Trans-cyclooctene bioorthogonal agent and its use in cancer and immunotherapy

Abstract

We provide a trans-cyclooctene conjugate of an immunomodulator that can be used for bioorthogonal delivery to a target site in a subject. [Solution] A conjugate comprising an immunomodulatory agent payload conjugated to one or more bioorthogonal portions, wherein the immunomodulatory agent payload is selected from the group consisting of therapeutic monoclonal antibodies, cytokines, chemokines, chemokine antagonists, and immune checkpoint inhibitor payloads; or a pharmaceutically acceptable salt thereof is provided.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 062,814, filed on 7 August 2020, which is incorporated herein by reference in its entirety.

[0002] This disclosure provides trans-cyclooctene derivatives and uses for orthogonal biodelivery to subjects for cancer and / or immunotherapy. [Background technology]

[0003] Immunotherapy to enhance the immune system's function against tumor growth and disseminated metastasis of cancer is clinically validated. Immunotherapy strategies utilize immune cells and include monoclonal antibodies against tumor antigens, immune checkpoint inhibitors, vaccinations, adoptive immunotherapy (e.g., CAR-T cells), and cytokine administration.

[0004] Bioorthogonal conjugated reactions or click reactions are selective and orthogonal (non-interacting) functionalities found in biological systems that have diverse applications in the fields of chemistry, chemical biology, molecular diagnostics, and medicine. In these fields, 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. Examples of these reactions include Staudinger ligation, azide-cyclooctin cyclization, and reverse electron-demand Diels-Alder reactions. [Overview of the project] [Means for solving the problem]

[0005] The present disclosure provides a conjugate comprising an immunomodulatory agent payload, a monoclonal antibody payload, or a therapeutic protein payload conjugated to one or more bioorthogonal moieties, wherein the immunomodulatory agent payload is selected from the group consisting of cytokines, chemokines, chemokine antagonists, and immune checkpoint inhibitor payloads; or a pharmaceutically acceptable salt thereof.

[0006] In one aspect, the present invention provides a conjugate of formula (I), or a pharmaceutically acceptable salt thereof

Chemical formula

Chemical formula

[0007] In another aspect, the present invention provides a pharmaceutical composition comprising a conjugate, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0008] In another aspect, the present invention provides a method of treating cancer or enhancing or inducing an immune response, the method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate, or a pharmaceutically acceptable salt or composition thereof, and a therapeutic support composition, the therapeutic support composition comprising a biocompatible support and a compound of the formula

Chemical formula

[0009] In another aspect, the present invention provides a pharmaceutical combination comprising a conjugate or pharmaceutically acceptable salt or composition thereof for use in treating cancer or enhancing or inducing an immune response; and a therapeutic support composition, wherein the therapeutic support composition comprises a biocompatible support as defined herein and a formula [ka] It contains a tetrazine-containing group.

[0010] In another aspect, the present invention provides the use of a combination comprising a conjugate, or a pharmaceutically acceptable salt or composition thereof, and a therapeutic support composition in the manufacture of an agent for treating cancer or enhancing or inducing an immune response, wherein the therapeutic support composition comprises a biocompatible support and a formula as defined herein. [ka] It contains a tetrazine-containing group.

[0011] Aspects of this disclosure include a method for delivering an effective amount of an immunomodulator to a target site in a subject, the method comprising the step of administering to the subject a therapeutic support composition and conjugate as defined herein.

[0012] Aspects of this disclosure also include kits comprising a conjugate, a therapeutic support composition as defined herein, and optionally, a compound of formula (III) as defined herein. [Brief explanation of the drawing]

[0013] [Figure 1] The following fluorescence images are shown of three mice that underwent the following treatments subcutaneously in the right flank: (1) SQL70 biopolymer was injected, followed 1 hour later by intravenous injection (IV) of 10 mg / kg of TCO-Fab-Cy5.5 (Cy-5.5-modified TCO-modified ranibizumab) via tail vein injection (TVI) (Group 1); (2) SQL70 biopolymer was injected, followed 1 hour later by intravenous injection (IV) of 10 mg / kg of Fab-Cy5.5 (Cy-5.5-modified ranibizumab) via tail vein injection (TVI) (Group 2); and (3) a control unmodified HA biopolymer was injected, followed 1 hour later by intravenous injection (IV) of 10 mg / kg of TCO-Fab-Cy5.5 (Cy-5.5-modified TCO-modified ranibizumab) via tail vein injection (TVI) (Group 3). [Modes for carrying out the invention]

[0014] It should be understood that, in relation to individual embodiments, certain features of the Invention described for clarity may also be provided in combination in a single embodiment. Conversely, in relation to a single embodiment, various features of the Invention described for simplification may also be provided individually or in any preferred partial combination. Any combination of embodiments relating to the Invention is specifically encompassed in the Invention and is disclosed herein in exactly the same way that each and all combinations are individually and expressly disclosed to the extent that such combinations encompass subject matter that is, for example, a stable compound (i.e., a compound that can be prepared, isolated, characterized, and tested for bioactivity). In addition, any partial combinations of various embodiments and their elements (e.g., elements of chemical groups listed in embodiments illustrating such variations) are also specifically encompassed in the Invention and are disclosed herein in exactly the same way that each and all such partial combinations are individually and expressly disclosed herein.

[0015] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. In the event of any conflict, including definitions, this specification shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in carrying out or testing the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and examples disclosed herein are illustrative and not intended to be limiting.

[0016] As used herein, "comprising," "including," "having," "has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases that do not preclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly recited.

[0017] The modifier "about" used in connection with a quantity includes the recited value and has the meaning required by the context (for example, it includes the degree of error associated with the measurement of at least a particular quantity). The modifier "about" should also be considered as disclosing a range defined by the absolute values of two endpoints. For example, the expression "about 2 to about 4" also discloses the range "2 to 4." The term "about" can refer to plus or minus 10% of the number being expressed. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" may become apparent from the context, such as rounding, etc., for example, "about 1" can also mean 0.5 to 1.4.

[0018] The conjunction "or" encompasses any combination of the one or more listed elements it connects. For example, the phrase "a device containing A or B" can refer to a device containing A but not B, a device containing B but not A, or a device containing both A and B. The phrases "at least one of A, B, ... and N" or "at least one of A, B, ... and N, or any combination thereof" are broadly defined to mean one or more elements selected from the group containing A, B, ... and N, or in other words, any combination of one or more elements A, B, ... or N, including the case of any single element alone, or a combination of any one or more elements that may also contain other elements not listed.

[0019] The definitions of specific functional groups and chemical terms are explained in more detail below. For the purposes of this disclosure, chemical elements are based on the periodic table, CAS versions, and the Handbook of Chemistry and Physics, 75th edition. th (ed.), identified according to the inside cover, and specific functional groups are generally defined as described therein. Furthermore, general principles of organic chemistry, as well as specific functional groups and reactivity, are as follows: Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; 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 rd Edi This is described in tion, Cambridge University Press, Cambridge, 1987; the entire contents of each of these are incorporated herein by reference.

[0020] As used herein, the term "alkoxy" refers to an alkyl group (as defined herein) 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.

[0021] As used herein, the term "alkyl" means a linear or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. The terms "lower alkyl" or "C1-C6-alkyl" mean a linear or branched hydrocarbon chain containing 1 to 6 carbon atoms. The term "C1-C3-alkyl" means a linear or branched hydrocarbon chain containing 1 to 3 carbon atoms. Representative examples of alkyls 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.

[0022] As used herein, the term "alkenyl" means a hydrocarbon chain containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond. The alkenyl group may or may not be substituted. For example, the alkenyl group may be substituted with an aryl group, such as phenyl.

[0023] As used herein, the term "alkynyl" refers to a linear or branched monovalent hydrocarbonyl group having 2 to 30 carbon atoms, e.g., 2 to 20 or 2 to 10 carbon atoms, and having at least one triple-bonded unsaturated moiety. The term "alkyne" also includes non-aromatic cycloalkyl groups having one or more rings and at least one triple bond, with 5 to 20 carbon atoms, e.g., 5 to 10 carbon atoms. Examples of such alkynyl groups include, but are not limited to, acetylenyl (-C≡CH) and propargyl (-CH2C≡CH), as well as cycloalkynyl moieties, e.g., substituted or unsubstituted cyclooctin moieties.

[0024] As used herein, the term "alkoxyalkyl" refers to an alkoxy group (as defined herein) attached to the parent molecule via an alkyl group.

[0025] 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-.

[0026] The term "amino acid" refers to both natural and unnatural amino acids, protected natural and unnatural amino acids, and amino acid analogs and mimetic compounds that function similarly to natural 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) and This includes pyrrolidine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., for example, hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group. Such analogs may have a modified R group (for example, norleucine) or may retain a modified peptide skeleton while maintaining the same basic chemical structure as natural amino acids. Non-limiting examples of amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0027] As used herein, the term "aryl" refers to a phenyl group, or a bicyclic aryl or tricyclic aryl fused ring system. An example of a bicyclic fused ring system is a phenyl group attached to a parent molecule and fused with one phenyl group. An example of a tricyclic fused ring system is a phenyl group attached to a parent molecule and fused with 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 are linked to the parent molecule via any carbon atoms contained within their rings and may be either unsubstituted or substituted.

[0028] As used herein, the term "azide" refers to the functional group -N3.

[0029] As used herein, the term “cycloalkyl” refers to a carbocyclic ring system containing 3 to 10 carbon atoms, 0 heteroatoms, and 0 double bonds. Typical examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. “Cycloalkyl” also encompasses carbocyclic ring systems in which a cycloalkyl is attached to a parent molecule and fused with an aryl group, a heteroaryl group, or a heterocycle as defined herein.

[0030] As used herein, the term "cycloalkenyl" means a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Examples of monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0031] As used herein, the term "cyclooctene" refers to an eight-carbon substituted or unsubstituted non-aromatic cyclic alkyl group having a monocyclic ring with a double bond. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).

[0032] As used herein, the term "fluoroalkyl" means an alkyl group (as defined herein) in which one, two, three, four, five, six, seven, or eight hydrogen atoms are substituted with 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.

[0033] As used herein, the term "alkoxyfluoroalkyl" refers to an alkoxy group (as defined herein) attached to the parent molecule via a fluoroalkyl group (as defined herein).

[0034] As used herein, the term "fluoroalkoxy" means a molecule in which at least one fluoroalkyl group (as defined herein) is attached to the parent molecule via an oxygen atom. This means that it is added. Typical examples of fluoroalkoxys include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0035] As used herein, the terms "halogen" or "halo" mean Cl, Br, I, or F.

[0036] 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 substituted with a halogen.

[0037] As used herein, the term "haloalkoxy" means that at least one haloalkyl group (as defined herein) is attached to the parent molecule via an oxygen atom.

[0038] As used herein, the term “heteroalkyl” means an alkyl group (as defined herein) in which one or more carbon atoms are substituted with a heteroatom selected from S, Si, O, P, and N. The heteroatom may be oxidized. Typical examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkylamines, and alkyl sulfides.

[0039] 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 five- or six-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., one, two, three, or four heteroatoms independently selected from O, S, and N). A five-membered aromatic monocyclic ring has two double bonds, and a six-membered aromatic monocyclic ring has three double bonds. Examples of bicyclic heteroaryl groups include monocyclic heteroaryl rings fused with a parent molecule and a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic heterocycle as defined herein. Examples of tricyclic heteroaryl groups include monocyclic heteroaryl rings that are attached to a parent molecule and fused with two of the following monocyclic cycloalkyl groups, monocyclic aryl groups, monocyclic heteroaryl groups, or monocyclic heterocycles as defined herein. 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, clomethyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienotienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryls are linked to the parent molecule via any carbon atom or nitrogen atom contained within their rings, and may be unsubstituted or substituted.

[0040] As used herein, the term “heterocycle” means monocyclic heterocycle, bicyclic heterocycle, and tricyclic heterocycle. 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. A 3-membered or 4-membered ring contains 0 or 1 double bonds and 1 heteroatom selected from the group consisting of O, N, and S. Each ring contains 1 or 1 heteroatoms. A 5-membered ring contains 0 or 1 double bond and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains 0, 1, or 2 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. 7- and 8-membered rings contain 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles, though not limited to them, 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, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolidinyl, oxazoli Examples include dinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiadinyl, 1,3-thiadinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianil. A bicyclic heterocycle is a ring system of a monocyclic heterocycle fused with a phenyl group, or a monocyclic heterocycle fused with a monocyclic cycloalkyl group, or a monocyclic heterocycle fused with a monocyclic cycloalkenyl group, or a monocyclic heterocycle fused with a monocyclic heterocycle, or a spiroheterocyclic group, or a bridged monocyclic heterocycle in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or by an alkenylene bridge of 2, 3, or 4 carbon atoms.Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranil, benzothiopyranil, chromaninil, 2,3-dihydrobenzofuranil, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinil. Examples of tricyclic heterocycles include bicyclic heterocycles fused to a phenyl group, bicyclic heterocycles fused to a monocyclic cycloalkyl group, bicyclic heterocycles fused to a monocyclic cycloalkenyl group, bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of a bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles, but not limited to, 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 Examples include decane. Monocyclic, bicyclic, and tricyclic heterocyclic rings are linked to the parent molecule via any carbon or nitrogen atom contained within the ring, and may be unsubstituted or substituted.

[0041] As used herein, the term "hydroxyl" means the -OH group.

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

[0043] In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is determined by the prefix "C x ~C y -" or "C x~y It is represented by (x is the minimum number of carbon atoms in the substituent, and y is the maximum number). Therefore, for example, "C1~C3-alkyl" and "C 1~3 "Alkyl" refers to an alkyl group containing 1 to 3 carbon atoms. This refers to the lukyl substituent. These two conventional usages are "C x ~C y -" and "C x~y These terms are interchangeable and have the same meaning.

[0044] The term "substituted" refers to a group that may be further substituted with one or more nonhydrogen substituents. Substituents include, but are not limited to, halogens, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, 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.

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

[0046] The term "selective delivery" refers to the delivery of a drug (e.g., payload) to the organ or tissue (or part thereof) that requires treatment or diagnosis, without significant binding to other non-target organs or tissues (or parts thereof).

[0047] The term "payload" refers to a drug intended for delivery to a target site in a subject. Payloads include therapeutic drugs.

[0048] As used herein, “payload portion” means payload D or D 1 From there, NH and NC are coupled to the linker. 1~4 This refers to the remaining portion of the payload, specifically the part after the nucleophilic groups of the payload, such as alkyl, O, or S, have been removed, or after the electrophilic groups of the payload, such as C(O), bonded to the linker, have been removed.

[0049] The term "therapeutic agent" refers to a drug that can treat and / or improve a subject's medical condition or disease, or one or more of its symptoms. The therapeutic agents of this disclosure also include prodrug forms of therapeutic agents.

[0050] The term "diagnostic agent" refers to an agent that aids in the diagnosis of a medical condition or disease. Representative diagnostic agents include contrast agents such as paramagnetic agents, optical probes, and radionuclides. A paramagnetic agent is a contrast agent that has magnetism under an externally applied field. Examples of paramagnetic agents include, but are not limited to, iron particles such as iron nanoparticles and iron microparticles. An optical probe is a fluorescent compound that can be detected by excitation with radiation of one wavelength and detection with radiation of a second different wavelength. Examples of the optical probes of the present disclosure include, but are not limited to, Cy5.5, Alexa680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate), and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. A radionuclide is an element that undergoes detectable radioactive decay. Examples of radionuclides useful in the embodiments of the present disclosure include, but are not limited to, 3 H, 11 C, 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 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 R e, 211 At, Rn, Ra, Th, U, Pu, and 241 Am.

[0051] The term "targeting agent" refers to a chemical or biopharmaceutical 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. "Stable association" means that one part binds to, or otherwise associates with, another part or structure under standard physiological conditions. Examples of such bonds include covalent and non-covalent interactions, and include, but are not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), and dipole-dipole force interactions. The targeting agent may be a member of a specific binding pair, and such members may include, but are not limited to, members of a receptor / ligand pair; the ligand-binding portion of a receptor; members of an antibody / antigen pair; the antigen-binding fragment of an antibody; haptens; members of a lectin / carbohydrate pair; members of an enzyme / substrate pair; biotin / avidin; biotin / streptavidin; digoxin / anti-digoxin; members of a DNA or RNA aptamer binding pair; members of a peptide aptamer binding pair, etc. The targeting agent includes a ligand that specifically (or substantially specifically) binds to a particular clinically relevant target receptor or cell surface target. The ligand may be an antibody, peptide, nucleic acid, phage, bacterium, virus, or other molecule that has a specific affinity for the target receptor or cell surface target. Examples of receptors and cell surface targets include, but are not limited to, PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, BCR-ABL, SRC, JAK2, MAP2K, EML4-ALK, BRAF V600E, 4-1BB, GITR, GSK3β, LT4-human mAb for inhibitory immune checkpoint receptor immunoglobulin-like transcript 4 (ILT4); leukocyte immunoglobulin-like receptor superfamily B member 2, LILRB2, lymphocyte immunoglobulin-like receptor 2, LIR2, monocyte / macrophage immunoglobulin-like receptor 10, MIR-10, CD85d, or other cell receptors or cell surface targets.

[0052] The term "target organ or tissue" refers to the organ or tissue targeted for payload delivery. Typical target organs and tissues 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.

[0053] The term "transplant" refers to the surgical insertion of a subject's body.

[0054] The terms "contact" or "contact" refer to a process of bringing at least two different species into contact so that they can interact with each other, for example, through non-covalent or covalent interactions or bonding reactions. However, it should be understood that the resulting complex or reaction product may be formed directly from the interactions or reactions between the added reagents, or from intermediates (which may be formed in the contact mixture) derived from one or more of the added reagents or components.

[0055] The term "binder" refers to a drug having a functional group that can form a covalent bond with the complementary functional group of another binder in a biological environment. The bonding of binders in a biological environment is sometimes called a bioconjugated reaction. Binders include bioorthogonal binders, which are binders having bioorthogonal functional groups. The bioorthogonal functional group of a bioorthogonal binder selectively reacts with the complementary bioorthogonal functional group of another bioorthogonal bonding partner. This selective reaction between bioorthogonal bonding partners minimizes side reactions with other binders, biocompounds, or other non-complementary bioorthogonal binders or non-complementary bioorthogonal functional groups. The bioorthogonal portion or functional group of a bioorthogonal binder can be, but is not limited to, a cripotent form. Examples of binders for triazole formation by a chemical reaction include azides and alkynes, trans-cyclooctene (TCO), and tetrazine (Tz) (e.g., 1,2,4,5-tetrazine). The binders useful in this disclosure may have high reactivity with the corresponding binder so that the reaction is rapid.

[0056] The term "functionalized" refers to the part to which a functional group is attached, for example, the part to which a binder functional group (e.g., a bioorthogonal functional group) is attached.

[0057] The term "administer" refers to any preferred route of administration to the subject, for example, but not limited to, oral administration, suppository administration, topical contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal or subcutaneous administration, intrathecal administration, or implantation of a sustained-release device, such as a mini osmotic pump.

[0058] As used herein, “parenteral” refers to methods of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0059] The term "leaving group" refers to an atom (or group of atoms) that, when substituted as a stable species, possesses the ability to accept bonding electrons and thereby acquire electron-withdrawing capabilities. Suitable examples of leaving groups include halides (e.g., Br, Cl, I), sulfonic acid esters (e.g., triflates, mesylates, tosylates, and brosylates), and nitrophenols.

[0060] The terms “pharmaceutically effective dose” and “therapeutic dose” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms, and / or to prevent or reduce the risk of the onset or recurrence of the disorder or disease or one or more of its symptoms. With respect to tumorigenic proliferative disorders, a pharmaceutically effective or therapeutic dose includes, in particular, an amount sufficient to shrink the tumor or reduce its growth rate.

[0061] As used herein, “subject,” “patient,” or “organism” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to whom the agents of this disclosure can be administered may include mammals, particularly primates, and especially humans. For veterinary use, suitable subjects may include livestock such as cattle, sheep, goats, cows, and pigs; poultry such as chickens, ducks, geese, and turkeys; and 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.

[0062] As used herein, the terms “to treat” or “treatment” mean treating or treating a disease or condition or symptoms thereof in a patient, such as a mammal (especially a human), which includes: (a) improving a disease or condition or symptoms thereof, for example, eliminating or reversing a patient’s disease or condition or symptoms; (b) suppressing a patient’s disease or condition or symptoms thereof, for example, by delaying or stopping the onset of the disease or condition or symptoms thereof; or (c) alleviating a patient’s disease or condition or symptoms thereof.

[0063] The term "physiological conditions" means conditions for cell survival and compatibility, primarily including aqueous conditions such as temperature, pH, and salinity.

[0064] In the case of the compounds described herein, the group and substituents are the permissible atoms and substituents. The atoms can be selected according to their valence, and as a result of this selection and substitution, stable compounds can be obtained that do not spontaneously undergo morphological changes such as rearrangement, cyclization, or exclusion.

[0065] Where a range of values ​​is described, it should be understood that the intervening value between the upper and lower limits of that range (up to 1 / 10 of the lower limit unit, unless the context clearly requires a different interpretation) and all other described values ​​or intervening values ​​within this described range are included within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included within these smaller ranges and may also be included within the invention subject to any specifically excluded limits within this described range. If the described range includes one or both limits, the range excluding one or both of these included limits is also included in this disclosure.

[0066] In the context of numerical ranges described herein, each of the intervening values ​​having a similar degree of accuracy is explicitly considered. For example, in the range of 6 to 9, the numbers 7 and 8 are considered in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

[0067] The compound may exist as a stereoisomer containing a chiral or asymmetric center. This stereoisomer is "R" or "S" depending on the configuration of substituents around the chiral carbon atom. The terms "R" and "S" as used herein refer to the configurations defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45:13-30. This disclosure considers various stereoisomers and mixtures thereof, which are explicitly within the scope of the invention. Stereoiomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of the compound can be prepared synthetically from commercially available starting materials containing a chiral or asymmetric center, or by the preparation of racemic mixtures and subsequent resolution methods well known to those skilled in the art. Such resolution methods include: (1) Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry 5 th (1) Conjugation of an enantiomer mixture with a chiral auxiliary, separation of the resulting diastereomer mixture by recrystallization or chromatography, and optical liberation of the optically pure product from the auxiliary, as described in edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England; (2) Direct separation of the optical enantiomer mixture by chiral column chromatography; or (3) Fractional recrystallization.

[0068] It should be understood that this compound may have tautomers and geometric isomers, and that these also constitute aspects of the present invention.

[0069] This disclosure also includes isotope-labeled compounds, which are identical to the compounds described herein except that one or more atoms are substituted with atoms having atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, for example, but not limited to, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 It is Cl. Deuterium (that is, 2 Substitution with heavier isotopes, such as H, can lead to greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), thus providing certain therapeutic benefits. Because it can be made possible, it may be preferable in some situations. This compound can incorporate positron-emitting isotopes for medical imaging and positron emission tomography (PET) tests to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated into the compound of formula (I), (II), or (III) are: 11 C, 13 N, 15 O, and 18 F is the isotope-labeled compounds disclosed herein can generally be prepared by conventional techniques well known to those skilled in the art, using appropriate isotope-labeled reagents instead of non-isotope-labeled reagents, or by methods similar to those described in the accompanying examples.

[0070] B. Conjugate Embodiments of the present invention are disclosed below. The first embodiment is indicated as E1, and subsequent embodiments are indicated as E1.1, E1.1A, E1.2, and so on.

[0071] E1. A conjugate comprising an immunomodulatory payload conjugated to one or more bioorthogonal moieties, wherein the immunomodulatory payload is selected from the group consisting of cytokines, chemokines, chemokine antagonists, therapeutic monoclonal antibodies, and immune checkpoint inhibitor payloads; or a pharmaceutically acceptable salt thereof. The bioorthogonal moieties comprise bioorthogonal functional groups such as trans-cyclooctene, tetrazine, azide, or alkyne. Bioorthogonal functional groups are well known in the art, as described in Randon, 2020, Bioconjugate Chem., 31(2):159~173).

[0072] E1.1. A conjugate comprising an immunomodulatory agent payload conjugated to one or more bioorthogonal moieties, wherein the immunomodulatory agent payload is an inhibitor of a cytokine payload, or a pharmaceutically acceptable salt thereof.

[0073] E1.1A. Conjugates described in E1.1, or pharmaceutically acceptable salts thereof, wherein the cytokine payload inhibitor is an inhibitor of TNF-α, infliximab, certolizumab, TGF-β, garnicertib, fresolimumbab, M7824, CSF-1, pexidartinib, or kabilizumab.

[0074] E1.2. A conjugate containing a monoclonal antibody payload conjugated to one or more bioorthogonal moieties, or a pharmaceutically acceptable salt thereof.

[0075] E1.3. Conjugates containing a therapeutic protein payload conjugated to one or more bioorthogonal moieties, or pharmaceutically acceptable salts thereof.

[0076] E1.3A. Conjugates or pharmaceutically acceptable salts thereof as described in E1.3, wherein the therapeutic protein payload is an antibody-based drug, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, manipulated protein scaffold, enzyme, growth factor, hormone, interferon, interleukin, or thrombolytic agent.

[0077] E1.3B. A conjugate as described in E1.3, or a pharmaceutically acceptable salt thereof, wherein the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen payload.

[0078] E1.3C. The therapeutic protein payload is erythropoietin (EPO, e.g., natural EPO or synthetic EPO (see, e.g., U.S. Patent Application Publication No. 2003 / 0191291)), e.g., but not limited to, PROCRIT®, EPREX®, or EPOGEN® (epoetin-α), ARANESP® (darbepoetin-α), NEORECORMON®, E POGIN® (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, daratumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mo Gamlizumab, nesitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, partuzumab, 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, Ultral) ente, etc.), insulin-like growth factors (e.g., IGF-I, IGF-II); blood factors (e.g., factor X, tissue plasminogen activator (TPA), etc., for example, but not limited to, ACTIVASE® (alteplase) tissue plasminogen activator, NOVOSEVEN® (recombinant human Vila factor), Vila factor, factor VIII (e.g., KOGENATE®), factor IX, β-globin, hemoglobin, etc.); colony-stimulating factors (e.g., granulocyte-CSF (G-C)); SFs (e.g., NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), Neulasta (pegfilgrastim), granulocyte-monocyte colony-stimulating factor, megakaryocyte colony-stimulating factor, etc.), transforming growth factors (e.g., TGF-β, TGF-α); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®), IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IE-12, etc.);Growth factors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF, e.g., REGRANEX® (becaprelmin)), 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., TNF-α-binding soluble receptor, e.g., ENBREL® (etanercept), soluble VEGF receptor, soluble interleukin receptor, soluble γ / δ T cell receptors, etc.); Enzymes (e.g., α-glucosidase, CERAZYME® (imiglucerase, β-glucocerebrosidase, CEREDASE® (alglucerase)); Enzyme activators (e.g., tissue plasminogen activator); Chemokines (e.g., IP-10, Mig, Groα / IL-8, regulatory normal T cell expression secretory protein (RANTES), MIP-1α, MIP-1ρ, MCP-1, PF-4, etc.); Angiogenic drugs (e.g., vascular endothelial growth factor (VEGF); Anti-angiogenic drugs (e.g., soluble VEGF receptor); Protein vaccines; Neuroactive peptides, e.g., bradykinin, cholecystokinin, gustin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostaglandin Thin, 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, etc.; other proteins, such as thrombolytic agents, atrial natriuretic peptide, bone morphogenetic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human α-1 antitrypsin, leukemia inhibitory factor, transforming growth factor, tissue factor, insulin-like growth factor, luteinizing hormone, follicle-stimulating hormone, macrophage activator, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, metalloproteinase tissue inhibitors;Vascular-active intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; white; Blood cell suppressants; or payloads such as IL-1 receptor antagonists (e.g., Kineret® (Anakinra)), the conjugates described in E1.3, or pharmaceutically acceptable salts thereof.

[0079] E2. A conjugate according to any of E1 to E1.3C, or a pharmaceutically acceptable salt thereof, wherein each bioorthogonal moiety independently contains trans-cyclooctene or tetrazine.

[0080] E3. A conjugate described in any of E1 to E2 of formula (I), or a pharmaceutically acceptable salt thereof. [ka] (In the formula, G is the bioorthogonal part, and each time G appears, it is independent. [ka] and; L 1 Each instance is an independent linker; m is an integer between 1 and 150; D 1 This includes immunomodulatory agent payloads, monoclonal antibody payloads, or therapeutic protein payloads; R 1A Each time it appears, C 1~4 Alkyl, C 1~4 Haloalkyl and C 1~4 Independently selected from the group consisting of alkoxys; q is 0, 1, or 2; q1 is either 0 or 1; R 1B Each time it appears, 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 Independently selected from the group consisting of alkylene-CO2H)2; R 1c and R 1d Each time it appears, it independently contains hydrogen or C 1~4 It is alkyl; R 1e Each time it appears, it is independently -C 1~4 Alkylene-CO2H, -C 1~4Alkylene-CONH2, or -C 1~4 It is alkylene-OH; R 1f Each time it appears, it independently produces hydrogen and C. 1~6 Alkyl, or C 1~4 It is alkylene-CO2H; Each instance of n is independently 0, 1, 2, or 3; L 2 Each time it appears, -C(O)- and C 1~3 Independently selected from the group consisting of alkylenes; G 1 Each instance is a heterocyclyl that is independently and arbitrarily substituted.

[0081] E3.1. The conjugate described in E3 of formula (I), or a pharmaceutically acceptable salt thereof. [ka] (In the formula, G is the bioorthogonal part, and each time G appears, it is independent. [ka] and; L 1 Each instance is an independent linker; m is an integer between 1 and 150; D 1 It is an immunomodulatory payload; R 1A Each time it appears, C 1~4 Alkyl, C 1~4 Haloalkyl and C 1~4 Independently selected from the group consisting of alkoxys; q is either 0 or 1; R 1B Each time it appears, G 1 , OH, -NR 1c -C 1~4 Alkilen-G 1 , -NR 1c -C 1~4 Alkylene-N(R) 1d)2, -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, and -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 Independently selected from the group consisting of alkyl)2; R 1c and R 1d Each time it appears, it independently contains hydrogen or C 1~4 It is alkyl; R 1e Each time it appears, it is independently -C 1~4 Alkylene-CO2H, -C 1~4 Alkylene-CONH2, or -C 1~4 It is alkylene-OH; R 1f Each time it appears, it independently produces hydrogen and C. 1~6 Alkyl, or C 1~4 It is alkylene-CO2H; Each instance of n is independently 0, 1, 2, or 3; L 2 Each time it appears, -C(O)- and C 1~3 Independently selected from the group consisting of alkylenes; G 1Each instance is a heterocyclyl that is independently and arbitrarily substituted.

[0082] Each time E3.1A.G appears, it is independent. [ka] The conjugate described in E3 or E3.1, or a pharmaceutically acceptable salt thereof.

[0083] Each time E3.1B.G appears, it is independent. [ka] The conjugate described in E3.1A, or a pharmaceutically acceptable salt thereof.

[0084] E3.2. An immunomodulatory payload is an antibody payload, a conjugate as described in E1 or E2-E3.1B, or a pharmaceutically acceptable salt thereof.

[0085] E4. A conjugate described in any of E1 or E2-E3.2, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory payload is an immune checkpoint inhibitor payload.

[0086] E4.1. Conjugates described in E4, or pharmaceutically acceptable salts thereof, in which the immune checkpoint inhibitor payload is pidilizumab, cintilimab, AMP-224, atezolizumab, durvalumab, BMS-936559, tremelimumab, indoximod, epacadostat, TIGIT inhibitors (e.g., LAG-3 such as anti-LAG-3 antibody; TIM-3 such as anti-TIM-3 antibody), B7 molecules, or BTLA pathway antagonists.

[0087] E5. The conjugate described in E4, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload.

[0088] E6. The conjugate described in E5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload.

[0089] E7. The conjugate described in E6, or a pharmaceutically acceptable salt thereof, wherein the PD-1 inhibitor payload is nivolumab, pembrolizumab, pidilizumab, cintilimab, or AMP-224 payload.

[0090] E8. The conjugate described in E5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload.

[0091] E9. PD-L1 inhibitor payloads include atezolizumab, avelumab, and durvalumab. , or the conjugate described in E8, or a pharmaceutically acceptable salt thereof, which is the BMS-936559 payload.

[0092] E10. The conjugate described in E5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload.

[0093] E11. The conjugate described in E10, or a pharmaceutically acceptable salt thereof, wherein the CTLA4 inhibitor payload is ipilimumab or tremelimumab payload.

[0094] E12. The conjugate described in E4, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor payload is an indoleamine 2,3-dioxygenase (IDO) inhibitor payload.

[0095] E13. The conjugate described in E12, or a pharmaceutically acceptable salt thereof, wherein the IDO inhibitor payload is indoximod or epacadostat payload.

[0096] E14. A conjugate described in E1 or any of E2-E3.2, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory payload is a cytokine payload.

[0097] E15. Conjugates described in E14, or pharmaceutically acceptable salts thereof, wherein 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.

[0098] E15.1. The interleukin payload is a conjugate or pharmaceutically acceptable salt thereof as described in E14, selected from IL-1 to IL-40.

[0099] E15.2. The conjugate described in E14, or a pharmaceutically acceptable salt thereof, wherein the interleukin payload is IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0100] E15.3. Conjugates described in E14, or pharmaceutically acceptable salts thereof, wherein the immunomodulatory payload is a type 1 cytokine (IL-2, IL-12, TNF-B, IFN-γ).

[0101] E15.4. The cytokine payload includes IFN-α, IFN-β, IFN-γ, pegylated IFN-α, and apolipoprotein AI fusion proteins with IFN-α, interleukins, IL-2, IL-2 covalently bound to immunoglobulins (e.g., cergutuzumab amunaleukin, 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 with the binding domain of IL-15Rα (e.g., RLI), triple fusion proteins containing human IL-15, the binding domain of IL-15Rα and apolipoprotein AI, and ALT-803 (IgG1 A conjugate as described in E14, selected from the group consisting of IL-15, IL-18, IL-21, tumor necrosis factor, TNF-α, TNF-β, erythropoietin (EPO), MIP3a, ICAM, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF, and tarimodine raherparepbec fused to the Fc domain, or That salt is pharmaceutically acceptable.

[0102] E16. A conjugate described in any of E1 or E2-E3.2, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory payload is a chemokine payload.

[0103] E17. The conjugate described in E16, or a pharmaceutically acceptable salt thereof, wherein the chemokine payload is a CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CXCL14 payload.

[0104] E18. A conjugate described in any of E1 or E2-E3.2, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory payload is a chemokine antagonist payload.

[0105] E19. The conjugate described in E18, or a pharmaceutically acceptable salt thereof, wherein the chemokine antagonist payload is the prelixafor payload.

[0106] E19.1. An immunomodulatory agent is a monoclonal antibody specific to a cytokine or cytokine receptor, as described in E1 or any of E2-E3.2, or a pharmaceutically acceptable salt thereof.

[0107] E20. The immunomodulatory payload is a conjugate containing a polypeptide as described in E1, E2-E18, or E19.1, or a pharmaceutically acceptable salt thereof.

[0108] E21. A polypeptide comprising one or more lysine residues, the conjugate described in E20, or a pharmaceutically acceptable salt thereof.

[0109] E21.1. A conjugate as described in E20, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.

[0110] E22. The conjugate described in E21, or a pharmaceutically acceptable salt thereof, wherein each bioorthogonal moiety is bound to one of one or more lysine residues.

[0111] E22.1. The conjugate described in E21.1, or a pharmaceutically acceptable salt thereof, wherein each bioorthogonal moiety is independently bound to one or more lysine, serine, threonine, or tyrosine residues.

[0112] E22.2. A conjugate according to any of E20-E22.1, or a pharmaceutically acceptable salt thereof, wherein the polypeptide contains an N-terminal amino acid, and the appearance of one of the bioorthogonal moieties is conjugated to the N-terminal amino acid.

[0113] A conjugate or pharmaceutically acceptable salt thereof, as described in any of E3 to E22.2, where E23.m is 1 to 20.

[0114] In the conjugates described herein, the linker L may have 1 to 100 linked atoms and may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, the 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 It may have approximately 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0115] The linker in formula (I) may include one or more (e.g., 1 to 10 or 1 to 5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1 to 10 or 1 to 5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties; where each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety is independently oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4 It can be optionally substituted with 1 to 5 substituents independently selected from the haloalkyl group.

[0116] In equation (I), the linker is: -Y 10 -(CH2) n’ -Y 20-(CH2) m’’ -Y 30 - (In the formula, Y 10 , Y 20 , and Y 30 These are, each independently, 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; where each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4 Optionally substituted with 1 to 5 substituents independently selected from the haloalkyl group; Each R 110 These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 It is a haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R 120 These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 It is a haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. It could be.

[0117] In certain embodiments, the linker is not a link. In certain embodiments, each R 110 These are, independently, hydrogen and C 1~4 Alkyl, C1~4 Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R 120 These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 These are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl compounds.

[0118] Typical linkers, though not limited to those listed below, include: [ka] These are some examples.

[0119] Typical linkers, though not limited to those listed below, include: [ka] These are some examples.

[0120] The linker in formula (I) 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. The linker in formula (I) is [ka] It may include.

[0121] In formula (I), the linker may contain one or more natural or non-natural amino acids, which may be called peptide linkers. 1 If the drug contains an amino portion, the linker may be attached to it using a peptide linker composed of carboxylic acid acyl units and one or more amino acids that constitute the protein or peptide sequence. The linker may also contain a self-sacrificing spacer that separates the drug and the protein peptide sequence.

[0122] In formula I, linker L may be a peptide linker represented by "AYZXW", where "A" is a carboxylic acid acyl unit, "Y" and "Z" are one or more natural or non-natural amino acids, together forming a peptide sequence, and "X" and "W" are peptides and drugs, D 1 or an optional additional linker having 1 to 50 linking atoms, or 5 to 10 linking atoms, or 1 to 10 linking atoms separating the bioorthogonal portion. In certain embodiments, one or more amino acids in the peptide linker are N-methylated.

[0123] In formula (I), Y may be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Y may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0124] In formula (I), Z is alanine, lysine, acetyl, or formyl-protected lysine. Z may be at least one amino acid selected from the group consisting of arginine, arginine protected with tosyl or a nitro group, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline. Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0125] Preferred YZ combinations include valine-citrulline; valine-alanine; and alanine-alanine.

[0126] In certain embodiments, A is -OC(O)-.

[0127] In certain embodiments, X is -OC(O)-.

[0128] In certain embodiments, W is -OC(O)-. In certain embodiments, X does not exist, and W is -OC(O)-.

[0129] In certain embodiments, -XW is [ka] That is the case.

[0130] In certain embodiments, -XW is [ka] That is the case.

[0131] In certain embodiments, the peptide linker is specially modified so that it is selectively cleaved (e.g., enzymatically cleaved) by one or more tumor-associated proteases to release a drug.

[0132] In certain embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., di-, tri-, or tetra-peptides). However, it will be understood that peptide linkers with up to 5, 6, 7, or 8 amino acid residues can also be suitably used.

[0133] In certain 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 -Nitro-Arg. In certain embodiments, pep 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.

[0134] In certain embodiments, the linker L in formula (I) is [ka] (for example, [ka] ), [ka] The linker mentioned above is D 1 On the right side of the amino acid side chain, such lysine or cysteine ​​(for example, [ka] ) can be bound together.

[0135] E24. L 1 is -OC(O)L 4 - or -OC 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 is halogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 A conjugate according to any of E3 to E23, or a pharmaceutically acceptable salt thereof, which is a phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0136] E25.GL 1 However, each time they appear, independently, [ka] The conjugate according to claim E24, or a pharmaceutically acceptable salt thereof.

[0137] E26.GL 1 However, each time they appear, independently, [ka] The conjugate described in E25, or a pharmaceutically acceptable salt thereof. When conjugated to a lysine residue, the conjugate is of the formula [ka] It can be represented as, where PPM is a polypeptide moiety having lysine residues and lysine side chains, and PPM is also a base [ka] It may have additional lysine or other amino acid side chains conjugated to it.

[0138] E27. R 1B G 1 , OH, -NR 1c -C 1~4 Alkilen-G 1 , -NR 1c -C 1~4 Alkylene-N(R) 1d )2, -N(R 1c )CHR 1e CO2H, -N(R) 1c )CH2CO2H, and -N(R 1f )-CH2CH2-(N(CH2CO2H)CH2CH2) n Selected from the group consisting of -N(CH2CO2H)2; R 1e is -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH; R 1f The conjugate is hydrogen or CH2CO2H, as described in any of E3 to E26, or a pharmaceutically acceptable salt thereof.

[0139] E27.1. R 1B -NR 1c -C 2~4 Alkylene-N(C) 1~4 Alkyl)3 + , -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 1~6A conjugate selected from the group consisting of alkylene-CO2H)2, as described in any of E3 to E26, or a pharmaceutically acceptable salt thereof.

[0140] E27.2. 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 A conjugate selected from the group consisting of )-CH(CH2O-CH2CH2-CO2H)2, or a pharmaceutically acceptable salt thereof, as described in E27.1.

[0141] E27.3.R 1A C 1~4 Alkyl conjugates as described in any of E27 to E27.2, or pharmaceutically acceptable salts thereof.

[0142] E27.4.R 1A A conjugate as described in E27.3, or a pharmaceutically acceptable salt thereof, wherein the compound is CH3.

[0143] E27.5.R 1c A conjugate or pharmaceutically acceptable salt thereof, as described in any of E27 to E27.4, wherein the hydrogen atom is hydrogen.

[0144] E28. R 1A C 1~4 It is alkyl; R 1B G 1 , OH, -NR 1c -C 1~4 Alkilen-G 1 , -NR 1c -C 1~4 Alkylene-N(R) 1d )2, -N(R 1c )CHR 1e CO2H, -N(R) 1c)CH2CO2H, and -N(R 1f )-CH2CH2-(N(CH2CO2H)CH2CH2) n Selected from the group consisting of -N(CH2CO2H)2; R 1e is -C 1~4 It is alkylene-CO2H; R 1f is hydrogen or C 1~4 It is alkylene-CO2H; G 1 It is a 4- to 8-membered monocyclic heterocycline containing a first nitrogen atom and, optionally, one additional heteroatom selected from nitrogen, oxygen, and sulfur, G 1 It is bonded to the first nitrogen, and optionally, C 1~4 Alkyl, C 1~4 Haloalkyl, halo, cyano, OH, -OC 1~4 It is substituted with 1 to 4 substituents independently selected from the group consisting of alkyl and oxo; n is 0, 1, or 2, and is a conjugate or pharmaceutically acceptable salt thereof as described in any of E3 to E26.

[0145] E29. R 1A It is CH3; R 1e It is -CH2CO2H; R 1f is hydrogen or CH2CO2H; G 1 It is bonded via a ring nitrogen atom, and optionally, C 1~4 Alkyl, C 1~4 Haloalkyl, halo, cyano, OH, -OC 1~4 A conjugate according to E28, or a pharmaceutically acceptable salt thereof, which is piperazinyl, morpholinyl, piperidinyl, azepanil, or pyrrolidinyl, substituted with 1 to 4 substituents independently selected from the group consisting of alkyl and oxo.

[0146] E30.L 2However, a conjugate described in any of E3 to E29, which is -C(O)-, or a pharmaceutically acceptable salt thereof.

[0147] E31. R 1B OH, N(H)CH2CO2H, -N(H)CHR 1e CO2H, -N(H)-CH2CH2-(N(CH2CO2H)CH2CH2) n Selected from the group consisting of -N(CH2CO2H)2 and -N(CH2CO2H)-CH2CH2-N(CH2CO2H)2; R 1e The conjugate described in E30, or a pharmaceutically acceptable salt thereof, is -CH2CO2H.

[0148] E32. The bio-orthogonal part is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0149] E32.1. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0150] E32.2. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0151] E32.3. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0152] E32.4. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0153] E32.5. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0154] E32.6. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0155] E32.7. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0156] E32.8. The bio-orthogonal portion is [ka] The conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof.

[0157] E32.9. The bio-orthogonal portion is [ka] A conjugate described in any of E1 to E31, or a pharmaceutically acceptable salt thereof, wherein (i.e., in formula (I), q is 0 and q1 is 0).

[0158] A pharmaceutical composition comprising a conjugate described in any of E33.E1 to E32.9, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0159] E34. A method for treating cancer or enhancing or inducing an immune response, wherein the method involves administering a therapeutically effective dose of any of the condyloma described in E1 to E32.9 to a subject in need. The treatment comprises administering a cegate, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in E33, and a therapeutic support composition, wherein the therapeutic support composition comprises a biocompatible support and a formula [ka] (In the formula, R 20 These are hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', C(= 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''; Each instance of R' and R'' is independently selected from hydrogen, aryl, and alkyl; Each instance of R''' is independently selected from aryl and alkyl groups; R 30These are halogens, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl; R a , R 31a and R 31b Each of these is independently hydrogen, a C1-C6 alkyl group, or a C1-C6 haloalkyl group; (t is 0, 1, 2, 3, or 4) A method comprising a tetrazine-containing group.

[0160] E35. The method according to E34, wherein the tetrazine-containing group is linked to or directly bound to the hyaluronic acid biocompatible support.

[0161] E36. Therapeutic support composition is formula (II) [ka] (In the formula, G 2 teeth, [ka] and; R 22 (It is a linker of 1 to 100 linked atoms.) The method according to E34 or E35, comprising a substituted hyaluronic acid unit.

[0162] E37.G 2 teeth, [ka] The method described in E36.

[0163] E38.G 2 teeth, [ka] and; R 20 is hydrogen or C 1~4The method described in E37, which is alkyl.

[0164] E39. A method which is a method of treating cancer, as described in any of E34-E38.

[0165] E39.1. The method described in E39 for cancer that is metastatic.

[0166] E40. Cancers include melanoma, kidney cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, stomach cancer, squamous cell carcinoma of the head and neck, and anal cancer. The method according to E39 or E39.1, which is vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

[0167] E40.1. The method according to E39 or E39.1, wherein the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer.

[0168] E41. The method described in any of E39-E40.1, wherein the cancer is a solid tumor.

[0169] E42. The cancer is a soft tissue sarcoma, as described in any of the methods in E39-E40.1.

[0170] E43. The method according to E42, wherein the soft tissue sarcoma is fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0171] E44. The method according to any of E34-E38, wherein the method is a method for enhancing or inducing an immune response.

[0172] E45. The method according to E44, wherein the immune response is an increase in one or more leukocytes, lymphocytes, monocytes, and eosinophils.

[0173] E46. The method according to any one of E34 to E45, further comprising administering a therapeutically effective dose of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, or a trans-cyclooctenprodrug thereof. The anticancer agents, immunomodulators, and their trans-cyclooctenprodrugs are known in the art, as described below herein and incorporated by reference in E46.

[0174] A kit comprising a conjugate described in any of E47.E1 to E32.9, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in E33, and instructions for use thereof.

[0175] The kit according to E45, further comprising the therapeutic support composition described in any of E34 to E38.

[0176] E49. A method for delivering an effective amount of an immunomodulatory payload according to any one of E1 to E22.2 to a target site of a subject, the method comprising administering a therapeutic support composition according to any one of E34 to E38 to the subject at the target site, and administering a conjugate, or a pharmaceutically acceptable salt or composition thereof, according to any one of E1 to E33 to the subject.

[0177] 1. Therapeutic proteins The therapeutic proteins for use in the conjugates disclosed herein are generally intended to be large molecules composed of long chains of amino acids that fold into a three-dimensional shape. Generally, therapeutic proteins can be classified based on their pharmacological activity, and they can be divided into five groups: (a) replacing deficient or abnormal proteins; (b) enhancing existing pathways; (c) providing novel functions or activities; (d) interfering with molecules or organisms; and (e) delivering other compounds or proteins, such as radionuclides, cytotoxic drugs, or effector proteins. Therapeutic proteins can also be classified based on their molecular type, hereinafter, as exemplary types, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, and manipulated proteins. Examples include sites, enzymes, growth factors, hormones, interferons, interleukins, or thrombolytic agents. Therapeutic proteins can also be classified based on the molecular mechanism of their activity, such as (a) non-covalent bonding to a target (e.g., mAbs); (b) affecting covalent bonding (e.g., enzymes); or (c) exerting activity without specific interactions (e.g., serum albumin).

[0178] Examples of therapeutic proteins include cytokines, chemokines, growth factors, hormones, antibodies, and antigens. Further examples, but not limited to, include: erythropoietin (EPO, e.g., natural or synthetic EPO (see, e.g., U.S. Patent Application Publication No. 2003 / 0191291), e.g., but not limited to PROCRIT®, EPREX®, or EPOGEN® (epoetin-α), ARANESP® (darbepoetin-α), NEORECORMON®, EPOGIN® (epoetin-β), etc.); Growth hormone (e.g., somatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®, SEROSTIM®, HUMATROPE®, etc.); therapeutic monoclonal antibodies (e.g., atezolizumab, avelumab, bevacizumab, semiprimab, cetuximab, daratumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, ne Citumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, panitumumab, pembrolizumab, partuzumab, 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, Lantu (e.g., s, Ultrarente), insulin-like growth factors (e.g., IGF-I, IGF-II); blood factors (e.g., factor X, tissue plasminogen activator (TPA), etc., for example, but not limited to, ACTIVASE® (alteplase) tissue plasminogen activator, NOVOSEVEN® (recombinant human factor Vila), factor Vila, factor VIII (e.g., KOGENATE®), factor IX, β-globin, hemoglobin, etc.);Colony-stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g., NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), Neulasta (pegfilgrastim), granulocyte-monocyte colony-stimulating factor, megakaryocyte colony-stimulating factor, etc.), transforming growth factors (e.g., TGF-β, TGF-α); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®), IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IE-12, etc.); growth factors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF, e.g., REGRANEX® (becaprelmin)), 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., TNF-α-binding soluble receptor, e.g., ENBREL® (Etanercept), soluble VEGF receptor, soluble interleukin receptor, soluble γ / δ T cell receptors, etc.); enzymes (e.g., α-glucosidase, CERAZYME® (imiglucerase, β-glucocerebrosidase, CEREDASE® (alglucerase)); enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10, Mig, Groα / IL-8, regulatory normal T cell expression secretory protein (RANTES), MIP-1α, MIP-1ρ, MCP-1, PF-4, etc.); angiogenic agents (e.g., vascular endothelial growth factor (VEGF)); anti-angiogenic agents (e.g., soluble; VEGF receptors; protein vaccines; neuroactive peptides, e.g., bradykinin, cholecystokinin, gustin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, galanin, growth hormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptides, sleep peptides, etc.; other proteins, e.g., thrombolytic peptides. Examples include agents, atrial natriuretic peptide, bone morphogenetic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human α-1 antitrypsin, leukemia inhibitor, transforming growth factor, tissue factor, insulin-like growth factor, luteinizing hormone, follicle-stimulating hormone, macrophage activator, tumor necrosis factor, neutrophil chemotactic factor, nerve growth factor, metalloproteinase tissue inhibitors; vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; leukemia inhibitor; IL-1 receptor antagonists (e.g., Kineret® (Anakinra)). It will be readily understood that the natural forms of the above therapeutic proteins are also targeted as payloads in this disclosure.

[0179] 2. Cytokines Cytokines can restrict tumor cell proliferation either directly through antiproliferative or pro-apoptotic activity, or indirectly by stimulating the cytotoxic activity of immune cells against tumor cells. Cytokines may be classified as type 1 or type 2, as described in Lucey et al., Clin Microbiol Rev. 1996;9(4):532-62. In this nomenclature, type 1 responses are defined as strong cellular immune responses accompanied by normal or elevated levels of IL-2, IFN-γ, TNF-β, and / or IL-12, while type 2 responses are defined as impaired cellular responses accompanied by increased activity of one or more B cells (e.g., hypergammaglobulinemia, autoantibody production, or high IgE) and elevated levels of IL-4, IL-5, IL-6, IL-10, and / or IL-13. Generally, type 1 cytokines have been shown to mediate antitumor responses. Cytokines that may be used as immunomodulators include, but are not limited to, IFN-α, IFN-β, and IFN-γ, interleukins (e.g., IL-1 to IL-40, in particular IL-7, IL-12, IL-15, IL-18, and IL-21), tumor necrosis factors (e.g., TNF-α and TNF-β), erythropoietin (EPO), MIP3a, ICAM, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (GCSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), as described in U.S. Patent Application Publication No. 2008 / 0014222.In embodiments of the present invention, the cytokines are IL-2, IL-2 covalently bound to immunoglobulin (e.g., sergutuzumabu amnaleukin, RO6874281) or PEG molecules (e.g., NKTR-214), IL-10, pegylated IL-10 (e.g., pegyrodecakin), IL-12, IL-15, recombinant aglycosylated IL-15, a fusion protein of IL-15 with a binding domain for IL-15Rα (e.g., RLI), a triple fusion protein containing human IL-15, a binding domain for IL-15Rα and apolipoprotein AI, ALT-803 (IL-15 fused to the IgG1 Fc domain), IL-21, GM-CSF, tarimodine laherpalepbec, IFN-α, pegylated IFN-α, and an apolipoprotein AI fusion protein with IFN-α.

[0180] In addition, inhibitors of specific cytokines, their alloreceptor agonists and / or antagonists may be used as cancer therapies. Cytokines are secreted or membrane-bound proteins that act as mediators of intracellular signaling to regulate the homeostasis of the immune system. They are produced by cells of the innate and adaptive immune systems in response to microorganisms, autoantigens, and tumor antigens. Inhibitors of TNF-α (e.g., infliximab, certolizumab) In particular, with regard to PD-1 pathway blockade, TGF-β (e.g., garnicertib, fresolimmab, M7824) and CSF-1 (e.g., pexidartinib, kabilizumab) can be used in the method of the present invention.

[0181] Immunotherapy using cytokines and cytokine inhibitors was developed by Berraondo et al. This is described in al., British Journal of Cancer (2019) 120, 6-15 (incorporated herein by reference).

[0182] 3. Chemokines Chemokines and / or chemokine receptor inhibitors may be used as immunomodulatory agents; these are chemotactic proteins that have the potential to attract macrophages, T cells, eosinophils, basophils, and other cells to sites of inflammation, infection, and / or tumor growth. These proteins are typically low molecular weight (7-9 kD). Chemokines form four main structural subclasses (C, CC, CXC, and CX3C) classified by their primary amino acid structure, which contain various combinations of conserved cysteine ​​residues.

[0183] Suitable immunomodulatory chemokines include CCL27 and CCL28, CC (CCL2, CCL3, CCL5), and CXC (CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXL12).

[0184] ELRCXC chemokines, including IL-8, GROα, GROβ, GROγ, NAP-2, and ENA-78 (Strieter, 1995, J Biol Chem, 270:27348-57), are also thought to be involved in inducing tumor angiogenesis (new blood vessel growth). This neovascularization is attributed to the binding of ELRCXC chemokines to CXCR2, and in the case of IL-8, probably CXCR1, expressed on the surface of vascular endothelial cells (ECs) in surrounding blood vessels, as well as their activation. Many different types of tumors have been shown to produce ELRCXC chemokines. Chemokine production is correlated with a more aggressive phenotype (Inoue, 2000, Clin Cancer Res, 6:2104-2119) and a poor prognosis (Yoneda, 1998, J Nat Cancer Inst, 90:447-54). Chemokines are potent chemotactic agents, and ELRCXC chemokines, in particular, have been shown to induce EC chemotaxis. Therefore, these chemokines are thought to induce endothelial cell chemotaxis towards their production sites within tumors. This may be a crucial step in the induction of angiogenesis by tumors. CXCR2 inhibitors or dual inhibitors of CXCR2 and CXCR1 would inhibit the angiogenesis of ELRCXC chemokines and thus inhibit tumor growth. This antitumor activity is similar to that of IL-8 (Arenberg, 1996, J Clin Invest, 97:2792-802), ENA-78 (Arenberg, 1998, J Clin Invest, 102:465-72), and GROα (Haghnegandar, 2000, J Leukoc). Antibodies against (Biology, 67:53-62) have been demonstrated. CXC chemokine inhibitors are described in U.S. Patent No. 10,046,002 (incorporated herein by reference) as follows: [ka] Includes.

[0185] 4. Chemokine Antagonists Suitable immunomodulatory agents for use in the method of the present invention include chemokines or chemokine receptor antagonists that inhibit the recruitment of suppressive immune cells into the tumor microenvironment. For example, but not limited to, the method of the present invention may use CCR1, CCR2, or CCR5 antagonists that reduce the infiltration of myeloid suppressor cells and regulatory T cells.

[0186] Suitable CCR, CXCR, and CCL inhibitors are described in Poeta et al., Frontiers in Immunology (2019), Article 379, doi:10.3389 / fimmu.2019.00379; Yu et al., Cancer Biol.Ther. (2008) 7:1037-43; and Chi et al., Int.J Clin Exp Pathol. (2015) 8:12533-40, including CCR1 (e.g., CCX721, BL5923), CCR2 (e.g., CCX9588, PF-04136309, CCX872, RDC018, 747, iCCR2), CCL2 (e.g., CNTO 888), and CCR4 (e.g., Affi Examples of inhibitors include those for 5, AF399 / 420 / 1802), CCR5 (e.g., Maraviroc), CCR7 (e.g., siRNA, MSM R707), CXCR2 (e.g., Navarixin, SB225002, Reparixin, SB265610, AZD5069), and CXCR4 (e.g., AMD3100, AMD3465, LY2510924, BKT140, BMS-936564, PF-06747143, PRX177561, POL5551, USL311, CTCE-9908).

[0187] 5. Immune checkpoint inhibitors As an immune checkpoint inhibitor, it is not limited to PD-1 inhibitors (e.g., Nivolt). Examples include mab (pembrolizumab, pidilizumab, cintilimab, AMP-224), PD-L1 inhibitors (e.g., atezolizumab, avelumab, durvalumab, BMS-936559), CTLA4 inhibitors (e.g., ipilimumab, tremelimumab), IDO inhibitors (e.g., indoximod, epacadostat), TIGIT inhibitors (e.g., LAG-3, e.g., anti-LAG-3 antibody as described in U.S. Patent Application Publication No. 2015 / 0259420, e.g., incorporated herein by reference; TIM-3, e.g., anti-TIM-3 antibody as described in U.S. Patent Application Publication No. 2015 / 0218274, e.g., incorporated herein by reference), and BTLA pathway antagonists.

[0188] 6. Cytokine inhibitors Cytokine inhibitors are a heterogeneous group of drugs that either 1) reduce cytokine synthesis; 2) decrease their concentration in their free active form; 3) block their interaction with specific receptors; or 4) interfere with cytokine receptor signaling. Examples of cytokine inhibitors include antagonists, soluble receptors, cytokine-binding proteins, and cytokines that block other cytokines. These drugs can range from small molecules and peptides to larger proteins such as mAbs, but are not limited to these.

[0189] In some embodiments, the immune response is regulated using xenobiotic agents, bioagents, naturally or artificially obtained adjuvants, cell therapies, and / or checkpoint inhibitors, including but not limited to PD-1, PD-L1, CTLA-4, B7 molecules, TIGIT, Tim-3 and / or Lag-3, and indoleamine 2,3-dioxygenase (IDO) inhibitors.

[0190] Additional therapeutic agents may include immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include PD-1 inhibitors (e.g., nivolumab, pidilizumab, cintilimab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, durvalumab, BMS-936559), CTLA4 inhibitors (e.g., ipilimumab, tremelimumab), or IDO inhibitors (e.g., indoximod, epacadostat).

[0191] C. Therapeutic Support Composition A therapeutic support composition includes a support. The support may be a biocompatible support composition, i.e., compatible with the subject's body. In some cases, the support is non-toxic to the subject and does not substantially react with the subject's tissues or biocompounds. For example, the support may be a hydrogel. The support can be implanted in the subject's body and can support a binder (e.g., a tetrazine-containing group) and a payload after binder conjugation. Typical supports, but not limited to, include polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, crosslinked polymer matrices, metals, ceramics, plastics, bone graft materials, alginates, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, and heparin. Supports may also include particles such as nanoparticles and microparticles.

[0192] The hydrogel may be a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, chondroitin sulfate, heparin, etc. Other suitable sugar-based biomaterials are those described in Polymer Advanced Technology, 2014, 25, 448-460. Polymers that can be used as supports include, but are not limited to, polyphosphazenes, polyanhydrides, polyacetals, poly(orthoesters), polyphosphoesters, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, as well as blends / composites / copolymers thereof. Typical polyethers include, but are not limited to, poly(ethylene glycol)(PEG ), polypropylene glycol (PPG), triblock pluronic (Pluronic [PEG] n -[PPG] m Examples include -[PEG]n), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA). Supports may also include proteins and other poly(amino acids), such as collagen, gelatin, elastin and elastin-like polypeptides, albumin, fibrin, poly(γ-glutamic acid), (L-lysine), poly(L-glutamic acid), and poly(aspartic acid).

[0193] In some embodiments, the support is a hydrogel. In some embodiments, the support is an alginate. In some embodiments, the support is chitin. In some embodiments, the support is hyaluronic acid (e.g., substantially non-crosslinked non-hydrogel hyaluronic acid). In some embodiments, the support is chitosine.

[0194] In certain embodiments, the support is a particle. The particles of this disclosure may have a diameter of 2 cm or less, for example, 1.5 cm or less, or 1 cm or less, or 0.5 cm or less. For example, the particles may be nanoparticles or microparticles. Nanoparticles include particles having an average size on the nanometer scale (e.g., 1000 nm or less). Microparticles are particles having an average size on the micrometer scale (e.g., 1000 μm or less). "Average" means the arithmetic mean. In some embodiments, the nanoparticles have a diameter in the range of 1 nm to 1 μm, for example, 10 nm to 1 μm, or 25 nm to 1 μm, or 50 nm to 1 μm, or 75 nm to 1 μm, or 100 nm to 1 μm, or 150 nm to 1 μm, or 200 nm to 1 μm, or 250 nm to 1 μm, or 300 nm to 1 μm, or 350 nm to 1 μm, or 400 nm to 1 μm, or 450 nm to 1 μm, or 500 nm to 1 μm. In other embodiments, the microparticles have diameters in the range of 1 μm to 1 mm, for example, 10 μm to 1 mm, or 25 μm to 1 mm, or 50 μm to 1 mm, or 75 μm to 1 mm, or 100 μm to 1 mm, or 150 μm to 1 mm, or 200 μm to 1 mm, or 250 μm to 1 mm, or 300 μm to 1 mm, or 350 μm to 1 mm, or 400 μm to 1 mm, or 450 μm to 1 mm, or 500 μm to 1 mm. In another embodiment, small particles with a diameter of about 10 to 100 nm can be aggregated to form larger composites (e.g., clusters or aggregates of about 1 to 10 μm). The particles of this disclosure may be substantially spherical, and as a result, these particles have a substantially circular cross-section. Other particle shapes may be used, for example, ellipsoids, cubes, cylinders, cones, needles, or other irregular shapes, though not limited to these.

[0195] "Particles" can take the form of any manufactured material, molecule, cryptophan, virus, phage, etc. Particles may be composed of materials such as metals, ceramics, plastics, glass, composites, polymers, and hydrogels, but are not limited to these. For example, particles can be made from inert materials such as alginates or iron oxides. In some examples, particles can be magnetic and may be formed from paramagnetic, superparamagnetic, or ferromagnetic materials, or other materials that respond to magnetic fields. Furthermore, particles may be of any shape, such as spheres, rods, or asymmetric shapes. Particles, or a group of particles in a complex, may be functionalized with receptors that have a specific affinity for or bind to clinically relevant substrates. The receptors may be inherent to the particles themselves. For example, the particles themselves may be viruses or phages that have a specific affinity for a particular substrate. In addition, or alternatively, particles can also be functionalized by covalently binding to, attaching to, or associating with receptors that specifically bind to, or otherwise recognize, a clinically relevant substrate. The functionalized receptor may be an antibody, peptide, nucleic acid, phage, bacterium, virus, or any other molecule having a specified affinity for the target substrate. Examples of materials that can be used for the "particles" and / or "support" include polylactic acid, polyglycolic acid, PLGA polymer, alginate and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin-rich gel, and Examples include galose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphatidines, poly(vinyl alcohol), poly(alkylene oxide), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pluronic polyols, polyoxomers, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of any of these. These examples do not limit their concentrations, crosslinking with other agents, methods of administration, design degradation profiles, and other features well known to those skilled in the art.

[0196] Particles, or a group of particles in a complex, may be functionalized with a targeting agent (e.g., ligand or antibody) that specifically (or substantially specifically) binds to a target (e.g., a target receptor or cell surface target, e.g., a clinically relevant receptor or cell surface target (e.g., an antigen)). The targeting agent may be directly attached to the particles themselves. The targeting agent may be an antibody, peptide, nucleic acid, phage, bacterium, virus, or any other molecule having specific affinity for the target receptor or cell surface target. In some cases, the receptor or cell surface target is PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, BCR-ABL, SRC, JAK2, MAP2K, EML4-ALK, BRAF V600E, 4-1BB, GITR, GSK3β, or other cell receptors or cell surface targets. These particles may be attached with other compounds or molecules (e.g., fluorescent dyes, autofluorescent markers, or luminescent markers) that may be useful for detecting the particles (e.g., in vivo detection). Ligands and / or detectable labels may be directly attached to these particles, or they may be attached via bioorthogonal functional groups as described herein.

[0197] In certain embodiments, the support is a bone graft material, such as a bone graft substitute. The bone graft substitute is a material whose structure is similar to bone. In some cases, the bone graft substitute is bioreabsorbable, thereby dissolving or being absorbed in the body over time. The bone graft substitute may also be osteoconductive, thereby promoting the formation of blood vessels and new bone into the bone graft substitute. In some cases, the bone graft substitute is osteoinducible, thereby promoting the formation of new bone through the active recruitment of mesenchymal stem cells from surrounding tissue. For example, the bone graft substitute may contain growth factors such as osteomorphic proteins. Examples of bone graft substitutes, but not limited to, include hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone fragments, and combinations thereof.

[0198] The therapeutic support compositions of the present disclosure comprise a support and a first binder covalently bonded to the support. The binder may be attached to the support on the surface of the support, for example, on the solvent-contactable surface of the support (e.g., the surface of the support in contact with the surrounding solvent). In some cases, the binder is attached directly to the support. For example, the binder can be covalently bonded to the surface of the support via covalent bonds such as amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. In some cases, the binder is covalently bonded to the support via amide bonds. In other cases, the binder may be linked to the support via a linker. Any suitable linker can be used to link the binder to the support. Typical linkers may have 1 to 100 linking atoms, such as ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, a linker may have 1 to 50 linking atoms, or 5 to 50 linking atoms, or 10 to 50 linking atoms. Representative linkers, though not limited to them, include the following: [ka]

[0199] In a particular embodiment, the therapeutic support composition includes a support and the formula: [ka] (In the formula, R 20These include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'"", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R"", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R"", C(=S)NR'R Selected from the group consisting of '', 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'', where R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl each time it appears; R 30 These are halogens, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl; R a , R 31a and R 31b (Each element is independently hydrogen, a C1-C6 alkyl group, or a C1-C6 haloalkyl group; t is 0, 1, 2, 3, or 4) It contains a tetrazine-containing group.

[0200] In a particular embodiment, the therapeutic support composition is: [ka] (In the formula, R 20These include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'"", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R"", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R"", C(=S)NR'R Selected from the group consisting of '', 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'', where R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R''' is independently selected from aryl and alkyl each time it appears; R 22 (This refers to a linker consisting of 1 to 100 linked atoms, which may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups.) It has, for example, 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0201] In a particular embodiment, the therapeutic support composition is: [ka] (In the formula, R 20This includes hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S( =O)R', S(=O)2R"', S(=O)2NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R'', NR'R”, NR'C(=O)R”, NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR”, N Selected from the group consisting of R'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R''; R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; R'''' is independently selected from aryl and alkyl each time it appears; R 30 These are halogens, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl; R a , R 31a and R 31b (Each element is independently hydrogen, a C1-C6 alkyl group, or a C1-C6 haloalkyl group; t is 0, 1, 2, 3, or 4) It holds.

[0202] In a particular embodiment, the therapeutic support composition is: [ka] (In the formula, R 20These include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'"", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R"", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R"", C(=S)NR'R Selected from the group consisting of '', 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'', where R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; where R'' is independently selected from aryl and alkyl each time it appears. Substituted alginates having units of or their salts Includes.

[0203] In a particular embodiment, the therapeutic support composition is: [ka] Includes units of.

[0204] In some embodiments, the therapeutic support composition, formula: [ka] Includes units of.

[0205] In some embodiments, the therapeutic support composition, formula: [ka] Includes units of.

[0206] In some embodiments, the therapeutic support composition, formula (II): [ka] (In the formula, G 2 teeth, [ka] And; R 22 R is a linker of 1 to 100 linked atoms; 20 (As defined herein) Contains substituted hyaluronic acid having units of .

[0207] In another embodiment, G 2 teeth, [ka] That is the case.

[0208] In another embodiment, G 2 teeth, [ka] And; R 20 is hydrogen or C 1~4 It is alkyl.

[0209] The compound of formula (II) is formula (II-A): [ka] (In the formula, R 20These include hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'"", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R"", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R"", C(=S)NR'R Selected from the group consisting of '', 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'', where R' and R'' are independently selected from hydrogen, aryl, and alkyl each time they appear; where R'' is independently selected from aryl and alkyl each time it appears. It contains the compound of (II-A). In another embodiment according to formula (II-A), R 20 is hydrogen or C 1~4 It is alkyl.

[0210] In some embodiments, the therapeutic support composition, [ka] Includes units of.

[0211] Other therapeutic support compositions are illustrated in International Publication No. 2017 / 044983, International Publication No. 2015 / 139025A1, and International Publication No. 2014 / 205126A1, the entire contents of each of these are incorporated herein by reference.

[0212] Hyaluronic acid derivatives include hyaluronic acid having multiple glucuronic acid units, and tetrazine-containing groups linked or directly bonded to the glucuronic acid units of hyaluronic acid. Hyaluronic acid may also have multiple N-acetylglucosamine units. In certain embodiments, the N-acetylglucosamine units of hyaluronic acid are not linked or conjugated to the tetrazine-containing groups.

[0213] The tetrazine-containing group can be linked or directly bonded via a carboxylic acid of a glucuronic acid unit. The tetrazine-containing group can be incorporated into hyaluronic acid at concentrations of approximately 0.1% to approximately 80%, for example, approximately 1% to approximately 75%, approximately 5% to approximately 75%, approximately 10% to approximately 50%, or approximately 40% to approximately 75%, depending on the percentage of carboxylic acid linked or conjugated to the tetrazine-containing group.

[0214] D. Treatment Method Aspects of this disclosure include methods for delivering a payload to a target site of a subject. In this context, the method includes selectively delivering a payload to a target site of a subject. Selective delivery of the payload includes delivering the payload to a target site (e.g., an organ or tissue or part thereof) without targeting other parts of the subject (e.g., other organs or tissues or parts thereof) that do not require the administration of the payload. Selective delivery of the payload can be achieved using the support compositions and functionalized payloads described herein.

[0215] In some cases, the support compositions of this disclosure can be localized to a desired target site in a subject. For example, the method of this disclosure may include administering the support composition described herein to a subject. The support composition may be administered to the subject at a desired target site in the subject. In some cases, the support composition may be implanted in the subject at a desired target site in the subject. In some embodiments, the support composition may be conjugated with a targeting agent described herein, and the method may include administering the support composition to a subject (e.g., systemically). In these embodiments, the support composition conjugated with the targeting agent may be localized to a desired target site in the subject by specific binding of the targeting agent to its target (e.g., antibody-antigen interaction), or may be localized on the surface of a desired target (e.g., a cell surface) by specific binding of the targeting agent to its target (e.g., antibody-antigen interaction).

[0216] As described herein, selective binding can occur between bioorthogonal binding partners (for example, between the tetrazine binder of the support composition and the complementary trans-cyclooctene binder of the functionalized payload). As previously stated, localized administration of the support composition to a desired site in a subject allows the selective binding between the binder of the support composition and the complementary binder of the functionalized payload to localize the payload to the desired target site. Therefore, in certain embodiments, the method includes administering the functionalized payload to a subject such that the functionalized payload binds to the support composition to form a support complex. For example, the functionalized payload may be administered systemically to a subject. Upon administration of the functionalized payload to a subject, contact occurs between the binder of the support composition and the complementary binder of the functionalized payload, causing the binder and its complementary binder to bind to each other to form a support complex, thereby allowing the payload to be selectively delivered to the target site in the subject. In some embodiments, selective delivery of the functionalized payload achieves a higher concentration of the payload at the target site than at other parts of the subject (e.g., non-target sites in the subject).

[0217] The indications for this approach include cancers of both the hematological and solid systems. 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 subtypes of soft tissue sarcomas and osteosarcomas. This composition can be used for the treatment and / or diagnosis of pigmented villonodular synovitis.

[0218] The compositions of this disclosure are useful for the treatment and / or diagnosis of medical conditions or diseases of subjects suitable for treatment or diagnosis by administration of a payload (e.g., a parent drug (i.e., a drug before conjugation with the composition)). “Treatment” means that at least improvement of the symptoms associated with the medical condition the subject suffers from is achieved, in which improvement is used broadly and means at least a reduction in the degree of a parameter (e.g., symptoms) associated with the medical condition being treated. Accordingly, treatment also includes the complete inhibition of the pathological condition, or at least the symptoms relating thereto, e.g., prevention or cessation of occurrence, e.g., termination, where the subject is no longer afflicted with the medical condition, or at least the symptoms characterizing the medical condition. Treatment may include inhibition, i.e., cessation of the occurrence or further occurrence of clinical symptoms, e.g., alleviation or complete inhibition of active disease. Treatment may include reduction, i.e., regression of clinical symptoms. For example, in relation to cancer, the term “treatment” includes: reduction of solid tumor growth, inhibition of cancer cell replication, and reduction of the overall tumor burden. This includes, but is not limited to, an increase in survival time, or any or all of the following: improvement of one or more cancer-related symptoms.

[0219] Subjects receiving treatment are those who require treatment and are suitable for treatment with the parent drug. Therefore, a variety of subjects are considered suitable for treatment with the compositions disclosed herein. Generally, such subjects are "mammals," and humans are included. Other subjects include pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., as animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

[0220] In certain embodiments, the functionalized payload, therapeutic support composition, additional therapeutic agents, and methods can be used for the treatment, prevention, and / or diagnosis of solid tumors, including, but not limited to, melanoma (e.g., unresectable, metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer such as metastatic epithelial ovarian cancer), endometrial cancer, breast cancer (e.g., Examples of cancers to be treated include triple-negative breast cancer, glioblastoma (e.g., glioblastoma multiforme), 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 cancer, pancreatic neuroendocrine tumors, prostate adenocarcinoma, nasopharyngeal cancer, and cutaneous T-cell lymphoma. The disclosed approach is suitable as an adjuvant / neoadjuvant system. For example, particles disclosed herein can be placed during a biopsy, and once the test results are returned, the physician can deliver the appropriate cocktail to the desired site in the body. This can minimize tumor size, particularly with respect to surgically resectable tumors. Next, at the end of the surgery, the surgeon can minimize the risk of any cancer cells that may have been missed at the resection margin by placing more particles around the resection cavity and treating the patient with further therapeutic agents (e.g., chemotherapy drugs of the disclosed approach).

[0221] In certain embodiments, the disclosed method allows for the placement of particles disclosed herein at the time of biopsy. Upon return of the results, the physician can 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 immunotherapy agents, via the biopsy site to enhance the immune system with fewer side effects. This combination approach may be beneficial to the patient. While the chemotherapeutic agent treats the solid tumor or specific site, the enhancement of the immunotherapy response may benefit distal metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can be used with or in combination with anthracyclines, taxanes, gemcitabine, and other agents to enhance the effects of one or more immunomodulatory agents such as ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer).

[0222] 1. Cancer The disclosed methods can be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that may include persistent growth signaling, evasion of growth inhibitors, resistance to cell death, achievement of replication immortality, induction of angiogenesis, and activation of invasion and metastasis. The disclosed methods can enhance or induce an immune response against cancer in a subject. The immune response may result in an increase in one or more of the following: leukocytes, lymphocytes, monocytes, and eosinophils.

[0223] Cancers that can be treated by the disclosed methods include, but are not limited to, astrocytoma, adrenocortical carcinoma, appendiceal carcinoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, brainstem cancer, brainstem glioma, Breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse pontine glioma, ductal carcinoma, endometrial cancer, ependymoma, Ewing's sarcoma, esophageal cancer, eye cancer, fibrosarcoma, bladder cancer, gastric cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer Examples of cancers include 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 tumors, squamous cell carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma, thyroid cancer, gestational trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0224] In some embodiments, cancers that can be treated by the disclosed method 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 pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0225] While not bound by any particular theory, local release of certain anticancer drugs using the compounds and methods of the present invention may induce or contribute to immunogenic cell death (ICD). For example, certain anticancer drugs (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 activating a specific T cell response. ICD is characterized by the secretion of damage-associated molecular patterns (DAMPs). During ICD, three key DAMPs are exposed on the cell surface. Calreticulin (CRT), one of the DAMP molecules, is normally located within the lumen of the endoplasmic reticulum (ER), but translocates to the surface of dead cells after induction of 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 exert immunostimulatory effects in response to interactions with several antigen-presenting cell (APC) surface receptors such as CD91 and CD40, and also promote cross-presentation of antigens derived from tumor cells on MHC class I molecules, which subsequently triggers a CD8+ T cell response. Other important DAMPs specific to ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is thought to be a late apoptosis marker, and its release into the extracellular space appears to be necessary for the optimal release and presentation of tumor antigens to dendritic cells. This binds to several pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) 2 and 4, which are expressed on APCs. The most recently identified DAMP released during immunogenic cell death is ATP, which, upon secretion, functions as a "find-me" signal to monocytes, inducing their attraction to apoptotic sites. Kroemer et al. Curr. Op. Immunol. 2008(20), 504-511.

[0226] Therefore, local release of ICD-inducing factors using the compounds and methods of the present invention may be beneficial when combined with one or more immunomodulators.

[0227] In certain embodiments, the functionalized payload, therapeutic support composition, and method can be used for the treatment, prevention, and / or diagnosis of solid tumors, including, but not limited to, melanoma (e.g., unresectable, metastatic melanoma), renal cancer (e.g., renal cell carcinoma), and other solid tumors. Examples include cancer, 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.

[0228] The disclosed approach is suitable as an adjuvant / neoadjuvant system. For example, during a biopsy, the therapeutic support composition disclosed herein can be placed, and once the test results are returned, the physician can administer an appropriate cocktail to deliver the therapeutic agent to the desired site in the body (a compound of formula (I) and, optionally, another therapeutic agent). The biopsy results may indicate the amount and type of therapeutic agent to be delivered to the site of the tumor. For example, chemokines (agents that attract cancer cells and / or immune cells) and adjuvants that enhance the immune system with fewer side effects, as well as chemotherapy agents, can be delivered and can be combined with immunotherapy agents.

[0229] The disclosed compounds and compositions may be administered before surgical resection. The disclosed methods may minimize tumor size before surgical resection. This may minimize tumor size, particularly with respect to surgically resectable tumors. The disclosed conjugates, compounds and compositions may be administered during surgical resection. The disclosed conjugates, compounds and compositions may be administered after surgical resection. At the end of surgical resection, the therapeutic support composition may be placed around the resection cavity, and the subject may subsequently be treated with an additional therapeutic agent to minimize the risk of any cancer cells that may have been missed at the resection margin.

[0230] The disclosed method may involve systemic administration of multiple doses of a functionalized payload concentrated at a single site. The second payload may be delivered using the disclosed method. If the tumor is resistant to the first payload, the second functionalized payload may be administered using the disclosed method. The second payload may be a TCO-labeled payload of gemcitabine or docetaxel. The TCO-labeled payload of gemcitabine or docetaxel may be administered in combination with doxorubicin. The second functionalized payload may be activated by a therapeutic support composition used for the first prodrug.

[0231] The functionalized payloads disclosed herein may function as adjuvants. This combination approach may be beneficial to patients. Chemotherapy agents may treat solid tumors or specific sites and enhance or induce immune responses, and enhancement of the immunotherapy response by the functionalized payload and / or other agents may be beneficial for distal metastatic sites. For example, in certain embodiments, the disclosed compositions and methods may be used with or in combination with anthracyclines, taxanes, gemcitabine, and other agents to enhance the effects of ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer).

[0232] The disclosed method may be used to treat diffuse pontine glioma. Diffuse pontine glioma (DIPG) is a childhood brainstem tumor that can be highly malignant and difficult to treat. There is no known treatment for DIPG, and the survival rate has remained dire 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 childhood brainstem tumors, with an estimated 200–300 children affected each year in the United States. Due to the rarity of this tragic disease and the lack of past experimental model systems, research has been hindered, and the survival rate has remained the same for the past 40 years. Diagnosis of DIPG begins with clinical symptoms and may be confirmed by MRI. The disease may begin with generalized symptoms over several months, such as altered and difficult behavior at school, double vision, abnormal or limited eye movements, asymmetrical smile, balance problems, and frailty. Alternatively, severe neurological dysfunction may occur rapidly, The symptoms are present for less than one month prior to diagnosis. Clinical examination may reveal three sets of symptoms: multiple cranial neuropathy, long-cordal symptoms such as hyperreflexia and clonus, and ataxia. Elongation of the pontine segment of the brainstem can lead to obstructive hydrocephalus and increased intracranial pressure.

[0233] The nerve nuclei that are crucial for life-sustaining functions such as respiration and heart rate are located in the pons, and if left untreated, respiration and heart rate can be damaged by DIPG.

[0234] The disclosed method may be used to deliver a molecular payload to the site of DIPG. The disclosed method may include a step of systemically administering a drug that is activated only at the tumor site. The disclosed method may be used as neoadjuvant or adjuvant therapy. Biomaterial may be placed during biopsy. The results of the biopsy may indicate the amount and type of therapeutic agent to be delivered to the site of the tumor. The disclosed compounds and compositions may be administered before surgical resection. The disclosed method may minimize the size of the tumor before surgical resection. The disclosed compounds and compositions may be administered during surgical resection. The disclosed compounds and compositions may be administered after surgical resection. At the end of surgical resection, biomaterial may be placed around the resection cavity, and the subject may subsequently be treated with further administration of another therapeutic agent. The disclosed biodegradable gel may be implanted during biopsy or surgery. The disclosed method does not require any additional invasive procedure to deliver additional doses of the disclosed compounds and compositions.

[0235] The disclosed method may involve systemic administration of multiple doses of a functionalized payload concentrated at a single site. The second payload may be delivered using the disclosed method. If the tumor is resistant to the first payload, the second functionalized payload may be administered using the disclosed method. The second payload may be a TCO-labeled payload of gemcitabine or docetaxel. The TCO-labeled payload of gemcitabine or docetaxel may be administered in combination with doxorubicin. The second functionalized payload may be activated by a therapeutic support composition used for the first prodrug.

[0236] 2. Method of administration The administration method may include any number of methods for administering the disclosed conjugate, compound, or composition. The administration method may include tablets, pills, sugar-coated tablets, hard and soft gel capsules, granules, pellets, skin patches, skin creams, skin gels, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixirs, solid emulsions, solid dispersions or dispersible powders. For the purpose of preparing pharmaceutical compositions for oral administration, the conjugates, compounds, or compositions disclosed herein may be mixed with adjuvants and excipients, such as gum arabic, talcum, starch, sugars (e.g., mannitose, methylcellulose, lactose), gelatin, surfactants, magnesium stearate, aqueous or non-aqueous solvents, paraffin derivatives, crosslinking agents, dispersants, emulsifiers, lubricants, preservatives, flavorings (e.g., ether oil), solubility enhancers (e.g., benzyl benzoate or benzyl alcohol), or bioability enhancers (e.g., Gelucire®). In pharmaceutical compositions, the conjugates, compounds, or compositions disclosed herein may be dispersed in microparticles, such as nanoparticle compositions.

[0237] For parenteral administration, the conjugates, compounds, or compositions disclosed herein may be dissolved or suspended in a physiologically acceptable diluent, such as water, a buffer, an oil 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 nano-suspensions.

[0238] As used herein, the term "parenteral" refers to methods of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0239] The therapeutic support composition is preferably administered locally to the tumor site by injection or transplantation. The functionalized payload, such as the conjugate or compound of formula (I) or (III), may be administered by any convenient route, taking into consideration the subject's condition and the judgment of the medical professional. Parenteral administration is a preferred means of administration of the conjugate or compound of formula (I) or (III).

[0240] The amount of composition administered to a subject can initially be determined based on the dose of the parent drug and / or the guidance of the drug regimen. Generally, the composition can enable targeted delivery of the conjugated drug and / or an increase in its serum half-life, and thus enable at least one of dose reduction or administration reduction in the drug regimen. Accordingly, the composition enables dose reduction and / or administration reduction in the drug regimen compared to the parent drug before conjugation in the composition of the present disclosure.

[0241] The compositions of this disclosure can be delivered by any preferred means, including oral, parenteral, and topical methods. For example, transdermal administration methods via topical routes can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, topical medications, powders, and aerosols.

[0242] Pharmaceutical preparations can be provided as unit dosage forms. In such dosage forms, the pharmaceutical preparation can be divided into unit doses containing an appropriate amount of the composition of this disclosure. A unit dosage form may be a packaged preparation containing individual amounts of the preparation, such as tablets, capsules, and powders packed in pouches, vials, or ampoules. Alternatively, a unit dosage form may be a capsule, tablet, sugar-coated tablet, cachet, or lozenge, or may contain an appropriate number of any of these in package form.

[0243] The compositions of this disclosure may be present in any preferred amount, which may vary depending on various factors, including, but not limited to, the subject's weight and age, disease status, etc. Preferred doses of 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 1000 mg.

[0244] In some embodiments, multiple doses of the composition are administered. The frequency of administration of the composition may vary depending on various factors, such as the severity of symptoms or the condition of the subject. For example, in some embodiments, the composition is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0245] The compositions of the Disclosure may be administered at any preferred frequency, interval, and duration. For example, the compositions of the Disclosure may be administered once every hour, or two or three times or more times every hour, once a day, or two or three times or more times a day, or for two, three, or four days, to achieve the subject's desired level of medication. It can be administered once every 1 day, 5 days, 6 days, or 7 days. When the composition of the present disclosure is administered more than once a day, typical intervals include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes, as well as 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, and 24 hours. The composition of the present disclosure can be administered once, twice, or three or more times over periods of 1 hour, 1 to 6 hours, 1 to 12 hours, 1 to 24 hours, 6 to 12 hours, 12 to 24 hours, 1 day, 1 to 7 days, 1 week, 1 to 4 weeks, 1 month, 1 to 12 months, more than 1 year, or indefinitely.

[0246] The compositions of this disclosure may be administered in combination with another active agent. Combination administration includes administering the compositions of this disclosure and the active agent at intervals of no more than 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours from each other. Combination administration also includes administering the compositions of this disclosure and the active agent simultaneously or nearly simultaneously (for example, at intervals of no more than about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes from each other), or consecutively in any order. In addition, the compositions of this disclosure and the active agents may each be administered once daily, two or three times daily, or more times, to achieve a desired daily dose level.

[0247] Concomitant administration may be accompanied by concomitant transplantation or concomitant injection.

[0248] In some embodiments, co-administration may involve co-formulation, for example, the preparation of a single pharmaceutical formulation containing both the composition of the Disclosure and the active agent. In other embodiments, the composition of the Disclosure and the active agent may be formulated separately and administered in combination to a subject.

[0249] The compositions and active agents of this disclosure may be present in formulations in any preferred 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 active agents of this disclosure may be present in any preferred weight ratio, for example, 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 doses and dose ratios of the compositions and active agents of this disclosure are also preferred in the formulations and methods described herein.

[0250] 3. Combination therapy In one embodiment, the present invention provides a method for treating cancer or enhancing or inducing an immune response, the method comprising administering to a subject in need a therapeutically effective amount of a conjugate of the present invention (e.g., formula (I)), or a pharmaceutically acceptable salt or composition thereof; a therapeutic support composition described herein; and an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, or a trans-cyclooctemprodrug thereof.

[0251] The present invention also provides pharmaceutical combinations comprising: a conjugate described herein, or a pharmaceutically acceptable salt or composition thereof, for use in the treatment or prevention of cancer, or for use in enhancing or inducing an immune response; a therapeutic support composition described herein; and an additional therapeutic agent selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctempordrugs thereof.

[0252] The present invention also provides the use of a pharmaceutical combination comprising: a conjugate described herein, or a pharmaceutically acceptable salt or composition thereof; a therapeutic support composition; and an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, or a trans-cyclooctemprodrug, for use in the treatment or prevention of cancer, or in enhancing or inducing an immune response.

[0253] In the methods and uses described herein, the components of a pharmaceutical combination may be administered / used simultaneously, individually, sequentially, and in any order, and the components may be administered individually or as a fixed combination. For example, slowing the progression of a disease or treating a disease according to the present invention may include the administration of a first active ingredient in free or pharmaceutically acceptable salt form and the administration of a second active ingredient in free or pharmaceutically acceptable salt form, simultaneously or sequentially in any order, in a combined therapeutically effective or effective amount, for example, in a daily dose equivalent to the amount described herein. The individual active ingredients of the combination may be administered individually or simultaneously in divided or single dosage forms at various points in time during the treatment period. The present invention should therefore be understood to encompass all such simultaneous or alternating regimens, and the term “administer” should be interpreted accordingly. Thus, when used herein, pharmaceutical combination defines either a fixed combination in one unit dosage form or in individual dosage forms for combination administration, in which case the combination administration may be administered simultaneously or independently at different points in time. As another example, the therapeutic support composition and conjugate may be administered simultaneously (e.g., by concomitant injection or concomitant transplantation), individually, or sequentially, followed by the administration of additional therapeutic agents selected from the group consisting of anticancer agents, immunomodulators, or their trans-cyclooctemprodrugs.

[0254] Methods and uses in the treatment of cancer include administering / localizing therapeutic support compositions to tumors. In the methods and uses disclosed herein, administration of conjugates, or pharmaceutically acceptable salts thereof, or compositions; therapeutic support compositions; and additional therapeutic agents may inhibit tumor growth.

[0255] Another therapeutic agent may be administered simultaneously with or sequentially to the disclosed conjugate and composition. Sequential administration includes administration before or after the disclosed conjugate and composition. Another therapeutic agent may be administered before the disclosed conjugate and composition. Another therapeutic agent may be administered after the disclosed conjugate and composition. Another therapeutic agent may be administered simultaneously with the disclosed conjugate and composition. In some embodiments, another therapeutic agent may be administered in the same composition as the disclosed conjugate. In other embodiments, there may be a time interval between the administration of the other therapeutic agent and the administration of the disclosed conjugate or composition. In some embodiments, the administration of the other therapeutic agent and the disclosed conjugate or composition may allow for lower doses of the other therapeutic agent and / or administration at less frequent intervals. When used in combination with one or more other active ingredients, the conjugate or composition of the present invention and the other active ingredients may be used in lower doses than when each is used alone. Accordingly, the pharmaceutical compositions of the present invention include those containing one or more active ingredients in addition to the conjugate of the present disclosure.

[0256] a. Anticancer drugs Examples of anticancer agents include, but are not limited to, 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), Adolucil (fluorouracil), afatinib dimaleate, Afinitor (everolimus), Aldara (imiquimod), and Aldes. Leukin, Alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), Ambochlorin (chlorambucil), Ambochlorin (chlorambucil), Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), Arsenic trioxide, Alzera (ofatumumab), Asparaginase Erwinia c hrysanthemi), Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexar (tositumomab and I131 iodine tositumomab), bicalutamide, bleomycin, bortezomib, bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, busulfex (busulfan), cabazitaxel, cabozantinib-S-malate, CAF, Campus (aremutuzumab), camptosar Calcium (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomib, Casodex (bicalutamide), CeeNU (lomustine), Seruvidine (daunorubicin hydrochloride), Celvarix (recombinant HPV bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, Kome Trik (cabozantinib-S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, cytarabine, liposome, Citosal-U (cytarabine), cytoxane (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin difutitox, denosumab, DepoCyt (li Liposome cytarabine, DepoFoam (liposome cytarabine), dexrazoxane hydrochloride, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Efdex (fluorouracil), Elitec (rasburicase), Elence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate,Elvege (bismodegib), erlotinib hydrochloride, erwinase (asparaginase, erwinia, chrysanthemis), etopophos (etoposide phosphate), etoposide, etoposide phosphate, evaset (doxorubicin hydrochloride liposome), everolimus, evista (raloxifene hydrochloride), exemestane, fareston (toremifene), faslodex (fulvestrant), FEC, femara (letrozole), filgrastim, fludarabine phosphate, fludarabine phosphate, fluoroplex (fluorouracil), Fluorouracil, Forex (methotrexate), Forex PFS (methotrexate), Forfiri, Forfiri-bevacizumab, Forfiri-cetuximab, Forfirinox, Forfox (leucovorin, fluorouracil, oxaliplatin), Forotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV tetravalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemt Zumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Glucarpidase, Goserelin acetate, Halaben (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine (recombinant), HPV quadrivalent vaccine (recombinant), Hycamtin (topotecan hydrochloride), HyperCVAD, Ibritumomab tiuxetan, Ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosf Ifosfamide, Ifosfamide, Ifosfamide (Ifosfamide), Imatinib mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A (Recombinant Interferon α-2b), Iodine-131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepyrone, Ixempra (Ixabepyrone), Jakafi (Ruxolitinibrate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine),Keoxifen (raloxifene hydrochloride), Kepivans (palifermin), Cyprolis (carfilzomib), lapatinib nitosylate, lenalidomide, letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Leblanc (aminolevulinic acid), lymphoridine (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), liposomal cytarabine, lomustine, Leupron (leuprolide acetate), Leupron Depot (leuprolide acetate), Leupron Depot-P ed (leuprolide acetate), Leupron Depot-3 months (leuprolide acetate), Leupron Depot-4 months (leuprolide acetate), Marquivo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechloretamine hydrochloride, Megace (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, Mesna, Mesnex (mesna), Metazolaston (temozolomide), methotrexate, methotrexate LPF (methotrexate), Me Xate (methotrexate), Mexate-AQ (methotrexate), Mitomycin C, Mitoditrex (mitomycin C), MOPP, Mozovir (plelixafor), Mustargen (mechloretamine hydrochloride), Mutamycin (mitomycin C), Mirelan (busulfan), Mirosal (azacitidine), Mirotalg (gemtuzumab ozogamicin), Nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle preparation), Navelbine (vinorelbine tartrate), Nelarabine, Neosal (cyclophosphamide), Newpogen ( Filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, ofatumumab, omasetaxin / mepesuccinate, Oncasper (peguasparagase), Ontac (denileukin / difutitox), OEPA, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle preparation, parifermin, palonosetron hydrochloride, disodium pamidronate, panitumumab, Paraplat (carboplatin),Paraplatin (carboplatin), pazopanib hydrochloride, pegasparagase, pegylated interferon α-2b, PEG-intron (pegylated interferon α-2b), pemetrexed disodium, perjeta (pertuzumab), pertumumab, platinol (cisplastin), platinol-AQ (cisplastin), prelixafor, pomalidomide, pomalist (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, proleukin (aldesleukin), prolia (denosumab), promacta (Eltrombopag Olamine), Provenge (Ciproisel-T), Prinetol (Mercaptopurine), Radium-223, Dichloride, Raloxifene Hydrochloride, Rasburicase, R-CHOP, R-CVP, Recombinant HPV Bivalent Vaccine, Recombinant HPV Quadravalent Vaccine, Recombinant Interferon α-2b, Regorafenib, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidespin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sclero Sole Intraplurral Aerosol (talc), Ciproisel-T, sorafenib tosylate, Sprycel (dasatinib), Stanford V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, stent (sunitinib malate), Sylatron (pegylated interferon α-2b), Synovir (thalidomide), Synribo (omasetaxin / 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, salomide (thalidomide), Toposal (etoposide), topotecan hydrochloride, toremifene, Tricel (temirolimus), tositumomab and I131 iodine tositumomab, Totect (dexrazoxane hydrochloride), trametinib, trastuzumab, Treanda (bendamustine hydrochloride),Trisenox (arsenic trioxide), Tykerb (lapatinib nitosylate), vandetanib, VAMP, Vectibix (panitumumab), VelP, Verban (vinblastine sulfate), Velcade (bortezomib), Versal (vinblastine sulfate), vemurafenib, Vepsid (etoposide), Viajul (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincaser PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, bismodegib, boraxase (glucarpidase), vorinostat, Botrient (pazopanib hydrochloride), Welcovorin (leucovorin) Luxium), Xalkori (crizotinib), Xeloda (capecitabine), Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-afibercept), Zelboraf (vemurafenib), Zevalin (ibritumomab / tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-afibercept, Zoladex (goserelin acetate), zoledronic acid, Zorinza (vorinostat), Zometa (zoledronic acid), and Zytiga (abiraterone acetate).

[0257] The anticancer drugs include PBD dimer, calitiamycin, speromycin, tubulisin B, rhizoxin, dorastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothyron B, taxol, cryptophycin, SN38, Velcade, bruseantine, DAVLBH, DM1, phyllanthoside, Alimta, T2 toxin, MMC, and vantaranib. (vantalanib), vinorelbine, breferzin, sunitinib, daunomycin, semaxanib, tarceva, ilessa, irinotecan, LY-541503, geldanomycin, gemcitabine, methotrexate, gleevec, topotecan, bleomycin, doxorubicin, cisplatin, N-mustards, etoposide, or 5-FU.

[0258] 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.

[0259] b. Immunomodulators Other therapeutic agents may be TLRs or STINGs. Other immunomodulators include cytokines, chemokines, chemokine antagonists, and immune checkpoint inhibitors.

[0260] i.TLR Agonist TLR agonists are immunomodulators. TLR-mediated signaling in response to pathogen-associated molecular patterns (PAMPs) is a continuous cascade of transcriptional regulatory events, which vary depending on the TLR agonist, associated cell type, and the pathogenicity of the antigen. Individual genes (particularly inflammatory cytokines, e.g., IL-1(α and β), IL-6, IL-18, TNF-C) are transiently induced, which represents the innate immune system's ability to interpret infection and direct appropriate responses while promoting cleavage (T. Ravasi, CA Wells, DA Hume, Bioessays 29, 1215 (Nov. 15, 2007); JCRoachet al., Proc Natl Acad Sci USA 104, 16245 (Oct. 9, 2007); M. Gilchrist et al.). al., Nature 441, 173 (May 11, 2006)).

[0261] Examples of TLR agonists, though not limited to them, include: TLR1 / 2 heterodimers (e.g., Pam3CSK4, i.e., trispalmitoylCysSerLysLysLysLys), TLR3 (e.g., polyI:C, polyICLC), TLR4 (e.g., monophosphoryl lipid A, lipopolysaccharide, GLA-SE, G100), TLR5 (e.g., flagellin), TLR2 / 6 heterodimers (e.g., diacyl lipopeptides of Gram-positive bacteria, mycopeptides) TLR7 (e.g., imidazo[4,5-c]quinoline-4-amine, e.g., imiquimod, and those incorporated herein by reference as described in U.S. Patent No. 4,689,338, and polyriboinosinate-polyribocytidylic acid (Poly I:C), etc.), TLR3 (polyadenylate-polyuridylic acid (Poly A:U)), TLR2 (peptidoglycan), TLR2 and TLR4 (e.g., Bacillus Calmette-Guerin (BCG)), [ka] TLR7 / 8 (e.g., loxoribine, imidazo[4,5-c]quinoline-4-amine, e.g., reximod (R848) and MEDI9197), [ka] This also includes TLR9 (e.g., CpG ODNs, e.g., ODN D-SL01, MGN1703, CPG7909, SD-101, EMD 1201081). CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides in specific sequence relationships (CpG motifs). CpG ODNs have a partially or completely phosphorothioated (PS) backbone, in contrast to the native phosphodiester (PO) backbone found in genomic bacterial DNA. Based on their structural features and activity against human peripheral blood mononuclear cells (PBMCs), particularly B cells and plasmacytoid dendritic cells (pDCs), three main classes of stimulated CpG ODNs have been identified. CpG-A ODNs are characterized by a PO-centered CpG-containing palindromic sequence motif and a PS-modified 3' poly-G chain. These induce high IFN-α production from pDCs but are weak stimulants of TLR9-dependent NF-κB signaling and inflammatory cytokine (e.g., IL-6) production. CpG-B ODNs contain a whole PS scaffold with one or more CpG dinucleotides. These potently activate B cells and TLR-dependent NF-κB signaling but have a weak effect on stimulating IFN-α secretion. CpG-C ODNs possess characteristics of both class A and B cells. They contain a complete PS scaffold and a CpG-containing palindromic sequence motif. Class C CpG ODNs also induce B cell stimulation along with strong IFN-α production from pDCs.

[0262] Further TLR9 agonists are described in International Publication No. 2019 / 115402, European Patent No. 2017281, U.S. Patent No. 2019 / 0160173, U.S. Patent No. 2019 / 0151345, U.S. Patent No. 2011 / 0311518, and U.S. Patent No. 2011 / 0293565, each incorporated herein by reference.

[0263] Single-stranded and double-stranded RNAs can function as TLR agonists, as described in Roers et al., Immunity (2016) 44, 739-754 (incorporated herein by reference).

[0264] ii. STING Agonist STING agonists are immunomodulators that recognize cyclic dinucleotides in the cytosol of cells and then regulate host defense genes of many inflammatory cytokines, including type I interferons and inflammatory cytokines. These signals can then stimulate the adaptive immune system through antigen cross-presentation and T cell priming, in conjunction with other mechanisms (Barber GN. STING: infection, inflammation and cancer. Nat Rev Immunol. 2015;15(12):760-70). TLRs and STING agonists can also promote antitumor immune responses in solid tumors and cancers treated with immunotherapy (Berger G, Marloye M, Lawler SE. Immunotherapy. Trends Mol). Med. 2019;25(5):412-427).

[0265] STING agonists are ADU-S100 and 2'3'-cG S A S Includes MP. STING agonists include: cyclic dinucleotides and their analogues, e.g., [ka] .

[0266] STING agonists further include modified cyclic dinucleotides. In some embodiments, the modified cyclic dinucleotide may not exist naturally or may be chemically synthesized. In some embodiments, the modified cyclic dinucleotide has the formula: [ka] It is a compound of the following. In some embodiments, R1 and R2 may each independently be 9-purine, 9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or 9-isoguanine, and their structures are as follows: [ka] As stated above, R1 and R2 may be the same or different. In some embodiments, the compound may be provided primarily in the form of stereoisomers of Rp.Rp or Rp.Sp., or as a prodrug, or as a pharmaceutically acceptable salt thereof, as described in U.S. Patent No. 2016 / 0287623 (incorporated herein by reference). In some embodiments, the compound may be provided primarily in the form of stereoisomers of Rp.Rp. In certain embodiments, the compound may be in the form of the following formula or primarily as a stereoisomer of Rp.Rp: [ka]

[0267] STING agonists are formulated as described in U.S. Patent No. 2017 / 0333552 (incorporated herein by reference). [ka] It may contain the following compounds.

[0268] STING agonists are formulated as described in U.S. Patent No. 2018 / 0064745 (incorporated herein by reference). [ka] It may contain the following compounds.

[0269] STING agonists are formulated as described in U.S. Patent No. 2019 / 0185511 (incorporated herein by reference). [ka] It may contain the following compounds.

[0270] The STING agonist is formulated as described in International Publication No. 2014 / 189806 (incorporated herein by reference). [ka] It may contain the following compounds.

[0271] STING agonists are formulated as described in U.S. Patent No. 2019 / 0062365 (incorporated herein by reference). [ka] It may contain the following compounds.

[0272] The STING agonist is formulated as described in International Publication No. 2018 / 198076 (incorporated herein by reference). [ka] It may contain the following compounds.

[0273] STING agonists are formulated as described in U.S. Patent No. 2018 / 0092937 (incorporated herein by reference). [ka] It may contain the following compounds.

[0274] STING agonists are formulated as described in U.S. Patent No. 2018 / 0273578 (incorporated herein by reference). [ka] It may contain the following compounds.

[0275] STING agonists are formulated as described in U.S. Patent No. 2019 / 0183917 (incorporated herein by reference). [ka] It may contain the following compounds.

[0276] STING agonists are formulated as described in U.S. Patent No. 2019 / 0185509 (incorporated herein by reference). [ka] It may contain the following compounds.

[0277] STING agonists are formulated as described in U.S. Patent No. 2019 / 0185510 (incorporated herein by reference). [ka] It may contain the following compounds.

[0278] STING agonists are formula as described in U.S. Patent No. 2017 / 0233430 (incorporated herein by reference). [ka] It may contain the following compounds.

[0279] STING agonists are formulated as described in U.S. Patent No. 2018 / 0002369 (incorporated herein by reference). [ka] It may contain the following compounds.

[0280] STING agonists are formulated as described in U.S. Patent No. 2018 / 0186828 (incorporated herein by reference). [ka] It may contain the following compounds.

[0281] STING agonists are formulated as described in U.S. Patent No. 2019 / 0016750 (incorporated herein by reference). [ka] It may contain the following compounds.

[0282] STING agonists are formulated as described in U.S. Patent No. 2018 / 0162899 (incorporated herein by reference). [ka] It may contain the following compounds.

[0283] STING Agonist is described in International Publication No. 2018 / 138684 (this document). As described in the book (which is incorporated by reference), formula [ka] It may contain the following compounds.

[0284] STING agonists are formulated as described in International Publication No. 2018 / 138685 (incorporated herein by reference). [ka] It may contain the following compounds.

[0285] STING agonists are formulated as described in International Publication No. 2019 / 118839 (incorporated herein by reference). [ka] It may contain the following compounds.

[0286] STING agonists are formulated as described in U.S. Patent No. 2017 / 0044206 (incorporated herein by reference). [ka] It may contain the following compounds.

[0287] STING agonists are formulated as described in International Publication No. 2018 / 118665 (incorporated herein by reference). [ka] It may contain the following compounds.

[0288] STING agonists are formulated as described in International Publication No. 2018 / 208667 (incorporated herein by reference). [ka] It may contain the following compounds.

[0289] STING Agonist is described in International Publication No. 2019 / 125974 (this document). As described in the book (which is incorporated by reference), formula [ka] It may contain the following compounds.

[0290] STING agonists are formulated as described in International Publication No. 2018 / 009648 (incorporated herein by reference). [ka] It may contain the following compounds.

[0291] STING agonists are formulated as described in International Publication No. 2018 / 009652 (incorporated herein by reference). [ka] It may contain the following compounds.

[0292] STING Agonist is described in International Publication No. 2018 / 013887 (this document). As described in the book (which is incorporated by reference), formula [ka] It may contain the following compounds.

[0293] STING agonists are formulated as described in International Publication No. 2018 / 013908 (incorporated herein by reference). [ka] It may contain the following compounds.

[0294] STING agonists are formulated as described in International Publication No. 2019 / 046511 (incorporated herein by reference). [ka] It may contain the following compounds.

[0295] STING Agonist is described in International Publication No. 2019 / 051488 (this document). As described in the book (which is incorporated by reference), formula [ka] It may contain the following compounds.

[0296] STING agonists are formulated as described in International Publication No. 2019 / 051489 (incorporated herein by reference). [ka] It may contain the following compounds.

[0297] STING agonists are formulated as described in U.S. Patent No. 2019 / 0192549 (incorporated herein by reference). [ka] It may contain the following compounds.

[0298] STING agonists are formulated as described in International Publication No. 2018 / 100558 (incorporated herein by reference). [ka] It may contain the following compounds.

[0299] STING agonists are formulated as described in International Publication No. 2019 / 092660 (incorporated herein by reference). [ka] It may contain the following compounds.

[0300] STING agonists are formulated as described in International Publication No. 2019 / 027858 (incorporated herein by reference). [ka] It may contain the following compounds.

[0301] STING agonists are formulated as described in U.S. Patent No. 2018 / 0093964 (incorporated herein by reference). [ka] It may contain the following compounds.

[0302] STING agonist, formula [ka] (In the formula, X, 1 ~X 3 , L, Q, Z, Y, n, and R 6 ~R 8This may include compounds as described in International Publication No. 2018 / 234805 (incorporated herein by reference).

[0303] STING agonist, formula [ka] (In the formula, X, 1 ~X 3 , L, Q, Y, and R 6 ~R 8 This may include compounds as described in International Publication No. 2018 / 234807 (which is incorporated herein by reference).

[0304] STING agonist, formula [ka] (In the formula, X, 1 ~X 3 , L, Q, Y, and R 6 ~R 11 This may include compounds as described in International Publication No. 2018 / 234808 (incorporated herein by reference).

[0305] STING agonists include, for example, the compound DMXAA: [ka] Includes.

[0306] STING agonists include diamidobenimidazole, for example, [ka] Includes.

[0307] c. Trans-cyclooctene functionalized prodrugs Trans-cyclooctene-functionalized prodrugs are known in the art and include prodrugs of anticancer agents, as described in International Publication No. 2018 / 187740, International Publication No. 2014 / 205126, International Publication No. 2015 / 139025, and International Publication No. 2017 / 044983 (incorporated herein by reference). Another embodiment using trans-cyclooctene-functionalized prodrugs will be described later.

[0308] E46.1A. Trans-cyclooctene functionalized prodrug, formula (III) [ka] (In the formula, R 1a Each time it appears, hydrogen, C 1~4 Alkyl and C 1~4 Independently selected from the group consisting of haloalkyls; R 1b Each time it appears, hydrogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C(O)OH, C(O)OC 1~4 Alkyl, C(O)N(R) 1c )CHR 1e CO2H, C(O) N(R 1c )CHR 1e C(O)OC 1~4 Alkyl, C(O)N(R) 1c )-C 1~6 Alkylene-CO2H and C(O)N(R) 1c )-C 1~6 Alkylene-C(O)OC 1~4 Independently selected from the group consisting of alkyl groups; R 1c Each time it appears, it independently contains hydrogen or C 1~4 It is alkyl; R 1e Each time it appears, it is independently -C 1~4 Alkylene-CO2H, -C 1~4 Alkylene-CONH2, or -C 1~4It is alkylene-OH; Each time D appears, it is independently selected from a group consisting of stimulants for anticancer drug payloads, Toll-like receptor (TLR) agonist payloads, and interferon gene (STING) agonist payloads; Each instance of L is an independent linker; Each time t appears, it is independently 1, 2, or 3; (Each instance of p is independently 0, 1, or 2.) The method described in E46, which is represented by or a pharmaceutically acceptable salt thereof.

[0309] E46.1B. Trans-cyclooctene functionalized prodrugs are given by formula (III). [ka] (In the formula, R 1a Each time it appears, hydrogen, C 1~4 Alkyl and C 1~4 Independently selected from the group consisting of haloalkyls; R 1b Each time it appears, C(O)N(R 1c )-C 1~6 Alkylene-SO3H, C(O)N(R) 1c )-C 2~6 Alkylene-N(C) 1~4 Alkyl)3 + , C(O)N(R 1c )-(CH2CH2O) 1~3 -CH2CH2N((CH2CH2O) 1~3 -C 1~6 Alkylene-CO2H)2 and C(O)N(R) 1c )-CH(CH2O-(CH2CH2O) 0~2 -C 1~6 Independently selected from the group consisting of alkylene-CO2H)2; R 1c Each time it appears, it independently contains hydrogen or C 1~4 It is alkyl; Each time D appears, it is independently selected from a group consisting of stimulants for anticancer drug payloads, Toll-like receptor (TLR) agonist payloads, and interferon gene (STING) agonist payloads; Each instance of L is an independent linker; Each time t appears, it is independently 1, 2, or 3; (Each instance of p is independently 0, 1, or 2.) A compound of formula (III), represented by or a pharmaceutically acceptable salt thereof, or a compound of formula (III) for use in the method described in E46.

[0310] E46.2.R 1a A compound or pharmaceutically acceptable salt described in the method described in E46.1A or E46.1B, or a compound or pharmaceutically acceptable salt for use in the method described in E46.1B, wherein the compound is hydrogen.

[0311] E46.3.R 1a C 1~4 A compound or pharmaceutically acceptable salt of an alkyl compound as described in E46.1A or E46.1B, or a compound or pharmaceutically acceptable salt for use in the method described in E46.1B.

[0312] E46.4.R 1a A compound or pharmaceutically acceptable salt described in the method described in E46.1A or E46.1B, or a compound or pharmaceutically acceptable salt for use in the method described in E46.1B, wherein the compound is CH3.

[0313] E46.5.R 1b However, 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 1~6Alkylene-CO2H and C(O)N(R) 1c )-C 1~6 Alkylene-C(O)OC 1~4 A method according to either E46.1A or E46.2 to E46.4, selected from the group consisting of alkyl groups.

[0314] E46.6A.R 1b However, C(O)OH, C(O)N(R) 1c )CHR 1e CO2H and C(O)N(R) 1c A method according to either E46.1A or E46.2 to E46.4, selected from the group consisting of CH2CO2H.

[0315] E46.6B.R 1b However, -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 A compound selected from the group consisting of )-CH(CH2O-CH2CH2-CO2H)2, as described in any of E46.1B to E46.4, or a pharmaceutically acceptable salt, or a compound or pharmaceutically acceptable salt for use in the method described in any of E46.1B to E46.4.

[0316] E46.6C. [ka] but, [ka] The compounds described in E46.1B, or pharmaceutically acceptable salts thereof, or compounds or pharmaceutically acceptable salts thereof for use in the method described in E46.1B.

[0317] E46.6D. [ka] but, [ka] The compounds described in E46.1B, or pharmaceutically acceptable salts thereof, or compounds or pharmaceutically acceptable salts thereof for use in the method described in E46.1B.

[0318] E46.6E. [ka] but, [ka] The compounds described in E46.1B, or pharmaceutically acceptable salts thereof, or compounds or pharmaceutically acceptable salts thereof for use in the method described in E46.1B.

[0319] E46.6F. [ka] but, [ka] The compounds described in E46.1B, or pharmaceutically acceptable salts thereof, or compounds or pharmaceutically acceptable salts thereof for use in the method described in E46.1B.

[0320] E46.7.R 1e The method according to E46.5 or E46.6A, wherein the compound is -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH.

[0321] E46.8.R 1e However, -C 1~4 The method described in E46.5 or E46.6A, wherein the material is alkylene-CO2H.

[0322] E46.9.R1e The method described in E46.5 or E46.6A, wherein the compound is -CH2CO2H.

[0323] E46.10.R 1b However, C(O)N(R 1c )-C 1~6 The method described in E46.1A or E46.2 to E46.4, wherein the material is alkylene-CO2H.

[0324] E46.11.R 1b However, C(O)N(R 1c The method described in E46.1A or E46.2 to E46.4, wherein the result is CH2CO2H.

[0325] E46.12.R 1c The method described in any of E46.1A to E46.11, wherein the hydrogen is hydrogen.

[0326] E46.13.R 1b The method according to either E46.1A or E46.2 to E46.4, wherein the hydrogen is hydrogen.

[0327] E46.14.R 1b The method according to either E46.1A or E46.2 to E46.4, wherein the function is C(O)OH.

[0328] In the compounds described herein, linker L may have 1 to 100 linked atoms and may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, 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.

[0329] The linker in formula (III) may include one or more (e.g., 1 to 10 or 1 to 5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1 to 10 or 1 to 5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties; where each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety is independently oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4 It can be optionally substituted with 1 to 5 substituents independently selected from the haloalkyl group.

[0330] In equation (III), the linker is: -Y 10 -(CH2) n’ -Y 20 -(CH2) m’’ -Y 30 - (In the formula, Y 10 , Y 20 , and Y 30 These are, each independently, 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, a These are alkennyrene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene; where each alkylene, alkennyrene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy, and C 1~4Optionally substituted with 1 to 5 substituents independently selected from the haloalkyl group; Each R 110 These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 It is a haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R 120 These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 It is a haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. It could be.

[0331] In certain embodiments, the linker is not a link. In certain embodiments, each R 110 These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R 120 These are, independently, hydrogen and C 1~4 Alkyl, C 1~4 These are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl compounds.

[0332] Typical linkers, though not limited to those listed below, include: [ka] These are some examples.

[0333] Typical linkers, though not limited to those listed below, include: [ka] These are some examples.

[0334] The linker in formula (III) is polyethylene glycol (e.g., PEG with an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diyrbis(III) (May contain one or more of methyl carbamates, arylenes (e.g., phenylene), ethylene-oxy, amines, esters, amides, carbamates, ketones (i.e., formyl), or carbonates. The linker in formula (III) is, [ka] It may include.

[0335] In formula (III), the linker may comprise one or more natural or non-natural amino acids, which may be called peptide linkers. If drug (D) contains an amino moiety, the linker may be bound to it using peptide linkers composed of carboxylic acid acyl units and one or more amino acids that constitute a protein or peptide sequence. The linker may also comprise a self-sacrificing spacer separating the drug and the protein peptide sequence.

[0336] In formula III, linker L may be a peptide linker represented by "AYZXW", where "A" is a carboxylic acid acyl 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, separating the peptide and drug, D, or bioorthogonal moieties. In certain embodiments, one or more amino acids in the peptide linker are N-methylated.

[0337] In formula (III), Y may be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Y may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0338] In formula (III), 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. Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0339] Preferred YZ combinations include valine-citrulline; valine-alanine; and alanine-alanine.

[0340] In certain embodiments, A is -OC(O)-.

[0341] In certain embodiments, X is -OC(O)-.

[0342] In certain embodiments, W is -OC(O)-. In certain embodiments, X does not exist, and W is -OC(O)-.

[0343] In certain embodiments, -XW is [ka] That is the case.

[0344] In certain embodiments, -XW is [ka] That is the case.

[0345] In certain embodiments, the peptide linker is specially modified so that it is selectively cleaved (e.g., enzymatically cleaved) by one or more tumor-associated proteases to release a drug.

[0346] In certain embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., di-, tri-, or tetra-peptides). However, it will be understood that peptide linkers with up to 5, 6, 7, or 8 amino acid residues can also be suitably used.

[0347] In certain 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.

[0348] In certain embodiments, the linker L in formula (III) is [ka] The linker described above is located on the right side of the amino acid side chain of D, such as lysine or cysteine ​​(for example, [ka] ) can be bound together.

[0349] In some cases, the therapeutic agent is covalently bonded to the linker via an amide bond; for example, the therapeutic agent contains an amine to bond the carbonyl group of the therapeutic agent to the linker. In some cases, the therapeutic agent may be a carboxyl-containing therapeutic agent to bond the amine group of the therapeutic agent to the linker. In some cases, the therapeutic agent and the linker together form a carbamate base; for example, the therapeutic agent may be an amine-containing therapeutic agent to bond the acyloxy group of the therapeutic agent and the linker. In some cases, the therapeutic agent and the linker together form a carbonate base; for example, the therapeutic agent may be a hydroxyl-containing therapeutic agent to bond the acyloxy group of the therapeutic agent and the linker.

[0350] E46.15A. L is [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 , R15 , 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 17 Each instance is independently either hydrogen or -CH2OC(O)-; G x is halogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 Optionally, 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro Compounds or pharmaceutically acceptable salts described in any of E46.1A, E46.2-E46.6, or E46.7-E46.14, E46.1B-E46.4, or E46.6B-E46.6F, or compounds or pharmaceutically acceptable salts for use in any of the methods described in E46.1B-E46.4, or E46.6B-E46.6F.

[0351] E46.15B. L is [ka] and; L 3a is a combination; L 4a teeth, [ka] and; R 12 and R 13 Each of them independently consists of hydrogen or C 1~4A compound or pharmaceutically acceptable salt described in any of E46.1A, E46.2-E46.6, or E46.7-E46.14, or any of E46.1B-E46.4 or E46.6B-E46.6F, which is alkyl, or a compound or pharmaceutically acceptable salt for use in any of E46.1B-E46.4 or E46.6B-E46.6F.

[0352] A compound or pharmaceutically acceptable salt described in any of E46.1A, E46.2-E46.6, or E46.7-E46.15B, E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B, where E46.16.t is 1, or a compound or pharmaceutically acceptable salt used in any of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B.

[0353] E46.17A. [ka] teeth, [ka] [ka] and; R 18 Each instance is independently either 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' is a payload portion (e.g., 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, an anticancer drug payload portion), the method described in E46.16, the compounds described in E46.16, or pharmaceutically acceptable salts, or compounds or pharmaceutically acceptable salts for use in the method described in E46.16.

[0354] E46.17B. [ka] teeth, [ka] and; R 12 and R 13 Each of them independently consists of hydrogen or C 1~4 It is alkyl; D' is the payload portion (e.g., the anticancer drug payload portion), which is the method described in E46.16, the compound described in E46.16, or a pharmaceutically acceptable salt, or a compound or a pharmaceutically acceptable salt for use in the method described in E46.16.

[0355] A compound or pharmaceutically acceptable salt described in any of the methods of E46.1A, E46.2-E46.6A, or E46.7-E46.17B, E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.17B, where E46.18.p is 0, or a compound or pharmaceutically acceptable salt used in any of the methods of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.17B.

[0356] E46.19.t is 2 or 3, the method described in E46.18, the compound described in E46.18, or a pharmaceutically acceptable salt, or a compound or a pharmaceutically acceptable salt for use in the method described in E46.18.

[0357] E46.20.t is 2, [ka] but, [ka] The method described in E46.19, the compounds described in E46.19, or pharmaceutically acceptable salts, or compounds or pharmaceutically acceptable salts for use in the method described in E46.19.

[0358] Those skilled in the art will recognize that the payload D bound to the linker refers not to the payload molecule itself, but to the portion of the payload molecule bound to the linker. The release of payload D from the prodrug releases the payload itself.

[0359] D may be an anticancer drug payload of any of the anticancer drugs described herein.

[0360] D may be a TLR agonist payload of any of the TLR agonists described herein. Preferably, D is imidazo[4,5-c]quinoline-4-amine, for example, [ka] , [ka] That is the case.

[0361] D may be a STING agonist payload of any of the STING agonists described herein. D may be a cyclic dinucleotide payload, for example, [ka] The formula may be as follows: where Y is a nucleic acid base and X is O or S, as shown below. Examples of nucleic acid bases include naturally occurring purines and pyrimidine bases, as well as modified purines and pyrimidine bases, and other modified heterocyclic bases. Examples of such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, etc. Examples of nucleic acid base modifiers include deazapurine, N-1-methylguanosine, isoguanine, 2-aminopurine, 1,3-diaza-2-oxofenoxazine, 1,3-diaza-2-oxofenoxazine, 7-nitro-1,3-diaza-2-oxofenoxazine, 2,6-diaminopurine, purine, 6-thioguanine, hypoxanthine, 2-pyrimidinone, 2-pyridone, 4-thiouridine, imidazole-4-carboxamide, N-substituted 5-(carboxyamide)uridine, for example, 5-(N-benzylcarboxyamide)uridine, or 5-fluorodeoxyuridine. Payload D is, [ka] It is possible.

[0362] E46.21A. [ka] but, [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] A compound or pharmaceutically acceptable salt described in any of E46.1A, E46.2-E46.6, or E46.7-E46.15B, or any of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B, or a compound or pharmaceutically acceptable salt for use in any of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B.

[0363] E46.21B. [ka] but, [ka] [ka] A compound or pharmaceutically acceptable salt described in any of E46.1A, E46.2-E46.6, or E46.7-E46.15B, or any of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B, or a compound or pharmaceutically acceptable salt for use in any of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B.

[0364] E46.22. [ka] but, [ka] These are E46.1A, E46.2~E46.6, or E46.7~E46.15B. A compound or pharmaceutically acceptable salt described in any of the methods described in E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B, or a compound or pharmaceutically acceptable salt for use in any of the methods described in E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B.

[0365] E46.23. [ka] but, [ka] The compounds or pharmaceutically acceptable salts described in any of E46.1A, E46.2-E46.6, or E46.7-E46.15B, or any of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B, or for use in any of the methods described in E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B. In E46.23, p is 0 and L is -OC(O)-.

[0366] E46.24. [ka] but, [ka] The compounds or pharmaceutically acceptable salts described in any of E46.1A, E46.2-E46.6, or E46.7-E46.15B, or any of E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B, or for use in any of the methods described in E46.1B-E46.4, E46.6B-E46.6F, or E46.15A-E46.15B. In E46.24, p is 0 and L is -OC(O)-.

[0367] E.46.25.t is 1; L is, [ka] The method described in E46.18, the compound described in E46.18, or a pharmaceutically acceptable salt thereof, wherein D is doxorubicin, or the compound or a pharmaceutically acceptable salt thereof for use in the method described in E46.18.

[0368] According to the definition of "payload portion," payload portion D' is payload D or D 1 NH, NC are linked to the linker. 1~4 This refers to the remaining part of the payload, which is obtained by removing the nucleophile group such as alkyl, O, or S, or by removing the electrophile group such as C(O) bonded to the linker. For example, formula [ka] The compound is a compound, for example, [ka] Contains; compound [ka] is a compound, for example, [ka] is a compound, for example, [ka] Includes, formula [ka] The compound is a compound, for example, [ka] Includes DH, NH2-D ’ HOOC-D ’ The payload molecule itself is released, either by the release of HO-D' or by the release of HO-D'. According to the definition of the payload portion mentioned above, the "cyclic dinucleotide payload portion" is a cyclic dinucleotide payload from which the nucleophile (typically O) that binds to the linker has been removed. For example, [ka] but, [ka] In this case, the cyclic dinucleotide payload portion is [ka] This may be the case. In the compounds described herein, [ka] teeth, [ka] This may be the case, where D' is the cyclic dinucleotide payload portion. According to the definition of the payload portion described above, the "imidazo[4,5-c]quinoline-4-amine payload portion" is imidazo[4,5-c]quinoline-4-amine with its nucleophilic group (typically O or N) attached to the linker removed. For example, [ka] but, [ka] When this is the case, the imidazo[4,5-c]quinoline-4-amine payload portion D' is, [ka] It is possible. For example, [ka] but, [ka] When this is the case, the imidazo[4,5-c]quinoline-4-amine payload portion D' is, [ka] It is possible.

[0369] The preferred compound of formula (III) is formula [ka] Compounds of which, for example, [ka] Includes.

[0370] The preferred compound of formula (III) is formula [ka] Compounds of which, for example, [ka] for example, [ka] Includes.

[0371] The compound of formula (III) is [ka] [ka] [ka] Includes.

[0372] The compound of formula (III) is [ka] Includes.

[0373] The compound of formula (III) is [ka] [ka] [ka] [ka] [ka] Includes.

[0374] E. Synthesis method The disclosed compounds and conjugates can be better understood in relation to the following synthetic schemes and methods that describe the means by which the compounds can be prepared.

[0375] In general, conjugates of formula (I) or compounds of formula (III) can be prepared by reacting a suitably activated linker with a primary amine, secondary amine, or hydroxyl group. It should be understood that the reactive group on the linker (e.g., esters, carbonates, acyl chlorides, carboxylic acids) can be positioned at any selected site of the linker group. Conversely, the linker may contain a nucleophilic amine or hydroxyl group that can react with a suitable group, such as an aldehyde, ketone, ester, carbonate, carboxylic acid, or acyl chloride.

[0376] In certain embodiments, as shown below, a trans-cyclooctene activated for nucleophilic addition is subjected to a suitable payload (D / D) in the presence of a base. 1 A functionalized payload can be obtained by reacting the TCO with a payload attached to the TCO or linker. This payload or linker may contain a primary amine, a secondary amine, or a hydroxyl group that reacts with the activated TCO. In certain embodiments, the leaving group (LG) is a chloro leaving group, a p-nitrophenol leaving group, or an N-hydroxysuccinimide leaving group. Exemplary bases used in this reaction include organic and inorganic bases, such as triethylamine, pyridine, sodium hydroxide, and sodium bicarbonate.

[0377] [ka] Scheme 1A is a (poly)peptide containing a protein (e.g., a monoclonal antibody). A general method for conjugating trans-cyclooctene to the lysine side chain of an antibody is described. Nitrophenol carbonate is reacted with an amino group under basic conditions to obtain a trans-cyclooctene conjugated (poly)peptide. More specifically, a solution of the antibody in aqueous buffer can be incubated with a molar excess of a carbonate reagent. The reaction mixture can be quenched by adding an excess of an amine (ethanolamine, taurine, etc.). The trans-cyclooctene-antibody conjugate can then be purified by gel filtration. The number of conjugated trans-cyclooctene molecules per antibody molecule can be determined by measurement using spectrophotometry.

[0378] [ka] Scheme 1B shows a two-step method for conjugating trans-cyclooctene to a protein or peptide: a reaction between the carbonate ester and the lysine side chain, followed by the reaction of the amino-containing group R under basic conditions. 2 -N(R 1cFurther coupling with H yields trans-cyclooctene functionalized proteins / peptides, where R 1c R is as defined herein. 2 is -C 1~6 Alkylene-CO2H,-CHR 1e CO2H, -C 1~6 Alkylene-C(O)OC 1~4 Alkyl, C(O)OC 1~4 Alkyl, or -CHR 1e C(O)OC 1~4 It is alkyl.

[0379] The alcohol-containing side chain and the N-terminal amino group of the (poly)peptide react in the same manner as in schemes 1A and 1B to obtain a trans-cyclooctene conjugate.

[0380] [ka] Scheme 2 involves a similar reaction between the carbonate ester and the ornithine side chain of daptomycin, followed by the reaction of the amino-containing group R under basic conditions. 2 -N(R 1c ) Further coupling with H (for example, R 2 is -C 1~6 Alkylene-CO2H,-CHR 1e CO2H, -C 1~6 Alkylene-C(O)OC 1~4 Alkyl, C(O)OC 1~4 Alkyl, or -CHR 1e C(O)OC 1~4 It indicates that it is alkyl.

[0381] [ka] Scheme 3 shows the conversion of 11 to carboxylic acid intermediates, which can be further converted to payload-supported products 13 and 14. In Scheme 3, D' represents the payload portion (e.g., immunomodulatory payload portion, anticancer drug payload portion, TLR agonist payload portion).

[0382] [ka] Other carboxylic acids that can be prepared using 11 are shown in Scheme 4.

[0383] [ka] Scheme 5 shows a synthesis method for preparing a typical STING agonist TCO conjugate.

[0384] [ka] Scheme 6 shows a synthesis method for preparing a typical STING agonist TCO conjugate.

[0385] [ka] Scheme 7 shows a general method for conjugating a cyclic dinucleotide to trans-cyclooctene as shown in formula (I). The illustrated method proceeds by reacting nitrophenyl carbonate-substituted trans-cyclooctene with a cyclic dinucleotide in the presence of a base, forming a mono- or bis-substituted cyclic dinucleotide depending on the amount of trans-cyclooctene.

[0386] [ka] Scheme 8 shows a general method for conjugating a cyclic dinucleotide with trans-cyclooctene, in which case R 2 is -C 1~6 Alkylene-CO2H,-CHR 1e CO2H, -C 1~6 Alkylene-C(O)OC 1~4 Alkyl, C(O)OC 1~4 Alkyl, or -CHR 1e C(O)OC 1~4 It is alkyl, and this is C(O)N(R 1c)- C 1~6 Alkylene-CO2H, C(O)OH, C(O)N(R) 1c )CHR 1e CO2H, C(O)N(R) 1c )-C 1~6 Alkylene-C(O)OC 1~4 Alkyl, C(O)OC 1~4 Alkyl, or C(O)N(R) 1c )CHR 1e C(O)OC 1~4 The R of formula (I) is alkyl. 1b This corresponds to the process shown in Scheme 8, which may be modified to obtain bis-conjugated cyclic dinucleotides using an excess of trans-cyclooctene reagent, similar to Scheme 7.

[0387] [ka] Schemes 9 and 10 show typical synthetic methods for conjugating imidazo[4,5-c]quinoline-4-amine to trans-cyclooctene as shown in formula (I), following a procedure similar to that of schemes 7 and 8. [ka]

[0388] The disclosed compounds can be prepared as racemates or as individual enantiomers or diastereomers by either stereospecific synthesis or resolution. These compounds can be resolved to their component enantiomers or diastereomers, for example, by standard techniques (e.g., formation of stereoisomer pairs by salt formation with an optically active base, followed by fractional crystallization and regeneration of the free acid). Alternatively, these compounds can be resolved by formation of stereoisomer esters or amides, followed by chromatographic separation and removal of chiral auxiliaries. Alternatively, these compounds can be resolved using a chiral HPLC column. Enantiomers can also be obtained by kinetic resolution of the racemate of the corresponding ester using a lipase enzyme.

[0389] The compounds described herein may be in the form of salts, for example, pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” includes salts of the active compound prepared using relatively non-toxic acids or bases, depending on the specific substituents present in the compounds described herein. By conventional methods, the neutral form of the compound can be regenerated by contacting this salt with a base or acid and then isolating the parent compound. The parent form of the compound has certain physical properties, such as solubility in polar solvents, that differ from the various salt forms, but otherwise Therefore, this salt is equivalent to the parent compound for the purposes of this disclosure. An example of a pharmaceutically acceptable salt is Berge et al, 1977, “Pharmaceutically This is discussed in "Acceptable Salts," J.Pharm.Sci. Vol.66, pp.1-19.

[0390] For example, if the compound is anionic, or if it has a functional group that can be anionic (e.g., -COOH is -COO - If it has (which may be), a salt can be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions (e.g., Na + and K + ), alkaline earth cations (for example, Ca 2+ and Mg 2+ Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4 + ) and substituted ammonium ions (e.g., NH3R1 + NH2R2 + NHR3 + NR4 +Examples of suitable substituted ammonium ions include: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids (e.g., lysine and arginine).

[0391] If this compound is cationic, or if it has a functional group that can be cationic (for example, -NH2 is -NH3), + If it has (which may be), a salt can be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrite, phosphoric acid, and phosphorous acid.

[0392] Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphor sulfonic acid, cinnamic acid, citric acid, EDTA, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, gluchep tonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucinic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Suitable examples of polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose.

[0393] It may be convenient or desirable to prepare, purify, and / or handle active compounds in a chemically protected form. The term “chemically protected form” is used herein in the conventional chemical sense and refers to compounds in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, etc.). In practice, known chemical methods are used to reversibly dereactive functional groups that would otherwise be reactive under specified conditions. In a chemically protected form, one or more reactive functional groups are in the form of protected or protecting groups (also known as masked or masking groups or blocked or blocking groups). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be carried out without affecting the protected group; the protecting group can usually be removed in a later step without substantially affecting the rest of the molecule. See, for example, Protective Groups in Organic Chemistry (T. Green and P. Wuts; 3rd Edition; John Wil). ey and Sons, 1999); JFW McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973; “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981; “Methoden der organischen Chemie”, Houben-Weyl, 4 th edition, Vol.15 / l, Georg Thieme Verlag, Stuttgart 1974; H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Protein”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982; Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate”, Georg Thieme Verlag, Stuttgart 1974.

[0394] In organic synthesis, a wide variety of such "protection," "blocking," or "masking" methods are widely used and well known. For example, a compound having two non-equivalent reactive functional groups (both potentially reactive under specified conditions) can be derivatized to "protect" one of the functional groups under specified conditions, thereby rendering it non-reactive; thus protected, the compound can be used as a reactant having effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, this protected group can be "deprotected" to restore its original functionality.

[0395] The hydroxyl group can be protected as an ether (-OR) or ester (-OC(O)R), for example, t-butyl ether; benzyl, benzhydryl(diphenylmethyl), or trityl(triphenylmethyl) ether; trimethylsilyl or t-butyldimethylsilyl ether; or acetyl ester (-OC(O)CH3,-OAc).

[0396] The aldehyde group or ketone group can be protected as an acetal (RCH(OR)2) or ketal (R2C(OR)2), respectively, by reaction with a primary alcohol, for example, in which the carbonyl group (R2C=O) is converted to a diether (R2C(OR)2). This aldehyde group or ketone group can be readily regenerated by hydrolysis using a large excess of water in the presence of an acid.

[0397] The amine group can be protected as, for example, an amide (-NRC(O)R) or a urethane (-NRC(O)OR), such as methylamide (-NHC(O)CH3); benzyloxyamide (-NHC(O)OCH2C6H5, -NH-Cbz); t-butoxyamide (-NHC(O)OC(CH3)3, -NH-Boc); 2-biphenyl-2-propoxyamide (-NHCO(O)C(CH3)2C6H4C6H5, -NH-Bpoc), and 9-fluorenylmethoxyamide. They can be protected as (-NH-Fmoc), as 6-nitroberatryloxyamide (-NH-Nvoc), as 2-trimethylsilylethyloxyamide (-NH-Teoc), as 2,2,2-trichloroethyloxyamide (-NH-Troc), as allyloxyamide (-NH-Alloc), as 2(-phenylsulfonyl)ethyloxyamide (-NH-Psec); or, where appropriate (e.g., cyclic amines), as nitroxide radicals (>NO≪).

[0398] Carboxylic acid groups can be protected as esters, for example, alkyl esters (e.g., methyl esters; t-butyl esters); haloalkyl esters (e.g., haloalkyl esters). They can be protected as arylalkyl esters; trialkylsilylalkyl esters; or arylalkyl esters (e.g., benzyl esters; nitrobenzyl esters); or as amides, for example, as methylamides.

[0399] The thiol group can be protected as a thioether (-SR), for example, benzyl thioether; or acetamidomethyl ether (-S-CH2NHC(O)CH3).

[0400] The compounds described herein can also be modified by adding appropriate functionalities to enhance their selective biological properties. Such modifications are known in the art and include those that increase biopenetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to enable administration by injection, modify metabolism, and / or modify elimination rates. Examples of these modifications, but not limited to, include: esterification with polyethylene glycol, derivatization with pivolate or fatty acid substituents, conversion to carbamate salts, hydroxylation of aromatic rings, and heteroatom substitution in aromatic rings.

[0401] In certain embodiments, the product may be further modified, for example, by the manipulation of substituents. Such manipulations include, but are not limited to, reduction, oxidation, organometallic cross-coupling, alkylation, acylation, and hydrolysis reactions, which are well known to those skilled in the art. In some cases, the order in which the reaction scheme described above is carried out may be changed to accelerate the reaction or to avoid unwanted reaction products.

[0402] The compounds described herein can be purified by any purification protocol known in the art, including chromatography such as HPLC, preparative thin-layer chromatography, flash column chromatography, and ion-exchange chromatography. Any suitable steady-state phase can be used, such as normal-phase and reverse-phase chromatography, and ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and / or alumina chromatography. See, for example, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. LRSnyder and JJ Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed. E. Stahl, Springer-Verlag, New York, 1969.

[0403] F. Pharmaceuticals The compositions (e.g., support compositions, conjugates, trans-cyclooctenoprodrugs) can be provided in any preferred form, e.g., in the form of a pharmaceutically acceptable formulation, and can be formulated for any preferred route of administration, e.g., oral, topical, or parenteral administration. When the compositions are provided as injection solutions (e.g., in embodiments where they are administered intravenously or directly to tissue), the compositions can be provided in a ready-to-use dosage form, or as a reconstituted, storage-stable powder or liquid that may contain pharmaceutically acceptable carriers and excipients.

[0404] "Pharmacologically acceptable excipients," "pharmaceutically acceptable diluents," "pharmaceutically acceptable carriers," or "pharmaceutically acceptable adjuvants" are excipients, diluents, carriers, and / or adjuvants useful in preparing pharmaceutical compositions, and which generally include excipients, diluents, carriers, and adjuvants that are safe, non-toxic, and not biologically or otherwise harmful, and are acceptable for veterinary and / or human pharmaceutical uses. As used in this document, "pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants" includes one or more excipients, diluents, carriers, and adjuvants.

[0405] Methods for formulating compositions can be modified from readily available ones. For example, a composition can be provided as a pharmaceutical formulation comprising a therapeutically effective amount of the composition and a pharmaceutically acceptable carrier (e.g., saline solution). The pharmaceutical formulation may optionally contain other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to mammals, for example, to humans.

[0406] The compositions of this disclosure can be prepared into a variety of oral, parenteral, and topical dosage forms. Oral formulations include tablets, pills, powders, sugar-coated tablets, capsules, liquids, lozenges, cachets, gels, syrups, slurries, and suspensions that are suitable for ingestion by subjects.

[0407] The compositions of this disclosure may also be administered by injection, which may be intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection. Examples of pharmaceutical formulations for parenteral administration include aqueous solutions of the active compound in a water-soluble form. Furthermore, suspensions of the active compound may be prepared as suitable oily suspensions for injection. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. The aqueous suspensions for injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to enable the preparation of highly concentrated solutions. For injection, the agents of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or buffered saline.

[0408] In some cases, the compositions described herein can be administered by inhalation, for example, intranasally.

[0409] In some cases, the compositions of this disclosure can be administered transdermally.

[0410] In some cases, the composition can be administered via intraocular, intravaginal, and intrarectal routes, such as suppositories, inhaled air, powders, and aerosol formulations (see, for example, steroid inhalants, Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995).

[0411] Accordingly, this disclosure also provides pharmaceutical formulations comprising the compositions described herein and pharmaceutically acceptable carriers or excipients.

[0412] For preparing pharmaceutical formulations from the compositions of this disclosure, the pharmaceutically acceptable carrier may be solid or liquid. Solid formulations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more substances, which may also act as diluents, flavorings, binders, preservatives, tablet disintegrants, or encapsulating agents. Further details regarding the techniques for formulation and administration can be found, for example, in Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

[0413] In some embodiments, the pharmaceutical composition of the present invention is a vaccine comprising a conjugate or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and optionally an antigen. The antigen used in the immunogenic composition provided herein is an effective amount (e.g., in a therapeutic or prophylactic method). It may be supplied in an amount effective for use in [specific context]. For example, the immunogenic compositions of the present invention can be used to treat or prevent diseases or conditions such as infectious diseases and cancer. Exemplary antigens include, but are not limited to, tumor antigens and infectious disease antigens. The antigens used in the immunogenic compositions provided herein are typically extracellular polymers (e.g., polypeptides, polysaccharides, polynucleotides) relative to the host. The antigen may be any target epitope, molecule (including biomolecules), molecular complex (including molecular complexes containing biomolecules), intracellular aggregate, or cells or tissues in which induction or enhancement of the subject's immunoreactivity is desired. Often, the term antigen may refer to the polypeptide antigen of interest. However, as used herein, the antigen may also refer to a recombinant construct (e.g., an expression construct) encoding the polypeptide antigen of interest. In certain preferred embodiments, the antigen may be an infectious pathogen and / or an epitope, biomolecule, cell or tissue associated with an infectious disease, cancer, autoimmune disease, allergy, asthma, or any other medical condition in which an antigen-specific immune response is desirable or beneficial, or may be derived from such pathogen or tissue, or may be immunologically cross-reactive with such pathogen or tissue.

[0414] In certain embodiments, a tumor antigen or cancer antigen is used in conjunction with the immunogenic compositions provided herein. In certain embodiments, the tumor antigen is a peptide-containing tumor antigen, e.g., a polypeptide tumor antigen or a glycoprotein tumor antigen. In certain embodiments, the tumor antigen is a sugar-containing tumor antigen, e.g., a glycolipid tumor antigen or a ganglioside tumor antigen. In certain embodiments, the tumor antigen is a polynucleotide-containing tumor antigen expressing a polypeptide-containing tumor antigen, e.g., a DNA vector construct such as an RNA vector construct or plasmid DNA. In certain embodiments, the tumor antigen is an entire living, dead, or permeable cancer cell. Tumor antigens suitable for use with the immunogenic compositions provided herein include a wide variety of molecules, e.g., (a) polypeptide-containing tumor antigens, e.g., polypeptides (e.g., 8 to 20 amino acid lengths, but lengths outside this range are also common), lipopolypeptides, and glycoproteins; (b) sugar-containing tumor antigens, e.g., polysaccharides, mucins, gangliosides, glycolipids, and glycoproteins; and (c) polynucleotides expressing antigenic polypeptides.

[0415] In certain embodiments, the tumor antigen is, for example, (a) a full-length molecule associated with cancer cells, (b) homologs and modified forms thereof, e.g., molecules containing deletions, additions, and / or substitutions, and (c) fragments thereof. In certain embodiments, the tumor antigen is provided in recombinant form. In certain embodiments, the tumor antigen includes, for example, a class I restriction antigen recognized by CD8+ lymphocytes or a class II restriction antigen recognized by CD4+ lymphocytes.

[0416] In certain embodiments, tumor antigens include, but are not limited to, the following: (a) cancer / testicular antigens, e.g., NYESO-1, SSX2, SCP1, and RAGE, BAGE, GAGE, and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (e.g., can be used to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors); (b) variant antigens, e.g., p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancers), p21 / Ras (e.g., associated with melanoma, pancreatic cancer, and colorectal cancer), CDK4 (e.g., associated with melanoma), MUM1 (e.g., associated with melanoma), caspase-8 (e.g., associated with head and neck cancer), CIA (c) Overexpressed antigens, e.g., 0205 (associated with bladder cancer), HLA-A2-Rl 701, β-catenin (associated with melanoma), TCR (associated with T-cell non-Hodgkin lymphoma), BCR-abl (associated with chronic myeloid leukemia), triose phosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT, (c) Overexpressed antigens, e.g., galectin 4 (associated with colorectal cancer), galectin 9 ( (d) Common antigens, e.g., proteinase 3 (e.g., associated with chronic myeloid leukemia), WT 1 (e.g., associated with various leukemias), carbonic anase (e.g., associated with kidney cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast, colon, lung, and ovarian cancers), α-fetoprotein (e.g., associated with hepatocellular carcinoma), KSA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic and gastric cancers), telomerase catalytic protein, MUC-1 (e.g., associated with breast and ovarian cancers), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast and colon cancers), and carcinoembryonic antigens (e.g., associated with gastrointestinal cancers such as breast, lung, and colorectal cancers), (d) common antigens, e.g., melanoma melanocyte differentiation antigens, e.g., MART-1 / Melan A, gp 100, MC1 (i) other tumor antigens such as polypeptides and sugar-containing antigens, including (i) glycoproteins, e.g., sialyl Tn and sialyl Tn, (ii) melanocyte-stimulating hormone receptors, tyrosinase, tyrosinase-related protein-1 / TRP1 and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma), (e.g., prostate-related antigens, e.g., PAP, PSA, PSMA, PSHP1, PSM-P1, PSM-P2), (f) immunoglobulin idiotypes (e.g., associated with melanoma and B-cell lymphoma), and (g) polypeptides and sugar-containing antigens, including (i) glycoproteins, e.g., sialyl Tn and sialyl (ii) Lex (e.g., associated with breast and colorectal cancer), and various mucins; glycoproteins bound to carrier proteins (e.g., MUC-1 is bound to KLH); (ii) lipopeptides (e.g., MUC-1 linked to the lipid portion); (iii) polysaccharides (e.g., globoH synthase hexasaccharide) bound to carrier proteins (e.g., KLH); (iv) gangliosides such as GM2, GM12, GD2, GD3 (e.g., associated with brain tumors, lung cancer, and melanoma) also bound to carrier proteins (e.g., KLH).

[0417] In certain embodiments, tumor antigens include, but are not limited to, the following: p15, Hom / MeI-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigen, TSP-180, p185erbB2, p180erbB-3, c-met, nm-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29 (BCAA), CA 195, CA 242, CA-50, CAM43, CD68 (KP1), C0-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophyllin C-related protein), TAAL6, TAG72, TLP, TPS, etc.

[0418] G. Kitt Aspects of this disclosure include kits having the compositions described herein.

[0419] The kit may include a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, and a therapeutic support composition. The kit may also include a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, and a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt or composition thereof.

[0420] The kit may comprise a compound of formula (II) or formula (III), or a pharmaceutically acceptable salt or composition thereof, one or more immunomodulators, or pharmaceutically acceptable salts or compositions thereof, and optionally a therapeutic support composition. The therapeutic support composition described in this document may include one or more immunomodulators, or pharmaceutically acceptable salts or compositions thereof.

[0421] The therapeutic support composition, one or more immunomodulators, and compounds of formula (I), (II), and / or formula (III) may be contained in separate containers within the packaging. The kit may provide one or more therapeutic support compositions.

[0422] The kits described herein may include packaging configured to contain a composition (e.g., a therapeutic support composition and / or one or more immunomodulators). Similarly, one or more compounds of formulas (I), (II), and / or (III) may be provided in the kit. The packaging may be a sealed package, such as a sterile sealed package. "Sterile" means that microorganisms (e.g., fungi, bacteria, viruses, spore forms, etc.) are substantially absent. In some cases, the packaging may be configured to be sealed, for example, a moisture-resistant package, optionally under airtight and / or vacuum sealing.

[0423] In certain embodiments, the kit includes a reagent that can be used as a release agent for the releaseable linker described herein. The release agent may be any of the release agents described herein, and may include, but are not limited to, chemical release agents (e.g., acids, bases, oxidizing agents, reducing agents), solvents, etc. The release agent in the kit may be provided in any convenient form, such as a gas, solution, solid, granules, powder, suspension, etc. The release agent may be packaged in a separate container from the composition in the kit.

[0424] In addition to the components described above, the subject kit may further include instructions for carrying out the subject method. These instructions may be present in the subject kit in a variety of forms, and one or more may be present in the kit. One possible form of these instructions is as information printed on one or more sheets of paper in a suitable medium or support, such as within the kit packaging or in a package insert. Another form of the instructions may be a computer-readable medium on which the information is recorded or stored, such as a CD, DVD, Blu-ray, or computer-readable memory (e.g., flash memory). Yet another possible form of instructions is a website address, which allows the information to be viewed on a site that has been removed via the internet. Any convenient means may be present in the kit. [Examples]

[0425] H. Examples This disclosure has numerous embodiments, illustrated by the following non-limiting embodiments. The following embodiments are provided to provide a complete disclosure and explanation to those skilled in the art of how the invention is made and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments constitute all experiments performed.

[0426] Abbreviation: ACN Acetonitrile Dapto (daptomycin) DCM Dichloromethane DIPEA Diisopropylethylamine DMF (N,N-dimethylformamide) DMSO (Dimethyl Sulfoxide) Doxo Doxorubicin Et ethyl HCl ethyl acetate FCC Flash Column Chromatography h or hr hours HA Hyaluronic Acid HAT tetrazine-modified hyaluronic acid HMT Hydrogel-Modified Tetrazine HOAt 1-hydroxy-7-azabenzotriazole LC-MS (Liquid Chromatography-Mass Spectrometry) Me methyl MeCN acetonitrile MeOH methanol MeTz methyltetrazine min MTD maximum tolerated dose NHS N-hydroxysuccinimide PBS (phosphate-buffered saline) Ph Phenyl ppm parts per million rt / RT room temperature Standard error of the sample mean in SEM TAG Tetrazin-Modified Activated Gel TCO trans-cyclooctene TEA (Triethylamine) THF (Tetrahydrofuran) TFA (Trifluoroacetic Acid)

[0427] Example 1 Trans-cyclooctene antibody conjugate (prophetic example without supporting data) A solution of antibody (2.5 mg / mL) in aqueous buffer (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM ethylenediaminetetraacetate disodium salt), pH 6.5, is incubated with a molar excess of trans-cyclooctene derivatized carbonate in dimethylacetamide (DMA). The reaction is allowed to proceed at ambient temperature, and after completion, the solution is purified by gel filtration column. The concentration of the conjugate is determined by spectrophotometry using the known extinction coefficient of the antibody.

[0428] Example 2 Synthesis of TCO-monomethyl auristatin E (TCO-MMAE) conjugate [ka] Preparation of TCO-MMAE conjugate Monomethyl auristatin E (170 mg, 0.24 mmol) was added to DMF (2 mL) of rt, to which TCO-bis-NHS (100 mg, 0.24 mmol) and DIPEA (93 mg, 0.72 mmol). The solution was stirred in rt for 20 hours, acetonitrile (ACN, 8 mL) was added, and the mixture was purified by prep-HPLC (ACN / water 0-100%, formic acid 0.1%) to obtain TCO-NHS-MMAE (88 mg, 36%). TCO-NHS-MMAE (78 mg, 0.076 mmol) was added to THF (2 mL) and H2O (2 mL) of rt, to which LiOH (9.2 mg, 0.38 mmol). The solution was stirred in rt for 20 hours. After removing the solvent, HCl (aq, 0.5 N) was added to bring the pH to approximately 3. The mixture was purified by prep-HPLC (ACN / water 0-100%, formic acid 0.1%) to obtain TCO-acid-MMAE (54 mg, 76%, two isomers). LC-MS: (ESI+)928[M+H].

[0429] The synthesis of TCO-acid-MMAE is representative of the functionalization of the N-terminal amino group of polypeptides.

[0430] Example 3 Dapto-TCO-amino acid synthesis [ka] Example protocol: Add daptomycin (100 mg, 0.062 mmol), TCO-bis-NHS (62.5 mg, 0.149 mmol), and triethylamine (62.5 μL, 45.3 mg, 0.448 mmol) to DMSO and stir overnight at RT to produce dapto-TCO-NHS. LCMS: (ESI-)1926.8 [MH]. Add aspartic acid (104.5 mg, 0.785 mmol) and 4-dimethylaminopyridine (150.9 mg, 1.235 mmol) to dapto-TCO-NHS (126.1 mg, 0.0654 mmol) and stir at 37°C for 18 hours. Purify by HPLC to obtain dapto-TCO-aspartic acid. Yield: 100 mg, 0.0514 mmol. LCMS: (ESI-) 1944.8 [MH].

[0431] Using this approach, glycine and aspartate-modified TCO-prodrugs can be generated, which can generally be applied to the incorporation of other amino acid cargoes as well.

[0432] Example 4 Daptomycin-TCO-glycine conjugate [ka] Daptomycin (537 mg, 0.33 mmol), TCO-bis-NHS (350 mg, 0.83 mmol), and triethylamine (0.350) in DMSO (11 mL) (mL, 2.51 mmol). Stir overnight at RT. Then heat to 37°C. Add glycine (300 mg, 4.00 mmol) and triethylamine (1.8 mL, 13 mmol) and stir for 18 hours. Add 8 mL of water and purify by HPLC. Yield: dapto-TCO-glycine-373 mg, 0.20 mmol, 59.6%.

[0433] The synthesis of dapto-TCO-glycine and dapto-TCO-aspartic acid is representative of the derivatization of the lysine side chain of polypeptides.

[0434] Example 5 MMAE-TCO-Asp conjugate [ka] Bis(2-(trimethylsilyl)ethyl)(tert-butoxycarbonyl)-L-aspartate (1.1). Step 1: EDC (23.0 g, 120.0 mmol, 2.8 equivalents) was added to a mixture of Boc-Asp-OH (10.0 g, 42.9 mmol, 1.0 equivalent, Combi-blocks QA-1348), DIEA (37.3 mL, 214.0 mmol, 5.0 equivalents), 2-(trimethylsilyl)ethane-1-ol (12.2 g, 103.0 mmol, 2.4 equivalents), and DMAP (1.1 g, 8.6 mmol, 0.2 equivalents) in DCM (200.0 mL) cooled in an ice bath. The mixture was stirred overnight at room temperature, diluted with DCM (100.0 mL), and washed with aqueous HCl (0.5 M) until the pH of the organic layer was neutral. The organic layer was further washed with saturated NaHCO3 solution, dried over Na2SO4, and filtered. The filtrate was concentrated and purified by flash chromatography (ISCO column, 220 g) using a gradient of 0-30% siRNA in hexane to obtain 13.0 g (70%) of bis(2-(trimethylsilyl)ethyl)(tert-butoxycarbonyl)-L-aspartate as a colorless oil. 1 H-NMR (300MHz, CDCl3):5.50(br d,J=8.79Hz, 1H), 4.61-4.48(m,1H), 4.30-4.14(m,4H), 3.04-2.93(m,1H), 2 .83-2.73(m,1H), 1.45(s,9H), 1.06-0.94(m,4H), 0.004(s,9H), 0.03(s,9H).

[0435] Bis(2-(trimethylsilyl)ethyl) L-aspartate hydrochloride (1.2). Step 2: To a solution of compound-1.1 (10.4 g, 24.0 mmol) in dry DCM (30 mL) cooled in an ice bath, HCl (100 mL, 2.0 M HCl in ether) was added. This mixture was stirred at room temperature for 90 minutes, after which further HCl (100 mL, 4.0 M HCl in dioxane) was added. After completion, the reaction was monitored by LC-MS, and the mixture was concentrated to dryness. It was suspended in RINKAN and concentrated again. 11.4 g (approximately 20 wt% solvent) of 2-(trimethylsilyl)ethane-1-ol was obtained as a tar-like residue. (+esi)[M+H] + = 334.

[0436] Bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-6-hydroxy-1-methylcycloocta-4-ene-1-carbonyl)-L-aspartate (1.4). Step 3: To a solution of compound-1.2 (11.0 g, 80% purity, 23.9 mmol, 2.0 equivalents) in DMF (100.0 mL), DIPEA (11.0 mL, 62.7 mmol, 5.3 equivalents), (1R,6R,E)-6-hydroxy-1-methylcycloocta-4-ene-1-carboxylic acid (1.3), (2.2 g, 11.9 mmol, 1.0 equivalent), and HATU (9.1 g, 23.9 mmol, 2.0 equivalents) were successively added. The mixture was stirred overnight at room temperature and diluted with SiO (400 mL) and water (400 mL). The aqueous layer was extracted once with siRNA (400 mL). The combined organic layer was dried over Na₂SO₄ and filtered. The filtrate was concentrated and purified by flash chromatography (220 g, ISCO column) with a gradient of siRNA in hexane (0-70%) and elution in siRNA at a constant composition in 70% siRNA in hexane to obtain 3.96 g (75% purity, 50% yield) of bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-6-hydroxy-1-methylcycloocta-4-en-1-carbonyl)-L-aspartate as a viscous oil. (+esi)[M+H] + = 500.4.

[0437] Bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-1-methyl-6-(((4-nitrophenoxy)carbonyl)oxy)cycloocta-4-ene-1-carbonyl)-L-aspartate (1.5). Step 4: To a solution of compound 1.4 (4.0 g, 7.9 mmol, 1.0 equivalent) in anhydrous DCM (113 mL), pyridine (1.88 g, 23.8 mmol, 3.0 equivalents) was added. To this mixture, cooled in an ice bath, a solution of p-nitrophenyl chloroformate (2.1 g, 10.3 mmol, 1.3 equivalents) in DCM (28 mL) was slowly added. The mixture was stirred at room temperature for 12 hours and separated with RINKAN and water. The organic phase was washed with aqueous sodium bicarbonate solution and water, then dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in a minimum amount of DCM and purified by flash chromatography on a 220 g silica gel column (ISCO) with a stepwise gradient of siRNA in hexane (0-40%) as the eluent to obtain 2.91 g (44% yield; the desired product was eluted with approximately 30% siRNA and co-eluted with p-nitrophenol) of viscous oil.

[0438] Bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-6-((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartate(1.6). Step 5: Bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-1-methyl-6-(((4-nitrophenoxy)carbonyl)oxy)cycloocta-4-ene-1-carbonyl)-L-aspar cooled to 0°C Tate (1.5 mg, 296 mg, 0.45 mmol, 1.0 equivalent) contains (2S)-N-((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (352 mg, 0.49 mmol, 1.1 equivalents, monomethyl auristatin E, Advanced ChemBlock, DIPEA (173 mg, 1.34 mmol, 3.0 equivalents), and HOBt (173 mg, 0.89 mmol, 2.0 equivalents) were added. The mixture was stirred overnight at room temperature and diluted with SiO2. The mixture was washed twice with water and with an aqueous NH4Cl solution, dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure and subjected to C18 flash chromatography using a gradient of acetonitrile and water (0-100%) (100 g, ISCO Purified by gold-capped C18 flash column, 499 mg (90%) of bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-6-((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2- Methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartate was obtained as an oil. (+esi)[M+H] + = 1244.3.

[0439] ((1R,6R,E)-6-((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartic acid (TCO(asp)-MMAE). Step 6: Bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-6-((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-meth Xy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartate (1.6, 500 mg, 0.4 mmol, 1.0 equivalent) was mixed with TBAF (1.0 M in THF). The mixture was stirred overnight at room temperature and directly purified by C18 flash chromatography (100 g, ISCO gold-capped C18 flash column) using a gradient of acetonitrile and water (0-100%) to obtain 300 mg of solid containing approximately 4 molar equivalents of tetrabutylammonium species. The mixture was dissolved in ELISA (20 mL), washed six times with water (pH adjusted to 3-5 with dilute HCl), dried over Na2SO4, and filtered. The filtrate was concentrated to obtain a glassy solid, which was then treated with ether and concentrated again to obtain a powdered solid free of tetrabutylammonium byproducts. (-esi)[MH] - = 1042.1.

[0440] Example 6 TCO(asp)-spacelink-etoposide [ka] 4-((5R,5aR,8aR)-9-(((2R,4aR,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofl[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenyl(4-nitrophenyl)carbonate (3.1). Step 1: Etoposide (1.5g, 2.5 mmol, 1.0 equivalent, etoposide, Combi-blocks QA-7668) was dissolved in dry THF. Triethylamine (1.1 mL, 7.6 mmol, 3.0 equivalents) and DMAP (31 mg, 0.25 mmol, 0.1 equivalents) were added. The reaction mixture was cooled to 0°C. Chloroformate PNP (0.62 g, 3.1 mmol, 1.2 equivalents) was dissolved in THF and added dropwise. The reaction mixture was stirred at room temperature. After evaluation by TLC was complete, 1 mL of acetic acid was added and the reaction mixture was stirred for 2 minutes. The precipitate was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography in silica gel using a gradient of 0-100% EtOAC in hexane to remove any unreacted etoposide still present. 1 When evaluated by 1H NMR, approximately 30% of the desired product (2.25 g, 82% yield, 70% purity) was obtained.

[0441] tert-butyl(4-((5R,5aR,8aR)-9-(((2R,4aR,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofl[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenyl)ethane-1,2-diyrbis(methylcarbamate)(3.2). Step 2: Dissolve compound 3.1 in DMF (10 mL) and mono-Boc protected N,N-dimethylethylene-diamine (532 mg, 2.82 mg) (+esi)M+NH3+H + = 820.8.

[0442] 4-((5R,5aR,8aR)-9-(((2R,4aR,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofl[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenylmethyl(2-(methylamino)ethyl)carbamate 2,2,2-trifluoroacetate (3.3). Step 2: Compound 3.2 (460 mg, 0.573 mmol) from the above step was dissolved in DCM (10 mL) and cooled to 0°C. TFA (3 mL) was added, and the reaction mixture was stirred for 1 hour and concentrated to dryness. The materials were used directly in the next step without further refinement.

[0443] Bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-6-(((2-(((4-((5R,5aR,8aR)-9-(((2R,4aR,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofl[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenoxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartate(3,4). Step 4: Compound 3.3 (464 mg, 0.579 mmol, 1.1 equivalents) was dissolved in DMF (3.0 mL). HOBT (202 mg, 1.05 mmol, 2 equivalents) and DIPEA (0.275 mL, 1.58 mmol, 3 equivalents) were added. Compound 1.5 (350 mg, 0.526 mmol, 1.0 equivalent) was dissolved in DCM (2.0 mL) and added to the above solution. The reaction mixture was stirred overnight at room temperature. The reaction product was separated into aqueous solutions of siRNA and ammonium chloride. The organic layer was washed with water, dried over sodium sulfate, filtered, and concentrated. The material was chromatographed four times, first on a hexane:acetone gradient, then a hexane:siRNA gradient, then a DCM:EtOAC gradient, and finally a DCM:ACN gradient. Final chromatography removed adjacent impurities, and the desired compound was eluted in approximately 70% ACN:30% DCM. The fractions were pooled and concentrated to obtain the desired target product (94 mg, 17% yield). (+esi)M+H + = 1228.1.

[0444] [ka] ((1R,6R,E)-6-(((2-(((4-((5R,5aR,8aR)-9-(((2R,4aR,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofloxacin[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenoxy)carbony (Methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-ene-1-carbonyl)-L-aspartic acid (TCO(asp)-spacelink-etoposide). Step 5: Compound 3.4 was dissolved in DMSO (1.0 ml). Tetraethylammonium fluoride × H2O (78.2 mg, 0.524 mmol) was added all at once. The reaction mixture was stirred for 105 minutes and work-up based on the preferred LC-MS profile. The reaction mixture was separated into ELISA and water and acidified to pH 4 with dilute HCl. The organic phase was washed twice with water, then dried over sodium sulfate, filtered, and concentrated. The residue was purified by preparative HPLC (gradient of ACN in H2O containing 0.1% formic acid). The fraction containing the pure compound was pooled and lyophilized to obtain a white lyophilized product (42 mg, 55% yield). (-esi)[MH] - = 1026.2.

[0445] Example 7 Ranibizumab-Cy5.5-TCO conjugate [ka] The pH of the ranibizumab solution in PBS was adjusted to pH 8.5 using bicarbonate buffer (1M). The fluorescent dye sulfonated-Cy5,5-bisuxinimidyl ester (BisSE) (AAT BioQuest Cat# 158) was dissolved in DMSO to a concentration of 10 mM and added to the protein solution (75 μg of dye per mg of protein, total 750 μg, approximately 2.5 equivalents). The reaction mixture was rotated at room temperature for 1 hour. Following the manufacturer's instructions, the product was purified by buffer exchange using a PD-10 desalting column and equilibrated in PBS. The product was collected, and the protein concentration and label ratio were calculated by measuring UV absorbance and using the Degree of Labeling Calculator (https: / / www.aatbio.com / tools / degree-of-labeling-calculator). The label ratio was 1.5. A 1 mg sample was retained, diluted to 3.1 mg / mL with PBS, and then filtered through a 0.22 μm syringe filter to sterilize the product. Endotoxin levels were measured at 3.8 EU / mL. The remaining material was used to prepare the TCO conjugate.

[0446] Ranibizumab-Cy5.5 conjugate solution in PBS (0.7 at 8.5 mg / mL) The pH of 1 mL of the solution was adjusted to pH 8.5 using bicarbonate buffer (1 M). Trans-cyclooctene-PEG3-NHS (SiChem #SC-8406) (10 mM in DMSO) was added to the solution (approximately 38 μg of reagent per mg of protein, totaling approximately 225 μg, approximately 3.8 equivalents). The reaction mixture was rotated at room temperature for 1 hour. Following the manufacturer's instructions, the product was purified by buffer exchange using a PD-10 desalting column and equilibrated in PBS. The product was collected. The conjugate sample was treated with Cy3-tetrazine (AAT Bioquest Cat# 910), and the protein concentration and labeling ratio were calculated by measuring UV absorbance and using the Degree of Labeling Calculator (https: / / www.aatbio.com / tools / degree-of-labeling-calculator). The labeling ratio was 1.8. The solution was sterilized by filtration through a 0.22 μm syringe filter. The final product was a 0.71 mL solution with a protein concentration of 5.4 mg / mL. Endotoxin was measured at 6.5 EU / mL. [ka]

[0447] Example 8 trans-cyclooctene-trastuzumab conjugate [ka] A solution of antibody, e.g., trastuzumab (2.51 mg / mL), in aqueous buffer (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM ethylenediaminetetraacetate disodium salt PBS), pH 7.4-6.5, is incubated with a 5-fold molar excess of trans-cyclooctene-NHS ester derivatized carbonate in dimethylacetamide (DMA). The reaction is allowed to proceed at ambient temperature for 1 hour. The reaction mixture is then applied to a NAP-10 column, equilibrated with PBS, then eluted with fresh PBS, pH 7.4, and purified by gel filtration column after completion. The concentration of the conjugate is determined by spectrophotometry using the known extinction coefficient of the antibody. The effective concentration of TCO can be determined by treatment of the conjugate sample with a suitable tetrazine-functionalized fluorescent dye, followed by separation by SDS-PAGE and measurement of the fluorescence signal associated with the protein band.

[0448] Example 9 TCO-spacelink-SN-38 [ka] (S)-tert-butyl(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)ethane-1,2-diyrbis(methylcarbamate). To a solution of 7-ethyl-10-hydroxycamptothecin (SN-38, 660 mg, 1.68 mmol) in DMF (30 mL), 4-nitrophenyl chloroformate (336 mg, 1.68 mmol) and Et3N (708 μL, 504 mg, 5.04 mmol) were added. The mixture was stirred at room temperature for 1.5 hours. Et3N (708 μL, 504 mg, 5.04 mmol) and 4-nitrophenyl chloroformate (336 mg, 1.68 mmol) were added to the mixture. The mixture was stirred for a further 20 minutes, and a solution of tert-butylmethyl[2-(methylamino)ethyl]carbamate (948 mg, 5.04 mmol) in DMF (10.68 mL) was added. The mixture was stirred at room temperature for 1 hour, and water (20 mL) was added. The reaction mixture was then mixed with toluene (4* Extracted with 50 ml. The organic phase was separated into water (3 * The solution was washed with 20 mL of silica gel, dried, and concentrated (Na2SO4). The residue was purified by silica gel chromatography (DCM to 10% MeOH in DCM) to obtain the desired product (343 mg, yield: 41%) as a pale yellow solid. LCMS: (m / z, C4H9N4O8) = 607.2 [M + H] + .

[0449] [ka] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-ylmethyl(2-(methylamino)ethyl)carbamate 2,2,2-trifluoroacetate. Compound 2 (343 mg, 0.57 mmol) in DCM (3 mL) was stirred, and TFA (0.5 mL) was added. The mixture was stirred at room temperature for 2 hours. The resulting mixture was concentrated and dried under reduced pressure to obtain crude compound 3 (280 mg) as a white solid. The residue was used in the next step without further purification. LCMS: (m / z, C 29 H 31 N4F3O8)=507.1[M+H] + .

[0450] [ka] TCO-spacelink-SN-38. To a stirred mixture of triphosgene (47.5 mg, 0.16 mmol) in DMF (4 mL), (1R,6R,E)-6-hydroxy-1-methylcycloocta-4-ene-1-carboxylic acid (82.8 mg, 0.45 mmol) and DIPEA (116.0 mg, 0.9 mmol) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was added to a mixture of compound 3 (280.0 mg, 0.45 mmol) and DIPEA (116.0 mg, 0.9 mmol) in DMF (3 mL). The resulting mixture was stirred at room temperature for 12 hours. The mixture was purified by preparative HPLC ((CH3CN / H2O(FA)) 0%~70%) to obtain the desired product (105 mg, yield 32%). LCMS: (m / z, C 39 H 46 N4O 12 ) = 717.4[M+H] + .

[0451] Example 10 TCO(asp)-spacelink-SN-38 [ka] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl(4-nitrophenyl)carbonate (2.1). Step 1: 4-nitrophenyl chloroformate (771 mg, 3.82 mmol) was added at 0°C to a mixture of SN-38 (1.25 g, 3.19 mmol, Advanced ChemBlocks Cat#10250, Lot 10602) and DIPEA (1.25 mL, 7.50 mmol) in DMF (20 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was completed by TLC, the mixture was diluted with siRNA, washed with water and brine, dried over Na2SO4, concentrated, and purified with silica gel using a gradient of 0-100% EtOAC in hexane to obtain the desired product.

[0452] (S)-tert-butyl(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)ethane-1,2-diyrbis(methylcarbamate)(2.2). Step 2: The compound from (2.1) above was dissolved in DMF (10 mL), and mono-Boc protected N,N-dimethylethylene-diamine (1.76 g, 9.37 mmol, 2.5 equivalents) was added. The reaction mixture was stirred for 30 minutes. After completion, the DMF was removed under reduced pressure, and the crude product was purified by flash chromatography (100% ethyl acetate) to obtain the desired product (1.3 g, 57% yield in 2 steps). (+esi)[M+H] + = 607.5.

[0453] (S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-ylmethyl(2-(methylamino)ethyl)carbamate 2,2,2-trifluoroacetate (2.3). Step 3: Compound 2.2 (514 mg, 0.847 mmol) from the above was dissolved in DCM (10 mL) and cooled to 0°C. TFA (2.0 mL) was added and the mixture was stirred for 20 minutes, then allowed to rise to room temperature for 1 hour. The mixture was concentrated to dryness, redissolved in dichloroethane, and concentrated again. The crude material was used in subsequent steps without further purification.

[0454] Bis(2-(trimethylsilyl)ethyl)((1R,6R,E)-6-(((2-(((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartate (2.4). Step 4: Compound 2.3 (513 mg, 0.827 mmol, 1.1 equivalents) was dissolved in DMF (5.0 mL). DIPEA (0.393 μL, 2.26 mmol, 3 equivalents) and HOBT (288 mg, 1.50 mmol, 2 equivalents) were added. Next, intermediate 1.5 (500 mg, 0.752 mmol, 1.0 equivalent) was added as a solution in the minimum volume of DCM. The reaction mixture was stirred overnight at room temperature, and then separated with siRNA and ammonium chloride aqueous solutions. The organic phase was washed with water, dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in the minimum volume of DCM and packed onto a 40 g silica gel column (ISCO) for purification by flash chromatography using a gradient of 0–100% siRNA in hexane. The desired fraction was eluted with 100% siRNA immediately after closely eluting impurities (M+H+=918.5, 156 mg, 23% yield) without a diamine spacer. The fractions containing the slowest eluting peak and the target compound were combined and concentrated to obtain the product (406 mg, 52.3% yield). (+esi)[M+H] + = 1032.4.

[0455] [ka] ((1R,6R,E)-6-(((2-(((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartic acid (TCO(asp)-spacelink-SN-38). Step 5: Compound 2.4 (272 mg, 0.263 mmol) was dissolved in DMSO (3.0 mL) in a 40 mL vial with a lined cap and diaphragm. Tetraethylammonium fluoride × H2O (393 mg, 2.63 mmol) was added. The reaction mixture immediately turned dark green. After 2 hours, the reaction mixture was diluted with acetonitrile containing 0.1% TFA and then packed into an equilibrated C18 flash gold column (ISCO, 15.5 g) and eluted under a gradient of 10–100% ACN (containing 0.1% TFA) in water (containing 0.1% TFA). The desired product was eluted at approximately 45% ACN. The two fractions containing the product were lyophilized over 2 days and characterized by HPLC, which revealed some other, closely eluting impurities. The product was further purified by preparative HPLC using the same mobile phase conditions. The pure fractions were combined and concentrated to obtain the desired product as an orange lyophilized product. 1 ¹H NMR indicated the presence of excess water, so the sample was suspended in acetonitrile, concentrated to dryness, and converted into an easily transferable orange powder (62m). g was obtained (in 25% yield as monotrifluoroacetate). 1 1H NMR revealed the presence of TFA. (-esi)[MH] - = 830.5.

[0456] Example 11 AB25409 [ka] General procedure for preparing compound 2. Compound 2 was prepared according to the procedure in the literature (Chem.Sci., 2021, 12, 1259).

[0457] General procedure for the preparation of compound 3. A flame-dried round-bottom flask was packed with sodium hydride (60%, 399 mg, 9.96 mmol, 3.0 equivalents) and dry THF (10 mL). The mixture was cooled to 0°C, and TMS-ethanol (3.33 mL, 23.2 mmol, 7.0 equivalents) was added dropwise at 0°C. The mixture was stirred for 30 minutes, and a solution of compound 2 (934 mg, 3.32 mmol, 1 equivalent) in anhydrous THF (3 mL) was slowly added via syringe at 0°C under a nitrogen atmosphere. The reaction mixture was warmed to room temperature and stirred for 8 hours. The reaction was monitored by TLC for consumption of starting materials. The reaction mixture was quenched by pouring it into ice water (20 mL) and extracted with ethyl acetate (3 × 50 mL). The combined extract was washed with saturated NH4Cl and dried over Na2SO4. After concentration, the residue was purified by flash chromatography using a gradient of ethyl acetate (0-20%) in hexane to obtain compound 3, which was then mixed with TMS-ethanol. Next, the compound was maintained under high vacuum at 40°C for 30 minutes to obtain compound 3 (405 mg, 26%). TLC: PE / EA = 1 / 4. Rf(compound 2) = 0.2. Rf(product, 3) = 0.5

[0458] General procedure for the preparation of compound 7. Compound 5 (63 mg, 0.38 mmol, 1.0 equivalent) was added to a solution of compound 4 (109 mg, 0.38 mmol, 1.0 equivalent) in dry DCM (2 mL). The reaction mixture was stirred for 1 hour. LC-MS analysis of the reaction mixture showed a peak at m / z = 381.90, confirming the formation of intermediate compound 6. To the above reaction mixture, DMAP (93 mg, 0.76 mmol, 2.0 equivalents) was added, followed by a solution of compound 3 (108 mg, 0.38 mmol, 1.0 equivalent) in THF (1 mL). The resulting reaction mixture was heated at 50°C for 24 hours. The reaction was monitored by HPLC and LC-MS. The reaction mixture was concentrated to obtain the crude product, which was purified by silica gel flash chromatography using a gradient of MeOH (0-5%) in DCM to obtain compound 7 (47 mg, 21%). TLC:MeOH / DCM=0.05 / 1. Rf(compound 4)=0.2. Rf(compound 6)=0.3. Rf(product, 7)=0.4. LCMS of compound 6: 381.90[M+H]+. LCMS of compound 7:596. 90[M+H]+

[0459] General procedure for the preparation of compound AB25409. To a solution of compound 7 (47 mg, 0.079 mmol, 1.0 equivalent) in THF (3 mL), TBAF (1.0 M in THF, 1.2 mL, 1.2 mmol, 15 equivalents) was added. The reaction mixture was stirred at room temperature for 15 hours. The reaction was monitored by LC-MS and HPLC. The solvent was removed. The crude mixture was purified by preparative HPLC using 5-100% ACN in water containing 0.1% TFA. After lyophilization, the desired compound AB25409 (21 mg, 54%) was obtained as a pale yellow solid. LC-MS: 497.0[M+H]+; 1H NMR(400MHz, CD3OD)δ 9.85(s,1H), 8.34(d,J=8.2Hz, 1H), 8.20(s,1H), 8.14(d,J=8.4Hz, 1H), 8.07(d,J=9.0Hz , 1H), 7.86(d,J=8.4Hz, 1H), 5.99-6.14(m,1H), 5.71-5.77(m,1H), 5.29(s,1H), 3.86(br t,J=5.9Hz, 2H), 3.45(br t,J=5.4Hz, 2H), 3.30~3.35(m,4H), 2.27-2.31(m,2H), 2.05-2.23(m,3H), 1.70-2.02(m,3H), 1.36(t,J=7.3Hz, 6H), 1.15(s,3H)ppm.

[0460] Example 12 TCO-spacelink-etoposide [ka] 4-((5R,5aR,8aR,9S)-9-(((2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1 [3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofloxacin[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenyl(4-nitrophenyl) carbonate. A solution of etoposide (1.0 g, 1.7 mmol) and TEA (2.5 g, 25 mmol) in anhydrous THF (35 mL) was mixed with a solution of 4-nitrophenyl chloroformate (0.39 g, 1.95 mmol) in anhydrous THF (15 mL). The reaction mixture was stirred overnight at room temperature. The mixture was filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, DCM / CH3CN 2 / 1) to obtain compound 1 (0.78 g, 60% yield). LCMS:(m / z, C36H35NO17)=754.2[M+H] + Measured value: 754.1.

[0461] [ka] tert-butyl(4-((5R,5aR,8aR,9S)-9-(((2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1,3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofl[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenyl)ethane-1,2-diylbis(methylcarbamate). To a solution of carbonate etoposide compound 1 (1.2 g, 1.6 mmol) in DMF (10 mL), N-Boc, N,N'-dimethylethylenediamine (450 mg, 2.37 mmol), DIPEA (0.68 mL, 3.98 mmol), and DMAP (250 mg, 2.04 mmol) were added, and the reaction mixture was stirred at 60°C for 3 hours. The mixture was purified by reverse-phase chromatography (C18, ACN / H2O(FA) 0%~100%) to obtain compound 2 (650 mg, yield 51%). LCMS: (m / z, C39H50N2O16) = 825.3 [M + Na] + Measured value: 826.1.

[0462] [ka] 4-((5R,5aR,8aR,9S)-9-(((2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methylhexahydropyrano[3,2-d][1 [3]dioxin-6-yl)oxy)-6-oxo-5,5a,6,8,8a,9-hexahydrofloxacin[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-2,6-dimethoxyphenylmethyl(2-(methylamino)ethyl)carbamate. To a solution of compound 2 (650 mg, 0.81 mmol) in DCM (10 mL) cooled in an ice bath, TFA (1 mL) was added. The mixture was stirred at 0°C for 6 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by reverse-phase chromatography (C18, ACN / H2O(FA) 0%~100%) to obtain compound 3 (350 mg, yield 61%). LCMS: (m / z, C34H42N2O14) = 703.3 [M + H] + Measured value: 703.1.

[0463] [ka] TCO-spacelink-etoposide. To a solution of TCO (80 mg, 0.434 mmol) in THF (5 mL), triphosgene (52.5 mg, 0.177 mmol) and DMAP (130 mg, 1.07 mmol) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was added to a solution of compound 3 (300 mg, 0.427 mmol) and DIPEA (117 mg, 0.9 mmol) in DMF (2 mL) and stirred at room temperature for 5 minutes. The mixture was heated to 50°C and stirred for a further 2 hours. Ether (20 mL) and HCl (10 mL, 1 N in H2O) were added to the mixture. The organic phase was separated and concentrated. The residue was purified by reverse-phase chromatography (C18, ACN / H2O(FA) 0%~100%) to obtain TCO-spacelink-etoposide (105 mg, yield 26.9%). LCMS: (m / z, C45H56N2O18) = 911.4 [MH] - Measured value: 911.3.

[0464] Example 13 TCO(asp)-Exatecan [ka] Bis(2-(trimethylsilyl)ethyl)((6R,E)-6-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)-1-methylcycloocta-4-en-1-carbonyl)-L-aspartate (4.1). Step 1: Compound 1.5 (250 mg, 0.376 mmol, 1.0 equivalent) was dissolved in DMF (approximately 5 mL) and cooled to 0°C in an ice bath. To the solution, exatecan mesylate (220 mg, 0.414 mmol, 1.1 equivalents, Advanced Chemblocks Cat#10484, Lot 100981), DIPEA (200 μL, 1.1 mmol, 3 equivalents), and wet HOBt (144 mg, 0.752 mmol, 2 equivalents, approximately 80% purity) were added. The resulting slurry was warmed to room temperature under ambient conditions. After 20 hours, analysis of the reaction by LC-MS showed almost complete consumption of the starting materials. The reactants were separated into SiO2 and water. The organic phase was washed with water (twice), aqueous ammonium chloride (once), and brine. Some insoluble solids were present and removed by filtration. The organic phase was dried over sodium sulfate, filtered, and concentrated. The resulting dark brown / black oily residue was dissolved in a minimum amount of DCM, packed into a hexane-flashed silica gel column (24 g, gold-capped ISCO), and eluted with 0-100% siRNA in hexane to obtain the desired compound as a sticky grayish-green solid (240 mg, 66% yield). (+ESI)[M+H] + = 962.2.

[0465] [ka] ((6R,E)-6-((((1S,9S)-5-fluoro-9-hydroxy-4,9-dimethyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)carbamoyl)oxy)-1-methylcycloocta-4-ene-1 -Carbonyl)-L-aspartic acid (TCO(asp)-exatecan). Step 2: Compound 4.1 (150 mg, 0.156 mmol, 1.0 equivalent) was dissolved in 1,4-dioxane (approximately 5 mL). A 2.0 M aqueous LiOH solution (500 μL, 1.00 mmol, 6.4 equivalents) was added to the solution. Analysis by LC-MS after 1 hour showed the presence of the starting material, monoester intermediate, product, and by-products. A further 500 μL of the same LiOH solution was added. After another 30 minutes, LC-MS showed that the starting material had been consumed and the reactants were the desired compound and by-products in nearly equal proportions. The reaction mixture was acidified first with acetic acid and then with dilute HCl to adjust the pH to 1-2. The product was extracted with ELISA (5 times). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated. The off-white solid was purified by preparative HPLC, thereby obtaining the same mass spectrum and similar 1 Two distinct compounds with 1H NMR spectra were obtained. The early eluting peak was shown as Peak 2 (16 mg, 13% yield). (-ESI)[MH]-=759.4. HPLC (tr=8.73 min). The late eluting peak was shown as Peak 1 (20 mg, 17% yield). (-ESI)[MH]-=759.4. HPLC (tr=8.96 min).

[0466] Example 14 [ka] TCO-Exatecan. (1R,6R,E)-6-hydroxy-1-methylcycloocta-4-ene-1-carboxylic acid (220 mg, 1.20 mmol) and DMAP (292 mg, 2.40 mmol) were added to a mixture of triphosgene (177 mg, 0.6 mmol) in THF (10 mL). The mixture was stirred at room temperature for 30 minutes. The mixture was added to a mixture of compound 1 (700 mg, 1.32 mmol) and DIPEA (510 mg, 3.96 mmol) in DMF (10 mL). The resulting mixture was stirred at room temperature for 12 hours. The mixture was purified by preparative HPLC (CH3CN / H2O(FA)) 0%~70% to obtain the desired product (110 mg, yield 14%). LCMS: (m / z, C35H36FN3O8) = 646.3 [M+H] + .

[0467] Example C1 In vivo imaging of SQL70-targeted Fab-TCO conjugates. Monoclonal antibodies and antibody-drug conjugates are widely prescribed for the treatment of various types of cancer. The therapeutic potential of these drugs is limited by their systemic toxicity. In this specification, we evaluate a targeted form of a biological agent called TCO-Fab, which is administered intravenously (IV) and is locally retained when it reacts with a targeted biopolymer that is administered subcutaneously (SC).

[0468] Purpose of the exam The objective of this study is to provide proof of concept for the use of biopolymer-targeting agents in combination with appropriately functionalized biological agents. The biological agents are tracked to the injection site of the biopolymer using in vivo imaging. It will probably happen.

[0469] Test design Six-week-old female nu / nu mice were subcutaneously injected (SC) into the right flank with 100 μL of SQL70 biopolymer (groups 1 and 2) or a control biopolymer (group 3, used as a negative control).

[0470] One hour after injection of SQL70 biopolymer, animals were administered intravenously (IV) via tail vein injection (TVI) with either 10 mg / kg of TCO-Fab-Cy5.5 (Group 1) or 10 mg / kg of Fab-Cy5.5 (Group 2, as a negative control).

[0471] One hour after administration of the control biopolymer, animals were administered 10 mg / kg of TCO-Fab-Cy5.5 (group 3) by TVI as a negative control. The three treatment groups are detailed in Table 1.

[0472] [Table 1]

[0473] Test substance a. TCO-Fab-Cy5.5-(ranibizumab-Cy5.5-TCO conjugate) Supplier: | Storage: 4°C, protected from light Description: A clear red solution. Preparation: Protein concentration indicated in 25 mM sodium citrate, 100 mM sodium chloride, pH 5.5; sterile; ready for injection.

[0474] b. Fab-Cy5.5-(ranibizumab-Cy5.5 conjugate) Supplier: | Storage: 4°C, protected from light Description: A clear blue solution. Preparation: Protein concentration indicated in 25 mM sodium citrate, 100 mM sodium chloride, pH 5.5; sterile; ready for injection.

[0475] c.SQL70 Biopolymer Formula with approximately 10-15 kD MW and approximately 30% modification, [ka] Sodium tetrazine-modified hyaluronate, as shown. Supplier: | Storage: 4°C, protected from light Description: A dark pink injectable liquid. Formulation: Sterilized and pre-formulated in 63.2 mg / mL sodium chloride / sodium phosphate buffer.

[0476] d. Control biopolymer (hyaluronic acid) Supplier: | Storage: 4℃ Description: A white, hygroscopic solid. Formulation: A single formulation (to a concentration of 63.2 mg / mL) in physiological saline at pH 7.4 will be required. The product will be filtered through a 0.2 μm syringe filter inside a biohazard hood under sterile conditions. After formulation, the control biopolymer is stable at 4°C. Prepare fresh or at least one day before use.

[0477] Administration The test substance and vehicle solution were administered according to the indicated doses, routes, and times, as shown in Table 1. The volume of administration was determined based on the actual body weight of each mouse on previous measurement days.

[0478] Live Imaging Mice were imaged after injection of biopolymers to determine baseline values. Mice were imaged 1 hour, 4 hours, 24 hours, and 72 hours after IV injection. (Note: Timing is based on IV injection of various test substances, not SC injection of biopolymers).

[0479] The fluorescence filter used for Cy5.5 was optimized for excitation at 680 nm and emission at 720 nm.

[0480] 72 hours after IV injection (after the final live imaging time point was completed), tissue was removed from the injection site for ex vivo imaging. The biopolymer injection site was defined as the target region for each animal.

[0481] The fluorescence filter used for Cy5.5 would likely be one that excites at 680 nm and emits at 720 nm.

[0482] The mice did not exhibit any adverse reactions to the treatment. All mice were healthy and active throughout the study. One mouse in Group 1 (10 mg / kg TCO-Fab-Cy5.5) showed a signal at the SQL70 injection site compared to subjects in Groups 2 and 3 (Figure 1). Fluorescent signals were observed in the liver, kidneys, and bladder as the drug was removed from the body. This specification includes the disclosure of the following inventions. [Item 1] A conjugate comprising an immunomodulatory agent payload conjugated to one or more bioorthogonal moieties, wherein the immunomodulatory agent payload is selected from the group consisting of therapeutic monoclonal antibodies, cytokines, chemokines, chemokine antagonists, and immune checkpoint inhibitor payloads; or a pharmaceutically acceptable salt thereof. [Item 2] A conjugate as described in Item 1, or a pharmaceutically acceptable salt thereof, wherein each bioorthogonal portion independently comprises trans-cyclooctene or tetrazine. [Item 3] Conjugates of formula (I) as described in item 1 or 2, or pharmaceutically acceptable salts thereof. [ka] (In the formula, G is the bioorthogonal part, and each time G appears, it is independent. [ka] and; L 1 Each instance is an independent linker; m is an integer between 1 and 150; D 1 It is an immunomodulatory payload; R 1A Each time it appears, C 1~4 Alkyl, C 1~4 Haloalkyl and C 1~4 Independently selected from the group consisting of alkoxys; q is 0, 1, or 2; q1 is either 0 or 1; R 1B Each time it appears, 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 Independently selected from the group consisting of alkylene-CO2H)2; R 1c and R 1d Each time it appears, it independently contains hydrogen or C 1~4 It is alkyl; R 1e Each time it appears, it is independently -C 1~4 Alkylene-CO2H, -C 1~4 Alkylene-CONH2, or -C 1~4 It is alkylene-OH; R 1f Each time it appears, it independently produces hydrogen and C. 1~6 Alkyl, or C 1~4 It is alkylene-CO2H; Each instance of n is independently 0, 1, 2, or 3; L 2 Each time it appears, -C(O)- and C 1~3 Independently selected from the group consisting of alkylenes; G 1 Each instance is a heterocyclyl that is independently and arbitrarily substituted. [Item 4] A conjugate or pharmaceutically acceptable salt thereof as described in any one of items 1 to 3, wherein the immunomodulatory agent payload is an immune checkpoint inhibitor payload. [Item 5] The conjugate described in Item 4, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload. [Item 6] The conjugate described in Item 5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload. [Item 7] The conjugate described in Item 6, or a pharmaceutically acceptable salt thereof, wherein the PD-1 inhibitor payload is nivolumab, pembrolizumab, pidilizumab, cintilimab, or AMP-224 payload. [Item 8] The conjugate described in Item 5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload. [Item 9] The conjugate described in Item 8, or a pharmaceutically acceptable salt thereof, wherein the PD-L1 inhibitor payload is atezolizumab, avelumab, durvalumab, or the BMS-936559 payload. [Item 10] The conjugate described in Item 5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload. [Item 11] The conjugate described in Item 10, or a pharmaceutically acceptable salt thereof, wherein the CTLA4 inhibitor payload is an ipilimumab or tremelimumab payload. [Item 12] The immune checkpoint inhibitor payload contains indoleamine 2,3 - A dioxygenase (IDO) inhibitor payload, such as the conjugate described in item 4, or a pharmaceutically acceptable salt thereof. [Item 13] The conjugate described in Item 12, or a pharmaceutically acceptable salt thereof, wherein the IDO inhibitor payload is indoximod or epacadostat payload. [Item 14] The immunomodulatory payload is a conjugate or a pharmaceutically acceptable salt thereof as described in any one of items 1 to 3, wherein the immunomodulatory payload is a cytokine payload. [Item 15] The conjugate described in Item 14, or a pharmaceutically acceptable salt thereof, wherein 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. [Item 16] A conjugate or pharmaceutically acceptable salt thereof as described in any one of items 1 to 3, wherein the immunomodulatory payload is a chemokine payload. [Item 17] The conjugate described in Item 16, or a pharmaceutically acceptable salt thereof, wherein the chemokine payload is a CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CXCL14 payload. [Item 18] A conjugate or pharmaceutically acceptable salt thereof as described in any one of items 1 to 3, wherein the immunomodulatory payload is a chemokine antagonist payload. [Item 19] The conjugate described in Item 18, or a pharmaceutically acceptable salt thereof, wherein the chemokine antagonist payload is a prelixafor payload. [Item 20] The immunomodulatory payload is a conjugate according to any one of items 1 to 18, comprising a polypeptide, or a pharmaceutically acceptable salt thereof. [Item 21] The polypeptide comprising one or more lysine residues, the conjugate described in Item 20, or a pharmaceutically acceptable salt thereof. [Item 22] The conjugate described in Item 21, or a pharmaceutically acceptable salt thereof, wherein each occurrence of the bioorthogonal portion is bound to one of the one or more lysine residues. [Item 23] A conjugate or pharmaceutically acceptable salt thereof as described in any one of items 3 to 22, wherein m is 1 to 20. [Item 24]L 1 is -OC(O)L 4 - or -OC 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(Gx )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 is halogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 A phenyl conjugate as described in any one of items 3 to 23, or a pharmaceutically acceptable salt thereof, which is a phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro. [Item 25] GL 1 Each time it appears, independently, [ka] The conjugates described in item 24, or pharmaceutically acceptable salts thereof. [Item 26] GL 1 Each time it appears, independently, [ka] The conjugates described in item 25, or pharmaceutically acceptable salts thereof. [Item 27]R 1B G 1 , OH, -NR 1c -C 1~4 Alkilen-G 1 , -NR 1c-C 1~4 Alkylene-N(R) 1d )2, -N(R 1c )CHR 1e CO2H, -N(R) 1c )CH2CO2H, and -N(R 1f )-CH2CH2-(N(CH2CO2H)CH2CH2) n Selected from the group consisting of -N(CH2CO2H)2; R 1e is -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH; R 1f is a conjugate, or a pharmaceutically acceptable salt thereof, as described in any one of items 3 to 26, which is hydrogen or CH2CO2H. [Item 28]R 1A C 1~4 It is alkyl; R 1B G 1 , OH, -NR 1c -C 1~4 Alkilen-G 1 , -NR 1c -C 1~4 Alkylene-N(R) 1d )2, -N(R 1c )CHR 1e CO2H, -N(R) 1c )CH2CO2H, and -N(R 1f )-CH2CH2-(N(CH2CO2H)CH2CH2) n Selected from the group consisting of -N(CH2CO2H)2; R 1e is -C 1~4 It is alkylene-CO2H; R 1f is hydrogen or C 1~4 It is alkylene-CO2H; G 1 It is a 4- to 8-membered monocyclic heterocycline containing a first nitrogen and, optionally, one other heteroatom selected from nitrogen, oxygen, and sulfur, G 1 It is bonded to the first nitrogen atom, and optionally, C 1~4 Alkyl, C1~4 Haloalkyl, halo, cyano, OH, -OC 1~4 It is substituted with 1 to 4 substituents independently selected from the group consisting of alkyl and oxo; n is 0, 1, or 2, and is a conjugate or pharmaceutically acceptable salt thereof as described in any one of items 3 to 26. [Item 29]R 1A It is CH3; R 1e It is -CH2CO2H; R 1f is hydrogen or CH2CO2H; G 1 It is bonded via a ring nitrogen atom, and optionally, C 1~4 Alkyl, C 1~4 Haloalkyl, halo, cyano, OH, -OC 1~4 The conjugate described in item 28, or a pharmaceutically acceptable salt thereof, is piperazinyl, morpholinyl, piperidinyl, azepanil, or pyrrolidinyl, substituted with 1 to 4 substituents independently selected from the group consisting of alkyl and oxo. [Item 30]L 2 is a conjugate, or a pharmaceutically acceptable salt thereof, as described in any one of items 3 to 29, which is -C(O)-. [Item 31] R 1B OH, N(H)CH2CO2H, -N(H)CHR 1e CO2H, -N(H)-CH2CH2-(N(CH2CO2H)CH2CH2) n Selected from the group consisting of -N(CH2CO2H)2 and -N(CH2CO2H)-CH2CH2-N(CH2CO2H)2; R 1e This refers to the conjugate described in item 30, which is -CH2CO2H, or a pharmaceutically acceptable salt thereof. [Item 32] The bio-orthogonal portion is [ka] The conjugate described in any one of items 1 through 31, or a pharmaceutically acceptable salt thereof. [Item 33] A pharmaceutical composition comprising a conjugate described in any one of items 1 to 32, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [Item 34] A method for treating cancer or enhancing or inducing an immune response, comprising administering to a subject in need a therapeutically effective amount of a conjugate described in any one of items 1 to 32, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in item 33, and a therapeutic support composition, wherein the therapeutic support composition comprises a biocompatible support and a formula [ka] (In the formula, R 20 These are hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', C(= 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''; Each instance of R' and R'' is independently selected from hydrogen, aryl, and alkyl; Each instance of R''' is independently selected from aryl and alkyl groups; R 30 These are halogens, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl; Ra , R 31a and R 31b Each of these is independently hydrogen, a C1-C6 alkyl group, or a C1-C6 haloalkyl group; (t is 0, 1, 2, 3, or 4) A method comprising a tetrazine-containing group. [Item 35] The method according to Item 34, wherein the tetrazine-containing group is linked to or directly bound to the hyaluronic acid biocompatible support. [Item 36] The therapeutic support composition is of formula (II) [ka] (In the formula, G 2 teeth, [ka] and; R 22 (It is a linker of 1 to 100 linked atoms.) The method according to item 34 or 35, comprising the substituted hyaluronic acid units. [Item 37]G 2 teeth, [ka] The method described in item 36. [Item 38]G 2 teeth, [ka] and; R 20 is hydrogen or C 1~4 The method described in item 37, wherein the alkyl group is alkyl. [Item 39] The method according to any one of items 34 to 38, wherein the method is a method for treating cancer. [Item 40] The method according to Item 39, wherein 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 / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma. [Item 41] The method according to Item 39 or 40, wherein the cancer is a solid tumor. [Item 42] The method according to Item 39 or 40, wherein the cancer is a soft tissue sarcoma. [Item 43] The method according to Item 42, wherein the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma. [Item 44] The method described above is a method for enhancing or inducing an immune response, as per items 34-38. The method described in any one of the items. [Item 45] The method according to Item 44, wherein the immune response is an increase in one or more of the leukocytes, lymphocytes, monocytes, and eosinophils. [Item 46] The method according to any one of items 34 to 45, further comprising administering a therapeutically effective dose of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, or a trans-cyclooctemprodrug thereof. [Item 47] A kit comprising a conjugate or pharmaceutically acceptable salt thereof as described in any one of items 1 through 32, or a pharmaceutical composition as described in item 33, and instructions for its use. [Item 48] The kit according to item 45, further comprising the therapeutic support composition described in any one of items 34 to 38.

Claims

1. A conjugate comprising an immunomodulatory agent payload conjugated to one or more bioorthogonal moieties, wherein the immunomodulatory agent payload is selected from the group consisting of therapeutic monoclonal antibodies, cytokines, chemokines, chemokine antagonists, and immune checkpoint inhibitor payloads; or a pharmaceutically acceptable salt thereof.

2. The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein each bioorthogonal portion independently comprises trans-cyclooctene or tetrazine.

3. A conjugate according to claim 1 or 2 of formula (I), or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 (In the formula, G is the bioorthogonal part, and each time G appears, it is independent. 【Chemistry 2】 And; L １ Each instance of it is an independent linker; m is an integer between 1 and 150; D １ This is an immunomodulatory payload; R １Ａ Each time it appears, C １～４ Alkyl, C １～４ Haloalkyl and C １～４ Independently selected from the group consisting of alkoxys; q is 0, 1, or 2; q1 is either 0 or 1; R １Ｂ Each time it appears, G １ , OH, -NR １ｃ -C １～４ alkylene - G １ , -NR １ｃ -C １～４ alkylene - N(R １ｄ ) ２ , -NR １ｃ -C １～６ alkylene - N(C １～４ alkyl) ３ ＋ , -N(R １ｃ ), CHR １ｅ CO ２ H, -N(R １ｃ ), -C １～６ alkylene - CO ２ H, -N(R １ｆ ), -C ２～４ alkylene - (N(C １～４ alkylene - CO ２ H), -C ２～４ alkylene) ｎ , -N(C １～４ alkylene - CO ２ H) ２ , -N(R １ｃ ), CHR １ｅ C(O)OC １～６ alkyl, -N(R １ｃ ), -C １～６ alkylene - C(O)OC １～６ alkyl, -N(R １ｆ ), -C ２～４ alkylene - (N(C １～４ alkylene - C(O)OC １～６ alkyl), -C ２～４ alkylene) ｎ , -N(C １～４ alkylene - C(O)OC １～６ alkyl) ２ , -N(R １ｃ ), -C １～６ alkylene - SO ３ H, -N(R １ｃ ), -(CH ２ CH ２ O) １～３ , -CH ２ CH ２ N((CH ２ CH ２ O) １～３ -C １～６ Alkylene-CO2 ２ H) ２ , and -N(R １ｃ )-CH(CH ２ O-(CH ２ CH ２ O) ０～２ -C １～６ Alkylene-CO2 ２ H) ２ Independently selected from the group consisting of; R １ｃ and R １ｄ Each time it appears, it independently contains hydrogen or C １～４ It is alkyl; R １ｅ Each time it appears, it is independently -C １～４ Alkylene-CO2 ２ H, -C １～４ Al Kiren-CONH ２ , or -C １～４ It is alkylene-OH; R １ｆ Each time it appears, hydrogen and C appear independently. １～６ Alkyl, or C １～４ Alkylene-CO2 ２ It is H; Each instance of n is independently 0, 1, 2, or 3; L ２ Each time it appears, -C(O)- and C １～３ Independently selected from the group consisting of alkylenes; G １ Each instance is a heterocyclyl that is independently and arbitrarily substituted.

4. The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory agent payload is an immune checkpoint inhibitor payload.

5. The conjugate according to claim 4, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload.

6. The conjugate according to claim 5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload.

7. The conjugate according to claim 6, or a pharmaceutically acceptable salt thereof, wherein the PD-1 inhibitor payload is nivolumab, pembrolizumab, pidilizumab, cintilimab, or AMP-224 payload.

8. The conjugate according to claim 5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload.

9. The conjugate according to claim 8, or a pharmaceutically acceptable salt thereof, wherein the PD-L1 inhibitor payload is atezolizumab, avelumab, durvalumab, or BMS-936559 payload.

10. The conjugate according to claim 5, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload.

11. The conjugate according to claim 10, or a pharmaceutically acceptable salt thereof, wherein the CTLA4 inhibitor payload is an ipilimumab or tremelimumab payload.

12. The conjugate according to claim 4, or a pharmaceutically acceptable salt thereof, wherein the immune checkpoint inhibitor payload is an indoleamine 2,3-dioxygenase (IDO) inhibitor payload.

13. The conjugate according to claim 12, or a pharmaceutically acceptable salt thereof, wherein the IDO inhibitor payload is indoximod or epacadostat payload.

14. The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory agent payload is a cytokine payload.

15. The conjugate according to claim 14, or a pharmaceutically acceptable salt thereof, wherein 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.

16. The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory agent payload is a chemokine payload.

17. The conjugate according to claim 16, or a pharmaceutically acceptable salt thereof, wherein the chemokine payload is a CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CXCL14 payload.

18. The conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the immunomodulatory agent payload is a chemokine antagonist payload.

19. The conjugate according to claim 18, or a pharmaceutically acceptable salt thereof, wherein the chemokine antagonist payload is a prelixafor payload.

20. The immunomodulatory payload comprises a polypeptide, the conjugate according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof.

21. The conjugate according to claim 20, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises one or more lysine residues.

22. The conjugate according to claim 21, or a pharmaceutically acceptable salt thereof, wherein each time the bioorthogonal portion appears, it is bound to one of the one or more lysine residues.

23. A conjugate according to any one of claims 3 to 22, or a pharmaceutically acceptable salt thereof, wherein m is 1 to 20.

24. L １ is -OC(O)L ４ - or -OC １～６ alkylene C(O)L ４ -; L ４ is a bond, -N(R １２ )-C ２～３ alkylene-N(R １３ ), -CH(NH C(O)R １４ ), C １～４ alkylene-S-S-C １～４ alkylene-OC(O)-, -NHNH C(O)CH(NH C(O)R １５ ), CH ２ C(O)-, -C １～６ alkylene-CH(G ｘ ), OC(O)-, 【Transformation 3】 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-CO2 ２ H, or -C １～４ Alkilen-CONH ２ And; G ｘ is halogen, C １～４ Alkyl, C １～４ Haloalkyl, C １～４ A conjugate according to any one of claims 3 to 23, or a pharmaceutically acceptable salt thereof, wherein the phenyl is optionally substituted with one to five substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

25. G-L １ Each time it appears, independently, 【Chemistry 4】 The conjugate according to claim 24, or a pharmaceutically acceptable salt thereof.

26. G-L １ Each time it appears, independently, 【Transformation 5】 The conjugate according to claim 25, or a pharmaceutically acceptable salt thereof.

27. R １Ｂ G １ ,OH,-NR １ｃ -C １～４ Alkilen-G １ , -NR １ｃ -C １～４ Alkylene-N(R) １ｄ ) ２ , -N(R １ｃ ) CHR １ｅ CO ２ H, -N(R １ｃ )CH ２ CO ２ H, and -N(R １ｆ ) - CH ２ CH ２ - (N(CH ２ CO ２ H) CH ２ CH ２ ) ｎ -N(CH ２ CO ２ H) ２ Selected from the group consisting of; R １ｅ is, -CH ２ CO ２ H, -CH ２ CH ２ CO ２ H, -CH ２ CONH ２ ien-CH ２ CH ２ CONH ２ ien-CH ２ OH, or -CH(CH ３ ) OH; R １ｆ is hydrogen or CH ２ CO ２ A conjugate according to any one of claims 3 to 26, or a pharmaceutically acceptable salt thereof, wherein H.

28. R １Ａ C １～４ It is alkyl; R １Ｂ G １ ,OH,-NR １ｃ -C １～４ Alkilen-G １ , -NR １ｃ -C １～４ Alkylene-N(R) １ｄ ) ２ , -N(R １ｃ ) CHR １ｅ CO ２ H, -N(R １ｃ )CH ２ CO ２ H, and -N(R １ｆ ) - CH ２ CH ２ - (N(CH ２ CO ２ H) CH ２ CH ２ ) ｎ -N(CH ２ CO ２ H) ２ Selected from the group consisting of; R １ｅ is, -C １～４ Alkylene-CO2 ２ It is H; R １ｆ is hydrogen or C １～４ Alkylene-CO2 ２ It is H; G １ This is a 4- to 8-membered monocyclic heterocycline containing a first nitrogen atom and, optionally, one other heteroatom selected from nitrogen, oxygen, and sulfur, G １ It is bonded to the first nitrogen, and optionally, C １～４ Alkyl, C １～４ Haloalkyl, halo, cyano, OH, -OC １～４ It is substituted with one to four substituents independently selected from the group consisting of alkyl and oxo; The conjugate according to any one of claims 3 to 26, or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, or 2.

29. R １Ａ CH ３ And; R １ｅ is, -CH ２ CO ２ It is H; R １ｆ is hydrogen or CH ２ CO ２ It is H; G １ It is bonded via a cyclic nitrogen atom, and optionally, C １～４ Alkyl, C １～４ Haloalkyl, halo, cyano, OH, -OC １～４ The conjugate according to claim 28, or a pharmaceutically acceptable salt thereof, is piperazinyl, morpholinyl, piperidinyl, azepanil, or pyrrolidinyl, substituted with one to four substituents independently selected from the group consisting of alkyl and oxo.

30. L ２ The conjugate according to any one of claims 3 to 29, or a pharmaceutically acceptable salt thereof, wherein is -C(O)-.

31. R １Ｂ is OH, N(H)CH ２ CO ２ H, -N(H)CHR １ｅ CO ２ H, -N(H)-CH ２ CH ２ - (N(CH ２ CO ２ H) CH ２ CH ２ ) ｎ -N(CH ２ CO ２ H) ２ , and -N(CH ２ CO ２ H)-CH ２ CH ２ -N(CH ２ CO ２ H) ２ Selected from the group consisting of; R １ｅ is, -CH ２ CO ２ The conjugate according to claim 30, or a pharmaceutically acceptable salt thereof, wherein H.

32. The aforementioned bio-orthogonal portion, 【Transformation 6】 A conjugate according to any one of claims 1 to 31, or a pharmaceutically acceptable salt thereof.

33. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

34. A method for treating cancer or enhancing or inducing an immune response, comprising administering to a subject in need a therapeutically effective amount of a conjugate according to any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 33, and a therapeutic support composition, wherein the therapeutic support composition comprises a biocompatible support and a formula 【Transformation 7】 (In the formula, R ２０ This includes hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, 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''; Each instance of R' and R'' is independently selected from hydrogen, aryl, and alkyl; Each instance of R''' is independently selected from aryl and alkyl groups; R ３０ These are halogens, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or cycloalkenyl; R ａ , R ３１ａ and R ３１ｂ These are, independently, hydrogen and C １ ~C ６ Alkyl, or C １ ~C ６ It is a haloalkyl; (t is 0, 1, 2, 3, or 4) A method comprising a tetrazine-containing group.

35. The method according to claim 34, wherein the tetrazine-containing group is linked to or directly bound to the hyaluronic acid biocompatible support.

36. The therapeutic support composition is of formula (II) 【Transformation 8】 (In the formula, G ２ teeth, 【Chemistry 9】 And; R ２２ (This is a linker consisting of 1 to 100 linked atoms.) The method according to claim 34 or 35, comprising a substituted hyaluronic acid unit.

37. G ２ teeth, 【Chemistry 10】 The method according to claim 36.

38. G ２ teeth, 【Chemistry 11】 And; R ２０ is hydrogen or C １～４ The method according to claim 37, wherein the alkyl group is used.

39. The method according to any one of claims 34 to 38, wherein the method is a method for treating cancer.

40. The method according to claim 39, wherein 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 / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

41. The method according to claim 39 or 40, wherein the cancer is a solid tumor.

42. The method according to claim 39 or 40, wherein the cancer is a soft tissue sarcoma.

43. The method according to claim 42, wherein the soft tissue sarcoma is fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

44. The method according to any one of claims 34 to 38, wherein the method is a method for enhancing or inducing an immune response.

45. The method according to claim 44, wherein the immune response is an increase in one or more of the leukocytes, lymphocytes, monocytes, and eosinophils.

46. The method according to any one of claims 34 to 45, further comprising administering a therapeutically effective dose of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulator, or a trans-cyclooctemprodrug thereof.

47. A kit comprising a conjugate according to any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 33, and instructions for use thereof.

48. The kit according to claim 45, further comprising the therapeutic support composition according to any one of claims 34 to 38.

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