cysteine. [ka] It can be attached to the right side.

[0565] In some embodiments, the payload is covalently bonded to the linker via an amide bond, and for example, the payload may be an amine-containing payload for bonding the payload to the carbonyl group of the linker, or in other cases, the payload may be a carboxyl-containing payload for bonding the payload to the amine group of the linker. In some examples, the payload and the linker together form a carbamate group, and for example, the payload may be an amine-containing payload for bonding the payload to the acyloxy group of the linker. In some examples, the payload and the linker together form a carbonate group, and for example, the payload may be a hydroxyl-containing payload for bonding the payload to the acyloxy group of the linker.

[0566] In some embodiments, L 1 teeth, [ka] or -O-, L 3a is a combination or C 1-6 It is alkylene, L 4a is a combination, -NHN:, -N(R 10 )-C 2-6 Alkylene-N(R) 11 )-,-N(R 12 )-C 2-3 Alkylene-N(R) 13 )C(O)-, -N(R 10 )-C 1-6 Alkylene-C(O)NHN:,-NHNHC(O)C 1-6 Alkylene-C(O)NHN:,-CH(NHC(O)R 14 )C 1-4 Alkylene-SSC 1-4 Alkylene-OC(O)-,-NHNHC(O)CH(NHC(O)R 15)CH2C(O)-, -C 1-6 Alkylene-CH(G x )OC(O)-, [ka] And, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 19 Each of them independently consists of hydrogen or C 1-4 It is alkyl, R 16 is hydrogen, C 1-4 Alkyl, -C 1-4 Alkylene-OH,-C 1-4 Alkylene-OC 1-4 Alkyl, -C 1-4 Alkylene-CO2H, or -C 1-4 It is alkylene-CONH2, R in each appearance 17 These are independently hydrogen or -CH2OC(O)-, G x is halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C 1-4 This is a phenyl molecule optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0567] In a particular embodiment, linker L 1 It is -OC(O)-.

[0568] In some embodiments, L 1 teeth, [ka] And, L 3a It is a combination, L 4a teeth, [ka] And, R 12 and R 13 Each of them independently consists of hydrogen or C 1-4 It is alkyl.

[0569] In some embodiments, p'' is 1. In some embodiments, p' is 1.

[0570] In some embodiments, [ka] [ka] And, R in each appearance 18 These are independently hydrogen or -CH2OC(O)NHD', R D This refers to hydrogen or C on the nitrogen atom of the payload. 1-4 It is alkyl, D and D' are independently payload portions.

[0571] 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 drug payload portion.

[0572] In some embodiments, [ka] And, R 12 and R 13 Each of them independently consists of hydrogen or C 1-4 It is alkyl, D and D' are independently payload portions (e.g., anticancer drug payload portions).

[0573] In some embodiments, p' is 0.

[0574] In some embodiments, p'' is 2 or 3.

[0575] In some embodiments, p is 2, [ka] That is the case.

[0576] Those skilled in the art will recognize that the payload (D or D') bound to the linker does not refer to the payload molecule itself, but rather to a portion of the payload molecule bound to the linker. The release of the payload (D or D') from the prodrug releases the payload itself.

[0577] The payload (D or D') may be any of the anticancer agents described herein.

[0578] In some embodiments, the payload includes a TLR7 / 8 agonist.

[0579] In some embodiments, the payload comprises a TLR7 / 8 agonist, where X is an antibody or antibody fragment that targets HER2, TROP2, nectin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM). In some embodiments, the payload comprises gardikimod, where X is an antibody or antibody fragment that targets HER2, TROP2, nectin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM).

[0580] In some embodiments, the payload comprises camptothecin or a derivative thereof. In some embodiments, the payload comprises exatecan. In some embodiments, the payload comprises camptothecin or a derivative thereof, where X is an antibody or antibody fragment that targets HER2, TROP2, nectin 4, or the extracellular matrix (ECM). In some embodiments, the payload comprises exatecan, where X is an antibody or antibody fragment that targets HER2, TROP2, nectin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM).

[0581] In some embodiments, the payload includes MMAE. In some embodiments, the payload includes MMAE or a derivative thereof, where X is an antibody or antibody fragment that targets HER2, TROP2, nectin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM).

[0582] In some embodiments, the payload comprises paclitaxel. In some embodiments, the payload comprises paclitaxel or a derivative thereof, where X is an antibody or antibody fragment that targets HER2, TROP2, nectin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM).

[0583] In some embodiments, the payload comprises docetaxel or a derivative thereof, where X is an antibody or antibody fragment that targets HER2, TROP2, nectin 4, CEACAM5, fibronectin, or the extracellular matrix (ECM).

[0584] In some embodiments, the payload-TCO conjugate has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0585] In a particular embodiment, the payload-TCO conjugate is [ka] [ka] [ka] Selected from.

[0586] In a particular embodiment, the payload-TCO conjugate is [ka] Selected from.

[0587] D. Combination therapy In one embodiment, a method is provided for treating cancer or enhancing or inducing an immune response, the method comprising administering to a subject in need a therapeutically effective amount of a targeted portion of the present disclosure or a pharmaceutically acceptable salt or composition thereof; a prodrug such as those described herein; and optionally, a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of anticancer agents or immunomodulators.

[0588] This disclosure also provides payload-TCO conjugates described herein, or pharmaceutically acceptable salts or compositions thereof, for use in the treatment or prevention of cancer, or for use in enhancing or inducing an immune response; and pharmaceutically acceptable combinations comprising additional therapeutic agents, such as anticancer agents or immunomodulators.

[0589] In the methods and uses described herein, the components of a pharmaceutically acceptable combination may be administered / used simultaneously, separately, sequentially, and in any order, and the components may be administered separately or as a fixed combination. For example, delaying or treating disease progression according to this disclosure may include administering a first active ingredient in the form of a free salt or pharmaceutically acceptable salt, in a joint therapeutic effective dose or effective dose, e.g., a daily dose corresponding to the amount described herein, and administering a second active ingredient in its free form or pharmaceutically acceptable salt form, simultaneously or sequentially in any order. The individual active ingredients of a combination may be administered separately at different times during the course of treatment, or simultaneously in divided or single dosage forms. Accordingly, this disclosure should be understood to encompass all such regimens of simultaneous or alternating treatment, and the term “administer” should be interpreted accordingly. Accordingly, as used herein, a pharmaceutically acceptable combination defines either a fixed combination of one unit dosage form or separate dosage forms for concomitant administration, and concomitant administration may be independently, simultaneously, or at different times.

[0590] Methods and uses in the treatment of cancer include administration / localization of the targeted portion in the tumor.

[0591] Additional therapeutic agents may be administered simultaneously with or sequentially to 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, additional therapeutic agents may be administered in the same composition as the payload-TCO conjugate. In other embodiments, there may be a time interval between the administration of the additional therapeutic agent and the administration of the payload-TCO conjugate or composition. In some embodiments, administration of an additional therapeutic agent with the payload-TCO conjugate or composition may allow for lower doses and / or less frequent administration of the other therapeutic agents. When used in combination with one or more other active ingredients, the payload-TCO conjugate or composition and the other active ingredients of this disclosure may be used in lower doses than when each is used alone. Therefore, the pharmaceutical compositions of this disclosure include those containing one or more other active ingredients in addition to the payload-TCO conjugate of this disclosure.

[0592] anticancer drugs Examples of anticancer drugs, though not limited to these, include abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib dimaleate, Afinitor (everolimus), Aldara (imiquimod), and Aldeslo Ikin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), Ambochlorin (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), arsenic trioxide, Arzerra (ofatumumab), asparaginase, erwinia chrysanthemum, Avastin (bevacizumab), axitinib, azacitidine, BEA COPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab and I131 iodine tositumomab), bicalutamide, bleomycin, bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib-S-malate, CAF, Campath (aremtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfil Zomib, Casodex (bicalutamide), CeeNU (lomustine), Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cometriq (cabozantinib-S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib,CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin difutitox, denosumab, DepoCyt (liposomal cytarabine) Bin), DepoFoam (liposomalcitarabine), dexrazoxane hydrochloride, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Efudex (fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Eloxa tin (oxaliplatin), eltrombopagolamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (bismodegib), erlotinib hydrochloride, Erwinaze (asparaginase erwinia chrysanthemum), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet ( Doxorubicin hydrochloride (liposome), everolimus, Evista (raloxifene hydrochloride), exemestane, Fareston (toremifene), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), Folfiri, Folfiri-bevacizumab, Folfiri-cetuximab, Folfirinox, Folfox (leucovorin, fluorouracil, oxaliplatin), Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV tetravalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin,Gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV tetravalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hyper-CV AD, ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Inlyta (axitinib), Intron A (recombinant interferon alpha-2b), iodine-131 tositumomab and tositumoma Mab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepyrone, Ixempra (ixabepyrone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Kypro lis (carfilzomib), lapatinib ditosylate, lenalidomide, letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), liposomalcitarabine, 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), mechloretamine hydrochloride, Megace (megestrol acetate), 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 (Prelixafor), Mustargen (Mechloretamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin Stabilized Nano (Particle formulation), Navelbine (vinorelbine tartrate), nelarabine, Neosar (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, ofatumumab, omasetaxin mepesuccinate, Oncaspar (peguaspar gauze), Ontak (deniroikin difutitox), OEPA, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin Stabilized nanoparticle formulations, Palifermin, Palonosetron hydrochloride, Pamidronate disodium, Panitumumab, Paraplat (carboplatin), Paraplatin (carboplatin), Pazopanib hydrochloride, Pegaspargaze, Peginterferon alfa-2b, PEG-Intron (pegylated interferon alfa-2b), Pemetrexed disodium, Perjeta (pertuzumab), Pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Prelixafor, Pomalidomide, Po malyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopagolamine), Provenge (ciproisel-T), Purinethol (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, rasburicase, R-CHOP, R-CVP, recombinant HPV bivalent vaccine, recombinant HPV tetravalent vaccine, recombinant interferon alpha-2b, regorafenib,Revlimid (lenalidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, Sclerosol intrapleural aerosol (talc), ciproisel-T, sorafenib tosylate, Sprycel (dasatinib), Stanford V, Sterilized talc powder (talc), Steritalc (talc), Stivarga (regorafenib), Sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alpha-2b), Synovir (thalidomide), Synribo (omacetaxin mepesuccinate), Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tocitumomab and 1 131 Iodine tocitumomab, Totect (dexrazoxane hydrochloride), trametinib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), vandetanib, VAMP, Vectibix (panitumumab), VelP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomes, vinorelbine tartrate, bismodegib, Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine),Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dex, Examples include lazoxane hydrochloride, Ziv-aflibercept, Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), and Zytiga (abiraterone acetate).

[0593] The anticancer agents may be PBD dimer, calicheamicin, speromycin, tubulisin B, rhizoxin, drastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothyron B, taxol, cryptophycin, SN38, Velcade, bruseantine, DAVLBH, DM1, filantoside, Alimta, T2 toxin, MMC, vantaranib, vinorelbine, breferdin, sunitinib, daunomycin, semacanib, tarceva, Iressa, irinotecan, LY-541503, geldanomycin, gemcitabine, methotrexate, gleevec, topotecan, bleomycin, doxorubicin, cisplatin, N-mustard, etoposide, or 5-FU.

[0594] In certain embodiments, the anticancer agent is an anthracycline. In certain embodiments, the anticancer agent is a taxane. In certain embodiments, the anticancer agent is gemcitabine. In certain embodiments, the anticancer agent is doxorubicin. In certain embodiments, the anticancer agent is docetaxel. In certain embodiments, the anticancer agent is SN38. In certain embodiments, the anticancer agent is monomethyl auristatin E.

[0595] Compound Synthesis Targeted moieties may be prepared using the methods disclosed herein and modifications thereof, as will be apparent from the consideration of the methods disclosed herein and methods well known in the art. In addition to the teachings herein, conventional and well known synthetic methods may be used. The synthesis of typical targeted moieties disclosed herein can be achieved as disclosed in the following examples. Where available, reagents and starting materials may be purchased commercially from, for example, Sigma Aldrich or other chemical suppliers.

[0596] Given typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.), it will be understood that 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 routine optimization procedures.

[0597] Furthermore, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. Suitable protecting groups for various functional groups, as well as preferred conditions for protecting and deprotecting specific functional groups, are well known in the art. For example, numerous protecting groups are described in Wuts, PGM, Greene, TW, & 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 leave as a stable species, accompanied by its bonding electrons. Examples of suitable leaving groups include halides (e.g., Br, Cl, I), sulfonic acid esters (e.g., triflates, mesylates, tosylates, and brosylates), and nitrophenols.

[0598] As shown in Scheme I, the compound of formula V (wherein the formula, dotted line, R 1 , R 2, R 3 , R 4 Each of rings A, t, L, p, and X is defined independently herein, and R 50 A synthetic handle for binding to L (e.g., a leaving group, e.g., a halo, or a portion of L that can be bound to X, or a further portion of L, e.g., hydroxy, amino, methylamino, etc.) can be prepared by coupling a compound of formula I-2 with a appropriately functionalized antibody fragment portion X. Suitable methods can be adapted from the literature (e.g., 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. A preferred coupling method, but is not limited thereto, involves the use of a succinimide functional group capable of forming an amide bond with a primary amine on the antibody fragment, or L can be functionalized with a group capable of forming a covalent bond with a cysteine ​​residue on the antibody fragment portion, such as pyrrole-2,5-dione. Scheme I [ka]

[0599] The compound of formula I-1 can be prepared according to scheme II, with each dotted line, R 1 , R 2 , R 3 , R 4 Rings A, t, and L are defined independently herein, and R 50 X is a synthetic handle for binding to L, such as a leaving group, such as a halo or thioether, or a part of L or a further part of L that can be bound to X, such as hydroxy, amino, methylamino, etc., and each LG is independently a leaving group, such as a halo. Scheme II [ka]

[0600] As shown in Scheme II, compound II-3 is obtained by coupling compound II-1 with compound II-2 in the presence of N2H4. Further modification of compound II-3 with compound II-4 and / or compound II-6 under standard coupling conditions yields compound II-5 and / or compound II-7. Alternatively, compound II-3 can be obtained by coupling compound II-8 with compound II-9 in the presence of N2H4. Compound II-10 can be obtained by contacting compound II-3 with a suitable oxidizing agent (e.g., NaNO2). Alternatively, compound II-3 can be obtained by contacting compound II-10 with thiourea dioxide.

[0601] After each reaction is complete, the intermediate or final compound can be recovered by conventional techniques such as neutralization, extraction, precipitation, chromatography, and filtration, and optionally purified.

[0602] 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. In addition, any of the intermediates or any products obtained by the processes outlined in Scheme I or II can be derivatized at any step to obtain a variety of compounds of formula V.

[0603] Exemplary payloads and their conjugates may 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 entirety of which is incorporated herein). Exemplary procedures for a particular payload are shown in the following examples, but these procedures can be adapted to prepare other payloads, such as those disclosed herein. [Examples]

[0604] The following embodiments are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that function well in the implementation of the Disclosure and can therefore be considered to constitute a particular form for its implementation. However, those skilled in the art will understand that, in view of the Disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the Disclosure to obtain similar or similar results.

[0605] LC-MS analysis method: Test specimens were subjected to PNGaseF(IgG) and DTT or DTT alone (Fab) in RapiGest according to the manufacturer's protocol. Samples were diluted to 100 μg / mL with water and centrifuged at 4°C at 16.1 k RCF for 10 minutes. The samples were then analyzed by LC-MS (LC-Q-TOF), and the mass spectrum was reconstructed from the charge ladder.

[0606] Example 1: Synthesis of tetrazine-trastuzumab targeting moiety Trastuzumab (22.1 mg / mL, 1.1 mL) in 0.01 M PBS was mixed with 20 equivalents of methyltetrazine-PEG4-NHS (Clickchemtools #1069-10). [ka]

[0607] The reactants were thoroughly mixed and aged at room temperature for 1 hour. At this point, the reactants were quenched by adding 1 volume of 0.1 M Tris buffer. The resulting solution was buffer-exchanged with 0.01 M PBS to remove excess reagent and buffer salts. The resulting targeted moiety solution (6.3 mg / mL, 1.6 mL) was analyzed by SDS-Page and LC-MS (data not shown) to confirm the formation of the targeted moiety and was therefore used for subsequent studies. Based on the analysis, it is assumed that up to 12 methyltetrazine-PEG4 units are covalently bound to the antibody.

[0608] Example 2: Tetrazin-Fab targeting moiety Fab was prepared from trastuzumab using a commercially available kit (Pierce® Fab Preparation Kit #44985) according to the manufacturer's protocol and purified 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 reactants were thoroughly mixed and aged at room temperature for 1 hour, at which point the reactants were quenched by adding 1 volume of 0.1 M Tris buffer. The resulting solution was buffer-exchanged with 0.01 M PBS to remove excess reagents and buffer salts. The resulting targeted moiety solution (1.0 mg / mL, 10.3 mL) was analyzed by SDS-Page and LC-MS (data not shown) to confirm the formation of the targeted moiety and was therefore used for subsequent studies. Based on the analysis, it is assumed that up to approximately 6 methyltetrazine-PEG4 units are covalently bound to the Fab.

[0609] Example 3: Tetrazin-Fab targeting moiety Fab was prepared from enfortumab using the following method: 0.1 mg of papain at a concentration of 0.5 mg / ml was pretreated by incubation with 1 mM DTT and 2 mM EDTA at 37°C for 30 minutes. 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 in a (1:100) molar ratio and incubated at 37°C for 2 hours. The digested mixture was placed on an anti-CH1 affinity column, washed with 25 mM Tris, 150 mM NaCl, pH 8.0, and eluted with 50 mM sodium citrate, 150 mM NaCl, pH 3.0. The filtrate containing the product Fab was dialyzed to PBS. The purified Fab was concentrated in PBS to 0.2 mg / mL. Fab was mixed with Me-Tet-PEG9-NHS (prepared to 10 mM in DMSO) in a 3:1 molar ratio and incubated at 37°C for 2 hours. The resulting conjugate was analyzed by LC-MS, and the DAR was calculated to be 2.66.

[0610] Example 4: Tetrazin-Fab targeting moiety Fab was prepared from brentuximab using the following method: 0.1 mg of papain at a concentration of 0.5 mg / ml was pretreated by incubation with 1 mM DTT and 2 mM EDTA at 37°C for 30 minutes. 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 in a (1:100) molar ratio and incubated at 37°C for 2 hours. The digested mixture was placed on an anti-CH1 affinity column, washed with 25 mM Tris, 150 mM NaCl, pH 8.0, and eluted with 50 mM sodium citrate, 150 mM NaCl, pH 3.0. The filtrate containing the product Fab was dialyzed to PBS. The purified Fab was concentrated in PBS to 0.2 mg / mL. Fab was mixed with Me-Tet-PEG9-NHS (prepared to 10 mM in DMSO) in a 3:1 molar ratio and incubated at 37°C for 2 hours. The resulting conjugate was analyzed by LC-MS, and the DAR was calculated to be 4.57.

[0611] Example 5A: Tetrazin-Fab targeting moiety Using a commercially available kit (Pierce® Fab Preparation Kit #44985) according to the manufacturer's protocol, Fab was prepared from sacituzumab and purified using Protein G resin (BioVision #6511-25). The purified Fab 10 mM Me-Tet-PEG9-NHS was prepared in DMSO. The two components were reacted at 25°C for 2 hours in a drug-to-protein molar ratio of 3:1, and then dialyzed against PBS, pH 7.4 to remove excess Me-Tet-PEG9-NHS compound from the protein component. The resulting targeted moiety solution was analyzed by SDS-Page and LC-MS to confirm the formation of the targeted moiety. As confirmed by LC-MS, it is expected that on average approximately two methyltetrazine molecules are covalently bound to each Fab.

[0612] Example 5B: Tetrazin-Fab targeted moiety (compound TM-2) [ka] Compound TM-2 was prepared and its characteristics were evaluated as follows.

[0613] Sacituzumab Fab amino acid sequence: Heavy chain: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHT (Sequence ID 9).

[0614] Light chain: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTKSFNRGEC (Sequence ID 10).

[0615] Sacituzumab Fab-tetrazine conjugate generation: A TROP2 Fab expression construct was generated using the coding sequences of the variable regions of the heavy and light chains of the sacituzumab (human anti-TROP2) antibody. The coding sequences were synthesized and subcloned into a PCDN3.4 expression vector. The constructed plasmid was transformed into E. coli for proliferation and scale-up. The purified plasmid was confirmed by sequencing. Constructs containing the heavy and light chains of TROP2 Fab were transfected into HEK293 cells (suspension culture) using polymer polyethyleneimine (PEI) reagent. Culture medium was collected 6-7 days after transfection. The culture medium containing TROP2 Fab was centrifuged, filtered, and then added to a KappaSelect affinity column (Mabselect Prism). The loading buffer was 25 mM Tris containing 150 mM NaCl, pH 8.0, and elution was performed with 100 mM sodium citrate buffer containing 150 mM NaCl, pH 2.5. The collected solution was neutralized with 1 M arginine, 400 mM succinate buffer, pH 9.0. Affinity-purified proteins were further purified by gel filtration using Superdex S-200 5 / 150GL column chromatography. The samples were eluted using 2X PBS mobile phase at pH 7.4. The purified TROP2 Fab was analyzed by SDS-PAGE and SEC-HPLC.

[0616] Tetrazine conjugation of TROP2 Fab: TROP2 Fab was buffered overnight with PBS pH 7.4. Methyltetrazine-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 in a molar ratio of 3:1 (methyltetrazine-PEG9-NHS to TROP2 Fab). The amount of Fab used for tetrazine conjugation varied from 30 to 100 mg, but the 3:1 ratio was always maintained in each conjugation procedure. The solution was then buffered with PBS pH 7.4 to remove excess methyltetrazine-PEG9-NHS and buffer salts.

[0617] Characterization of sacituzumab Fab-Tz conjugate (compound TM-2): Samples of the prepared Fab-Tz conjugate were analyzed by SDS-PAGE and Q Exactive HF-X LC-MS (Thermo Scientific) using an Acquity UPLC protein BEH C4 column (300A, 1.7 μm, 2.1 × 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 × 300 mm, Waters Corp). Proteins were eluted with PBS containing 15% isopropanol, pH 7.4. Size exclusion chromatograms showed 99% monomer species of the Fab-tetrazine conjugate with minimal aggregation (1.0%). The tetrazine-to-antibody ratio was calculated to be 2.3. [Table 7]

[0618] Example 6: HER2 Fab-tetrazine targeting moiety (compound TM-1) Compound TM-1 and its isotype Fab-Tz were prepared and their characteristics were evaluated as follows.

[0619] Trastuzumab Fab amino acid sequence: Heavy chain:EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKV (Sequence ID 3).

[0620] Light chain:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 4).

[0621] Isotype Fab-Tz amino acid sequence: Heavy chain: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHT (Sequence ID 5).

[0622] Light chain: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTKSFNRGEC (Sequence ID 6).

[0623] HER2 Fab Generation: An HER2 Fab expression construct was generated using the coding sequences of the variable regions of the heavy and light chains of the trastuzumab (human anti-HER2) antibody. The coding sequences were synthesized and subcloned into a PCDN3.4 expression vector. The constructed plasmid was transformed into E. coli for proliferation and scale-up. The purified plasmid was confirmed by sequencing. The construct containing the heavy and light chains of HER2 Fab was transfected into HEK293 cells (suspension culture) using polymer polyethyleneimine (PEI) reagent. 6 liters of culture medium were collected 6-7 days after transfection. The culture medium containing HER2 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 100 mM sodium citrate buffer containing 150 mM NaCl, pH 2.5. The collected solution was neutralized with 1 M arginine, 400 mM succinate buffer, pH 9.0. Affinity-purified proteins were further purified by gel filtration using Superdex S-200 5 / 150GL column chromatography. Sample injection was 20 mL at a flow rate of 0.3 mL / min, with a mobile phase of 2×PBS at pH 7.4. The purified HER2 Fab was analyzed by SDS-PAGE and SEC-HPLC. The final yield was 742 mg, which was stored at -80°C for long-term storage.

[0624] Isotype Fab generation: The coding sequence was synthesized and subcloned into a pTT5 expression vector. The constructed plasmid was transformed into E. coli for growth and scale-up. The purified plasmid was confirmed by sequencing. Constructs containing the heavy and light chains of the isotype Fab were transfected into HEK293 cells (suspension culture) using polymer polyethyleneimine (PEI) reagent. Two liters of culture medium were collected seven days after 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, 400 mM succinate buffer, pH 9.0. Affinity-purified proteins were further purified by gel filtration using Superdex S-200 5 / 150GL column chromatography. Sample injection was 3 mL at a flow rate of 0.3 mL / min, with a mobile phase of 2×PBS at pH 7.4. Purified isotype Fab was analyzed by SDS-PAGE and SEC-HPLC. The final yield was 200 mg, which was stored at -80°C for long-term storage.

[0625] Tetrazine conjugation of HER2 Fab and isotype Fab: HER2 Fab and isotype Fab were buffered overnight in PBS pH 7.4. Methyltetrazine-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 in a molar ratio of 3:1 (methyltetrazine-PEG9-NHS to HER2 Fab). The amount of Fab for tetrazine conjugation varied in the range of 30–100 mg, but the 3:1 ratio was always maintained in each conjugation procedure. The solution was then buffered in PBS pH 7.4 to remove excess methyltetrazine-PEG9-NHS and buffer salts. [ka]

[0626] Characterization of compound TM-1 and isotype Fab-Tz: Samples of prepared compound TM-1 or isotype Fab-Tz were analyzed by SDS-PAGE and Q Exactive HF-X LC-MS (Thermo Scientific) equipped with an Acquity UPLC protein BEH C4 column (300A, 1.7 μm, 2.1 × 50 mm) to confirm the formation of compound TM-1 and determine the tetrazine vs. antibody ratio. HPLC-SEC analysis was performed using UltiMate 3000 HPLC (Thermo Scientific) equipped with an xBridge BEH 200A SEC column (7.8 × 300 mm, Waters Corp). Proteins were eluted with PBS containing 15% isopropanol, pH 7.4. Size exclusion chromatograms of compound TM-1 and isotype Fab-Tz showed more than 97% monomer species of the Fab-tetrazine conjugate with minimal aggregation (≤3.0%). The tetrazine-to-antibody ratio was calculated as 2.2 for compound TM-1 and 1.8 for isotype Fab-Tz.

[0627] Flow cytometry analysis of compound TM-1 and isotype Fab-Tz: Cell binding analysis by flow cytometry using NCI-N87 (HER2-positive) cells was tested with either unconjugated HER2Fab, compound TM-1, or isotype Fab-Tz control. 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 in 96-well plates (200,000 cells per well) and centrifuged at 400×g for 5 minutes. The supernatant was removed, and cells were incubated at 4°C for 1 hour with 10-fold titration starting with 400 nM compound TM-1, unconjugated HER2Fab, or isotype control (IgG). For isotype Fab-Tz cell binding experiments, titration was 3-fold starting with 300 nM. Plates were centrifuged, washed three times with FACS buffer, and resuspended in 100 mL of secondary antibody either goat anti-human IgG Alexa 488 (catalog #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). The cells were incubated at 4°C in the dark for 1 hour. The supernatant was removed, cells were washed twice with PBS, and analyzed by CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, USA) or Attune NxT (Thermo Scientific, MA, USA). Mean fluorescence intensity (MFI) values ​​were calculated and plotted against antibody concentration. Binding of compound TM-1 to HER2-positive cells was observed to be equivalent to that of unconjugate HER2 Fab, while no binding was detected for isotype Fab-Tz.

[0628] Example 7: General procedure for preparing compound A [ka] Compound 2 (800 mg, 1.79 mmol) in DMF (4.00 mL) was added at 0°C to a solution of MMAE (1.40 g, 1.95 mmol) and DIEA (690 mg, 5.34 mmol) in DMF (4.00 mL), and the mixture was stirred at 25°C for 16 hours. Then, HOBt (480 mg, 3.56 mmol) in DMF (0.50 mL) was added to the reaction mixture at 0°C, and the reaction mixture was stirred at 25°C for 1.0 hour. After the reaction mixture was cooled to 0°C, TBAF (1 M, 4.45 mL in THF) was added. After stirring the mixture at 25°C for 2.0 hours, another batch of TBAF (1 M, 4.45 mL in THF) was added at 0°C, and the reaction mixture was continued to stir at 25°C for 12.0 hours. LC-MS showed that one major peak was at the desired mass. The resulting reaction mixture was purified by preparative HPLC (column: Welch XB-C18 7μm 110 A 250*50mm; mobile phase: [water (0.1% TFA)IN]; B%: 50~70% - 40 min. Number of injections: 2, retention time: 37 min, flow rate: 60 mL / min) to obtain compound A (450 mg, purity 99.0%; 64.6 mg, 99.2%, yield 31.2%). LCMS (m / z): 928.6 [M+H] + . 1 HNMR:(400MHz,DMSO-d6):δ 8-46-8.28(m,1H),8-03-7.84(m,1H),7.64(d,J=8.8Hz,1H),7-35-7.23(m,4H),7-21-7.13(m,1 H),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).

[0629] Example 8: General procedure for preparing compound B [ka] General procedure for the preparation of compound 4 [ka] To a solution of compound 2 (1.00 g, 5.43 mmol) in DCM (10 mL), DIEA (2.10 g, 16.3 mmol), EDCI (2.08 g, 10.9 mmol), DMAP (1.33 g, 10.9 mmol), and compound 3 (1.61 g, 8.14 mmol) were added. The mixture was stirred at 25°C for 16 hours. TLC showed that compound 2 was completely consumed and one new spot was formed. The reaction mixture was partitioned into DCM (20 mL) and H2O (10 mL). The organic phase was separated, washed with saturated citric acid aqueous solution (3 mL) and brine (20 mL), then 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 / 1-1 / 1) to obtain compound 4 (700 mg, yield 39.4%). 1 HNMR(400MHz,CDCl3):δ ppm 1.12(s,3H),1.60(dd,J=15.45,6.19Hz,1H),1-79-1.87(m,2H),1.92(br d,J=5.88Hz,1H),1.95(s,1H),1-98-2.00(m,1H),2.02(br d,J=4.13Hz,1H),2.26(dd,J=11.63,3.88Hz,1H),2-30-2.36(m,1H),2-77-2.89(m,1H),2-88-2.88(m,1H),3.00(dd,J=16.95,4 .57Hz,1H),3.70(s,4H),3.75(s,3H),4.80(dt,J=8.00,4.50Hz,1H),5.66(dd,J=16.63,2.38Hz,1H),6-02-6.12(m,1H),6.54(br d,J=7.88Hz,2H).

[0630] General procedure for the preparation of compound 6 [ka] To a solution of compound 4 (700 mg, 2.14 mmol) in DCM (5 mL), Py (846 mg, 10.7 mmol) and compound 5 (1.72 g, 8.55 mmol) in DCM (5 mL) were added. The mixture was stirred at 25°C for 1 hour. TLC showed that compound 4 was completely consumed and a new spot was formed. The reaction mixture was partitioned into DCM (20 mL) and H2O (10 mL). The organic phase was separated, washed with saturated 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 / 1-1 / 1) to obtain compound 6 (490 mg, yield 46.5%). 1 HNMR(400MHz,CDCl3):δ ppm 1.17(s,3H),1-55-1.61(m,1H),1.58(br s,1H),1.76(dd,J=14.76,6.25Hz,1H),1-87-2.03(m,3H),2-06-2.15(m,1H),2-19-2.41(m,3H),2.82(dd,J=17.13,4.50Hz,1H),3.0 3(dd,J=17.07,4.44Hz,1H),3.72(s,3H),3.77(s,3H),4-78-4.86(m,1H),5.67(dd,J=16.70,2.44Hz,1H),6-03-6.14(m,1H),6.58(br d,J=7.88Hz,1H),7-40-7.45(m,2H),8-27-8.33(m,2H).

[0631] General procedure for the preparation of compound 7 [ka] To a solution of compound 6 (490 mg, 995 μmol) and MMAE (714 mg, 995 μmol) in DMF (4 mL), DIEA (64.3 mg, 497 μmol) and HOBt (202 mg, 1.49 mmol) were added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that compound 6 was completely consumed and a single major peak with the desired mass was detected. The residue was purified by preparative HPLC (under 0.1% TFA conditions) to obtain compound 7 (500 mg, 46.9% yield). 1 HNMR(400MHz,CDCl3):δ ppm 0.84(br d,J=6.75Hz,4H),0.89(br d,J=4.50Hz,5H),0.92(br d,J=6.63Hz,4H),0.98(br d,J=6.25Hz,3H),1.04(br d,J=6.88Hz,3H),1.16(s,3H),1-25-1.27(m,3H),1-59-1.74(m,3H),1.88(br d,J=9.38Hz,4H),2.07(br d,J=8.38Hz,5H),2.27(br s,4H),2-36-2.43(m,2H),2-45-2.53(m,1H),2.89(br s,6H),2-95-3.01(m,4H),3.04(br s,2H),3-29-3.34(m,3H),3-36-3.47(m,5H),3-67-3.73(m,4H),3.76(s,3H),3-82-3.89(m,1H),4-05-4.19(m,3H),4.28(br s,1H),4-63-4.86(m,3H),4.96(d,J=2.50Hz,1H),5.24(br s,1H),5.63(br d,J=18.14Hz,1H),5.82(br s,1H),6-53-6.74(m,3H),7-30-7.41(m,5H).

[0632] General procedure for the preparation of compound B [ka] 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 single major peak with the desired mass was detected. The residue was adjusted to pH approximately 2 with saturated citric acid aqueous solution and then purified by preparative HPLC (0.1% TFA conditions) to obtain compound B (265 mg, yield 53.4%). 1 HNMR(400MHz,CDCl3):δ ppm 0-80-1.04(m,25H),1.09(s,3H),1.24(d,J=6.88Hz,3H),1-69-1.82(m,2H),1-88-1.94(m,3H),2-02-2.11(m,4H),2.16(br d,J=18.64Hz,1H),2-11-2.24(m,2H),2.32(br d,J=5.25Hz,2H),2-40-2.45(m,1H),2.52(br d,J=5.50Hz,2H),2.81(br dd,J=14.01,4.88Hz,1H),2-94-3.04(m,2H),3.08(s,2H),3-14-3.27(m,6H),3.33(s,1H),3.39(s,3H),3-48-3.57(m,2H),3.94(br d,J=1.25Hz,1H),4-05-4.18(m,4H),4.30(br dd,J=6.19,4.82Hz,2H),4-54-4.67(m,4H),4.91(br d,J=2.00Hz,2H),5.30(br s,1H),5-64-5.73(m,1H),5-79-5.89(m,1H),6.61(br d,J=7.38Hz,1H),7-30-7.42(m,5H),7-55-7.64(m,1H).

[0633] Example 9: Alternative route to compound B and synthesis of compound C. [ka] General procedure for the preparation of compound 2 [ka] To a solution of compound 1 (20.0 g, 83.2 mmol) in MeOH (80 mL), KOH (8.19 g, 124 mmol) in H2O (80 mL) was added. The mixture was stirred at 25°C for 24 hours. The reaction was monitored by TLC (compound 1, PE / SiO=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 obtain the undesirable ester. The aqueous layer was acidified with 1 M HCl to pH=4 while cooling in an ice 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 obtain compound 2 (5.50 g, yield 35.9%). The crude product was used in the next step without further purification. 1 H NMR:(400MHz,DMSO-d6):δ ppm 11.9(br s,1H),5-81-5.94(m,1H),5.58(dd,J=16.45,2.31Hz,1H),4.65(br s,1H),4.24(br s,1H),2-04-2.24(m,2H),1-87-2.03(m,1H),1-61-1.86(m,4H),1-36-1.46(m,1H),0.97(s,3H).

[0634] General procedure for the preparation of compound 3 [ka] To a solution of compound 2 (8.00 g, 43.4 mmol) in MeCN (160 mL), DIEA (39.3 g, 304 mmol) and DSC (47.8 g, 186.4 mmol) were added. The mixture was stirred at 40°C for 14 hours. Completion of the reaction was confirmed by TLC (compound 2, DCM / MeOH = 10 / 1, R). f(=0.5). The reaction mixture was poured into water (400 mL), and the temperature was then raised from 20°C to 27°C. After 15 minutes, the mixture was cooled to 17°C in an ice bath and stirred for 15 minutes. The solid was filtered, washed with water (3 × 20 mL), and dried under vacuum at 35°C for 4 hours to obtain 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 hour using mechanical stirring. Heating was stopped, and the mixture was cooled to 8°C in an ice bath for 15 minutes. The solid was filtered, washed with acetonitrile (2 × 10 mL), and dried under vacuum at 35°C for 3 hours to obtain compound 3 (6.65 g, yield 36.3%). 1 H NMR:(400MHz,CDCl3):δ 6-03-6.14(m,1H),5-60-5.67(m,1H),5.29(br s,1H),2-80-2.88(m,8H),2-25-2.47(m,4H),1-94-2.18(m,4H),1.29(s,3H).

[0635] General procedure for the preparation of compound 4 [ka] To a solution of compound 3 (100 mg, 0.24 mmol) in DMF (20 mL), MMAE (136 mg, 0.19 mmol) and DIEA (61.2 mg, 0.47 mmol) were added. The mixture was stirred at 25°C for 16 hours. LC-MS showed the detection of one major peak with the desired mass. The residue was purified by preparative HPLC (water (0.1% I-ACN)) to obtain compound 4 (41.0 mg, yield 16.9%). LC-MS (m / z): 1025.6 (M+H) + .

[0636] General procedure for the preparation of compound B [ka] To a solution of compound 4 (500 mg, 0.49 mmol) and compound 4-1 (519 mg, 3.90 mmol) in DMF (10 mL), DIEA (378 mg, 2.93 mmol) and DMAP (119 mg, 0.97 mmol) were added. The mixture was stirred at 25°C for 12 hours. LC-MS showed the detection of one major peak with the desired mass. The residue was purified by preparative HPLC (water (0.1 IFA)-ACN) to obtain compound B (161 mg, yield 31.6%). 1 H NMR:(400MHz,MeOD-:δ 7.73-8.00(m,1-H),7.20-7.39(m,4-H),5.78-5.96(m,1H),5.73(br s,1-H),5.20-5.28(m,1-H),5.13-5.20(m,1-H),4.49-4.74(m,3-H),4.17-4.28(m,2-H), 4.04-4.10(m,1-H),3.85-3.90(m,1-H),3.50-3.80(m,2-H),3.31-3.50(m,9-H),3.28-3.3 0(m,3-H),2.77-3.12(m,6-H),2.44-2.58(m,2-H),1.78-2.36(m,13-H),1.56-1.72(m,2- H),1.22-1.48(m,3-H),1.08-1.23(m,9-H),0.80-1.07(m,18H).LCMS(m / z):1043.62(M+H) + ;1065.61(M+Na) + .

[0637] General procedure for the preparation of compound C [ka] 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), DIEA (242 mg, 1.87 mmol) and DMAP (76.3 mg, 0.62 mmol) were added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that compound 4 was completely consumed and a single major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain compound C (92.0 mg, yield 29.9%). 1 H NMR(400MHz,MeOD-:δ 7.86-8.00(m,1-H),7.15-7.45(m,5-H),5.67-5.98(m,2H),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,5H),3.30(br s,4H),3.20(dt,J=11.57,7.47Hz,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,18H).LCMS(m / z):985.6(M+H) + .

[0638] Example 10: Synthesis of Compound D [ka] [ka] General procedure for the preparation of compound 6 [ka] 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), (2,5-dioxopyrrolidine-1-yl)2,2,2-trichloroethyl carbonate (210 g, 723 mmol) in dioxane (1 L) was added. The mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to remove the dioxane. The residue was extracted with MTBE (5 L), and then the aqueous phase was adjusted to pH approximately 4 with saturated KHSO4 aqueous solution and extracted with RINKAN (5 L). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. To a solution of the above crude product in MeOH (2 L), SOCl2 (90.2 g, 758 mmol) was added, and the mixture was stirred at 25°C for 2 hours. LC-MS indicated that the reaction was complete and that a single major peak with the desired mass was detected. The reaction mixture was adjusted to a pH of approximately 9-10 with saturated NaHCO3 aqueous solution and then extracted with toluene (5 L). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was precipitated with PE (10 vol) to obtain compound 6 (190 g, yield 74.4%). 1 H NMR:(400MHz,CDCl3):δ 3.25(br s,1H)3.85(s,-H)4.64-4.83(m,2H)5.30(dd,J=9.51,1.13Hz,1H)5.92(br d,J=9.38Hz,-H)7.30-7.45(m,5H).LCMS(m / z):391.9 / 393.9(M+H) + .

[0639] General procedure for the preparation of compound 7 [ka] To a solution of compound 6 (185 g, 499 mmol) in toluene (1.9 L), 4-methylbenzenesulfonic acid pyridine (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 hours. LC-MS showed the detection of one major peak with the desired mass. The reaction mixture was then cooled to 25°C, and the reaction mixture was concentrated under reduced pressure to remove toluene. The residue was diluted with H2O (500 mL) and then extracted with siRNA (500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 7 (285 g, crude), which was carried over as is.

[0640] General procedure for the preparation of compound 8 [ka] 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 one major 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 layer was diluted with saturated KHSO4 (1 L) aqueous solution and extracted with siRNA (5 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was precipitated with PE (10 vol) to obtain compound 8 (95.0 g, yield 34.3%). 1 H NMR(400MHz,MeOD):δ 3.82(s,-H)4.41-4.47(m,-H)4.50-4.56(m,1H)4.60(d,J=4.88Hz,1H)5.47(d,J=4. 75Hz,1H)6.46(s,-H)6.86-6.94(m,-H)7.34-7.46(m,7H).LCMS(m / z):495.9(M+Na) + .

[0641] General procedure for the preparation of 7-Troc-Baccatin III [ka] To a solution of baccatin III (30.0 g, 51.1 mmol) in DCM (300 mL), DMAP (625 mg, 5.11 mmol), pyridine (14.2 g, 179 mmol), and 2,2,2-trichloroethyl carbonochloride (15.2 g, 71.6 mmol) were added. The mixture was stirred at 25°C for 0.5 hours. LC-MS showed that baccatin III was completely consumed and a single major peak with the desired mass was detected. The residue was diluted with water (300 mL), extracted with DCM (300 mL), washed with water (200 mL) and brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 7-Troc-baccatin III (45.0 g, yield 34.3%). LCMS (m / z): 761.5 / 763.5 (M+Na) + .

[0642] General procedure for the preparation of compound 9 [ka] To a solution of 7-Troc-baccatin III (26.0 g, 34.1 mmol) and compound 8 (32.4 g, 68.2 mmol) in DCM (1000 mL), DMAP (4.20 g, 34.1 mmol) and DCC (21.1 g, 102 mmol) were added. The mixture was stirred at 0°C for 1 hour. LC-MS showed that compound 8 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was filtered. The crude product was washed with saturated NH4Cl aqueous solution (100 mL) and water (1000 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 9 (35.0 g, crude). LCMS (m / z): 1240.0 / 1242.0 (M+Na) + .

[0643] General procedure for the preparation of compound 10 [ka] To a solution of compound 9 (80.0 g, 65.6 mmol) in MeOH (350 mL), 4-methylbenzenesulfonic acid hydrate (24.9 g, 131 mmol) was added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that approximately 50% of compound 9 remained, and one major peak with the desired mass was detected. The reaction mixture was filtered and concentrated, and the residue was purified by preparative HPLC (water (0.1% TFA)-ACN). The eluate was concentrated under reduced pressure to remove the solvent, and then extracted with siRNA (500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 10 (13.0 g, yield 17.9%). LC-MS (m / z): 1120.2 (M+Na) + .

[0644] General procedure for the preparation of compound 11 [ka] Compound 10 (13.0 g, 11.8 mmol) was mixed with a solution of DMAP (722 mg, 5.90 mmol), EDCI (2.70 g, 14.2 mmol), and benzoic acid (1.70 g, 14.2 mmol) in DCM (260 mL). The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 10 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was washed with saturated citric acid aqueous solution (100 mL), saturated NaHCO3 aqueous solution (100 mL), and water (200 mL), dried over NaSO4, filtered, and concentrated under reduced pressure to obtain compound 11 (11.0 g, yield 77.3%). LC-MS (m / z): 1204.1 (M+H) + .

[0645] General procedure for the preparation of compound 12 [ka] To a solution of compound 11 (20.0 g, 16.6 mmol) in MeOH (200 mL) and AcOH (200 mL), Zn dust (21.6 g, 331 mmol) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 11 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was filtered, diluted with H2O (500 mL), and then extracted with RINKAN (100 mL × 3). The combined organic layer was washed with saturated NaHCO3 aqueous solution (200 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain compound 12 (5.0 g, yield 21%). LC-MS (m / z): 854.3 (M+H) + .

[0646] General procedure for the preparation of compound 13 [ka] Compound 12 (5.00 g, 5.90 mmol), DIEA (1.50 g, 11.7 mmol), and Compound 3 (3.90 g, 8.80 mmol) were dissolved in DMF (50 mL). The mixture was stirred at 25°C for 16 hours. LC-MS showed that approximately 50% of Compound 12 remained, and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain Compound 13 (505 mg, yield 7.4%). LC-MS (m / z): 1161.4 (M+H) + .

[0647] General procedure for the preparation of compound D [ka] To a solution of compound 13 (150 mg, 0.13 mmol) in DMF (1.50 mL), DMAP (94.7 mg, 0.78 mmol), compound 13-1 (66.7 mg, 0.65 mmol), and DIEA (100 mg, 0.78 mmol) were added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that compound 13 was completely consumed and a single major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain compound D (75.0 mg, yield 50.5%). LC-MS (m / z): 1148.5 (M) + .

[0648] Example 11: General procedure for the preparation of compound E [ka] Compound 13 (350 mg, 0.30 μmol), DMAP (221 mg, 1.81 mmol), and Compound 14 (249 mg, 0.39 mmol, HCl) were dissolved in DMF (0.3 mL). The mixture was stirred at 25°C for 16 hours. LC-MS showed that Compound 13 was completely consumed and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain Compound E (205 mg, yield 41.3%). LC-MS (m / z): 1646.5 (M+H) + .

[0649] Example 12: Synthesis of Exatecan Prodrug Compound F [ka] Procedure for the preparation of compound 2 [ka] To a solution of methyl compound 1 (25.0 g, 104 mmol, 1.0 equivalent) in MeOH (125 mL), NaOMe (5.40 M, 105 mL, 5.5 equivalents) in H2O (125 mL) was added. The mixture was stirred at 25°C for 24 hours. The reaction was monitored by TLC, and TLC (PE:EA = 2:1, product R) was obtained. f =0.20) is reactant (R f This indicated that 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 to pH=4 while cooling in an ice bath (T < 7°C). The aqueous layer was extracted with MTBE (5 × 500 mL). The combined MTBE layers were dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the residue, and evaporated three times over MeCN to obtain compound 2 (9.1 g, 49.3 mmol, yield 47.4%). 1 H NMR:400MHz,CDCl3δ ppm 1.11(s,3H),1.65(br dd,J=15.82,6.19Hz,1-H),1.78-2.02(m,4-H),2.05-2.42(m,4H),4.49(br s,1H),5.64(dd,J=16.63,2.00Hz,1-H),6.00-6.14(m,1H).

[0650] Procedure for the preparation of compound 3 [ka] To a solution of compound 2 (6.6 g, 35.8 mmol, 1.0 equivalent) in MeCN (130 mL), DIEA (32.4 g, 250 mmol, 43.6 mL, 7.0 equivalents) and DSC (39.4 g, 154 mmol, 4.3 equivalents) were added. The mixture was stirred at 25°C for 16 hours. The reaction was monitored by TLC, and TLC (DCM:MeOH = 10:1, product R) was obtained. f =0.60) is reactant (R fThis indicated that 0.20) was consumed. The reaction mixture was purified by recrystallization from H2O (350 mL) at 25°C, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was evaporated three times over MeCN to obtain compound 3 (10.0 g, 21.6 mmol, yield 60.4%, purity 91.4%). HPLC:R t =2.14 minutes, purity: 91.4%. 1 H NMR:400MHz, CDCl3δ ppm 1.28(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,1H),5.63(dd,J=16.70,2.19Hz,1-H),6.02-6.15(m,1H).

[0651] Procedure for the preparation of compound 5 [ka] To a solution of compound 3 (6.6 g, 15.6 mmol, 1.0 equivalent) in DMF (70 mL), DIEA (4.04 g, 31.2 mmol, 5.44 mL, 2.0 equivalents) and exatecan (6.23 g, 11.7 mmol, 0.75 equivalents) were added. The mixture was stirred at 25°C for 1 hour. LC-MS (EC17183-7-P1A1) showed the detection of one major peak (RT=0.44 min) with the desired mass. The reaction mixture was filtered and purified by preparative HPLC (column: Phenomenex luna C18 (250 × 70 mm, 10 μm); mobile phase: [wIr (FA)-ACN]; gradient: 40%-70% B over 15 minutes) to obtain compound 5 (6.0 g, 7.76 mmol, yield 49.6%, purity 96.1%).

[0652] LCMS (Monitor): R t =0.44 minutes, MS calculated value: 742.2, MS measured value: [M+H] + = 743.1.

[0653] LCMS:R t =0.44 minutes, MS calculated value: 742.2, MS measured value: [M+H] += 743.3.

[0654] HPLC:R t =3.04 minutes, purity: 96.1%.

[0655] Procedure for preparing compound F [ka] To a solution of compound 5-1 (9.95 g, 16.5 mmol, 1.5 equivalents) in DMSO (40 mL), DIEA (2.85 g, 22.0 mmol, 3.85 mL, 2.0 equivalents), DMAP (2.70 g, 22.08 mmol, 2.0 equivalents), and compound 5 (8.2 g, 11.04 mmol, 1 equivalent) were added. The mixture was stirred at 25°C for 16 hours. LC-MS (EC17183-21-P1A11) showed a single major peak (R) with the desired mass. t The detection of compound F (=0.39 min) was indicated. The reaction mixture was filtered, purified by preparative HPLC (FA conditions), and replaced with the AcOH salt to obtain compound F (7.5 g, 5.89 mmol, yield 53.3%, purity 96.5%).

[0656] LCMS (Monitor): R t =0.39 minutes, MS calculated value: 1227.5, MS measured value: [M+H] + = 1228.8.

[0657] LCMS:R t =0.38 mins, MS calculated value: 1227.5, MS measured value: [M+H] + = 1228.7.

[0658] HPLC:R t =2.28 minutes, purity: 96.5%.

[0659] Example 13: Synthesis of MMAE-TCO compound G [ka] To a solution of Compound 1-2 (100 mg, 97.5 μmol) in DMF (1.00 mL) were added DMAP (95.3 mg, 780 μmol), Compound 1-3 (175 mg, 292 μmol), and DIEA (129 μL, 780 μmol). The mixture was stirred at 25°C for 24 hours. LC-MS showed that one major peak with the desired mass (calculated MS: 1510.91, found MS: [M+H] + =1512.1) was detected. The mixture was filtered and purified by preparative HPLC (AcOH conditions) to afford Compound G (44 mg, yield 29.8%) as a white solid.

[0660] LCMS: calculated MS: 1510.91, found MS: [M+H] + =1511.1.

[0661] HPLC: R t =6.981 min, purity: 95.3%

[0662] Example 14: Synthesis of MMAE-TCO Compound H ##STR## To a solution of Compound 1-2 (100 mg, 97.5 μmol) in DMF (1.00 mL) were added DMAP (95.3 mg, 780 μmol), Compound 2-1 (97.6 mg, 780 μmol), and DIEA (129 μL, 780 μmol). The mixture was stirred at 25°C for 16 hours. LC-MS showed that one major peak with the desired mass (calculated MS: 1035.3, found MS: [M+H] + =1036.6) was detected. The mixture was filtered and purified by preparative HPLC to afford Compound H (20 mg, yield 19.8%) as a white solid.

[0663] LCMS: calculated MS: 1034.6, found MS: [M+H] + =1035.8.

[0664] HPLC: R t =4.566 min, purity: 95.06%

[0665] Example 15: Synthesis of MMAE-TCO Compound I

Chemical Formula

[0666] LCMS: calculated MS: 1033.6, found MS: [M+H] + =1034.8.

[0667] HPLC: R t =5.863 min, purity: 96.39%

[0668] Example 16: Synthesis of MMAE-TCO Compound J

Chemical Formula

[0669] LCMS: MS calculated value: 1034.6, MS measured value: [M+H] + = 1035.8.

[0670] HPLC:R t =7.609 minutes, purity: 95.48%

[0671] Example 17: General procedure for preparing intermediate 5-1B [ka] To a solution of tert-butyl 3-hydroxypropanoate (0.50 g, 3.42 mmol) and pyridine (552 μL, 6.84 mmol) in THF (10.0 mL), triphosgene (405 mg, 1.36 mmol) was gradually added over 5 minutes at 0°C under an N2 atmosphere. The mixture was stirred at 0°C for 1 hour. TLC showed that compound 1 was completely consumed and one new spot was formed. Aliquots of the reaction mixture were quenched with benzylamine, and the desired mass was detected by LC-MS (MS calculated value: 279.15, MS measured 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 obtain the residue. Intermediate 5-1B (520 mg, 2.49 mmol, yield 72.8%) was obtained as a colorless oil. The residue was used directly in the next step without purification.

[0672] Example 18: General procedure for preparing intermediate 5-3B [ka] To a solution of 5-1B (321 mg, 1.24 mmol) in THF (10 mL), DMAP (304 mg, 2.49 mmol) and 5-2C (520 mg, 2.49 mmol) were added at 0°C. The mixture was stirred at 25°C for 2 hours. LC-MS was performed to determine the complete consumption of 5-2C and the desired mass (MS calculated value: 430.1, MS measured value: [M+Na]). +The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (12 g silica gel, PE / EA = 0%~100%) to obtain the product. Intermediate 5-3B (502 mg, 1.16 mmol, yield 93.5%) was obtained as a colorless oil. LCMS: MS calculated value: 430.1, MS measured value: [M+Na] + = 453.1. 1 H NMR:(400MHz,CDC-3)δ:7.35-7.22(m,5H),5.38(br d,J=6.3Hz,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=6.1Hz,2H),1.38(s,9H).

[0673] Example 19: General procedure for preparing intermediate 5-3C [ka] A mixture of 5-3B (500 mg, 1.16 mmol) was mixed with HCl / dioxane (5 mL, 2 M) at 0°C, and the mixture was stirred at 25°C for 48 hours. LC-MS was performed to determine the complete consumption of 5-3B and the desired mass (MS calculated value: 374.1, MS measured value: [M+Na]). + The reaction mixture was filtered and concentrated under reduced pressure to produce a colorless oil. Intermediate 5-3C (400 mg, 1.06 mmol, yield 91.9%) was obtained as a colorless oil. LCMS: MS calculated value: 374.1, MS measured value: [M+Na] + =397.0

[0674] Example 20: General procedure for preparing intermediates 5-4 [ka] Pd(OH)2 (68.9 mg, 245 μmol) was added to a dry hydrogenation bottle under an Ar atmosphere, followed by the addition of MeOH (5 mL) which completely permeated the Pd(OH)2. Subsequently, under an Ar atmosphere, a solution of 5-3C (400 mg, 1.06 mmol) in MeOH (5 mL) was slowly added. The mixture was degassed and purged three times with H2, and then stirred under an H2 atmosphere (15 psi) at 20-25°C for 12 hours. LC-MS showed that 5-3C was completely consumed. Under an Ar atmosphere, the reaction mixture was carefully filtered under reduced pressure, and the organic layer was concentrated under reduced pressure to obtain the crude compound. Intermediate 5-4 (324 mg, crude) was obtained as a...

Claims

1. A method for forming an antibody-payload conjugate in vivo in a subject requiring an antibody-payload conjugate, wherein the method is Administering an effective amount of a targeted portion to the subject, wherein the targeted portion comprises at least one antibody or fragment thereof having at least one tetrazine portion covalently bound thereto. Administering a single dose of a therapeutically effective payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene (TCO) moiety covalently bound thereto. A method comprising the antibody or a fragment thereof having binding affinity to a receptor on a tumor, and further comprising the ratio of the antibody-payload conjugate at the tumor site to the plasma being greater than about 2:

1.

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

1.

3. The method according to claim 1 or 2, wherein the administration is performed sequentially.

4. The method according to any one of the prior claims, wherein a single dose of the payload-TCO conjugate is administered to the subject about 2 to 48 hours, or about 3 to 48 hours, or about 4 to 48 hours after the targeting portion is administered to the subject.

5. The method according to any one of the prior claims, wherein the targeted portion is administered at least about 8 to about 24 hours before the payload-TCO conjugate is administered.

6. A method of administering a payload, wherein the method is a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion comprises Fab having at least one tetrazine portion covalently bound thereto, b) Administering a single dose of a therapeutically effective payload-TCO conjugate to the subject, wherein the payload-TCO conjugate comprises a payload having at least one trans-cyclooctene moiety covalently bound thereto, A method comprising the above, wherein the single dose of the payload-TCO conjugate is administered to the subject approximately 2 to 48 hours, or approximately 3 to 48 hours, or approximately 4 to 48 hours after the targeting portion is administered to the subject.

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

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

9. The method according to claim 7 or 8, wherein a single dose of the payload-TCO conjugate is administered to the subject about 8 to 22 hours, about 8 to 16 hours, about 8 to 22 hours, about 12 to 16 hours, or about 16 to 20 hours after the targeting portion is administered to the subject.

10. The targeting portion is formula I, formula II, or formula V, 【Chemistry 1】 During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R ３ and R ４ If both are absent, then tetrazine, or R ３ and R ４ R represents an additional bond that forms dihydrotetrazine when both are present, however, when ring A is aryl. ３ and R ４ Assuming that both exist, X is the antibody fragment portion, p is between 1 and 20. In each appearance, L is independently a linker. R at each occurrence １ is independently selected from hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -OR', -SR', -C(=O)R', -C(=S)R', -OC(=O)R''', -SC(=O)R''', -OC(=S)R''', -SC(=S)R''', -S(=O)R', -S(=O) ２ R''', -S(=O) ２ NR'R'', -C(=O)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, heterocyclyl, or cycloalkyl is optionally substituted with 1 to 3 Z １ ​ R in each appearance ２ Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl has 1 to 3 Z of any choice. １ It has been replaced with, R ３ and R ４ Both are absent, or R ３ and R ４ Each of these is an independent group that can be removed after hydrogen or a trigger event. R in each appearance ２０ These independently include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, and -CF ３ , -CF ２ R', -NO ２ , -OR', -SR', -C(=O)R', -C(=S)R', -OC(=O)R''', -SC(=O)R''', -OC(=S)R''', -SC(=S)R''', -S(=O)R', -S(=O) ２ R''', -S (=O) ２ Selected from the group consisting of NR'R'', -C(=O)O-R', -C(=O)S-R', -C(=S)O-R', -C(=S)S-R', -C(=O)NR'R'', -C(=S)NR'R'', -NR'R'', -NR'C(=O)R'', -NR'C(=S)R'', -NR'C(=O)OR'', -NR'C(=S)OR'', -NR'C(=O)SR'', -NR'C(=S)SR'', -OC(=O)NR'R'', -SC(=O)NR'R'', -OC(=S)R'R''', -SC(=S)R'R'', -NR'C(=O)NR''R'', and -NR'C(=S)NR''R'', R in each appearance ２２ The linker is independently and optionally comprises one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups, and consists of 1 to 100 linked atoms. R in each appearance ３０ These are independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R ａ , R ３１ａ , and R ３１ｂ Each of them independently produces hydrogen and C １ -C ６ - Alkyl, or C １ -C ６ - It is a haloalkyl, Each Z １ These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -OR', -SR', -C(=O)R', -C(=S)R', -OC(=O)R''', -SC(=O)R''', -OC(=S)R''', -SC(=S)R''', -S(=O)R', -S(=O) ２ R''', -S (=O) ２ Selected from NR'R'', -C(=O)O-R', -C(=O)S-R', -C(=S)O-R', -C(=S)S-R', -C(=O)NR'R'', -C(=S)NR'R'', NR'R'', -NR'C(=O)R'', -NR'C(=S)R'', -NR'C(=O)OR'', -NR'C(=S)OR'', -NR'C(=O)SR'', -NR'C(=S)SR'', -OC(=O)NR'R'', -SC(=O)NR'R'', -OC(=S)R'R''', -SC(=S)R'R'', -NR'C(=O)NR''R'', and -NR'C(=S)NR''R'', In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. The method according to any one of the prior claims, wherein t in each occurrence is independently 0, 1, 2, 3, or 4.

11. The targeting portion is formula I or II, 【Chemistry 2】 During the ceremony, X is the antibody fragment portion, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance ２０ These independently include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, and CF. ３ CF ２ -R', NO ２ , OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ Selected from the group consisting of NR'R'', C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', R in each appearance ２２ The linker is independently and optionally comprises one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups, and consists of 1 to 100 linked atoms. R in each appearance ３０ These are independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R ａ , R ３１ａ , and R ３１ｂ Each of them independently produces hydrogen and C １ -C ６ - Alkyl, or C １ -C ６ - It is a haloalkyl, In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl, and in each occurrence, R''' is independently selected from aryl and alkyl. The method according to any one of the prior claims, wherein t in each occurrence is independently 0, 1, 2, 3, or 4.

12. The method according to any one of the prior claims, wherein the targeting portion is formula IIA. 【Transformation 3】

13. Each R ２０ The method according to any one of claims 10 to 12, wherein is independently hydrogen or alkyl.

14. The method according to claim 10, wherein the targeting portion is formula VII. 【Chemistry 4】

15. The method according to claim 10 or 11, wherein ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl.

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. Each R １ The method according to any one of claims 10 or 14-17, wherein is independently hydrogen or alkyl.

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

20. The method according to any one of claims 14 to 18, wherein t is 0 in each occurrence.

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

22. X is daclizumab, RG6292, basiliximab, HuMax-TAC, rabetsuzumab, 15-1-32, PR1A3, cT84.66, tusamithiamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matsuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, GC1118, imugatuzumab Panitumumab, Artumumab, Tomzotuximab, Laprituximab, Oportuzumab, Sitatuzumab, Tucotsuzumab, Katumakisomab, Edrecolomab, Adecatumumab, Ramucizumab, Ramucirumab, Valinacimab, Oralatumab, Ramucirumab, Sacituzumab, Pr1E11, Enfortumab, J591, MLN591, Verantama Moxetumomab, Inotuzumab, Epratuzumab, Pinatuzumab, Ubrituzumab, Ofatumumab, Rituzumab, Obinutuzumab, Tositumomab, Ibritumomab, Ronkastuzumab, XMAB-5574, MOR208, Coltuzumab, Denintuzumab, Tapritumomab, MDX-1342, Polatuzumab, Isatuzumab, Daratumumab, MOR202 TAK-079, I-131-BC8, Iomab-B, carotuximab, bemarituzumab, appletuzumab, lupalzumab, zolbetuximab, clodiximab, andecaliximab, milbetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, lovalpituzumab, indatuximab, rorbotuzumab, promiximab, BI 836826, otreltuzumab, naratuximab, milatuzumab, anetuzumab, amatsuximab, MMOT-0530A, sarilumab, elotuzumab, belimumab, KL-6, MY. 1E12, hMUC1-1H7, TAB004, huC242, cribatuzumab, 8HuDS6, gatipotuzumab, AR20.5, cantuzumab, codlituzumab, ECT204, MDX-1414, pertuzumab, trastuzumab, margetuximab, patrizumab, cerivantuzumab, lumuletuzumab, elgemzumab, AV-203, CDX-3379, GSK284933, brentuximab, gemtuzumab, BI835858, vadasutuximab, lintuzumab, KHK2823, tacrotuzumab, G4723A, grembatumumab, terisotuzumab, onarutuzumab, SAIT301, tisotuzumab, rifastuzumab, indusatuzumab, bundletuzumab, sofituzumab, borsetuzumab, vibatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, bovalilizumab, LCAR-B38M, BI The method according to any one of claims 10 to 21, wherein the antibody fragment portion is derived from 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, cibrotuzumab, F16, R6N, datopotamab, 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 according to claim 23, wherein the imaging contrast agent is a protein.

25. The method according to any one of claims 10 to 24, wherein L in each occurrence independently binds to X via a cystine or lysine residue on X.

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 linked atoms, 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

29. Each L independently contains 5 to 50 linked atoms, each comprising one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, or heteroarylene atoms, and each alkylene, alkenylene, alkynylene, arylene, or heteroarylene is independently and optionally oxo, halo, or C. １－４ Alkyl, C １－４ Alkoxy, and C １－４ The method according to any one of claims 10 to 28, which may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

30. X is an antibody fragment that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, Mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5, and each L independently 【Transformation 5】 A method according to any one of claims 10 to 29, selected from the group consisting of the following.

31. X is the antibody fragment portion that targets HER2, p is 1 to 5, and each L is independent. 【Transformation 6】 The method according to any one of claims 10 to 14, selected from the group consisting of the following.

32. The method according to any one of the prior claims, wherein the targeting portion is formula IIF. 【Transformation 7】

33. The method according to claim 32, wherein X is an antibody fragment portion that targets HER2, and p is 1 to 5.

34. The payload-TCO conjugate is formula VIII or a pharmaceutically acceptable salt thereof. 【Transformation 8】 During the ceremony, In each occurrence, G is independent. 【Chemistry 9】 And, L in each appearance １ It is independent and is a linker, m is an integer between 1 and 150. D is the payload, R in each appearance １Ａ C １－４ Alkyl, C １－４ Haloalkyl and C １－４ Selected from the group consisting of alkoxys, q is 0, 1, or 2, q1 is 0 or 1, R at each occurrence １Ｂ is independently, G １ , -OH, -NR １ｃ -C １－４ alkylene-G １ , -NR １ｃ -C １－４ alkylene-N(R １ｄ ) ２ , -NR １ｃ -C １－６ alkylene-N(C １－４ alkyl) ３ ＋ , -N(R １ｃ )CHR １ｅ CO ２ H, -N(R １ｃ )-C １－６ alkylene-CO ２ H, -N(R １ｃ )CHR １ｅ C(O)OC １－６ alkyl, -N(R １ｆ )-C ２－４ alkylene-(N(C １－４ alkylene-CO ２ H)-C ２－４ alkylene) ｎ -N(C １－４ alkylene-CO ２ H) ２ , -N(R １ｆ )-C ２－４ alkylene-(N(C １－４ alkylene-C(O)OC １－６ alkyl)-C ２－４ alkylene) ｎ -N(C １－４ alkylene-C(O)OC １－６ alkyl) ２ , -N(R １ｃ )-C １－６ alkylene-SO ３ H, -N(R １ｃ )-(CH ２ CH ２ O) １－３ -CH ２ CH ２ N((CH ２ CH ２ O) １－３ -C １－６ alkylene-CO ２ H) ２ , -N(R １ｃ )-C １－６ Alkylene-C(O)OC １－６ Alkyl and -N(R １ｃ )-CH(CH ２ O-(CH ２ CH ２ O) ０－２ -C １－６ Alkylene-CO2 ２ H) ２ Selected from the group consisting of, R in each appearance １ｃ and R １ｄ Independently, hydrogen or C １－４ It is alkyl, R in each appearance １ｅ -C １－４ Alkylene-CO2 ２ H, -C １－４ Alkilen-CONH ２ , or -C １－４ It is an alkylene-OH group. R in each appearance １ｆ Hydrogen, C １－６ Alkyl, or C １－４ Alkylene-CO2 ２ It is H, n in each occurrence is independently 0, 1, 2, or 3. L in each appearance ２ These independently consist of -C(O)- and C １－３ Selected from the group consisting of alkylenes, G in each appearance １ The method according to any one of the prior claims, wherein is independently a heterocyclil that has been optionally substituted.

35. The method according to any one of the prior claims, wherein the payload is paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepyrone, patupyrone, platinum, exatecan, deruxtecan, dorastatin 10, MMAE, MMAD, MMAF, mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterin, seco-DUBA, duocalmycin, cyclosporine A, or rapamycin.

36. The aforementioned payload-TCO conjugate, 【Chemistry 10-1】 【Chemistry 10-2】 【Chemistry 10-3】 A method according to any one of the prior claims, selected from the above.

37. A method for administering a therapeutically effective dose of monomethyl auristatin E (MMAE) to a subject with cancer, wherein the method is a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is of formula IIF, 【Chemistry 11】 In the formula, X is a trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), or X is a sacituzumab Fab containing (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5, and the subject is administered the above. b) Administering a single therapeutic dose of an effective amount of MMAE-TCO conjugate having the following structure to the subject, 【Chemistry 12】 A method comprising administering the single dose of the MMAE-TCO conjugate to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

38. A method for treating cancer in a subject with cancer, or for enhancing or inducing an immune response, wherein the method is a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is of formula IIF, 【Chemistry 13】 In the formula, X is a trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), or X is a sacituzumab Fab containing (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5, and the subject is administered the above. b) Administering a single therapeutic dose of an effective amount of MMAE-TCO conjugate having the following structure to the subject, 【Chemistry 14】 A method comprising administering the single dose of the MMAE-TCO conjugate to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

39. A method for reducing tumor volume in a subject having a tumor, wherein the method is a) Administering an effective amount of the targeted portion to the subject, wherein the targeted portion is of formula IIF, 【Chemistry 15】 In the formula, X is a trastuzumab Fab containing (SEQ ID NO: 3) and (SEQ ID NO: 4), or X is a sacituzumab Fab containing (SEQ ID NO: 9) and (SEQ ID NO: 10), and p is 1 to 5, and the subject is administered the above. b) Administering a single therapeutic dose of an effective amount of MMAE-TCO conjugate having the following structure to the subject, 【Chemistry 16】 A method comprising administering the single dose of the MMAE-TCO conjugate to the subject approximately 2 to 48 hours, or approximately 8 to 24 hours, after the targeted portion has been administered to the subject.

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 approximately 8 to 24 hours after the targeted portion is administered 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 about 8 to 12 hours, about 8 to 16 hours, about 8 to 22 hours, about 12 to 16 hours, or about 16 to 20 hours after the targeting portion is administered to the subject.

42. The method according to any one of the prior claims, wherein the subject has cancer, and 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, 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 lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

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

44. The method according to 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 hematological malignancy such as myelodysplastic syndrome, acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammaglobulinemia, plasmacytomyeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, or nodular lymphocyte-predominant Hodgkin lymphoma.

46. The method according to any one of the prior claims, further comprising administering an additional therapeutically effective amount of the therapeutic agent.

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

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