Tetrazine conjugates for in VIVO targeted delivery of a payload

Abstract

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

Description

Atty Docket No. 63XT-401927-WO TETRAZINE CONJUGATES FOR IN VIVO TARGETED DELIVERY OF A PAYLOAD CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U. S. C. § 119(e) to U. S. Provisional Application Numbers 63 / 791,193, filed April 18, 2025, which is incorporated by reference in its entirety.FIELD

[0001] The present disclosure relates generally to tetrazine conjugates, including antibody-tetrazine conjugates, for bioorthogonal delivery of a payload to a targeted location in a subject, which conjugates have applications, e.g., in the treatment of cancer, tumor growth, and immunotherapy.BACKGROUND

[0002] Bioorthogonal conjugation or click reactions are selective and orthogonal (non-interacting with) functionalities found in biological systems, and have found use in various applications in the fields of chemistry, chemical biology, molecular diagnostics, and medicine, where they can be used to facilitate the selective manipulation of molecules, cells, particles and surfaces, and the tagging and tracking of biomolecules in vitro and in vivo. These reactions include the Staudinger ligation, the azide-cyclooctyne cycloaddition, and the inverse-electron-demand Diels-Alder reaction.SUMMARY

[0003] Provided herein are targeting moieties which comprise an antibody or antibody fragment moiety covalently bonded to one or more tetrazine moieties. It is contemplated that the targeting moieties described herein achieve a desirable payload release that is not achievable with other targeting moieties known in the art. The targeting moieties described herein are designed to, once administered to a subject, localize at a target site within the subject. The targeting moieties can be administered locally or systemically. Once administered, a prodrug comprising a payload or therapeutic agent and one or more complimentary bioorthogonal components (i.e., a trans-cyclooctene moiety) can be administered, which when in contact with the targeting moiety in vivo, allows for targeted delivery of the payload or therapeutic agent. In some embodiments, the targeting moiety is a therapeutic targeting moiety. In some embodiments, the targeting moieties described herein comprise a diagnostic agent such that the targeting moieties described herein can be used in diagnosing conditions or diseases, with or without administering a payload or therapeutic agent.

[0004] In some embodiments, provided is a method for treating cancer, comprising administering to a subject in need thereof, a support composition as described herein to a target location, and administering to the subject a conjugate, or the pharmaceutically acceptable salt or composition thereof, as described herein.

[0005] In some embodiments, the cancer is metastatic. In some embodiments the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, squamousAtty Docket No. 63XT-401927-WO cell carcinoma of head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi’s sarcoma, Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma Wilms’ tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

[0006] In some embodiments, the cancer is a melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue carcinoma, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a lymphoma or leukemia. In some embodiments, the cancer is a hematologic malignancy.DETAILED DESCRIPTION

[0007] The following description sets forth exemplary embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0008] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (i.e.. compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.A. Definitions

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of tire present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0010] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include pluralAtty Docket No. 63XT-401927-WO references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0011] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10% ” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0012] The conjunctive term “or” includes any and all combinations of one or more listed elements associated by the conjunctive term. For example, the phrase “an apparatus comprising A or B” may refer to an apparatus including A where B is not present, an apparatus including B where A is not present, or an apparatus where both A and B are present. The phrases “at least one of A, B,... and N” or “at least one of A, B,... N, or combinations thereof’ are defined in the broadest sense to mean one or more elements selected from the group comprising A, B,... and N, that is to say, any combination of one or more of the elements A, B,... or N including any one element alone or in combination with one or more of the other elements which may also include, in combination, additional elements not listed.

[0013] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0014] The term “alkyl” as used herein, means a straight or branched, saturated hydrocarbon chain containing from 1 to 30 carbon atoms. The term “lower alkyl” or “Ci-Ce-alkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “C1-C3- alkyl” means a straight or branched chain hydrocarbon containing from 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tertbutyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3 -methylhexyl, 2,2-dimethylpentyl, 2, 3 -dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.Atty Docket No. 63XT-401927-WO

[0015] The term “alkoxy” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0016] The term “alkenyl” as used herein, means a hydrocarbon chain containing from 2 to 30 carbon atoms with at least one carbon-carbon double bond. The alkenyl group may be substituted or unsubstituted. For example, the alkenyl group may be substituted with an aryl group, such as a phenyl.

[0017] The term “alkynyl.” as used herein, refers to straight or branched monovalent hydrocarbyl groups having from 2 to 30 carbon atoms, such as 2 to 20, or 2 to 10 carbon atoms and having at least 1 site of triple bond unsaturation. The term “alkyne” also includes non-aromatic cycloalkyl groups of from 5 to 20 carbon atoms, such as from 5 to 10 carbon atoms, having single or multiple rings and having at least one triple bond. Examples of such alkynyl groups include, but are not limited to acetylenyl (-C=CH), and propargyl (-CH2C=CH), and cycloalkynyl moieties, such as, but not limited to, substituted or unsubstituted cyclooctyne moieties.

[0018] The term “alkoxyalkyl” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0019] The term “alkylene” as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 30 carbon atoms, for example, of 2 to 10 carbon atoms. Representative examples of alkylene 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-.

[0020] The term “amino acid” refers to both natural and unnatural amino acids, protected natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids include 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and pyrrolidine and selenocysteine. Non-natural amino acids refer to amino acid analogs having the same basic chemical structure as a naturally occurring amino acid, i.e., by way of example only, an a-carbon attached to a hydrogen, carboxyl group, amino group, and R group. Such analogs can have a modified R group (e.g., norleucine as an example) or retain a modified peptide backbone while retaining the same basic chemical structure as a natural amino acid. Non-limiting examples of non-natural amino acids or amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, homophenylalanine, ornithine, formyl glycine, phenyl glycine, para-azidophenyl glycine, para-azidophenylalanine, para-acetophenylalanine, 4-(3-methyl-( 1.2,4,5-tetrazine))-phenylglyine, and4-(3-methyl-(l,2,4,5-tetrazine))-phenylalanine.

[0021] The term “aryl” as used herein, refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or multiple rings (e.g., bicyclic or tricyclic) including fused systems. RepresentativeAtty Docket No. 63XT-401927-WO examples of aryls include, but are not limited to, phenyl, naphthyl, and anthracenyl. The monocyclic, bicyclic, and tricyclic aryls are connected to the parent molecular moiety through any carbon atom contained within tire rings, and can be unsubstituted or substituted. The aromatic bicyclic ring system or aromatic tricyclic ring system does not contain non-aromatic rings. Thus, if a bicyclic ring system or tricyclic ring system contains a non-aromatic ring, tire ring system is a cycloalkyl or heterocyclyl, depending on whether a heteroatom is present in the non-aromatic ring, regardless of the point of attachment to tire remainder of the molecule.

[0022] In some embodiments, the term “aryl” as used herein, refers to a phenyl group, or bicyclic aryl or tricyclic aryl fused ring systems. Bicyclic fused ring systems are exemplified by a phenyl group appended to tire parent molecular moiety and fused to a phenyl group. Tricyclic fused ring systems are exemplified by a phenyl group appended to the parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryls include, but arc not limited to, naphthyl.Representative examples of tricyclic aryls include, but are not limited to, anthracenyl. The monocyclic, bicyclic, and tricyclic aryls are connected to tire parent molecular moiety through any carbon atom contained within the rings, and can be unsubstituted or substituted.

[0023] The term “azide” as used herein, refers to the functional group -N3.

[0024] The term “cycloalkyl” as used herein, refers to a non-aromatic carbocyclic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10, carbon atoms and zero heteroatoms. Cycloalkyl ring systems may contain one or more double bonds, so long as the ring is not aromatic; and thus, the term cycloalkyl includes cycloalkenyl ring systems. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl. “Cycloalkyl” also includes carbocyclic ring systems in which a cycloalkyl group is fused to an aryl or heteroaryl as defined herein, regardless of tire point of attachment to the remainder of the molecule.

[0025] In some embodiments, the term “cycloalkyl” as used herein, refers to a carbocyclic ring system containing three to ten carbon atoms, zero heteroatoms and zero double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl. “Cycloalkyl” also includes carbocyclic ring systems in which a cycloalkyl group is appended to the parent molecular moiety and is fused to an aryl group as defined herein, a heteroaryl group as defined herein, or a heterocycle as defined herein.

[0026] The term “cycloalkenyl” as used herein, means a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl orcycloheptenyl.Atty Docket No. 63XT-401927-WO

[0027] The term “cyclooctene” as used herein, refers to a substituted or unsubstituted non-aromatic cyclic alkyl group of 8 carbon atoms, having a single ring with a double bond. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).

[0028] The term “fluoroalkyl” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by fluorine. Representative examples of fluoroalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl such as 3,3,3-trifluoropropyl.

[0029] The term “alkoxyfluoroalkyl” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.

[0030] The term “fluoroalkoxy” as used herein, means at least one fluoroalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom. Representative examples of fluoroalkyloxy include, but are not limited to, difluoromethoxy, trifluoromethoxy and 2.2,2-trifluoroethoxy.

[0031] The term “halogen” or “halo” as used herein, means Cl, Br, I, or F.

[0032] The term “haloalkyl” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen.

[0033] The term “haloalkoxy” as used herein, means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.

[0034] The term “heteroalkyl” as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms has been replaced by a heteroatom selected from S, Si, O, P and N. The heteroatom may be oxidized. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, and alkyl sulfides.

[0035] The term “hctcroaryl” as used herein, refers to an aromatic group having a single ring, multiple rings or multiple fused rings, with one or more ring heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments, the term “heteroaryl” as used herein, refers to an aromatic monocyclic ring or an aromatic bicyclic ring system or an aromatic tricyclic ring system. The aromatic monocyclic rings are five or six membered rings containing al least one heteroatom independently selected from the group consisting of N, O and S (e.g.. 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). The five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. Representative examples of monocyclic heteroaryl include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl. pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-l,2-dihydropyridinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl. indolyl. quinolinyl, imidazopyridine, benzooxadiazolyl,Atty Docket No. 63XT-401927-WO and benzopyrazolyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. The monocyclic, bicyclic, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings, and can be unsubstituted or substituted. In some embodiments, the aromatic bicyclic ring system or aromatic tricyclic ring system does not contain non-aromatic rings. Thus, if a bicyclic ring system or tricyclic ring system contains a non-aromatic ring, the ring system is a cycloalkyl or heterocyclyl, depending on whether a heteroatom is present in the non-aromatic ring, regardless of the point of attachment to the remainder of the molecule.

[0036] In some embodiments, the five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. In some embodiments, exemplary bicyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring appended to the parent molecular moiety and fused to a monocyclic cycloalkyl group, as defined herein, a monocyclic aryl group, as defined herein, a monocyclic heteroaryl group, as defined herein, or a monocyclic heterocycle, as defined herein. In some embodiments, the tricyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring appended to the parent molecular moiety and fused to two of a monocyclic cycloalkyl group, as defined herein, a monocyclic aryl group, as defined herein, a monocyclic heteroaryl group, as defined herein, or a monocyclic heterocycle, as defined herein.

[0037] The terms “heterocyclyl,” “heterocycle,” or “heterocyclic” as used herein, refers to a non-aromatic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10, carbon atoms and at least one (c.g., 1-5, 1-4, 1-3, 1-2, or 1) hctcroatom, and optionally one or more oxo and / or double bonds. The terms “heterocyclyl”, “heterocycle” or “heterocyclic” include monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridged ring systems, provided that at least one non-aromatic ring system containing at least one heteroatom is present. In some embodiments, the monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, the three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. In some embodiments, the five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. In some embodiments, the six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to. azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, l,3-dimethylpyrimidine-2,4(lH,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl,Atty Docket No. 63XT-401927-WO thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring system in which two non- adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl). 2,3-dihydro-lH-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5 -epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-lH-l,4-methanocyclopenta[c]furan, aza-adamantane (l-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings, and can be unsubstituted or substituted.[0038J The term “hydroxyl” as used herein, means an -OH group.

[0039] The term “hydroxyalkyl” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a hydroxyl group.

[0040] In some instances, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix “Cx-Cy-” or “Cx.y,” wherein x is the minimum and y is the maximum number of carbon atoms in the substituent. Thus, for example, “Ci-Cs-alkyl” and “Cualkyl” refer to an alkyl substituent containing from 1 to 3 carbon atoms. The two conventions “Cx-Cy-” and “Cx.y” arc used interchangeably and have the same meaning.

[0041] The term “substituted” refers to a group that may be further substituted with one or more nonhydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =0, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.Atty Docket No. 63XT-401927-WO

[0042] The term “tetrazine” refers to a substituted or unsubstituted aromatic cyclic group of 2 carbon atoms and 4 nitrogen atoms, having a single ring with three double bonds. Examples of tetrazine groups include 1,2,3,4-tetrazine and 1,2,4,5-tetrazine. As used herein, 1,2, 4, 5 -tetrazine is referred to as a “Tz” group.

[0043] The term “selectively delivering” refers to delivering an agent (e.g., a payload) to an organ or tissue (or portion thereof) in need of treatment or diagnosis, without significant binding to other nontarget organs or tissues (or portions thereof). In some embodiments, the targeting moieties, or therapeutic targeting moiety, described herein do not themselves have a therapeutic effect, but rather are designed to allow the selective or targeted delivery of a therapeutic agent. However, it may be that the targeting moiety does have a therapeutic effect, and thus, such constructs are not excluded by the present disclosure.

[0044] The term “payload” refers to an agent for delivery to a target site in a subject. Payloads include therapeutic agents.

[0045] The term “therapeutic agent” refers to an agent capable of treating and / or ameliorating a condition or disease, or one or more symptoms thereof, in a subject. Therapeutic agents of the present disclosure also include prodrug forms of therapeutic agents.

[0046] The term “diagnostic agent” refers to agents dial assist in diagnosing conditions or diseases. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, radionuclides, and the like. Paramagnetic agents are imaging agents that are magnetic under an externally applied field. Examples of paramagnetic agents include, but are not limited to, iron particles including iron nanoparticles and iron microparticles. Optical probes are fluorescent compounds that can be detected by excitation at one wavelength of radiation and detection at a second, different, wavelength of radiation. Optical probes of the present disclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1 ’-dioctadecyl-3,3,3 ’,3 ’-tetramethylindodicarbocyanine perchlorate) and DiR (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include, but are not limited to,3H,nC,13N,18F,19F,60Co,64Cu,67Cu,68Ga,82Rb,89Zr,90Sr,90Y,99Tc,99mTc,111In,1231,1241,1251,1291,1311,137Cs,177Lu,186Re,188Re,211At,212Pb,225Ac, Rn, Ra, Th, U, Pu, and241Am.

[0047] The term “targeting agent” refers to a chemical or biological agent that specifically binds to a target (e.g., a targeted organ or tissue), thereby forming a stable association between the targeting agent and the specific target. By “stably associated” or “stable association” is meant that a moiety is bound to or otherwise associated with another moiety or structure under standard physiological conditions. Bonds may include covalent bonds and non-covalent interactions, such as, but not limited to. ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipoledipole interactions, and the like. A targeting agent may be a member of a specific binding pair, such as,Atty Docket No. 63XT-401927-WO but are not limited to: a member of a receptor / ligand pair; a ligand-binding portion of a receptor; a member of an antibody / antigen pair; an antigen-binding fragment of an antibody; a hapten; a member of a lectin / carbohydrate pair; a member of an enzyme / substrate pair; biotin / avidin; biotin / streptavidin; digoxin / antidigoxin; a member of a DNA or RNA aptamer binding pair; a member of a peptide aptamer binding pair; and the like. Targeting agents include ligands that specifically bind (or substantially specifically bind) a particular clinically-relevant target receptor or cell surface target. The ligand can be an antibody, peptide, nucleic acid, phage, bacteria, virus, or other molecule with a specific affinity for a target receptor or cell surface target. Examples of receptors and cell surface targets include, but are not limited to. PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, LT4 - human mAh directed against the inhibitory immune checkpoint receptor immunoglobulin-like transcript 4 (ILT4; leukocyte immunoglobulin-like receptor subfamily B member 2. LILRB2, lymphocyte immunoglobulin-like receptor 2, LIR2, monocyte / macrophage immunoglobulin-like receptor 10, MIR-10, CD85d, or other cellular receptors or cell surface targets. Additional examples are included in various embodiments disclosed herein.

[0048] The term “targeted organ or tissue’’ refers to an organ or tissue that is being targeted for delivery of the payload. Representative organs and tissues for targeting include those that can be targeted by chemical or biological targeting agents, as well as those organs and tissues that cannot be targeted by chemical or biological targeting agents.

[0049] The term “implanting” refers to surgical implantation into a subject’s body.

[0050] The term “contacting” or “contact” refers to the process of bringing into contact at least two distinct species such that they can interact with each other, such as in a non-covalent or covalent binding interaction or binding reaction. It should be appreciated, however, the resulting complex or reaction product can be produced directly from an interaction or a reaction between the added reagents or from an intermediate from one or more of tire added reagents or moieties, which can be produced in the contacting mixture.

[0051] The term “binding agent” refers to an agent having a functional group capable of forming a covalent bond to a complementary functional group of another binding agent in a biological environment. Binding between binding agents in a biological environment may also be referred to as bioconjugation. Binding agents include bioorthogonal binding agents, which are binding agents having bioorthogonal functional groups. Bioorthogonal functional groups of bioorthogonal binding agents selectively react with a complementary bioorthogonal functional group of another bioorthogonal binding partner.Selective reaction between bioorthogonal binding partners can minimize side reactions with other binding agents, biological compounds, or other non-complementary bioorthogonal binding agents or non-complementary bioorthogonal functional groups. Bioorthogonal moieties or functional groups of bioorthogonal binding agents include, but are not limited to, an azide and alkyne for formation of a triazole via Click-chemistry reactions, trans-cyclooctene (TCO) and tetrazine (Tz) (c.g., 1, 2,4,5-Atty Docket No. 63XT-401927-WO tetrazine), and others. The binding agents useful in the present disclosure may have a high reactivity with the corresponding binding agent so that the reaction is rapid.

[0052] The term “functionalized” refers to a moiety having a functional group attached to the moiety, such as for example a moiety having a binding agent functional group (e.g., a bioorthogonal functional group) attached thereto.

[0053] The term “administering” refers to any suitable route of administration to a subject, such as, but not limited to, oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional. intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to the subject.

[0054] The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0055] The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides (e.g., Br, Cl, I), sulfonate esters (e.g., triflate, mesylate, tosylate, and brosylate), and nitrophenols.

[0056] The term “pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[0057] As used herein, the term “subject,” “patient,” or “organism” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which an agent(s) of the present disclosure may be administered may include mammals, particularly primates, especially humans. For veterinary applications, suitable subjects may include, for example, livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, suitable subjects may include mammals, such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.

[0058] The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition or symptom(s) thereof in a patient, such as a mammal (particularly a human) that includes: (a) ameliorating the disease or medical condition or symptom(s) thereof, such as, eliminating or causing regression of the disease or medical condition or symptom) s) thereof in a patient; (b) suppressing the disease or medical condition or symptom(s) thereof, for example by, slowing or arresting theAtty Docket No. 63XT-401927-WO development of the disease or medical condition or symptom(s) thereof in a patient; or (c) alleviating a symptom of the disease or medical condition or symptom(s) thereof in a patient.

[0059] The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

[0060] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0061] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0062] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 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 contemplated.

[0063] The compounds may exist as stereoisomers wherein asymmetric or chiral centers are present. The stereoisomers are “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The disclosure contemplates various stereoisomers and mixtures thereof, and these are specifically included within the scope of this disclosure. Stereoisomers include enantiomers and diastereomers and mixtures of enantiomers or diastereomers. Individual stereoisomers of tire compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography, and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel’s Textbook of Practical Organic Chemistry,” 5thedition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.Atty Docket No. 63XT-401927-WO

[0064] It should be understood that the compounds may possess tautomeric forms as well as geometric isomers, and that these also constitute an aspect of the disclosure.

[0065] The present disclosure also includes isotopically-labeled compounds, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the disclosure are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to,2H,3H,13C,14C,15N,18O,170,31P,32P,35S,18F, and36C1, respectively. Substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and, hence, may be preferred in some circumstances. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron-emitting isotopes that can be incorporated arenC,13N,15O, and18F. Isotopically-labeled compounds disclosed herein can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent.B. Targeting Moieties

[0066] Provided herein are targeting moieties which comprise a biocompatible support, an antibody or antibody fragment moiety, or in certain embodiments an antibody or antibody fragment moiety, covalently bonded to one or more tetrazine moieties. The targeting moieties described herein are designed to, once administered to a subject, localize at a target site within the subject. The targeting moieties can be administered locally or systemically. In some embodiments, the targeting moiety is a therapeutic targeting moiety. Once administered, a prodrug comprising a complimentary bioorthogonal component (i.e., a trans-cyclooctene moiety) can be administered, which when in contact with the targeting moiety in vivo, allows for targeted drug delivery of a payload or therapeutic agent. In some embodiments, the targeting moieties described herein comprise a diagnostic agent such that the targeting moieties described herein can be used in diagnosing conditions or diseases, with or without administering a payload or therapeutic agent.

[0067] Provided herein is a targeting moiety of Formula I, Formula II, or Formula III:Atty Docket No. 63XT-401927-WOwherein:X is a biocompatible support, antibody, antibody fragment moiety;p is 1-150;L, at each occurrence, is independently a linker;R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Ra, R31a, and R31bare each independently hydrogen, Ci-Ce-alkyl, or Ci-Ce-haloalkyl;ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl;R2, at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl. or cycloalkyl is optionally substituted with one to three Z1; each Z1is independently selected from halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl. alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR'. C(=O)R', C(=S)R', OC(=O)R'", SC(=O)R'". OC(=S)R'". SC(=S)R'", S(=O)R', S(=O)2R"', S(=O)2NR' R". C(=O)O-R'. C(=O)S-R', C(=S)O-R', C(=S)S-R'.C(=0)NR'R". C(=S)NR’R", NR'R". NR'C(=0)R". NR’C(=S)R”, NR'C(=0)0R". NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR". 0C(=0)NR’R", SC(=O)NR’R". OC(=S)R’R"’, SC(=S)R’R".NR'C(=0)NR" R", and NR’C(=S)NR" R";R' and R". at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R"’, at each occurrence, is independently selected from aryl and alkyl; andt, at each occurrence, is independently 0, 1, 2, 3, or 4.Atty Docket No. 63XT-401927-WO

[0068] In some embodiments, p is 1-16, or 4-12, or 2-10, or 5-10, or 1-8, or 3-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 0-4.

[0069] In some embodiments, the targeting moiety is of Formula I or II:wherein:X is an antibody or antibody fragment moiety;p is 1-16;L, at each occurrence, is independently a linker;R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Ra, R31aand R31bare each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl;R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; R’" at each occurrence is independently selected from aryl and alkyl; andt, at each occurrence, is independently 0, 1, 2, 3, or 4.

[0070] In some embodiments, the moiety is represented by Formula IIA:

[0071] In some embodiments, the moiety is represented by Formula III:Atty Docket No. 63XT-401927-WO

[0072] In some embodiments, ring A is phenyl, pyrimidinyl, triazinyl. oxazolyl, isoxazole, imidazolyl, oxadiazolyl.6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5.6.7, 8-tetrahydropyrido[4.3-d]pyrimidinyl. or 5.6,7,8-tetrahydropyrido [3,4-d]pyrimidinyl.

[0073] In some embodiments, at least one of the moiety:

[0074] In some embodiments, at least one of the moiety:is represented by a formula selected from:Atty Docket No. 63XT-401927-WOwherein the R1is dihydropyran.Atty Docket No. 63XT-401927-WO

[0075] In some embodiments, R2, at each occurrence, is independently halo, alkyl, or haloalkyl.

[0076] In some embodiments, t, at each occurrence, is 0.

[0077] In some embodiments, R2, at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl. In some embodiments, R2, at each occurrence, is independently halo, alkyl, or haloalkyl. In some embodiments, R2, at each occurrence, is independently halo or alkyl.

[0078] In some embodiments, X is a biocompatible support.

[0079] In some embodiments, ring A is other than pyridyl. In some embodiments, ring A is other than aryl. In some embodiments, ring A is other than phenyl.

[0080] In some embodiments, the targeting moiety is of the formula:Atty Docket No. 63XT-401927-WO

[0081] In some embodiments, p is 1-10, 1-5, 2-8, 4-10, 1-4, or 1-3.

[0082] In some embodiments, p is an average loading on X and is about 1, about 2. about 3. about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0083] In some embodiments, the targeting moiety is of Formula 1-1 or Formula 1-2:Atty Docket No. 63XT-401927-WO

[0084] In some embodiments, X is a biocompatible support which comprises a particle, polymer, viscous or non-viscous liquid material, gel, hydrogel, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, or a protein.

[0085] In some embodiments, X is a biocompatible support which comprises a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, chondroitin sulfate, heparin, a suitable sugar-based biomaterial, a polyphosphazene, polyanhydride, polyacetal, poly(ortho ester), polyphosphoester, polycaprolactone, polyurethane, polylactide, polycarbonate, polyamide, polyether, a blend / composites / or co-polymer thereof, collagen, gelatin, elastin, an elastin-like polypeptide, albumin, fibrin, poly(gamma-glutamic acid), poly(L-lysine), poly(L-glutamic acid), or poly(aspartic acid). In some embodiments, X is a biocompatible support comprising hyaluronic acid with a molecular weight of about 5-25 kD, or 26-75 kD, or 76-200 kD, or >201 kD.

[0086] In some embodiments, X is an antibody or antibody fragment moiety. In some embodiments X is a peptide, including, but not limited to cyclic peptides.Antibody and Antibody Fragment Moieties

[0087] In certain embodiments, die targeting agent, or X, is an antibody, or antibody fragment moiety, that targets one or more of CD25 (NCBI Gene ID 3559). CEA (NCBI Gene ID 634), CEACAM5 (NCBI Gene ID 1048), ASPH (NCBI Gene ID 444), EGFR (NCBI Gene ID 1956). EPCAM (NCBI Gene ID 4072), VEGFR (NCBI Gene ID 3791), PDGFR (NCBI Gene ID 5159). TROP2 (NCBI Gene ID 4070), Nectin4 (NCBI Gene ID 81607), PSMA (NCBI Gene ID 2346), BCMA (NCBI Gene ID 608), CD22 (NCBI Gene ID 933), CD20 (NCBI Gene ID 920), CD19 (NCBI Gene ID 930), CD79b (NCBI Gene ID 974), CD38 (NCBI Gene ID 952), CD45 (NCBI Gene ID 5788), Endoglin (NCBI Gene ID 2022), FGFR2 (NCBI Gene ID 14183), C4.4A (NCBI Gene ID 27076), Claudin-18.2 (NCBI Gene ID 51208), MMP9 (NCBI Gene ID 4318), Folate receptor (NCBI Gene ID 2348), DLL3 (NCBI Gene ID 10683), CD138 (NCBI Gene ID 6382). CD56 (NCBI Gene ID 4684), CD37 (NCBI Gene ID 951), CD74 (NCBIAtty Docket No. 63XT-401927-WO Gene ID 972), mesothelin (NCBI Gene ID 10232), IL-6R (NCBI Gene ID 3570), SLAMF7 (NCBI Gene ID 57823), BAFF (NCBI Gene ID 10673), CSPG4 (NCBI Gene ID 1464), MUC1 (NCBI Gene ID 4582), GPC3 (NCBI Gene ID 2719), HER2 (NCBI Gene ID 2064), HER3 (NCBI Gene ID 2065), CD30 (NCBI Gene ID 943), CD33 (NCBI Gene ID 945), CD123 (NCBI Gene ID 3563), GPNMB (NCBI Gene ID 10457), cMET (NCBI Gene ID 4233), CD142 (NCBI Gene ID 2152), NaPi2B (NCBI Gene ID 10568), GCC (NCBI Gene ID 2984), STEAP1 (NCBI Gene ID 26872), MUC16 (NCBI Gene ID 94025), CD70 (NCBI Gene ID 970), CD44 (NCBI Gene ID 960), (NCBI Gene ID ), Antibody fragments (NCBI Gene ID ), vWF (NCBI Gene ID 7450), TNF (NCBI Gene ID 7124). IL-6R (NCBI Gene ID 3570), BCMA (NCBI Gene ID 608), ADAMTS5 (NCBI Gene ID 11096), CX3CR1 (NCBI Gene ID 1524), CXCR4 (NCBI Gene ID 7852), TfRl (NCBI Gene ID 7037), VEGFR (NCBI Gene ID 3791), or PSMA (NCBI Gene ID 2346).

[0088] In some embodiments, X is an antibody or antibody fragment moiety which targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, CD25, CLDN4 (NCBI Gene ID 1364), TNC (NCBI Gene ID 3371), FN1 (NCBI Gene ID 2335), ITGAV (NCBI Gene ID 3685), TACSTD2 (NCBI Gene ID 4070). CSPG4 (NCBI Gene ID 1464), CD174 (NCBI Gene ID 2525), GPNMB (NCBI Gene ID 10457), GPC1 (NCBI Gene ID 2817), ITGB6 (NCBI Gene ID 3694), SEZ6 (NCBI Gene ID 124925), SLITRK6 (NCBI Gene ID 84189), NaPi-2b (NCBI Gene ID 20531), ZIP6 (NCBI Gene ID 25800), ROR1 (NCBI Gene ID 4919), or ROR2 (NCBI Gene ID 4920).

[0089] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets one or more of CD25 (NCBI Gene ID 3559), CEA (NCBI Gene ID 634), CEACAM5 (NCBI Gene ID 1048), ASPH (NCBI Gene ID 444), EGFR (NCBI Gene ID 1956), EPCAM (NCBI Gene ID 4072), VEGFR (NCBI Gene ID 3791), PDGFR (NCBI Gene ID 5159), TROP2 (NCBI Gene ID 4070). Nectin4 (NCBI Gene ID 81607), PSMA (NCBI Gene ID 2346), CD22 (NCBI Gene ID 933), CD20 (NCBI Gene ID 920). CD19 (NCBI Gene ID 930), CD79b (NCBI Gene ID 974), CD38 (NCBI Gene ID 952), CD45 (NCBI Gene ID 5788), Endoglin (NCBI Gene ID 2022), FGFR2 (NCBI Gene ID 14183), C4.4A (NCBI Gene ID 27076), Claudin-18.2 (NCBI Gene ID 51208), MMP9 (NCBI Gene ID 4318), Folate receptor (NCBI Gene ID 2348), DLL3 (NCBI Gene ID 10683), CD138 (NCBI Gene ID 6382), CD56 (NCBI Gene ID 4684), CD37 (NCBI Gene ID 951), CD74 (NCBI Gene ID 972), mesothelin (NCBI Gene ID 10232), IL-6R (NCBI Gene ID 3570), SLAMF7 (NCBI Gene ID 57823), BAFF (NCBI Gene ID 10673). MUC1 (NCBI Gene ID 4582), GPC3 (NCBI Gene ID 2719). HER2 (NCBI Gene ID 2064), HER3 (NCBI Gene ID 2065), CD30 (NCBI Gene ID 943). CD33 (NCBI Gene ID 945). CD123 (NCBI Gene ID 3563), GPNMB (NCBI Gene ID 10457). cMET (NCBI Gene ID 4233), CD 142 (NCBI Gene ID 2152), NaPi2B (NCBI Gene ID 10568), GCC (NCBI Gene ID 2984), STEAP1 (NCBI Gene ID 26872), MUC16 (NCBI Gene ID 94025), CD70 (NCBI Gene ID 970), CD44 (NCBI Gene ID 960), (NCBI Gene ID), Antibody fragments (NCBI Gene ID), vWF (NCBI Gene ID 7450), TNF (NCBI Gene ID 7124), IE-6R (NCBI Gene ID 3570), CSPG4 (NCBI Gene ID 1464), BCMA (NCBI Gene ID 608), ADAMTS5 (NCBI Gene ID 11096), CX3CR1 (NCBI Gene ID 1524), CXCR4 (NCBI Gene ID 7852), TfRl (NCBI Gene IDAtty Docket No. 63XT-401927-WO 7037), VEGFR (NCBI Gene ID 3791), PSMA (NCBI Gene ID 2346), ANTXR1 (NCBI Gene ID 84168), or FAP (NCBI Gene ID 2191).

[0090] In some embodiments, X is an antibody or antibody fragment moiety which targets TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, CD25, ANTXR1, CSPG4, or FAP.

[0091] In some embodiments, X is an antibody or antibody fragment moiety which targets CEA, CEACAM5, ASPH, EGFR, EPCAM. VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, CD25, ANTXR1, or FAP.

[0092] In some embodiments, X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CEA, or CEACAM5.

[0093] In some embodiments, X is an antibody or antibody fragment moiety which targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, or CD25.

[0094] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD25, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment moiety derived therefrom.

[0095] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CEA, such as labetuzumab, 15-1-32, PR1A3, cT84.66, C17, or C44 or an antibody fragment moiety derived therefrom.

[0096] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CEACAM5, such as Tusamitamab or CC4, or an antibody fragment moiety derived therefrom.

[0097] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets ASPH, such as PAN-622, or an antibody fragment moiety derived therefrom.

[0098] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets EGFR, such as cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, duligotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, or laprituximab, or an antibody fragment moiety derived therefrom.

[0099] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets EPCAM, such as oportuzumab, citatuzumab. tucotuzumab, catumaxomab. edrecolomab, or adecatumumab, or an antibody fragment moiety derived therefrom.

[0100] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets VEGFR, such as ramucizumab, ramucirumab, or vulinacimab, or an antibody fragment moiety derived therefrom.

[0101] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment moiety derived therefrom.Atty Docket No. 63XT-401927-WO

[0102] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TROP2, such as Sacituzumab or Pr1E11, or an antibody fragment moiety derived therefrom.

[0103] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets Nectin4, such as enfortumab, or an antibody fragment moiety derived therefrom.

[0104] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets PSMA, such as J591 or MLN591. or an antibody fragment moiety derived therefrom.

[0105] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets BCMA, such as Belantamab, or an antibody fragment moiety derived therefrom.

[0106] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD22, such as moxetumomab, inotuzumab, epratuzumab, or pinatuzumab, or an antibody fragment moiety derived therefrom.

[0107] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD20, such as ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, or ibritumomab, or an antibody fragment moiety derived therefrom.

[0108] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD19. such as loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab. or MDX-1342, or an antibody fragment moiety derived therefrom.

[0109] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD79b, such as polatuzumab, or an antibody fragment moiety derived therefrom.

[0110] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD38, such as isatuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment moiety derived therefrom.

[0111] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD45, such as I-131-BC8, or lomab-B, or an antibody fragment moiety derived therefrom.

[0112] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets endoglin, such as carotuximab, or an antibody fragment moiety derived therefrom.

[0113] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets FGFR2, such as bemarituzumab or aprutumab, or an antibody fragment moiety derived therefrom.

[0114] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets C4.4A, such as lupartumab, or an antibody fragment moiety derived therefrom.

[0115] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets Claudin-18.2, such as zolbetuximab, or claudiximab, or an antibody fragment moiety derived therefrom.

[0116] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets MMP9, such as andecaliximab, or an antibody fragment moiety derived therefrom.Atty Docket No. 63XT-401927-WO

[0117] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets folate receptor, such as mirvetuximab, farletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment moiety derived therefrom.

[0118] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets DLL3, such as rovalpituzumab, or an antibody fragment moiety derived therefrom.

[0119] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD 138, such as indatuximab, or an antibody fragment moiety derived therefrom.

[0120] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment moiety derived therefrom.

[0121] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD37, such as BI 836826, otlertuzumab, or naratuximab, or an antibody fragment moiety derived therefrom.

[0122] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD74, such as milatuzumab, or an antibody fragment moiety derived therefrom.

[0123] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets mesothelin, such as anetumab, amatuximab, or MMOT-0530A, or an antibody fragment moiety derived therefrom.

[0124] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets IL-6R, such as tocilizumab or sarilumab, or an antibody fragment moiety derived there from.

[0125] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets SLAMF7, such as elotuzumab, or an antibody fragment moiety derived therefrom.

[0126] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets BAFF, such as belimumab, or an antibody fragment moiety derived therefrom.

[0127] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, or cantuzumab, or an antibody fragment moiety derived therefrom.

[0128] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets GPC3, such as codrituzumab, ECT204, or MDX-1414, or an antibody fragment moiety derived therefrom.

[0129] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment moiety derived therefrom.

[0130] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets HER3, such as patritumab, seribantumab, lumretuzumab, elgemtumab, AV-203, CDX-3379. or GSK284933, or an antibody fragment moiety derived therefrom.

[0131] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD30, such as brentuximab, or an antibody fragment moiety derived therefrom.Atty Docket No. 63XT-401927-WO

[0132] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD33, such as gemtuzumab, BI 835858, vadastuximab, or lintuzumab, or an antibody fragment moiety derived therefrom.

[0133] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment moiety derived therefrom.

[0134] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets GPNMB, such as glembatumumab, or an antibody fragment moiety derived therefrom.

[0135] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment moiety derived therefrom.

[0136] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD 142, such as tisotumab, or an antibody fragment moiety derived therefrom.

[0137] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets NaPi2B, such as lifastuzumab, or an antibody fragment moiety derived therefrom.

[0138] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets GCC, such as indusatumab, or an antibody fragment moiety derived therefrom.

[0139] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets STEAP1, such as vandortuzumab, or an antibody fragment moiety derived therefrom.

[0140] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets MUC16, such as sofituzumab, or an antibody fragment moiety derived therefrom.

[0141] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD70, such as vorsetuzumab, or an antibody fragment moiety derived therefrom.

[0142] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CD44. such as bivatuzumab, or an antibody fragment moiety derived therefrom.

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

[0144] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TNF, such as ozoralizumab, V565, or PF-05230905, or an antibody fragment moiety derived therefrom.

[0145] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets IE-6R, such as vobarilizumab, or an antibody fragment moiety derived therefrom.

[0146] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets BCMA, such as LCAR-B38M, or an antibody fragment moiety derived therefrom.

[0147] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets ADAMTS5, such as M6495, or an antibody fragment moiety derived therefrom.Atty Docket No. 63XT-401927-WO

[0148] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CX3CR1, such as BI 655088, or an antibody fragment moiety derived therefrom.

[0149] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CXCR4, such as AD-214 or ALX-0651, or an antibody fragment moiety derived therefrom.

[0150] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TfRl, such as TXB4, or an antibody fragment moiety derived therefrom.

[0151] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets VEGFR, such as CDP791, or an antibody fragment moiety derived therefrom.

[0152] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets PSMA, such as GY 1, or an antibody fragment moiety derived therefrom.

[0153] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets FN1, such as L19 or NJB2, or an antibody fragment moiety derived therefrom.

[0154] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets FAP, such as F19, OMTX005 or sibrotuzumab, or an antibody fragment moiety derived therefrom.

[0155] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TNG, such as F16 or R6N or an antibody fragment moiety derived therefrom.

[0156] In some embodiments, X is an antibody.

[0157] In some embodiments, the antibody is daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32. PR1A3, cT84.66, tusamitamab, CC4. PAN-622, cetuximab, necitumumab, nimotuzumab. matuzumab, AMG595, depatuxizumab. dapatuxizumab. duligotuzumab, futuximab. GC1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, laprituximab, oportuzumab, citatuzumab, tucotuzumab, catumaxomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, vulinacimab, olaratumab, ramucirumab, sacituzumab, Pr1E11, enfortumab, J591, MLN591, belantamab, moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MOR202, TAK-079, 1-131-BC8, lomab-B, carotuximab, bemarituzumab, aprutumab, lupartumab, zolbetuximab, claudiximab, andecaliximab, mirvetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, rovalpituzumab, indatuximab, lorvotuzumab, promiximab, BI 836826, otlertuzumab, naratuximab, milatuzumab, anetumab, amatuximab, MMOT-0530A, sarilumab, elotuzumab, belimumab, KL-6, MY.1E12, hMUCl-lH7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, cantuzumab, codrituzumab, ECT204, MDX-1414, pertuzumab, trastuzumab, margetuximab, patritumab, seribantumab, lumretuzumab, elgemtumab, AV-203, CDX-3379, GSK284933, brentuximab, gemtuzumab, BI 835858, vadastuximab, lintuzumab, KHK2823, taclotuzumab, G4723A, glembatumumab, telisotuzumab, onartuzumab, SAIT301, tisotumab, lifastuzumab, indusatumab.Atty Docket No. 63XT-401927-WO vandortuzumab, sofituzumab, vorsetuzumab, bivatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, vobarilizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, OMTX005, sibrotuzumab, C17, C44, F16 or R6N.

[0158] In some embodiments, X is an antibody selected from atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, and trastuzumab.

[0159] In some embodiments, X is an antibody fragment moiety.

[0160] In some embodiments, X or tire antibody fragment moiety is selected from tire group consisting of a single-chain variable fragment (scFv), a divalent (or bivalent) single -chain variable fragment (di-scFvs, bi-scFvs), an antigen-binding fragment (Fab), a single-domain antibody (sdAb), a VHH, an antigen-binding protein, a DotBody, an affibody, a DARPin, a DART, a TandAb, a diabody. a ribobody, a centyrin, a knottin. an affilin, an affimer, an alphabody, an anticalin, an atrimer, an avimer, a fynomer, a kunitz domain, an obody, a pronectin, a repebody, and a bicyclic peptide or a Humabody.

[0161] In some embodiments, X is an antibody fragment moiety selected from the group consisting of a single-chain variable fragment (scFv), a divalent (or bivalent) single-chain variable fragment (di-scFvs, bi-scFvs), an antigen-binding fragment (Fab), a single-domain antibody (sdAb), and a VHH.

[0162] In some embodiments, the antibody fragment moiety is an antigen-binding protein a DotBody, affibody, DARPin, DART, TandAb, diabody, ribobody, centyrin, knottin, affilin, affimer, alphabody, anticalin, atrimer, avimer, fynomer, kunitz domain, obody, pronectin, repebody, bicyclic peptide, or Humabody.

[0163] In some embodiments, X or the antibody fragment moiety is an antigen-binding fragment (Fab). The Fab is a region on an antibody that binds to antigens, and is comprised of one constant and one variable domain of each of the heavy and the light chain. In some embodiments, the Fab comprises four domains: VH, CHI, VL and CL1. In some embodiments, the Fab comprises 400-500 amino acids, or 440-480 amino acids. In some embodiments, the Fab has a molecular weight of about 50 kDa, or 40-55 kDa, or 45-50 kDa, or 45-55 kDa.

[0164] In some embodiments, the Fab of trastuzumab, enfortumab, brentuximab, sacituzumab, L19 -binding to FN-1 (Gene ID 2335), or F16 - binding to TNC (Gene ID 3371).

[0165] In some embodiments, the antibody fragment moiety comprises one or more PEG units, which may enhance circulation life.

[0166] In some embodiments, tire antibody fragment moiety is an antigen-binding protein. Antigenbinding proteins are proteins which are designed to be antibody-mimetics, exhibiting a high affinity and specificity for a given target. In some embodiments, tire antigen-binding protein is a single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-Atty Docket No. 63XT-401927-WO chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to die amino terminus of the VH sequence.

[0167] In some embodiments, the antigen-binding protein is about 5-10 kDa, or about 7 kDa. In some embodiments, the antigen-binding protein is about are about 50-80, or 60-70, or 66 amino acids in length. In some embodiments, the antigen-binding protein comprises a cysteine only at the N- or C-terminus. In some embodiments, the antigen-binding protein comprises a cysteine only at the N-terminus. In some embodiments, the antigen-binding protein comprises a cysteine only at the C-terminus.

[0168] In some embodiments, the antibody fragment moiety is an antigen-binding protein that targets TNC, FN1, CLDN4. MMP9. EpCAM, ITGAV. CEA. CEACAM5. ASPH, EGFR, EPCAM. VEGFR, PDGFR, CSPG4, TROP2, Nectin4, PSMA, BCMA, HER2, or CD25. In some embodiments, the antibody fragment moiety is an antigen-binding protein that targets HER2. Antigen-binding proteins can be prepared and tested according to standard methods or purchased from commercial sources (e.g., Affilogic).

[0169] In some embodiments, the antibody fragment moiety is derived from daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, CT84.66, tusamitamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, duligotuzumab, futuximab. GC1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, laprituximab, oportuzumab, citatuzumab, tucotuzumab, catumaxomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, vulinacimab, olaratumab, ramucirumab, sacituzumab, Pr1E11, enfortumab, J591, MLN591, belantamab, moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MOR202, TAK-079, I-131-BC8, lomab-B, carotuximab, bemarituzumab, aprutumab, lupartumab, zolbetuximab, claudiximab, andecaliximab, mirvetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, rovalpituzumab, indatuximab, lorvotuzumab, promiximab, BI 836826, otlertuzumab, naratuximab, milatuzumab, anetumab, amatuximab, MMOT-0530A, sarilumab, elotuzumab, belimumab, KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, cantuzumab, codrituzumab, ECT204, MDX-1414, pertuzumab, trastuzumab, margetuximab, patritumab, seribantumab, lumretuzumab, elgemtumab, AV-203, CDX-3379, GSK284933, brentuximab, gemtuzumab, BI 835858, vadastuximab, lintuzumab, KHK2823, taclotuzumab, G4723A, glembatumumab, telisotuzumab, onartuzumab, SAIT301, tisotumab, lifastuzumab, indusatumab, vandortuzumab, sofituzumab, vorsetuzumab, bivatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, vobarilizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1. L19, NJB2, F19, OMTX005, sibrotuzumab, C17, C44, F16 or R6N.

[0170] In some embodiments, X is an antibody fragment moiety derived from atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab,Atty Docket No. 63XT-401927-WO ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, or trastuzumab.

[0171] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets vWF, such as Caplacizumab.

[0172] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TNF, such as Ozoralizumab, V565, or PF-05230905.

[0173] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets IL-6R, such as Vobarilizumab.

[0174] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets BCMA, such as LCAR-B38M.

[0175] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets ADAMTS5, such as M6495.

[0176] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CX3CR1, such as BI 655088.

[0177] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CXCR4, such as AD-214 or ALX-0651.

[0178] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TfR1, such as TXB4.

[0179] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets VEGFR, such as CDP791.

[0180] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets PSMA, such as GYl.

[0181] In some embodiments, the antibody fragment moiety is caplacizumab, ozoralizumab, V565, PF-05230905, vobarilizumab, LCAR-B38M, M6495, BI 655088, AD-214, ALX-0651, TXB4, CDP791, or GY1.

[0182] In some embodiments, X further comprises an imaging contrast agent. In some embodiments, the imaging contrast agent is a protein.Linker Moieties

[0183] In some embodiments, L is bonded to X via a cystine or lysine residue on X.

[0184] In some embodiments, L is a non-cleavable linker.

[0185] In some embodiments, L is a cleavable linker.

[0186] In some embodiments, L comprises one or more amino acids.Atty Docket No. 63XT-401927-WO

[0187] In some embodiments, L comprises a polypeptide.

[0188] In some embodiments, L comprises one or more of a hydrazone, a hydrazide, a disulfide, a N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), a N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), a 4-(4’-acetylphenoxy)butanoic acid (AcBut), one or more linear or branched, natural or unnatural amino acid, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety.

[0189] In some embodiments, L comprises 1 to 100 linking atoms, from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms, or from 1 to 40 linking atoms, or from 1 to 30 linking atoms, or from 1 to 20 linking atoms, or from 1 to 10 linking atoms, or from 1 to 5 linking atoms, or from 5 to 30 linking atoms, or from 10 to 30 linking atoms, or from 5 to 40 linking atoms, or from 10 to 40 linking atoms.

[0190] In some embodiments, L comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moieties; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, CM alkoxy, and CM haloalkyl.

[0191] In some embodiments, L is an alkylene linker optionally comprising one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.

[0192] In some embodiments, linker L is of the formula:-Y10-(CHR130)n-Y20-(CHR140)n"-Y30-(CHR150)ni”-Y40- wherein:each of Y10, Y20, Y30, and Y40are independently a bond, -NR110-, -O-, -S(0)o-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-, -OC(O)O-, -(CH2CH2O)1-5-, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, arylene, or heteroarylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, CM alkoxy, and C haloalkyl;each R110is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;each R120is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;each R130is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain;each R140is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain;Atty Docket No. 63XT-401927-WO each R150is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain; andn’, n", and m" are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0193] In some embodiments, L is of the formula:-Y10-(CH2)n-Y20-(CH2)m-Y30- wherein:each of Y10, Y20, and Y30are independently a bond, -NR110-, -O-, -S(0)o-2-, -NR110C(O)-.-C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, C alkyl, C alkoxy, and C haloalkyl;each R110is independently hydrogen, Cu alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;each R120is independently hydrogen, C alkyl, CM haloalkyl, aryl, hctcroaryl, cycloalkyl or heterocyclyl; andn' and m" are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0194] In certain embodiments, each R11" is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R120is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. In certain embodiments, the linker is not a bond.

[0195] The linker L may comprise one or more of polyethylene glycol (e.g., PEG having an average molecular weight of from 300 g / mol to 10,000 g / mol). ethylene- l,2-diylbis(methylcarbamate, an arylene (e.g., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.

[0196] In some embodiments, the linker comprises one or more of:Atty Docket No. 63XT-401927-WO

[0197] In some embodiments, the linker comprises one or more of:[01981 In some embodiments, the linker comprises one or more of:Atty Docket No. 63XT-401927-WO

[0199] In some embodiments, the linker comprises one or more of:

[0200] In some embodiments, the linker comprises one or more H. In someembodiments, the linker comprises one or more

[0201] In some embodiments, the linker is, or comprises one or more:

[0202] In some embodiments, the linker is, or comprises one or more:Atty Docket No. 63XT-401927-WO

[0203] In some embodiments, the linker comprises one or more natural or unnatural amino acids, which may be referred to as a peptide linker. The linker may be a peptide linker made up of a carboxylic acyl unit, and one or more amino acids making up a protein or peptide sequence. The linker may also contain a self-immolating spacer which spaces the drug and the protein peptide sequence.

[0204] In some embodiments, the linker may be a peptide containing linker represented by “A — Y — Z — X2— W” in which “A” is the carboxylic acyl unit, “Y” and “Z” are each one or more natural or unnatural amino acids and together form a peptide sequence, and “X2” and “W” are optional additional linkers having from 1 to 50 linking atoms, or from 5 to 10 linking atoms, or from 1 to 10 linking atoms which spaces the peptide and the payload, D, or the bioorthogonal moiety. In certain embodiments, one or more of the amino acids in tire peptide linker is N-methylated.

[0205] In some embodiments, Y may be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In some embodiments Y may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0206] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline. In some embodiments Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0207] Exemplary Y-Z combinations include Valine-Citrulline; Valine- Alanine; and Alanine-Alanine.

[0208] In some embodiments, A is -OC(O)-.

[0209] In some embodiments, X2is -OC(O)-.

[0210] In some embodiments, W is -OC(O)-. In some embodiments, X2is absent and W is -OC(O)-.

[0211] In some embodiments, the moiety — X2— W comprises H. In someembodiments, the moiety — X2is HAtty Docket No. 63XT-401927-WO

[0212] In some embodiments,

[0213] In some embodiments,

[0214] In some embodiments, die peptide linker is specifically tailored so that it will be selectively cleaved (e.g., enzymatically cleaved) releasing the drug, such as by one or more of the tumor-associated proteases.

[0215] In some embodiments, the peptide linker has a chain length of two to four amino acid residues (i.e., a di-, tri-, or tetra-peptide). It will be understood, however, that peptide linkers up to five, six, seven, or eight amino acid residues may also suitably be employed.

[0216] In some embodiments, the peptide linker is Phe-Lys, Val-Lys, Vai- 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-N9-tosyl-Arg, or Phe-N9-Nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, VaLVal, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0217] In some embodiments, the linker L is, or comprises one or more of:Atty Docket No. 63XT-401927-WOMypocket No- 63XT-40V‘m «Atty Docket No. 63XT-401927-WO

[0218] In some embodiments, the linker L comprises one or more of:Atty Docket No. 63XT-401927-WO

[0219] In some embodiments, the linker L comprises one or more of:Atty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOR12, R13, R14, R15, and R19are each independently hydrogen or C1-4alkyl;R16is hydrogen, C1-4alkyl, –C1-4alkylene–OH, –C1-4alkylene–OC1-4alkyl, –C1-4alkylene–CO2H, or –C1-4alkylene–CONH2; andGxis phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, cyano, and nitro.

[0222] In some embodiments, the linker L comprises a carbonyl moiety for conjugating the tetrazine moiety to the linker or X. For example, the linker may comprise a polypeptide moiety (PPM) having the lysine residue and lysine side chain and the PPM may also have additional lysines, or other amino acid side chains conjugated to die carbonyl moiety. In some embodiments, the linker L may comprisepolypeptidelysine side chain

[0223] In some embodiments, the linker L is, or comprises one or more of:Atty Docket No. 63XT-401927-WO

[0224] In some embodiments, the linker L is, or comprises one or more of:Atty Docket No. 63XT-401927-WO

[0225] In some embodiments, tire linker L is, or comprises one or more of:

[0226] In some embodiments, the linker L is:Atty Docket No. 63XT-401927-WO

[0227] In some embodiments, the linker L is:Atty Docket No. 63XT-401927-WO

[0228] In some embodiments, the linker L is, or comprises one or more of

[0229] In some embodiments, the linker L is, or comprises one or more of:

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

[0231] In some embodiments, the linker L is, or comprises one or more of:

[0232] In some embodiments, the linker L is. or comprises one or more of:Atty Docket No. 63XT-401927-WO

[0233] In some embodiments, the linker L is, or comprises one or more of:

[0235] In some embodiments, the linker L is, or comprises one or more of:In one embodiment, provided is a targeting moiety oA targeting moiety of Formula I or Formula II or Formula III:wherein:X is a biocompatible support, antibody, antibody fragment moiety;p is 1-150;L, at each occurrence, is independently a linker;Atty Docket No. 63XT-401927-WO R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CSPG4, CEA, or CEACAM5; and L. at each occurrence, is independently a linker selected from the group consisting of:

[0239] In one embodiment, provided is a targeting moiety of Formula II:wherein:X is an antibody or antibody fragment moiety;p is 1-16;L, at each occurrence, is independently a linker;Atty Docket No. 63XT-401927-WO R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Ra, R31aand R31bare each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl;t, at each occurrence, is independently 0, 1, 2, 3, or 4;X is an antibody or antibody fragment moiety diat targets HER2, TROP2, Nectin-4, Claudin- 18.2, MMP9, mesothelin, FN1, FAP, TNG, or ECM, EPCAM, CSPG4, CEA, or CEACAM5; and L, at each occurrence, is independently a linker selected from the group consisting of:

[0240] In one embodiment, provided is a targeting moiety of Formula III:wherein:ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl;Atty Docket No. 63XT-401927-WO X is a biocompatible support, antibody, or antibody fragment moiety;p is 1-15;L. at each occurrence, is independently a linker;R2, at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, hctcroalkyl, aryl, hctcroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O) -cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;t, at each occurrence, is independently 0, 1, 2, 3, or 4;X is an antibody or antibody fragment moiety that targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNG, or ECM, EPCAM, CSPG4, CEA, or CEACAM5; and L. at each occurrence, is independently a linker selected from the group consisting of:

[0241] In some embodiments, ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl; wherein each may be optionally substituted.

[0242] In some embodiments, X is an antibody, or antibody fragment moiety, that targets HER2, TROP2, Nectin-4, FN1, CSPG4, FAP, TNC, or ECM.Atty Docket No. 63XT-401927-WO

[0243] In some embodiments, X is zolbetuximab, claudiximab, andecaliximab, anetumab, amatuximab, MMOT-0530A, L19, NJB2, F19, OMTX005, sibrotuzumab, F16, or R6N, trastuzumab, enfortumab, or sacituzumab, or an antibody fragment moiety derived therefrom.

[0244] In some embodiments, X is L19, NJB2, F19, C17, C44, OMTXG05, sibrotuzumab, F16, or R6N, trastuzumab, enfortumab, or sacituzumab, or an antibody fragment moiety derived therefrom.

[0245] In some embodiments, p is I- 100. In some embodiments, p is 1-75. In some embodiments, p is 1-50. In some embodiments, p is 1-30. In some embodiments, p is 1-20. In some embodiments, p is 1-10. In some embodiments, p is 5-10. In some embodiments, p is 1-15. In some embodiments, p is 8-12. In some embodiments, p is 1-8. 1-16, 1-25, 8-16, or 16-25.

[0246] In some embodiments, p is 1-12. In some embodiments, X is an antibody. In some embodiments, p is 1-6, or 5-6. In some embodiments, X is an antibody fragment moiety (e.g., Fab).

[0247] In some embodiments, p is 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 2-10, or 2-9, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 2-3, or 3-10, or 3-9, or 3-8, or 3-7, or 3-6, or 3-5, or 3-4, or 4-10, or 4-9, or 4-8, or 4-7, or 4-6, or 4-5, or 5-10, or 5-9, or 5-8, or 5-7, or 5-6, 6-10, or 6-9, or 6-8, or 6-7, 7-10, or 7-9, or 7-8, 8-10, or 8-9, or 9-10. In some embodiments, X is an antibody or an antibody fragment moiety.

[0248] In some embodiments, p is 1-16, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2. In some embodiments, X is an antibody fragment moiety (e.g., Fab).

[0249] In certain embodiments, p is 1-10, or 1-9, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 2-10, or 2-9, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 2-3, or 3-10, or 3-9, or 3-8, or 3-7, or 3-6, or 3-5, or 3-4, or 4-10, or 4-9, or 4-8, or 4-7, or 4-6, or 4-5, or 5-10, or 5-9, or 5-8, or 5-7, or 5-6, 6-10, or 6-9, or 6-8, or 6-7, 7-10, or 7-9, or 7-8, 8-10, or 8-9, or 9-10, or 1-8, or 1-16, or 1-25, or 8-16, or 16-25, and X is an antibody or an antibody fragment moiety of from 10 KDa to 30 KDa, or 15 KDa to 75 KDa, or 25-75 KDa, or 45-55 KDa, or less than 25 KDa, or less than 35 KDa, or less than 45 KDa, or about 50 KDa, or less than 55 KDa, or less than 65 KDa, or less than 75 KDa, or greater than 75 KDa.

[0250] In certain embodiments, p is dependent on the size and / or number of available binding sites on X for forming a covalent bond to L. In certain embodiments, when X is an antibody or an antibody fragment moiety greater than 75 KDa, p is 2-16. In certain embodiments, when X is an antibody or an antibody fragment moiety between 25-75 KDa, p is 1-10. In certain embodiments, when X is an antibody or an antibody fragment moiety between 45-55 KDa, p is 1 -10. In certain embodiments, when X is an antibody or an antibody fragment moiety of less than 25 KDa, p is 1-6, or 2-6, or 2-5, or about 1, about 2, about 3, about 4, about 5, or about 6.Atty Docket No. 63XT-401927-WO

[0251] In certain embodiments, provided is a compound selected from:Atty Docket No. 63XT-401927-WO C. Support Compositions

[0252] The support composition comprises a support. In certain embodiments, the support composition is a therapeutic support composition. Supports may be biocompatible supports compositions, i.e„ compatible with the subject’s body. In some instances, a support is non-toxic to the subject and does not substantially react with tissue or biological compounds in the subject. For example, the support can be a hydrogel, among others. A support is capable of implantation into a subject’s body and supporting binding agents (e.g., tetrazine-containing group), as well as payloads after the binding agents conjugate. Representative supports include, but are not limited to polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and the like. Supports also include particles, such as nanoparticles, microparticles, and the like.

[0253] Hydrogels may be polysaccharide hydrogels, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, chondroitin sulfate, heparin, and the like. Other suitable sugar -based biomaterials include those described in Polymer Advanced Technology, 2014, 25, 448-460. Polymers that may be used as the support can include, but are not limited to, polyphosphazenes, poly anhydrides, polyacetals, poly(ortho esters), polyphosphoesters, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, and blends / composites / co-polymers thereof. Representative polyethers include, but are not limited to, poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), triblock Pluronic ([PEG]n-[PPG]m-[PEG]n), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA). The support can also include proteins and other poly(amino acids), such as collagen, gelatin, elastin and elastin-like polypeptides, albumin, fibrin, poly(gamma- glutamic acid), poly(L-lysine), poly(L-glutamic acid), poly(aspartic acid), and the like.

[0254] 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 a hyaluronic acid (e.g., a non-hydrogel hyaluronic acid substantially without crosslinks). In some embodiments, the support is chitosin.

[0255] In certain embodiments, the support is a particle. Particles of the present disclosure can have a diameter that is 2 cm or less, such as 1.5 cm or less, or 1 cm or less, or 0.5 cm or less. For example, the particles can be nanoparticles or microparticles. Nanoparticles include particles having average dimensions in the nanometer scale (e.g., 1000 nm or less). Microparticles are particles having average dimensions in the micrometer scale (e.g., 1000 pm or less). By “average” is meant the arithmetic mean. In some embodiments, the nanoparticles have a diameter ranging from 1 nm to 1 pm, such as from 10 nm to 1 pm, or 25 nm to 1 pm, or 50 nm to 1 pm, or 75 nm to 1 pm, or 100 nm to 1 pm, or 150 nm to 1 pm, or 200 nm to 1 pm, or 250 nm to 1 pm, or 300 nm to 1 pm, or 350 nm to 1 pm, or 400 nm to 1 pm, or 450 nm to 1 pm, or 500 nm to 1 pm. In other embodiments, the microparticles have a diameter ranging from 1 pm to 1 mm, such as from 10 pm to 1 mm, or 25 µm to 1 mm, or 50 pm to 1 mm, or 75 pm to 1 mm, or 100 pm to 1 mm, or 150 pm to 1 mm, or 200 pm to 1 mm, or 250 pm to 1 mm, or 300 pm to 1 mm, orAtty Docket No. 63XT-401927-WO 350 pm to 1 mm, or 400 pm to 1 mm, or 450 pm to 1 mm, or 500 pm to 1 mm. In further embodiments, small particles on the order of 10-100 nm in diameter may be assembled to form larger complexes, such as clusters or assemblies on the order of 1-10 pm. Particles of the present disclosure may be substantially spherical, such that the particles have a substantially circular cross-section. Other particle shapes may also be used, such as, but not limited to, ellipsoid, cubic, cylindrical, conical, needle, or other irregular shapes.

[0256] A “particle” may take the form of any fabricated material, a molecule, cryptophan, a virus, a phage, etc. The particle may be composed of a material, such as, but not limited to, a metal, a ceramic, a plastic, a glass, a composite, a polymer, a hydrogel, and the like. For example, the particles may be made of an inert material, such as alginate or iron oxide. In some examples, the particles may be magnetic and can be formed from a paramagnetic, super-paramagnetic or ferromagnetic material, or other material that responds to a magnetic field. Further, a particle may be of any shape, for example, spheres, rods, non-symmetrical shapes, etc. The particles, or a group of several particles in a complex, may be functionalized with a receptor that has a specific affinity to bind to or interact with a clinically relevant substrate. The receptor may be inherent to the particle itself. For example, the particle itself may be a virus or a phage with an inherent affinity for certain substrates. Additionally or alternatively, the particles can be functionalized by covalently or otherwise attaching or associating a receptor that specifically binds or otherwise recognizes a particular clinically relevant substrate. The functionalized receptor can be an antibody, peptide, nucleic acid, phage, bacteria, virus, or any other molecule with a defined affinity for a target substrate. Examples of material diat may be used for die “particles” and / or “carrier” include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, poly anhydrides, polyphosphazines, poly( vinyl alcohols), polylalkylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers, pluronic polyols, polyoxamers, polyluronic acids), poly(vinylpyrrolidone) and copolymers or graft copolymers of any of the above. These examples do not limit their concentration, their cross-linking with different agents, their method of administration, their tailored degradation profiles and other characteristics known to those skilled in the art.

[0257] The particles, or a group of several particles in a complex, may be functionalized with a targeting agent (e.g., a ligand or antibody) that specifically binds (or substantially specifically binds) to a target (e.g., a target receptor or a cell surface target, such as a clinically relevant receptor or cell surface target (e.g., antigen)). The targeting agent may be attached directly to the particle itself. The targeting agent can be an antibody, peptide, nucleic acid, phage, bacteria, virus, or any other molecule with a specific affinity for a target receptor or cell surface target. In some instances, the receptor or cell surface target is PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, or other cellular receptors or cell surface targets. Other compounds or molecules, such as fluorophores or autofluorescent or luminescent markers, which may assist in detecting the particles (e.g., in vivo detection), may also be attached to theAtty Docket No. 63XT-401927-WO particles. The ligands and / or detectable labels may be attached directly to the particle or attached to the particle through bioorthogonal functional groups as described herein.

[0258] In certain embodiments, the support is a bone graft material, such as a bone graft substitute material. A bone graft substitute material is a material structurally similar to bone. In some instances, a bone graft substitute material is bioresorbable such that the bone graft substitute material can dissolve or be absorbed in the body over time. A bone graft substitute material can be osteoconductive, such that it facilitates blood vessel and new bone formation into the bone graft substitute material. In some instances, the bone graft substitute material is osteoinductive, such that facilitates the formation of new bone through active recruitment of mesenchymal stem cells from the surrounding tissue. For example, growth factors, such as bone morphogenetic proteins, may be included in the bone graft substitute material. Bone graft substitute materials include, but are not limited to, hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone chips, and the like, and combinations thereof.

[0259] In certain embodiments, the support compositions comprise substituted alginate having units of formula:salt thereof. wherein the dashed line represents a bond to L.

[0260] In some embodiments, the support compositions comprise substituted hyaluronic acid having units of formula:salt thereof, wherein the dashed line represents a bond to L.

[0261] The hyaluronic acid derivative includes a hyaluronic acid having a plurality of glucuronic acid units and a tetrazine-containing group linked or directly bonded to a glucuronic acid unit of the hyaluronic acid. The hyaluronic acid may also have a plurality of N-acetylglucosamine units. In certain embodiments, the N-acetylglucosamine units of the hyaluronic acid are not linked or conjugated to the tetrazine-containing group.

[0262] The tetrazine-containing group can be linked or directly bonded through a carboxylic acid of a glucuronic acid unit. The tetrazine-containing group can be incorporated into the hyaluronic acid from about 0.1% to about 80% as measured by the % of carboxylic acids being linked or conjugated to theAtty Docket No. 63XT-401927-WO tetrazine -containing group, such as about 1% to about 75%, about 5% to about 75%, about 10% to about 50%, or about 40% to about 75% as measured by the % of carboxylic acids being linked or conjugated to L of the tetrazine-containing group.

[0263] Additional support compositions are exemplified in WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126, the entire contents of each of which is incorporated herein by reference in their entirety.D. Trans-Cyclooctene Functionalized Prodrugs

[0264] Trans-cyclooctene functionalized prodrugs are known in the art, including prodrugs of anticancer agents, as described in WO2023 / 077129, WO2018 / 187740, WO2014 / 205126, WO2015 / 139025, and WO2017 / 044983, which are incorporated herein by reference. Further embodiments using trans-cyclooctene functionalized prodrugs follow.

[0265] In some embodiments, the trans-cyclooctene functionalized prodrugs is a conjugate comprised of a payload linked to one or more trans-cyclooctene moieties.

[0266] In some embodiments, the conjugate (or trans-cyclooctene functionalized prodrug) comprises an immunomodulatory agent payload, such as for example, an immunomodulatory agent payload selected from the group consisting of a cytokine, chemokine, chemokine antagonist, therapeutic monoclonal antibody, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.

[0267] In some embodiments, the immunomodulatory agent payload is an inhibitor of a cytokine payload, or a pharmaceutically acceptable salt thereof.

[0268] In some embodiments, the inhibitor of a cytokine payload is an inhibitor of TNF-a, infliximab, certolizumab, TGF-0, galunisertib, fresolimumab, M7824, CSF-1, pexidartinib, or cabiralizumab.

[0269] In some embodiments, the conjugate comprises a monoclonal antibody, or a pharmaceutically acceptable salt thereof.

[0270] In some embodiments, the conjugate comprises a therapeutic protein payload, or a pharmaceutically acceptable salt thereof.

[0271] In some embodiments, the therapeutic protein payload is an antibody-based drug, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin, or thrombolytic.

[0272] In some embodiments, the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen.

[0273] In some embodiments, the therapeutic protein payload is a payload of erythropoietin (EPO, e.g., native EPO or synthetic EPO (see, e.g., US 2003 / 0191291), such as, but not limited to, e.g., PROCRIT®, EPREX®, or EPOGEN® (epoetin-a), ARANESP® (darbepoietin-a), NEORECORMON®, EPOGIN® (epoetin-0), and the like); a growth hormone (e.g., a somatotropin, e.g., GENOTROPIN®,Atty Docket No. 63XT-401927-WO NUTROPIN®, NORDITROPIN®, SAIZEN®, SEROSTIM®, HUMATROPE®, etc.); therapeutic monoclonal antibody (e.g. Atezolizumab, Avelumab, Bevacizumab, Cemiplimab, Cetuximab, Daratumumab, Dinutuximab, Durvalumab, Elotuzumab, Ipilimumab, Isatuximab, Mogamulizumab, Necitumumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Panitumumab, Pembrolizumab, Pertuzumab, Ramucirumab, Rituximab, Trastuzumab etc.); human growth hormone (hGH); bovine growth hormone (bGH); follicle stimulating hormone (FSH); interferon (e.g., IFN-y, IFN-a, IFN-, IFN-co; IFN-r, consensus interferon, and the like); insulin (e.g., Novolin, Humulin. Humalog. Lantus, Ultralente, etc.), insulin-like growth factor (e.g., IGF-I, IGF-II); blood factors (e.g., Factor X, tissue plasminogen activator (TPA), and the like, such as, but not limited to, e.g., ACTIVASE® (alteplase) tissue plasminogen activator. NOVOSEVEN® (recombinant human factor Vila), Factor Vila, Factor VIII (e.g., KOGENATE®), Factor IX, β-globin. hemoglobin, and the like); 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, and the like), transforming growth factors (e.g., TGF-beta, TGF-alpha); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®), IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, and the like); a growth factor (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF, e.g., REGRANEX® (beclapermin)), fibroblast growth factors (FGFs, e.g., aFGF, bFGF, such as FIBLAST® (trafermin)), glial cell line -derived growth factor (GDNF), nerve growth factor (NGF), stem cell factor (e.g., STEMGEN® (ancestim)), keratinocyte growth factor, a hepatocyte growth factor, and the like); a soluble receptor (e.g., a TNF-a-binding soluble receptor such as ENBREL® (etanercept), a soluble VEGF receptor, a soluble interleukin receptor, a soluble y / 5 T cell receptor, and the like); an enzyme (e.g., a-glucosidase, CERAZYME® (imiglucarase, -glucocerebrosidase, CEREDASE® (alglucerase); an enzyme activator (e.g., tissue plasminogen activator); a chemokine (e.g., IP-10, Mig, Groa / IL-8, regulated and normal T cell expressed and secreted (RANTES), MIP-la, MIP-lp, MCP-1, PF-4, and the like); an angiogenic agent (e.g., vascular endothelial growth factor (VEGF); an anti-angiogenic agent (e.g., a soluble VEGF receptor); a protein vaccine; a neuroactive peptide such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropinreleasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, galanin, growth hormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, etc.; other proteins such as a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha- 1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, a tissue factor, an insulin-like growth factor, a luteinizing hormone, a follicle stimulating hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor, a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiogenin, angiotropin. fibrin; hirudin; a leukemia inhibitory factor; or an IL-1 receptor antagonist (e.g.. Kineret® (anakinra)); and the likeAtty Docket No. 63XT-401927-WO

[0274] In some embodiments, the conjugate is of Formula X, or a pharmaceutically acceptable salt thereof:whereinm is an integer from 1-150;G, at each occurrence, is independently an optionally substituted trans-cyclooctene moiety; D is a payload;L1, at each occurrence, is independently a linker.

[0275] In some embodiments of the conjugates described herein, each trans-cyclooctene moiety is independently:wherein:RIA, at each occurrence, is independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, and C1-4alkoxy;q is 0, 1, or 2;ql is 0 or 1;RIB, at each occurrence, is independently selected from the group consisting of G1, -OH, -NRlc-Ci.4alkylene-G1, -NRlc-Ci.4alkylene-N(Rld)2, -NRlc-Ci.6alkylene-N(Ci.4alkyl)3+, -N(Rlc)CHRleCO2H, -N(Rlc)-Ci-6alkylene-CO2H, -N(Rlf)-C2-4alkylene-(N(Ci.4alkylene-CO2H)-C2-4alkylene)n-N(Ci.4alkylene-CO2H)2, -N(Rlc)CHRleC(O)OCi.6alkyl, -N(Rlc)-Ci-6alkylene-C(O)OCi. ealkyl, -N(Rlf)-C2-4alkylene-(N(Ci.4alkylene-C(O)OCi.6alkyl)-C2-4alkylene)n-N(Ci.4alkylene-C(O)OCi.6alkyl)2, -N(Rlc)-Ci.6alkylene-SO3H, -N(Rlc)-(CH2CH2O)i.3-CH2CH2N((CH2CH2O)i.3-Ci.6alkylene- CO2H)2, and -N(Rlc)-CH(CH20-(CH2CH20)o-2-Ci.6alkylcnc-C02H)2;Rlcand Rld, at each occurrence, are independently hydrogen or Ci.4alkyl;Rle, at each occurrence, is independently -C1-4alkylene-CO2H, -Ci-4alkylene-CONH2, or -Ci-4alkylene-OH;Rlf, at each occurrence, is independently hydrogen, Ci-ealkyl, or Ci-4alkylene-CO2H;Atty Docket No. 63XT-401927-WO n, at each occurrence, is independently 0, 1, 2, or 3;L2, at each occurrence, is independently selected from the group consisting of –C(O)– and C1-3 alkylene; andG1, at each occurrence, is independently an optionally substituted heterocyclyl.

[0276] In some embodiments, die conjugate is of Formula X, or a pharmaceutically acceptable salt thereof,whereinG is the trans-cyclooctene moiety, and G, at each occurrence, is independentlyL1, at each occurrence, is independently a linker;m is an integer from 1-150;D is a payload;RIA, at each occurrence, is independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, and C1-4alkoxy;q is 0, 1 or 2;ql is 0 or 1;RIB, at each occurrence, is independently selected from the group consisting of G1, OH, -NRlc-Ci 4alkylene-G1, -NRlc-Ci-4alkylene-N(Rld)2, -NRlc-Ci-6alkylene-N(Ci-4alkyl)3+, -N(Rlc)CHRleCO2H, -N(Rlc)-Ci.6alkylene-CO2H, -N(Rlf)-C2.4alkylene-(N(Ci.4alkylene-CO2H)-C2- 4alkylene)„-N(Ci.4alkylene-CO2H)2, -N(Rlc)CHRleC(O)OCi.6alkyl, -N(Rlc)-Ci.6alkylene-C(O)OCi. ealkyl, -N(Rlf)-C2-4alkylene-(N(Ci.4alkylene-C(O)OCi.ealkyl)-C2-4alkylene)n-N(Ci.4alkylene-C(O)OCi.6alkyl)2, -N(Rlc)-Ci.6alkylene-SO3H, -N(Rlc)-(CH2CH2O)i.3-CH2CH2N((CH2CH2O)i.3-Ci.6alkylene-CO2H)2, and -N(Rlc)-CH(CH20-(CH2CH20)o-2-Ci.6alkylene-C02H)2;Rlcand Rld, at each occurrence, are independently hydrogen or Ci-4alkyl;Rle, at each occurrence, is independently -C1-4alkylene-CO2H, -Ci-4alkylene-CONH2, or -Ci-4alkylene-OH;Atty Docket No. 63XT-401927-WO Rlf, at each occurrence, is independently hydrogen, C1-6alkyl, or C1-4alkylene-CO2H;n, at each occurrence, is independently 0, 1, 2, or 3;L2, at each occurrence, is independently selected from the group consisting of –C(O)– and C1-3alkylene; andG1, at each occurrence, is independently an optionally substituted heterocyclyl.

[0277] In some embodiments, ql is 1.

[0278] In some embodiments, the payload is an immunomodulatory agent payload.

[0279] In some embodiments, the payload is a therapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.

[0280] In some embodiments, the payload is selected from a therapeutic agent for treating cancer (e.g., doxorubicin, daunorubicin, PNU-159682, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, baccatin III, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, exatecan, camptothecin, auristatin (dolastatin 10, MMAE, MMAD, MMAF), duocarmycin, pyrrolobcnzodiazapcnc dimer, mitomycin C, bleomycin, duocarmycin, PBD, calichcamicin, staurosporine, hemiasterlin), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., Amphotericin, and the like), an antibiotic (e.g., vancomycin, daptomycin. doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, and the like), a matrix metalloproteinase (MMP) inhibitor, L-dopa, oseltamivir, cefalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic-adenosine monophosphatidyl (c-AMP).

[0281] In some embodiments, the payload is selected from a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, exatecan, camptothecin, auristatin (dolastatin 10, MMAE, MMAD, MMAF) mitomycin C, bleomycin, duocarmycin, PBD, calicheamicin, staurosporine, hemiasterlin, and the like), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., Amphotericin, and the like), an antibiotic (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, and the like), lurbinectedin, gardiquimod, a matrix metalloproteinase (MMP) inhibitor, L-dopa, oseltamivir, cefalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin. and cyclic-adenosine monophosphatidyl (c-AMP).

[0282] Reference to a payload, means that one or more atoms, including hydrogen or non-hydrogen atoms, of the original, unmodified payload is replaced by a covalent bond to one or more linker. The payloads are derived from the known nuclear payload and are modified to be covalently bonded to atAtty Docket No. 63XT-401927-WO least one optionally substituted trans-cyclooctene via a linker. It is contemplated that the payload has reduced activity (e.g., <50%, <40%, <30%, <20%, <10% or less) when linked to the TCO compared with its activity when unmodified. When the payload it released from tire TCO, the activity of the payload will be recovered and equivalent to the unmodified payload.

[0283] In certain embodiments, a hydrogen atom bound to a heteroatom (e.g., N, O, or S) of the original, unmodified payload is replaced by a covalent bond to a linker. In certain embodiments, a halogen atom on a payload is replaced for attachment to the remainder of the compound. In certain embodiments, a hydrogen atom on a payload is replaced for attachment to the remainder of the compound. In certain embodiments, the hydrogen atom is on a heteroatom. In certain embodiments, the hydrogen atom is on a nitrogen. In certain embodiments, the hydrogen atom is on an oxygen. In certain embodiments, the hydrogen atom is on a carbon.

[0284] In some embodiments, G, at each occurrence, is independently

[0285] In some embodiments, G, at each occurrence, is independently

[0286] In some embodiments, the payload is a monoclonal antibody payload. A monoclonal antibody for use herein as a payload can be an entire monoclonal antibody, or a fragment thereof (e.g., antigenbinding fragment (Fab)). In some embodiments, the antibody is an immune cell engager, and as such would induce or elicit an immune response. In some embodiments, tire monoclonal antibody, or fragment thereof, targets one or more of CD3 (NCBI Gene ID 916), CD28 (NCBI Gene ID 940). CD137 (4-1BB) (NCBI Gene ID 3604), CD16 (NCBI Gene ID 2214), NKG2D (NCBI Gene ID 22914), CD64 (NCBI Gene ID 2209), GITR / TNFRSF18 (NCBI Gene ID 8487), CD25 (NCBI Gene ID 3559), CD40 (NCBI Gene ID 958), CD4 (NCBI Gene ID 920), CXCR4 (NCBI Gene ID 7852), G-CSFR (NCBI Gene ID 1441), GM-CSFR (NCBI Gene ID 1438), CD122 (NCBI Gene ID 3560), PD1 (NCBI Gene ID 5133), CTLA4 (NCBI Gene ID 1493), LAG3 (NCBI Gene ID 3902), TIGIT (NCBI Gene ID 201633), NCR1 (NCBI Gene ID 9437), TIM3 (NCBI Gene ID 84868), VISTA (NCBI Gene ID 64115), CD134 (NCBI Gene ID 7293), CD27 (NCBI Gene ID 939), CD40L (NCBI Gene ID 959), ICOS (NCBI Gene ID 29851), BAFFR (NCBI Gene ID 115650), LFA-1 (NCBI Gene ID 3689), or BTLA (NCBI Gene ID 151888).

[0287] In certain embodiments, the payload is an antibody or antibody fragment which targets CD3, such as OKT3, SP34, UCHT1, Teplizumab, Otelixizumab, Visilizumab, or Foralumab, or an antibody fragment derived therefrom.Atty Docket No. 63XT-401927-WO

[0288] In certain embodiments, the payload is an antibody or antibody fragment which targets CD28, such as Theralizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.

[0289] In certain embodiments, the payload is an antibody or antibody fragment which targets CD 137 (4-1BB), such as Utomilumab, Urelumab, LVGN6051, or AGEN2373, or an antibody fragment derived therefrom.

[0290] In certain embodiments, the payload is an antibody or antibody fragment which targets CD 16, such as AFM13, or an antibody fragment derived therefrom.

[0291] In certain embodiments, the payload is an antibody or antibody fragment which targets NKG2D, such as NNC0152-0002 orJNJ-64304500. or an antibody fragment derived therefrom.

[0292] In certain embodiments, the payload is an antibody or antibody fragment which targets CD64, such as H22, or an antibody fragment derived therefrom.

[0293] In certain embodiments, the payload is an antibody or antibody fragment which targets GITR / TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.

[0294] In certain embodiments, the payload is an antibody or antibody fragment which targets CD25, such as Daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0295] In certain embodiments, the payload is an antibody or antibody fragment which targets CD40, such as Iscalimab, ABBV-323, bleselumab (ASKP-1240), BI-655064, FFP-104, BMS986090, Dacetuzumab, or Lucatumumab, or an antibody fragment derived therefrom.

[0296] In certain embodiments, the payload is an antibody or antibody fragment which targets CD4, such as MAX.16H5, IT1208, Zanolimumab (HuMax-CD4), UB-421, or MTRX1011A, or an antibody fragment derived therefrom.

[0297] In certain embodiments, the payload is an antibody or antibody fragment which targets CXCR4, such as F50067, or an antibody fragment derived therefrom.

[0298] In certain embodiments, the payload is an antibody or antibody fragment which targets G-CSFR. such as CSL324, or an antibody fragment derived therefrom.

[0299] In certain embodiments, the payload is an antibody or antibody fragment which targets GM-CSFR, such as Mavrilimumab, or an antibody fragment derived therefrom.

[0300] In certain embodiments, the payload is an antibody or antibody fragment which targets CD122, such as Hu-Mik(beta)l, or an antibody fragment derived therefrom.

[0301] In certain embodiments, the payload is an antibody or antibody fragment which targets PD-1, such as CC-90006, Cemiplimab, Camrelizumab, or TSR-042, or an antibody fragment derived therefrom.

[0302] In certain embodiments, the payload is an antibody or antibody fragment which targets CTLA4, such as Tremelimumab or ipilimumab, or an antibody fragment derived therefrom.Atty Docket No. 63XT-401927-WO

[0303] In certain embodiments, the payload is an antibody or antibody fragment which targets LAG3, such as Relatlimab (BMS-986016), GSK2831781, Cemiplimab (REGN3767), Favezelimab, leramilimab, or Mavezelimab, or an antibody fragment derived therefrom.

[0304] In certain embodiments, the payload is an antibody or antibody fragment which targets TIGIT, such as BMS-986207, Tiragolumab, Vibostolimab, Etigilimab, Domvanalimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.

[0305] In certain embodiments, the payload is an antibody or antibody fragment which targets NCR1, such as hNKp46.02. or an antibody fragment derived therefrom.

[0306] In certain embodiments, the payload is an antibody or antibody fragment which targets TIM3, such as Cobolimab, Sym023, LY3321367, BMS-986258, SHR-1702. Sabatolimab, or INCAGN02390, or an antibody fragment derived therefrom.

[0307] In certain embodiments, the payload is an antibody or antibody fragment which targets VISTA, such as SG7, K01401-020, CI-8993, or JNJ-61610588, or an antibody fragment derived therefrom.

[0308] In certain embodiments, the payload is an antibody or antibody fragment which targets CD134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.

[0309] In certain embodiments, the payload is an antibody or antibody fragment which targets CD27, such as Varlilumab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.

[0310] In certain embodiments, the payload is an antibody or antibody fragment which targets CD40L, such as Dapirolizumab, or an antibody fragment derived therefrom.

[0311] In certain embodiments, the payload is an antibody or antibody fragment which targets ICOS, such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.

[0312] In certain embodiments, the payload is an antibody or antibody fragment which targets B AFFR, such as lanalumab, or an antibody fragment derived therefrom.

[0313] In certain embodiments, the payload is an antibody or antibody fragment which targets LFA-1. such as Efalizumab, or an antibody fragment therefrom.

[0314] In certain embodiments, the payload is an antibody or antibody fragment which targets BTLA, such as Icatolimab, or an antibody fragment derived therefrom.

[0315] In some embodiments, the payload is an anti-CD3 (aCD3) monoclonal antibody, or a derivative, or analog thereof. In some embodiments, the anti-CD3 (aCD3) monoclonal antibody is SP34, UCHT1, or OKT3, or a derivative, or analog thereof.

[0316] In some embodiments, at least one payload is selected from an inhibitor of poly (ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, an anti-CD3 (aCD3) monoclonal antibody, lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, Paclitaxel, Gemcitabine, Mitomycin C, Etoposide, exatecan, and MMAE, or a derivative, or analog thereof.Atty Docket No. 63XT-401927-WO

[0317] In some embodiments, D is a payload selected from an inhibitor of poly (ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, and an anti-CD3 (aCD3) monoclonal antibody, or a derivative, or analog thereof.

[0318] In some embodiments, at least one payload is selected from lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, Paclitaxel, Gemcitabine, Mitomycin C, Etoposide, exatecan, Seco-Duocarmycin SA, and MMAE, or a derivative, or analog thereof.

[0319] In some embodiments, a payload is an inhibitor of poly (ADP-ribose) polymerase (PARP), or a derivative, or analog thereof. In some embodiments, the inhibitor of poly (ADP-ribose) polymerase (PARP inhibitor) is niraparib, talazoparib, olaparib, pamiparib, rucaparib, veliparib, iniparib, 3-aminobenzamide, CEP-9722, E7016, or a derivative, or analog thereof.

[0320] In some embodiments, a payload is:Atty Docket No. 63XT-401927-WO

[0321] In some embodiments, a payload is a duocarmycin, or a derivative, or analog thereof. In some embodiments, the duocarmycin is Duocarmycin A, Duocarmycin Bl, Duocarmycin B2, Duocarmycin Cl, Duocarmycin C2, Duocarmycin D, Duocarmycin SA, CC-1065, adozelesin, carzelesin, bizelesin, or a derivative, or analog thereof.

[0322] In some embodiments, a payload is:Atty Docket No. 63XT-401927-WO

[0323] In some embodiments, a payload is a pyrrolobenzodiazepine (PBD), or a derivative, or analog thereof. In some embodiments, tire pyrrolobenzodiazepine (PBD) is [l,2]diazepino[3,4-e]indole, or a derivative, or analog thereof.

[0324] In some embodiments, a payload is:Atty Docket No. 63XT-401927-WO

[0325] In some embodiments, a payload is an inhibitor of tubulin polymerization. In some embodiments, a payload is hemiasterlin, HTI-286, or a derivative, or analog thereof.

[0327] In some embodiments, a payload is:Atty Docket No. 63XT-401927-WO

[0328] In some embodiments, the payload comprises a topoisomerase inhibitor. In some embodiments, the payload comprises camptothecin, or a derivative, or analog thereof. In some embodiments, the payload comprises topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, or rubitecan.

[0329] In some embodiments, the payload comprises:

[0330] In some embodiments, die payload comprises:

[0331] In some embodiments, die payload comprisesAtty Docket No. 63XT-401927-WO

[0332] In some embodiments, the payload comprises

[0333] In some embodiments, the payload comprises

[0334] In some embodiments, the payload comprises

[0335] In some embodiments, die payload comprises

[0336] In some embodiments, the payload comprises a polypeptide. In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues. In some embodiments, the linker L1is covalently bonded to a lysine, serine, threonine, or tyrosine residue present on the payload. In some embodiments, the polypeptide comprises one or more lysine residues. In some embodiments, the linker L1is covalently bonded to a lysine residue present on the payload.

[0337] In some embodiments, the payload comprises an N-terminal amino acid, wherein the linker L1is covalently bonded to a N-terminal amino acid.

[0338] In some embodiments, m is 1-20.

[0339] In some embodiments, the payload is an immunomodulatory agent payload.

[0340] In some embodiments, the immunomodulatory agent payload is an antibody payload.

[0341] In some embodiments, the immunomodulatory agent payload is the immune checkpoint inhibitor payload. In some embodiments, the immune checkpoint inhibitor payload is a payload of pidilizumab,Atty Docket No. 63XT-401927-WO sintilimab, AMP-224, atezolizumab, durvalumab, BMS-936559, tremelimumab, indoximod, epacadostat, a TIGIT inhibitor (e.g., LAG-3, such as an anti-LAG-3 antibody; TIM-3, such as an anti-TIM-3 antibody), a B7 molecule, or a BTLA pathway antagonist.

[0342] In some embodiments, the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload. In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload. In some embodiments, the PD-1 inhibitor payload is a nivolumab, pembrolizumab, pidilizumab, sintilimab, or AMP-224 payload.

[0343] In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload. In some embodiments, the PD-L1 inhibitor payload is an atezolizumab, avelumab, durvalumab, or BMS-936559 payload.

[0344] In some embodiments, the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload. In some embodiments, the CTLA4 inhibitor payload is an ipilimumab or tremelimumab payload.

[0345] In some embodiments, die immune checkpoint inhibitor payload is an indoleamine 2,3-dioxygenase (IDO) inhibitor payload. In some embodiments, the IDO inhibitor payload is an indoximod or epacadostat payload.

[0346] In some embodiments, the immunomodulatory agent payload is a cytokine payload.

[0347] In some embodiments, the cytokine payload is an interferon, interleukin, tumor necrosis factor, erythropoietin, MIP3a, ICAM, macrophage colony stimulating factor, Erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), or granulocyte-macrophage colony stimulating factor payload.

[0348] In some embodiments, the interleukin payload is chosen from IL-1 to IL -40. In some embodiments, the interleukin payload is IL -2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0349] In some embodiments, die immunomodulatory agent payload is a type 1 cytokine (IL-2, IL- 12, TNF-B, IFN-g).

[0350] In some embodiments, the cytokine payload is selected from the group consisting of IFN-alpha, IFN-beta, IFN-gamma, pegylated IFN-a, and apolipoprotein A-I fusion protein with IFN-a, interleukin, 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., pegilodecakin), IL-7, IL-12, IL-15, recombinant aglycosylated IL-15, fusion protein of IL-15 with the binding domain of IL-15Ra (e.g., RLI), triple fusion protein comprising human IL-15, the binding domain of IL-15Ra and apolipoprotein A-I, ALT-803 (IL-15 fused to IgGl Fc domain), IL-18, IL-21, tumor necrosis factor, TNF-alpha, TNF-beta), erytiiropoietin (EPO), MIP3a, ICAM, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (GCSF), granulocyte-macrophage colony stimulating factor (GM-CSF), GM-CSF, and talimogene laherparepvec.

[0351] In some embodiments, the immunomodulatory agent payload is the chemokine payload.Atty Docket No. 63XT-401927-WO

[0352] In some embodiments, the chemokine payload is a CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CX CL 14 payload.

[0353] In some embodiments, the immunomodulatory agent payload is the chemokine antagonist payload. In some embodiments, the chemokine antagonist payload is a plerixafor payload.

[0354] In some embodiments, the immunomodulatory agent is a monoclonal antibody specific to a cytokine or a cytokine receptor.

[0355] In some embodiments, die immunomodulatory agent payload comprises a polypeptide.

[0356] In some embodiments, the polypeptide comprises one or more lysine residues.

[0357] In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.

[0358] In some embodiments, the trans-cyclooctene is linked to one of the one or more lysine residues.

[0359] In some embodiments, the trans-cyclooctene is independently linked to one or more lysine, serine, threonine, or tyrosine residues.

[0360] In some embodiments, the polypeptide comprises an N-terminal amino acid, wherein an occurrence of the bioorthogonal moiety is linked to the N-terminal amino acid.

[0361] In some embodiments, m is 1-20. In some embodiments, m is 1-10. In some embodiments, m is 1-5. In some embodiments, m is 20, 19. 18, 17, 16, 15. 14. 13, 12, 11, 10. 9, 8, 7, 6, 5.4. 3, 2, or 1. In some embodiments, m is 1.

[0362] In some embodiments, die trans-cyclooctene functionalized prodrug of Formula XI:or a pharmaceutically acceptable salt thereof, wherein:Rla, at each occurrence, is independently selected from the group consisting of hydrogen, Ci-4alkyl, and C1-4haloalkyl:Rlb, at each occurrence, is independently selected from die group consisting of hydrogen, Ci-4alkyl, Ci-4haloalkyl, C(O)OH, C(O)OCi-4alkyl, C(O)N(Rlc)CHRleCO2H, C(O)N(Rlc)CHRleC(O)OCi-4alkyl, C(O)N(Rlc)-Ci-6alkylene-CO2lI, and C(0)N(Rlc)-Ci-6alkylene-C(0)0Ci alkyl;Atty Docket No. 63XT-401927-WO Rlc, at each occurrence, is independently hydrogen or Ci^alkyl;Rle, at each occurrence, is independently -Ci^alkylene-COjH, -Ci^alkylene-CONHj, or -Ci-4alkylene-OH;D, at each occurrence, is independently a payload;L1, at each occurrence, is independently a linker;p’, at each occurrence, is independently 0, 1, or 2; andp", at each occurrence, is independently 1. 2, or 3.

[0363] In some embodiments, D, at each occurrence, is independently selected from the group consisting of an anticancer agent payload, a toll-like receptor (TLR) agonist payload and a stimulator of interferon genes (STING) agonist payload.

[0364] In some embodiments, Rlais hydrogen.

[0365] In some embodiments, Rlais Cwalkyl.

[0366] In some embodiments, Rlais CH3.

[0367] In some embodiments, Rlbis selected from the group consisting of C(O)OH, C(O)OCi-4alkyl, C(O)N(Rlc)CHRleCO2H, C(O)N(Rlc)CHRleC(O)OCi-4alkyl, C(O)N(Rlc)-Ci-6alkylene-CO2H, and C(O)N(Rlc)-Ci.6alkylene-C(O)OCwalkyl.

[0368] In some embodiments, Rlbis selected from the group consisting of C(O)OH, C(O)N(Rlc)CHRleCO2H, and C(O)N(R1C)CH2CO2H.

[0369] In some embodiments, Rlbis selected from the group consisting of -NR1C-CH2CH2-N(CH3)3+. -N(R1C)-CH2CH2-SO3H, -N(R1C)-(CH2CH2O)3-CH2CH2N((CH2CH2O)3-CH2CH2-CO2H)2. and -N(R1C)-CH(CH2O-CH2CH2-CO2H)2.

[0370] In some embodiments, the trans-cyclooctene moiety (G) is:Atty Docket No. 63XT-401927-WO

[0371] In some embodiments, the trans-cyclooctene moiety is:

[0372] In some embodiments, the trans-cyclooctene moiety is. In some embodiments, the trans-cyclooctene moietyAtty Docket No. 63XT-401927-WO

[0374] In some embodiments, the trans-cyclooctene moiety

[0375] In some embodiments, the trans-cyclooctene moiety

[0378] In some embodiments, the trans-cyclooctene moiety-OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine; wherein R2may be optionally further substituted with a polyether.

[0379] In some embodiments, the trans-cyclooctene moiety comprises

[0380] In some embodiments, the trans-cyclooctene moiety comprises

[0381] In some embodiments, the trans-cyclooctene moiety of comprisesAtty Docket No. 63XT-401927-WO

[0382] In some embodiments, the trans-cyclooctene moiety of comprises

[0383] In some embodiments, Rleis -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH.

[0384] In some embodiments, Rleis -C1-4alkylene-CO2H.

[0385] In some embodiments, Rleis -CH2CO2H.

[0386] In some embodiments, Rlbis -C(O)N(Rlc)-Ci-6alkylene-CO2H.

[0387] In some embodiments, Rlbis -C(O)N(R1C)CH2CO2H.

[0388] In some embodiments, Rlcis hydrogen.

[0389] In some embodiments, Rlbis hydrogen.

[0390] In some embodiments, Rlbis C(O)OH.

[0391] In some embodiments, linker L1may have 1 to 100 linking atoms, and may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, linkers may have from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms, or from 1 to 40 linking atoms, or from 1 to 30 linking atoms, or from 1 to 20 linking atoms, or from 1 to 10 linking atoms, or from 1 to 5 linking atoms, or from 5 to 30 linking atoms, or from 10 to 30 linking atoms, or from 5 to 40 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms.

[0392] In some embodiments, linker L1may comprise one or more (e.g., 1-10 or 1-5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1 -10 or 1 -5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moieties; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, C1.4 alkyl, C1.4 alkoxy, and C1-4 haloalkyl.

[0393] In some embodiments, linker L1may be of the formula:-Y10-(CH2)„.-Y20-(CH2)m.-Y30- wherein:each of Y10, Y20, and Y30are independently a bond, -NR110-, -O-, -S(0)o-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene orAtty Docket No. 63XT-401927-WO heterocycloalkylene; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from oxo, halo,Ci-4 alkyl, Ci-4 alkoxy, and C haloalkyl;each R110is independently hydrogen, C alkyl, C haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;each R120is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; andn' and m" are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0394] In certain embodiments, the linker is a bond.

[0395] In certain embodiments, the linker is not a bond. In certain embodiments, each R110is independently hydrogen, C alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R120is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl.

[0396] Representative linkers include, but are not limited to, those shown below:

[0397] Representative linkers include, but are not limited to, those shown below:

[0398] In some embodiments, linker L1may comprise one or more of polyethylene glycol (e.g., PEG having an average molecular weight of from 300 g / mol to 10,000 g / mol), ethylene-1, 2-Atty Docket No. 63XT-401927-WO diylbis(methylcarbamate, an arylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. In some embodiments, linker L1may comprise

[0399] In some embodiments, linker L1may comprise one or more natural or unnatural amino acids, which may be referred to as a peptide linker. Where the drug (D) comprises an amino moiety, the linker may be bound thereto using a peptide linker made up of a carboxylic acyl unit, and one or more amino acids making up a protein or peptide sequence. In some embodiments, linker L1may also contain a selfimmolating spacer which spaces the drug and the protein peptide sequence.

[0400] In some embodiments, linker L1may be a peptide linker represented by “A — Y — Z — X — W” in which “A” is the carboxylic acyl unit, “Y” and “Z” are each one or more natural or unnatural amino acids and together form a peptide sequence, and “X” and “W” are optional additional linkers having from 1 to 50 linking atoms, or from 5 to 10 linking atoms, or from 1 to 10 linking atoms which spaces the peptide and the drug, D, or the bioorthogonal moiety. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated.

[0401] In some embodiments, Y may be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline. In some embodiments, Y may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0402] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline. In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine and citrulline.

[0403] In some embodiments, exemplary Y-Z combinations include Valine-Citrulline; Valine- Alanine; and Alanine- Alanine.

[0404] In certain embodiments, A is -OC(O)-.

[0405] In certain embodiments, X is -OC(O)-.

[0406] In certain embodiments, W is -OC(O)-. In certain embodiments, X is absent and W is -OC(O)-.Atty Docket No. 63XT-401927-WO

[0407] In certain embodiments,

[0408] In certain embodiments,

[0409] In certain embodiments, the peptide linker is specifically tailored so that it will be selectively cleaved (e.g., enzymatically cleaved) releasing the drug, such as by one or more of the tumor-associated proteases.

[0410] In certain embodiments, the peptide linker has a chain length of two to four amino acid residues (i.e., a di-, tri-, or tetra-peptide). It will be understood, however, that peptide linkers up to five, six, seven, or eight amino acid residues may also suitably be employed.

[0411] 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: ], Ala-Leu-Ala-Leu [SEQ ID NO: ], Phe-N9-tosyl-Arg, or Phe-N9-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.Atty Docket No. 63XT-401927-WO

[0413] The foregoing linkers may attach on the right-hand side to amino acid side chains of D such

[0414] In some embodiments, the payload is covalently bonded to the linker through an amide bond; e.g., die payload may be an amine-containing payload for attachment of the payload to a carbonyl group of the linker, or. in other cases, the payload may be a carboxyl-containing payload for attachment of the payload to an amine group of the linker. In some instances, the payload and linker, together form a carbamate group; e.g., the payload may be an amine-containing payload for attachment of the payload to an acyloxy group of the linker. In some instances, the payload and linker, together form a carbonate group; e.g., the payload may be a hydroxy-containing payload for attachment of the payload to an acyloxy group of the linker.

[0415] In some embodiments,L3ais a bond or Ci-ealkylene;Atty Docket No. 63XT-401927-WO L4ais a bond, –NHN:, -N(R10)-C2.6alkylene-N(R11)-, -N(R12)-C2.3alkylene-N(R13)C(O)-,R10, R11, R12, R13, R14, R15, and R19are each independently hydrogen or Ci.4alkyl;R16is hydrogen, Ci.4alkyl, -Ci.4alkylene-OH, -Ci.4alkylene-OCi.4alkyl, -Ci.4alkylene-CO2H, or — C i.4alkylene-CONH2;R17,at each occurrence, is independently hydrogen or -CH2OC(O)-; andG' is phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, Ci.4alkyl, C1-4haloalkyl. Ci.4alkoxy, cyano, and nitro.

[0416] In certain embodiments, linker L1is -OC(O)-.

[0417] In some embodiments,L3ais a bond;R12and R13are each independently hydrogen or Ci.4alkyl.

[0418] In some embodiments, p” is 1. In some embodiments, p’ is 1.Atty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOR18, at each occurrence, is independently hydrogen or -CH2OC(O)NHD’;RDis hydrogen or C1-4alkyl on a nitrogen atom of a payload; andD and D’ are independently a payload moiety.

[0420] In some embodiments, D or D’ is a cyclic dinucleotide payload moiety, imidazo[4,5-c]quinolin-4-amine payload moiety, TLR agonist payload moiety, STING agonist payload moiety, or anticancer agent payload moiety.R12and R13are each independently hydrogen or C1-4alkyl; andD and D’ are independently a payload moiety (e.g., anticancer agent payload moiety).

[0422] In some embodiments, p’ is 0.Atty Docket No. 63XT-401927-WO

[0423] In some embodiments, p” is 2 or 3.

[0424] In some embodiments,

[0425] The person skilled in the art will recognize that a payload (D or D’) bonded to a linker does not refer to a payload molecule per se, but refers to the portion of the payload molecule bonded to the linker. Release of the payload (D or D’) from a prodrug, releases the payload per se.

[0426] A payload (D or D’) may be an anticancer agent payload of any of the anticancer agents described herein.

[0427] In some embodiments, the payload comprises a TLR7 / 8 agonist, and X is a biocompatible support. In some embodiments, the payload comprises gardiquimod, and X is a biocompatible support.

[0428] In some embodiments, the payload comprises a TLR7 / 8 agonist, and X is an antibody or antibody fragment moiety which targets HER2, TROP2, CSPG4, FAP, CEA, CEACAM5, Nectin4, or extracellular matrix (ECM). In some embodiments, the payload comprises gardiquimod, and X is an antibody or antibody fragment moiety which targets HER2, TROP2, Nectin4, or extracellular matrix (ECM).

[0429] In some embodiments, the payload comprises camptothecin, or derivative thereof, and X is a biocompatible support. In some embodiments, the payload comprises exatecan, and X is a biocompatible support.

[0430] In some embodiments, the payload comprises camptothecin, or derivative thereof, and X is an antibody or antibody fragment moiety which targets HER2, TROP2, CSPG4, FAP, CEA, CEACAM5, Nectin4, or extracellular matrix (ECM). In some embodiments, the payload comprises exatecan, and X is an antibody or antibody fragment moiety which targets HER2, TROP2, Nectin4, or extracellular matrix (ECM).

[0431] In some embodiments, the payload comprises MMAE. and X is a biocompatible support.

[0432] In some embodiments, the payload comprises MMAE, or derivative thereof, and X is an antibody or antibody fragment moiety which targets HER2, TROP2, CSPG4, FAP, CEA, CEACAM5, Nectin4, or extracellular matrix (ECM).

[0433] In some embodiments, the payload comprises paclitaxel, or derivative thereof, and X is a biocompatible support.

[0434] In some embodiments, the payload comprises paclitaxel, or derivative thereof, and X is an antibody or antibody fragment moiety which targets HER2, TROP2, CSPG4, FAP, CEA, CEACAM5, Nectin4, or extracellular matrix (ECM).Atty Docket No. 63XT-401927-WO

[0435] In some embodiments, the payload comprises docetaxel, or derivative thereof, and X is a biocompatible support.

[0436] In some embodiments, the payload comprises docetaxel, or derivative thereof, and X is an antibody or antibody fragment moiety which targets HER2, TROP2, CSPG4, FAP, CEA, CEACAM5, Nectin4, or extracellular matrix (ECM).

[0437] In some embodiments, the payload has the structure:Atty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WO

[0438] In certain embodiments, the trans-cyclooctene functionalized prodrug is selected from:Atty Docket No. 63XT-401927-WOE. Methods of Treatment

[0439] Aspects of the present disclosure include methods for delivering a payload to a target location in a subject. In certain embodiments, the method includes selectively delivering a payload to tire target location in a subject. Selective delivery of the payload includes delivering the payload to the target location (e.g., an organ or tissue, or portion thereof), without targeting other locations in the subject (e.g., other organs or tissues, or portions thereof) that do not need administration of the payload. Selective delivery of tire payload may be achieved through use of the targeting moiety and the functionalized payloads described herein.

[0440] In some instances, a targeting moiety of tire present disclosure may be localized to a desired target location in a subject. For example, methods of the present disclosure may include administering to a subject a targeting moiety as described herein. The targeting moiety may be administered to die subject at a desired target location in die subject. In some instances, die targeting moiety may be injected locally into the subject at die desired target location in die subject. In some embodiments, die targeting moiety isAtty Docket No. 63XT-401927-WO administered systemically. In these embodiments, die targeting moiety may localize at a desired target location in the subject through specific binding of the targeting agent to its target (e.g., antibody-antigen interaction, and the like), or may localize on die surface of a desired target (e.g., a cell surface) through specific binding of die targeting agent to its target (e.g., antibody-antigen interaction, and the like).

[0441] As described herein, selective binding between bioorthogonal binding partners (e.g., between a tetrazine of the targeting moiety and its complementary trans-cyclooctene) of a prodrug may occur. Due to die localized administration of the targeting moiety to a desired location in the subject as described above, the selective binding between the trans-cyclooctene and its complementary binding agent of the prodrug will localize the payload to die desired target location.

[0442] Provided herein is a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a targeting moiety as described herein, or a pharmaceutically acceptable salt thereof, and a trans-cyclooctene prodrug.

[0443] In some embodiments, the cancer is metastatic. In some embodiments the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, colorectal cancer, squamous cell carcinoma of head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma Wilms’ tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

[0444] In some embodiments, the cancer is a melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, gastric cancer, colorectal cancer, soft tissue carcinoma, soft tissue sarcoma, osteosarcoma, or pancreatic cancer.

[0445] In some embodiments, tire cancer is a solid tumor.

[0446] In some embodiments, the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing’s sarcoma.

[0447] In some embodiments, the method also comprises enhancing or eliciting an immune response. In some embodiments the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0448] In some embodiments, the method further comprising administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof. Anticancer agents, immunomodulatory agents, and their trans-cyclooctene prodrugs are known in the art.

[0449] Indications for this approach include cancer, both hematological and solid cancers. In certain embodiments, tire approach can be used for the treatment and / or diagnosis of soft tissue sarcomas:Atty Docket No. 63XT-401927-WO rhabdomyosarcoma, fibrosarcoma, Ewing’s sarcoma, and all the different subtypes of soft tissue sarcoma as well as osteosarcoma. The compositions can be for tire treatment and / or diagnosis of pigmented villonodular synovitis.

[0450] In certain embodiments, die approach can be used for the treatment and / or diagnosis of hematological malignancies such as myelodysplastic syndromes, acute myeloid leukemia, myelodisplastic syndromes, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin’s lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy -cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte predominant Hodgkin’s lymphoma, as well as others.

[0451] The compositions of the present disclosure find use in treatment and / or diagnosis of a condition or disease in a subject that is amenable to treatment or diagnosis by administration of the payload (e.g., the parent drug (i.e., the drug prior to conjugation to the composition)). By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the subject is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where tire pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from tire condition, or at least the symptoms that characterize the condition. Treatment may include inhibition, that is, arresting tire development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease. Treatment may include relief, that is, causing tire regression of clinical symptoms. For example, in tire context of cancer, tlie term “treating” includes any or all of: reducing growth of a solid tumor, inhibiting replication of cancer cells, reducing overall tumor burden, prolonged survival and ameliorating one or more symptoms associated with a cancer.

[0452] The subject to be treated can be one that is in need of therapy, where the subject to be treated is one amenable to treatment using the parent drug. Accordingly, a variety of subjects may be amenable to treatment using the compositions disclosed herein. Generally, such subjects are “mammals,” with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys).

[0453] In certain embodiments, 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 carcinoma, breast cancer (e.g., triple negative breastAtty Docket No. 63XT-401927-WO cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, squamous cell carcinoma of head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi’s sarcoma, Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma Wilms’ tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, cutaneous T-cell lymphoma, among others. The disclosed approach lends itself well as an adjuvant / neoadjuvant system. For example, particles as disclosed herein could be placed during the biopsy, once the results from the study come back, the practitioner could deliver the appropriate cocktail to the desired site in the body. This would minimize the size of the tumor particularly in the context of a surgically resectable tumor. Then at the end of the surgery, the surgeon could administer additional targeting moiety to the subject to target the surgical cavity and treat the patient with further doses of treatment (e.g., chemotherapy through the disclosed approach) to minimize the risk of any cancer cells that may have been missed in the surgical margins.

[0454] In certain embodiments, a targeting moiety as disclosed herein could be administered and the practitioner could deliver the appropriate cocktail to the desired site in lire body. This would minimize the size of the tumor particularly in the context of a surgically resectable tumor. Then at the end of the surgery, the surgeon could administer additional targeting moiety to the subject to target the surgical cavity and treat the patient with further doses of treatment (e.g., chemotherapy through the disclosed approach) to minimize the risk of any cancer cells that may have been missed in the surgical margins.

[0455] In certain embodiments, the disclosed methods provide the ability to place particles as disclosed herein at the time of the biopsy. When the biopsy results return, the practitioner can deliver immunomodulatory agents through to the biopsy site.

[0456] In certain embodiments, the disclosed methods provide the ability for a practitioner to deliver immunomodulatory agents, such as TLR agonists, STING agonists, chemokines (agents that attract cancerous cells and / or immune cells) and adjuvants to enhance the immune system with fewer side effects as well as the chemotherapeutics agents combined with immunotherapy agents. This combination approach would be beneficial to patients. The chemotherapy agent would treat the solid tumor or specific location, while the enhanced response of the immunotherapy would help with distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods could employ or be used with an thracy clines, taxanes, gemcitabine and other agents to enhance the efficacy of one or more immunomodulatory agents such as ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).Cancer

[0457] The disclosed methods may be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that may include sustained proliferative signaling, evasion of growthAtty Docket No. 63XT-401927-WO suppressors, resistance to cell death, enablement of replicative immortality, induction of angiogenesis, and tire activation of invasion and metastasis. The disclosed methods may enhance or elicits an immune response against a cancer in the subject. The immune response may lead to an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0458] Cancer that may be treated by the disclosed methods, includes, but is not limited to, astrocytoma, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, brain stem cancer, brain stem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse intrinsic pontine glioma, ductal cancer, endometrial cancer, ependymoma, Ewing’s sarcoma, esophageal cancer, eye cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular cancer, histiocytosis, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, macroglobulincmia, melanoma, mesothelioma, mouth cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, nonHodgkin’s lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue carcinoma, soft tissue sarcoma, solid tumor, squamous cell carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thyroid cancer, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms tumor, non-small cell lung cancer (NSCLC), diffuse large B-cell lymphoma (DLBCL), or oral tongue squamous cell carcinoma (OTSCC).

[0459] In some embodiments, tire cancer that may be treated by the disclosed methods is melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue carcinoma, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, (lie cancer is a solid tumor. In some embodiments, the cancer is a soft tissue carcinoma. In some embodiments, the cancer is a fibrosarcoma. In some embodiments, the cancer is diffuse intrinsic pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0460] In some embodiments, tire cancer that may be treated by the disclosed methods is a hematological malignancy, such as myelodysplastic syndromes, acute myeloid leukemia, myelodisplastic syndromes, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin’s lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B -lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy-cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte predominant Hodgkin’s lymphoma, as well as others.Atty Docket No. 63XT-401927-WO

[0461] Without being bound by a particular theory, local release of certain anti-cancer agents using the compounds and methods of the disclosure may produce or contribute to immunogenic cell death (ICD). For example, certain anti-cancer agents (e.g., anthracyclines, cyclophosphamide, oxaliplatin) have been reported to induce ICD. Kroemer et al. Annu. Rev. Immunol. 2013 (31), 51-72. Immunogenic apoptosis of cancer cells can induce an effective antitumor immune response through activation of dendritic cells (DCs) and consequent activation of specific T cell response. ICD is characterized by secretion of damage-associated molecular patterns (DAMPs). Three important DAMPs which are exposed to the cell surface during ICD. Calreticulin (CRT), one of the DAMP molecules, which is normally in the lumen of endoplasmic reticulum (ER), is translocated after the induction of immunogenic apoptosis to the surface of dying cell where it functions as an "eat me" signal for professional phagocytes. Other important surface exposed DAMPs are heat-shock proteins (HSPs), namely HSP70 and HSP90, which are under stress condition also translocated to the plasma membrane. On the cell surface they have an immunostimulatory effect, based on their interaction with number of antigen-presenting cell (APC) surface receptors like CD91 and CD40 and also facilitate cross presentation of antigens derived from tumor cells on MHC class I molecule, which than leads to the CD8+ T cell response. Other important DAMPs, characteristic for ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is considered to be late apoptotic marker and its release to the extracellular space seems to be required for the optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs) such as Toll-like receptor (TLR) 2 and 4, which are expressed on APCs. The most recently found DAMP released during immunogenic cell death is ATP, which functions as a "find-me" signal for monocytes when secreted and induces their attraction to the site of apoptosis. Kroemer et. al. Curr. Op. Immunol. 2008 (20), 504-511.

[0462] Thus, local release of ICD inducers using the compounds and methods of the disclosure may be beneficially combined with one or more immunomodulatory agents.

[0463] In certain embodiments, the targeting moiety 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), breast cancer (e.g., triple negative breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, among others.

[0464] The disclosed approach lends itself well as an adjuvant I neoadjuvant system. For example, targeting moieties as disclosed herein could be placed during the biopsy, once the results from the study come back, the practitioner could administer the appropriate cocktail to deliver treatment to the desired site in the body (compounds as disclosed herein and optional additional therapeutic agent(s)). The results of tire biopsy may indicate the amount and type of treatment to deliver to the site of a tumor. For example, chemokines (agents that attract cancerous cells and / or immune cells) and adjuvants to enhanceAtty Docket No. 63XT-401927-WO the immune system with fewer side effects as well as the chemotherapeutics agents could be delivered and combined with immunotherapy agents.

[0465] The disclosed methods may include one or multiple systemic doses of targeting moieties that focus at one location or more locations. The disclosed methods may be used to deliver a functionalized payload to these location through systemic or local administration. In some embodiments, the targeting moiety is delivered systemically. In some embodiments, the targeting moiety and the payload (i.e., a TCO-labeled payload) are both delivered systemically. In some embodiments, the targeting moiety is delivered locally.

[0466] The disclosed compounds and compositions may be administered prior to surgical resection. The disclosed methods may minimize the size of die tumor prior to surgical resection. This would minimize the size of the tumor particularly in the context of a surgically resectable tumor. The disclosed conjugates, compounds and compositions may be administered during surgical resection. The disclosed conjugates, compounds and compositions may be administered after surgical resection. The targeting moiety may be placed around the surgical cavity at the end of surgical resection and the subject may then be treated with further doses of a treatment to minimize the risk of any cancer cells that may have been missed in the surgical margins.

[0467] The disclosed methods may include multiple systemic doses of functionalized payload that focus at one location. The disclosed methods may be used to deliver a second payload. The disclosed methods may be used to administer a second functionalized payload if the tumor is resistant to the first payload. A second payload may be a TCO-labeled payload of gemcitabine or docetaxel. The TCO-labeled payload of gemcitabine, paclitaxel, or docetaxel may be administered in combination with doxorubicin. The second functionalized payload may be activated by the targeting moiety used for the first prodrug.

[0468] The functionalized payloads disclosed herein may function as adjuvants. This combination approach would be beneficial to patients. The chemotherapy agent would treat the solid tumor or specific location and may enhance or elicit an immune response, while the enhanced response of the immunotherapy of the functionalized payload and / or separate agent may help with distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods could employ or be used with anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, camptothecin analogues and other agents to enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).

[0469] The disclosed methods may be used to treat diffuse intrinsic pontine gliomas. Diffuse intrinsic pontine gliomas (DIPG) are pediatric brainstem tumors that may be highly malignant and may be difficult to treat. There is no known curative treatment for DIPG, and survival odds have remained dismal over the past four decades. DIPG patients have a median overall survival of just 11 months, with a two-year survival rate below 10%. DIPG account for 75-80% of brainstem tumors in children, affecting an estimated 200-300 children in the U. S. each year. The rarity of this devastating disease andAtty Docket No. 63XT-401927-WO previous lack of experimental model systems has impeded research, and over the past four decades survival odds have remained the same. Diagnosis of DIPG may begin with clinical symptoms and may be confirmed by MRI. The disease may begin with several months of generalized symptoms, including behavioral changes and difficulties in school, double vision, abnormal or limited eye movements, an asymmetric smile, loss of balance, and weakness. Alternately, severe neurologic deterioration may happen more quickly, with symptoms present for less than a month prior to diagnosis. Clinical examination may reveal tire triad of multiple cranial neuropathies, long tract signs such as hyperreflexia and clonus, as well as ataxia. Expansion of tire pons section of the brainstem may cause obstructive hydrocephalus and increased intracranial pressure.

[0470] Nuclei critical for life-sustaining function such as breathing and heartbeat in are located in the pons and without treatment, breathing and heartbeat may be damaged by DIPG.

[0471] The disclosed methods may include multiple systemic doses of functionalized payload that focus at one location. The disclosed methods may be used to deliver a second payload. The disclosed methods may be used to administer a second functionalized payload if the tumor is resistant to the first payload. A 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 the targeting moiety used for the first prodrug.Modes of Administration

[0472] Methods of treatment may include any number of modes of administering a disclosed conjugate, compound or composition. Modes of administration may include tablets, pills, dragees, 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. In the pharmaceutical composition, the conjugate, compound or compositions disclosed herein may also be dispersed in a microparticle, e.g.. a nanoparticulate composition.

[0473] For parenteral administration, the conjugates, compounds or compositions disclosed herein may be dissolved or suspended in a physiologically acceptable diluent, such as water, buffer, oils with or without solubilizers, surface-active agents, dispersants or emulsifiers. Suitable oils may 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 an aqueous, lipid, oily or other kind of solution or suspension, or even administered in the form of liposomes or nano-suspensions.

[0474] The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion.Atty Docket No. 63XT-401927-WO

[0475] The amount of composition administered to a subject can be initially determined based on guidance of a dose and / or dosage regimen of the parent drug. In general, the compositions can provide for targeted delivery and / or enhanced serum half-life of the bound drug, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen. Thus, the compositions can provide for reduced dose and / or reduced administration in a dosage regimen relative to the parent drug prior to being conjugated in a composition of the present disclosure.

[0476] The pharmaceutical formulation may be provided in unit dosage form. In such form the pharmaceutical formulation may be subdivided into unit doses containing appropriate quantities of the compositions of the present disclosure. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, such as packeted tablets, capsules, and powders in pouches, vials or ampoules.

[0477] In some embodiments, provided is a kit comprising a targeting moiety, or a pharmaceutically acceptable salt thereof, as described herein, or the pharmaceutical composition comprising the same, and instructions for use thereof.

[0478] In some embodiments, the kit further comprising a prodrug.

[0479] Compositions of the present disclosure can be present in any suitable amount, and can depend on various factors including, but not limited to. weight and age of the subject, slate of the disease, etc. Suitable dosage ranges for the composition of the present disclosure include from 0.1 mg to 10,000 mg, or 1 mg to 1000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For instance, suitable dosages for the composition of the present disclosure include 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg. 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg. 250 mg, 300 mg, 350 mg, 400 mg. 450 mg, 500 mg, 550 mg, 600 mg, 650 mg. 700 mg. 750 mg, 800 mg, 850 mg, 900 mg, 950 mg. or 1000 mg.

[0480] In some embodiments, multiple doses of a composition are administered. The frequency of administration of a composition can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc. For example, in some embodiments, a composition is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (bid), or three times a day (tid).

[0481] The compositions of the present disclosure can be administered at any suitable frequency, interval and duration. For example, the composition of the present disclosure can be administered once an hour, or two, three or more times an hour, once a day, or two, three, or more times per day, or once every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, so as to provide the desired dosage level to the subject. When the composition of the present disclosure is administered more than once a day, representative intervals include 5 min, 10 min, 15 min, 20 min. 30 min. 45 min and 60 minutes, as well as 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, and 24 hours. The composition of the presentAtty Docket No. 63XT-401927-WO disclosure can be administered once, twice, or three or more times, for an hour, for 1 to 6 hours, for 1 to 12 hours, for 1 to 24 hours, for 6 to 12 hours, for 12 to 24 hours, for a single day, for 1 to 7 days, for a single week, for 1 to 4 weeks, for a month, for 1 to 12 months, for a year or more, or even indefinitely.

[0482] The compositions of the present disclosure can be co-administered with another active agent. Co-administration includes administering the composition of the present disclosure and active agent within 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, or 24 hours of each other. Coadministration also includes administering the composition of the present disclosure and active agent simultaneously or approximately simultaneously (e.g., within about 1 min, 5 min, 10 min, 15 min, 20 min, or 30 minutes of each other), or sequentially in any order. In addition, the composition of the present disclosure and the active agent can each be administered once a day, or two, three, or more times per day so as to provide the desired dosage level per day.

[0483] Co-administration can be accomplished by coimplantation or coinjection.

[0484] In some embodiments, co-administration can be accomplished by co-formulation, e.g., preparing a single pharmaceutical formulation including both the composition of the present disclosure and the active agent. In other embodiments, the composition of the present disclosure and the active agent can be formulated separately and co-administered to the subject.

[0485] The composition of the present disclosure and the active agent can be present in a formulation in any suitable weight ratio, such as from 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 composition of the present disclosure and the other active agent can be present in any suitable weight ratio, such as l:100 (w / w), 1:75, 1:50, 1:25, 1:10. 1:5, 1:4, 1:3, 1:2, 1:1, 2:1. 3:1, 4:1, 5:1, 10:1. 25:1, 50:1, 75:1, or 100:1 (w / w). Other dosages and dosage ratios of the composition of the present disclosure and the active agent are suitable in the formulations and methods described herein.Combination Therapies

[0486] In one aspect, provided is a method of treating cancer or enhancing or eliciting an immune response comprising administering to a subject in need thereof: a therapeutically effective amount of a targeting moiety of the disclosure, or a pharmaceutically acceptable salt or composition thereof; and a prodrug, such as those as described herein; and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0487] The disclosure also provides a pharmaceutical combination comprising a targeting moiety described herein, or a pharmaceutically acceptable salt, or composition thereof; a prodrug as described herein; and optionally an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, for use in the treatment or prevention of a cancer or for use in enhancing or eliciting an immune response.Atty Docket No. 63XT-401927-WO

[0488] The disclosure also provides the use of a pharmaceutical combination comprising a targeting moiety as described herein, or a pharmaceutically acceptable salt, or composition thereof; a prodrug, such as those described herein; and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a transcyclooctene prodrug thereof for the treatment or prevention of a cancer or for use in enhancing or eliciting an immune response.

[0489] In the methods and uses described herein, the components of the pharmaceutical combinations may be administered / used simultaneously, separately, or sequentially, and in any order, and the components may be administered separately or as a fixed combination. For example, the delay of progression or treatment of diseases according to the disclosure may comprise administration of the first active ingredient in free or pharmaceutically acceptable salt form and administration of the second active ingredient in free or pharmaceutically acceptable salt form, simultaneously or sequentially in any order, in jointly therapeutically effective amounts or effective amounts, e.g., in daily dosages corresponding to tire amounts described herein. The individual active ingredients of the combination can be administered separately at different times during the course of therapy or concurrently in divided or single dosage forms. The instant disclosure is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and tire term "administering" is to be interpreted accordingly. Thus, a pharmaceutical combination, as used herein, defines either a fixed combination in one dosage unit form or separate dosages forms for the combined administration where the combined administration may be independently at the same time or at different times. As a further example, the targeting moiety (or therapeutic targeting moiety) and prodrug may be administered / used simultaneously (e.g., through coinjection or coimplantation), separately, or sequentially, followed by administration of die additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0490] The methods and uses in treating cancer include administering / localizing the targeting moiety at a tumor. In the methods and uses disclosed herein, the administration of the prodrug, or a pharmaceutically acceptable salt, or composition thereof; die targeting moiety; and optionally an additional therapeutic agent may inhibit the growth of the tumor.

[0491] Additional therapeutic agent(s) may be administered simultaneously or sequentially with the disclosed conjugates and compositions. Sequential administration includes administration before or after the disclosed conjugates and compositions. An additional therapeutic agent may be administered before the disclosed conjugates and compositions. An additional therapeutic agent may be administered after the disclosed conjugates and compositions. An additional therapeutic agent may be administered at the same time as the disclosed conjugates and compositions. In some embodiments, the additional therapeutic agent or agents may be administered in the same composition as the disclosed conjugates. In other embodiments, there may be an interval of time between administration of the additional therapeutic agent and the disclosed conjugates or compositions. In some embodiments, administration of an additionalAtty Docket No. 63XT-401927-WO therapeutic agent with a disclosed conjugate or composition may allow lower doses of tire other therapeutic agents and / or administration at less frequent intervals. When used in combination with one or more other active ingredients, the conjugates or compositions of tire present disclosure and the other active ingredients may be used in lower doses than when each is used singly. Accordingly, the pharmaceutical compositions of the present disclosure include those that contain one or more other active ingredients, in addition to a conjugates of tire present disclosure.Anticancer agents

[0492] Exemplary anti-cancer 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), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and I 131 Iodine Tositumomab), Bicalutamide, Bleomycin, Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabozantinib- S-Malate, CAF, Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carboplatin, Carboplatin-Taxol, Carfilzomib, Casodcx (Bicalutamide), CeeNU (Lomus tine), Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, Chlorambucil, Chlorambucil-Prednisone, CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clof arabine), Clolar (Clofarabine), CMF, Cometriq (Cabozantinib-S-Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine. Cytarabine liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib. Daunorubicin Hydrochloride, Decitabine. Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine). Dexrazoxane Hydrochloride. Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome). Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil). Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride). Eloxatin (Oxaliplatin). Eltrombopag Olamine. Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride. EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil),Atty Docket No. 63XT-401927-WO Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), Folfiri, Folfiri- Bevacizumab, Folfiri-Cetuximab, Folfirinox, Folfox (Leucovorin, Fluorouracil, Oxaliplatin), Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hyper-CVAD. Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Ilex (Ifosfamide), Ifosf amide, Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A (Recombinant Interferon Alfa- 2b), Iodine 131 Tositumomab and Tositumomab. Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepilone. Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin).Kyprolis (Carfilzomib), Lapatinib Ditosylate, Lenalidomide, Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Levulan (Aminolevulinic Acid). Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome). Liposomal Cytarabine, Lomustine. Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lupron Depot- 3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace (Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin- stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Ofatumumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox), OEPA, OPPA, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Panitumumab, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor. Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone. Procarbazine Hydrochloride. Proleukin (Aldesleukin). Prolia (Denosumab), Promacta (Eltrombopag Olamine), Provenge (Sipuleucel-T). Purinethol (Mercaptopurine). Radium 223 Dichloride, Raloxifene Hydrochloride, Rasburicase. R-CHOP, R-CVP. Recombinant HPV Bivalent Vaccine, Recombinant HPV Quadrivalent Vaccine, Recombinant Interferon Alfa- 2b. Regorafenib, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab). Rituximab, Romidepsin,Atty Docket No. 63XT-401927-WO Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol (Talc), Sipuleucel-T, Sorafenib Tosylate, Sprycel (Dasatinib), Stanford V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa- 2b), Synovir (Thalidomide), Synribo (Omacetaxine Mepesuccinate), Tafinlar (Dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and I 131 Iodine Tositumomab, Totect (Dexrazoxane Hydrochloride), Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Vandetanib. VAMP. Vectibix (Panitumumab), VelP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), Xelox. Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide). Yervoy (Ipilimumab), Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), and Zytiga (Abiraterone Acetate).

[0493] The anticancer agent may be a PBD dimer, calicheamicin, speromycin, tubulysin B, rhizoxin, dolastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothilone B. taxol, cryptophycin, SN38, velcade, bruceantin. DAVLBH, DM1, Phyllanthoside. Alimta, T2 Toxin. MMC, vantalanib, vinorelbine, brefeldin, sunitinib, daunomycin, semaxanib, tarceva, iressa, irinotecan. LY-541503. geldanomycin, gemcitabine, methotrexate, gleevec. topotecan. bleomycin, doxorubicin, cisplatin, N-mustards, etoposide, or 5-FU.

[0494] In certain embodiments, an anticancer agent is an anthracycline. In certain embodiments, anticancer agent is a taxane. In certain embodiments, anticancer agent is gemcitabine. In certain embodiments, anticancer agent is doxorubicin. In certain embodiments, anticancer agent is docetaxel. In certain embodiments, anticancer agent is SN38. In certain embodiments, anticancer agent is monomethyl auristatin E.Synthesis of the Compounds

[0495] The targeting moieties may be prepared using the methods disclosed herein and routine modifications thereof, which will be apparent given the disclosure herein and methods well known in the art. Conventional and well-known synthetic methods may be used in addition to the teachings herein. The synthesis of typical targeting moieties described herein may be accomplished as described in the following examples. If available, reagents and starting materials may be purchased commercially, c.g., from Sigma Aldrich or other chemical suppliers.Atty Docket No. 63XT-401927-WO

[0496] It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0497] Additionally, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in Wuts, P. G. M., Greene, T. W., & Greene, T. W. (2006). Greene's protective groups in organic synthesis. Hoboken, N. J., Wiley-Interscience, and references cited therein.

[0498] As shown in Scheme I, compounds of Formula III (wherein R2, ring A, t, L, p, and X are independently defined herein, and R50is a synthetic handle for bonding to L, such as a leaving group, e.g., halo, or a portion of L capable of linking to X or a further portion of L, e.g., hydroxy, amino, methylamino, etc.) can be prepared by coupling a compound of Formula 1-2 with a suitably functionalized biocompatible support, antibody, or antibody fragment moiety X. Suitable methods can be adapted from the literature (see, e.g., W02020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and W02014 / 205126). Compounds of Formula 1-2 can be prepared by reacting compound 1-1 with a precursor to L under suitable coupling reaction conditions. In some embodiments, X is an antibody or antibody fragment. Suitable coupling methods include, but arc not limited to, use of a succinimide functional group which is capable of forming an amide bond with a primary amine on the antibody or antibody fragment, or L can be functionalized with a group capable of forming a covalent bond to a cysteine residue on the antibody or antibody fragment, such as a pyrrole -2, 5-dione.Scheme I

[0499] Upon each reaction completion, each of the intermediate or final compounds can be recovered, and optionally purified, by conventional techniques such as neutralization, extraction, precipitation, chromatography, filtration and the like.Atty Docket No. 63XT-401927-WO

[0500] It should be understood that any of the compounds or intermediates shown in Scheme I or II may be prepared using traditional methods or purchased from commercial sources. In addition, any of the intermediates or any product obtained by the process outlined in Scheme I or II can be derivatized at any step to provide various compounds of Formula V.

[0501] Exemplary payloads can be prepared can be prepared according to methods adapted from the literature (see, e.g., WO2022 / 032191, W02021 / 007160, W02020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126, which methods are incorporated herein in their entirety). Exemplary procedures for MMAE payloads are shown in Examples A, B, and C, which procedures can be adapted to prepare other payloads such as those disclosed herein.EXAMPLES

[0502] The following examples are included to demonstrate specific embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques to function well in tine practice of the disclosure, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Atty Docket No. 63XT-401927-WO

[0503] The following abbreviations are used throughout.DIPEA N-ethyl-N-propan-2-ylpropan-2-amineDCM DichloromethaneHATU [bis(dimethylamino)methylidene]({3H-[l,2,3]triazolo[4,5-b]pyridin-3- yl })oxidanium; hexafluoro-λ5-phosphanuideDMF N, N-dimethylformamideDMAP N, N-dimethylpyridin-4-aminePIDA [bis(acetoxy)iodo]benzenePy pyridineTHF tetrahydrofuranDCC dicyclohexyl-carbodiimideTFA hydron;2,2,2-trifluoroacetateEDCI 3-(ethyliminomethylideneamino)propyl-dimethylazanium;chlorideCuTc cuprio thiophene-2-carboxylateLC-MS or HPLC Method:• LCMS Method 1: (5-95AB, 3.0 min)Mobile phase: Ramp from 5% ACN (0.01875%TFA) in water (0.0375%TFA) to 95% ACN in water in 0.60 min. Flow rate is set at 2.0 mL / min; then hold at 95% ACN for 0.18 min. Flow rate is set at 2.0 mL / min; return back to 5% ACN in water and hold for 0.02 min. Flow rate is set at 2.0 mL / min.Column temperature at 50 °C. The column is of Kinetex® EVO Cl 82.1 x 30 mm 5 um.• LCMS Method 2: (5-95 AB, 1 min)Mobile phase: Ramp from 5% ACN (0.01875%TFA) in water (0.0375%TFA) to 95% ACN in 3.40 min. Flow rate is set at 0.9 mL / min, then hold at 95% ACN for 0.30 min. Flow rate is set at 0.9 mL / min, return back to 5% ACN in water and hold for 0.30 min. Flow rate is set at 1.2 mL / min. Column temperature at 50 °C. The column is of Kinetex® EVO Cl 83.0 x 50 mm 2.6 um.• HPLC Method 1: ( 10- 80 AB, 4 min)Mobile phase: Ramp from 10% ACN (0.018%TFA) in water (0.037%TFA) to 80% ACN in water in 3.00 min. Flow rate is set at 1.5 mL / min; then hold at 80% ACN for 0.70 min. Flow rate is set at 1.5 mL / min; return back to 10% ACN in water and hold for 0.29 min. Flow rate is set at 2.0 mL / min. Column temperature at 50 °C. The column is of Kinetex Cl 8 LC Column 4.6 x 50 mm, 5 um.Atty Docket No. 63XT-401927-WO Example 1: Targeting Moiety PrecursorGeneral procedure for preparation of Cpd.2step 1: 85.5% yield 2Atty Docket No. 63XT-401927-WO

[0504] To a solution of Cpd. 1 (20.0 g, 188 mmol, 1.00 eq) in EtOH (200 mL) was added Mel (54.7 g, 385 mmol, 24 mL, 2.05 eq) slowly at 20 °C under N2. The mixture was stirred at 65 °C under N2 for 16 hrs. TLC (Dichloro methane / Methanol = 5 / 1) indicated no starting material remained and one major new spot with larger polarity was detected. The mixture was filtered and the cake was concentrated in vacuum. The crude product was triturated with dioxane (250 mL) at 25 °C for 0.5 hr. The mixture was filtered and tire cake was concentrated in vacuum. Cpd.2 (40.0 g, 161 mmol, 85.5% yield) was obtained as a white solid and used into next step directly.General procedure for preparation of Cpd.7a

[0505] To a mixture of Cpd.6 (7.00 g, 68.5 mmol, 6.77 mL, 1.00 eq), EDCI (14.4 g, 75.1 mmol, 1.10 eq) and Cpd. E-3 (20.0 g, 79.6 mmol, 1.16 eq) in DMF (200 mL) was added DMAP (840 mg, 6.88 mmol, 0.10 eq) in one portion at 20 °C under N2. The mixture was stirred at 20 °C for 12 hrs. About 52% of the desired mass was detected by LCMS. The mixture was poured into ice-water (200 mL) and stirred for 5 mins. The mixture was extracted with ethyl acetate (100 mL x 3). The combined organic phase was washed with brine (100 mL x 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The mixture was purified by column chromatography (SiO2, hexanes / Ethyl acetate = 50 / 1 to 1 / 1, TLC: Petroleum ether / Ethyl acetate = 1 / 1, Rf = 0.43). Cpd.7a (11.0 g. 32.8 mmol, 100% purity, 47.8% yield) was obtained as a white solid and confirmed by 1H-NMR and LCMS.

[0506] LCMS (Method 2): 220 nm, RT = 0.506 min, m / z = 358.3 (M+Na)+.

[0507] 1H NMR: (400 MHz, CDCl3). δ 7.99 - 8.03 (m, 2H), 7.35 - 7.38 (m, 2H), 4.95 (s, 1H), 4.61 - 4.68 (m, 2H), 4.45 - 4.54 (m, 2H), 4.35 - 4.41 (m, 4H), 1.47 (s, 9H), 1.43 (s, 3H).Atty Docket No. 63XT-401927-WO General procedure for preparation of Cpd.3

[0508] To a solution of Cpd.7a (6.80 g, 20.2 mmol, 1.26 eq) in DCM (20 mL) was added BF3. Et2O (10 M, 4.00 mL, 2.48 eq) drop-wised at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 hr. Py (5.10 g, 64.4 mmol, 5.20 mL, 4.00 eq) was added drop-wised to the mixture, then Cpd.2 (4.00 g, 16.1 mmol, 1.00 eq) in DMF (20 mL) was added drop-wised to the mixture at 0 °C under N2. DCM was removed by vacuum carefully at 5 °C and the mixture was continually stirred at 80 °C for 1 hr. TLC indicated no starting materials remained. After cooling to 25 °C, PIDA (5.20 g, 16.1 mmol, 1.00 eq) was added portion wise and the mixture was stirred at 25 °C for 0.5 hr. About 18% of the desired mass was detected by LCMS. MeOH (10 mL) was added drop-wised to the mixture at 20 °C under N2and stirred for 30 mins. The mixture was poured into ice-water ( 100 mL) and stirred for 5 mins. 1’he mixture was extracted with ethyl acetate ( 100 mL x 3). The combined organic phase was washed with brine (100 mL x 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The mixture was purified by column chromatography (SiO2, hexane / Ethyl acetate = 50 / 1 to 1 / 1, TLC: Petroleum ether / Ethyl acetate=l / l, Rf= 0.43). Cpd.3 (1.50, 4.50 mmol, 100% purity, 27.9% yield) was obtained as a red solid and confirmed by 1H-NMR and LCMS.

[0509] LCMS (Method 2): 220 nm, RT = 0.570 min. m / z = 334.3 (M+H)+.

[0510] 1H NMR: (400 MHz, CDCl3). δ 8.50 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 4.97 (s, 1H), 4.44 (d, J = 5.6 Hz, 2H), 2.80 (s, 3H), 1.49 (s, 9H)General procedure for preparation of Cpd.9Bu3SnCI, t-BuLiTHF, -78-0 °C, 4 hrsstep 4: 36.1% yield

[0511] To a solution of Cpd. 8 (5.00 g, 59.4 mmol, 5.43 mL, 1.00 eq) in dry THF (100 mL) was added t-BuLi (1.3 M, 50.0 mL, 1.09 eq) drop-wised at -78 °C under N2and the resulting solution was stirred at 0 °C for 1 hr. The mixture was cooled to -78 °C and tributyl(chloro)stannane (13.9 g, 42.7 mmol, 11.5Atty Docket No. 63XT-401927-WO mL, 0.719 eq) was added drop-wised and stirring was continued for 1 hr at -78 °C under N₂. TLC (Petroleum ether: Ethyl acetate = 10: 1) indicated Cpd. 8 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phase was washed with brine (200 mL x 2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The mixture was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether / Ethyl acetate = 100 / 0) to afford Cpd. 9 (8.00 g, 21.4 mmol, 36.0% yield) as a lightyellow oil. It was succeeded and confirmed by ¹H-NMR.

[0512] ¹H NMR: (400 MHz, CDCl₃).84.67 - 4.79 (m, 1H), 3.85 - 3.95 (m, 2H), 1.97 - 2.06 (m, 2H), 1.80 -1.90 (m, 2H), 1.49 - 1.54 (m, 4H), 1.27 - 1.38 (m, 8H), 0.87 - 0.95 (m, 15H)General procedure for preparation of Cpd. 4

[0513] To a mixture of Cpd. 3 (1.30 g, 3.90 mmol, 1.00 eq), CuTc (650 mg, 7.97 mmol, 2.04 eq) and Cpd. 9 (1.90 g, 5.09 mmol, 1.31 eq) in dioxane (25 mL) was added Pd(PPh3)4(455 mg, 393 pmol, 1.01e-1 eq) in one portion at 20 °C under N₂. The mixture was stirred at 60 °C under N₂ for 0.5 hr. About 40% of the desired mass was detected by LCMS. The mixture was poured into ice-water (150 mL) and stirred for 5 mins. The mixture was extracted with ethyl acetate (150 mL x 3). The combined organic phase was washed with brine (150 mL x 2), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The mixture was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column. Eluent of 0-60% Ethyl acetate / hexanes gradient @ 80 mL / min. TLC: Petroleum ether / Ethyl acetate = 3 / 1, Rf = 0.4). Cpd. 4 (0.80 g, 2.17 mmol, 100% purity, 55.5% yield) was obtained as a red solid and confirmed by ¹H-NMR and LCMS.

[0514] LCMS (Method 2): 220 nm, RT = 0.573 min, m / z = 370.1 (M+H)+.

[0515] ¹H NMR: (400 MHz, CDCl3). δ 8.58 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 6.77 (t, J = 4.4 Hz, 1H), 4.97 (s, 1H), 4.28 - 4.50 (m, 4H), 2.35 - 2.54 (m, 2H), 2.02 - 2.12 (m, 2H), 1.49 (s, 9H)Atty Docket No. 63XT-401927-WO General procedure for preparation of Cpd.5

[0516] To a solution of Cpd. 4 (0.80 g, 2.17 mmol, 1.00 eq) in DCM (20 mL) was added TFA (3.22 g, 28.27 mmol, 2.10 mL, 13.0 eq) in one portion at 20 °C under N₂. The mixture was stirred at 20 °C under N₂ for 2 hrs. About 85% of the desired mass was detected by LCMS. The mixture was concentrated in vacuum. The mixture was purified by Prep-HPLC (column: CD03-Welch Xtimate C18 150*25mm*5um; mobile phase: [H₂O (0.225% FA)-ACN]; gradient:3%-33% B over 10.0 min). Cpd.5 (0.40 g, 1.49 mmol, 100% purity, 68.6% yield, FA salt) was obtained as a red solid and confirmed by ¹H-NMR and LCMS.

[0517] LCMS (Method 1): 220 nm, RT = 0.881 min, m / z = 270.0 (M+H)+.

[0518] ¹H NMR: (400 MHz, DMSO-d₆). 58.52 (d, J = 8.4 Hz, 2H), 8.28 (s, 3H), 7.73 (d, J = 8.0 Hz, 2H), 6.64 (t, J = 4.8 Hz. 1H), 4.24 - 4.31 (m, 2H), 4.19 (s. 2H), 2.35 - 2.41 (m, 2H), 1.90 - 1.99 (m, 2H) General procedure for preparation of Cpd.7

[0519] To a 40-ml vial containing Cpd. 5b (1.00 g. 1.94 mmol, 2.09 eq) was added DMF (12 mL) and the mixture was cooled to 0 °C in an ice / water bath. HATU (370 mg, 973 pmol, 1.05 eq) and DIPEA (500 mg, 3.87 mmol, 673 pL, 4.17 eq) were added to the mixture. The mixture was stirred in the cold bath for 0.25 hr. Then Cpd. 5 (250 mg, 928 μmol, 1.00 eq) in DMF (3 mL) was added into the mixtureAtty Docket No. 63XT-401927-WO slowly under N2. The reaction mixture was allowed to 20 °C and stirred for 1 hr under N2. About 60% of the desired mass was detected by LCMS. The mixture was filtered and the organic phase was concentrated in vacuum. The mixture was purified by prep-HPLC (colunm: CD05-Phenomenex Luna C18 100*40mm*10um; mobile phase: [H2O (0.225% FA)-ACN]; gradient:24%-54% B over 13.0 min).Cpd. 7 (0.40 g, 522 pmol, 100% purity, 56.2% yield) was obtained as a red solid and confirmed by ¹H-NMR and LCMS.

[0520] LCMS (Method 1): 220 nm, RT = 1.334 min, m / z = 766.4 (M+H)+.

[0521] ¹H NMR: (400 MHz, MeOD). δ 8.54 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 6.76 (t, J = 4.0 Hz, 1H), 4.53 (s, 2H), 4.31 - 4.37 (m, 2H), 3.79 (t, J = 6.0 Hz, 2H), 3.72 (t, J = 6.4 Hz, 2H), 3.57 -3.64 (m, 32H), 2.52 - 2.56 (m, 4H), 2.40 - 2.47 (m, 2H), 2.01 - 2.10 (m, 2H)General procedure for preparation of Cpd. 10

[0522] To a mixture of Cpd.7 (300 mg, 381 pmol, 1.00 eq) and 1 -hydroxypyrrolidine-2, 5-dione (180 mg, 1.56 mmol, 4.10 eq) in DCM (30 mL) was added DIPEA (180 mg, 1.39 mmol, 242 pL, 3.65 eq) in one portion at 20 °C under N2. The mixture was cooled to 0 °C. DCC (180 mg, 872 pmol, 176 pL, 2.28 eq) in DCM (10 mL) was added drop-wised to the mixture at 0 °C under N2. The mixture was stirred at 0 °C under N2 for 2 hrs. About 73% of the desired mass was detected by LCMS. Reaction mixture was combined second reaction mixture (330 mg scale). The mixture was filtered and the organic phase was concentrated in vacuum. The mixture was purified by prep-HPLC (column: CD03-Welch Xtimate C18 150*25mm*5um; mobile phase: [H2O (0.225% FA)-ACN]; gradient:28%-58% B over 10.0 min). Cpd. 10 (301.32 mg, 336 pmol, 96.2% purity, 43.5% yield) was obtained as a red solid. The product was confirmed by ¹H-NMR, LCMS and HPLC.

[0523] LCMS (Method 2): 220 nm, RT = 0.455 min, m / z = 863.7 (M+H)+

[0524] HPLC (Method 1): 220 nm, RT = 2.213 min, 96.2% purity.

[0525] ¹H NMR: (400 MHz, CDCl₃). δ 8.56 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.20 (t, J = 5.2 Hz, 1H), 6.76 (t, J = 4.0 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H), 4.33 - 4.41 (m. 2H), 3.79 - 3.87 (m.4H), 3.51 - 3.68 (m, 32H), 2.81 - 2.94 (m, 6H), 2.58 (t, J = 6.0 Hz, 2H), 2.40 - 2.46 (m, 2H), 2.04 - 2.09 (m, 2H).Atty Docket No. 63XT-401927-WO Example 2: Synthesis of Mal-Glu-Tz-DHPAtty Docket No. 63XT-401927-WO

[0527] General procedure for preparation of compound 17

[0528] To a mixture of compound 4A (400 mg, 3.666 mmol, 1 eq), compound 16 (3.406 g, 14.66 mmol, 4 eq) and Ni(OTf)2(654 mg, 1.83 mmol, 0.5 eq) was added N2H4•H2O (5.872 g, 115 mmol, 5.69 mL, 98% purity, 31.4 eq) under atmosphere, the mixture was stirred 60 °C for 24 hrs. Then 1 N HC1 was added to adjust pH to 3, followed by the addition of H2O (20 mL) and NaNO2(2.023 g, 29.3 mmol, 1.59 mL, 8 eq) slowly under the condition of an ice-water bath, and the resulting mixture will be stirred at 0 °C for another 2 hrs. LCMS analysis showed compound 4A was consumed completely, and one main peak with desired mass-to-charge ratio. The reaction mixture was extracted with EtOAc (60 mL x 3). The combined organic layers were washed with saturated Na2S2O3aqueous solution (60 mL x 3) and brine (60 mL x 3), dried over Na2SC>4, filtered and concentrated under reduced pressure to give the residue. The residue was purified by prep-HPLC (FA condition) to give compound 17 (320 mg, 764 pmol, 20.8% yield, 88.2% purity) as a purple solid, confirmed by 1H NMR, LCMS and HPLC. LCMS: Rt= 0.571 min, MS cal.: 369.4, MS observed: [M+H]+= 370.1. HPLC: Rt= 4.373 min, purity: 88.2%. II NMR: 400 MHz, CDCl3δ: 8.57 (d, J = 8.4 Hz, 2 H), 7.50 (d, J = 8.1 Hz, 2 H), 6.76 (t, J = 4.3 Hz, 1 H), 4.98 (br s, 1 H), 4.44 (br d, J = 5.6 Hz, 2 H), 4.37 - 4.40 (m, 2 H), 2.41 - 2.47 (m, 2 H), 2.04 - 2.11 (m, 2 H), 1.49 (s, 9 H).

[0529] General procedure for preparation of compound 18

[0530] A solution of compound 17 (320 mg, 764 μmol, 1 eq) in DCM (3.2 mL) and TFA (1.232 g, 10.8 mmol, 0.8 mL, 14.1 eq) was stirred at 20-25 °C for 2 hrs. LCMS analysis showed the starting material was consumed completely, and one main peak with desired mass-to-charge ratio. The reaction mixture was concentrated under reduced pressure to give compound 18 (300 mg, 726 pmol, 95.0% yield, 92.5% purity, TFA salt) as a purple solid, confirmed by 1H NMR, LCMS and HPLC. LCMS: Rt= 0.319 min, MS cal.: 269.3, MS observed: [M+H]+= 269.9. HPLC: Rt = 1.944 min, purity: 92.5%. II NMR: 400 MHz, DMSO-d₆ δ: 8.51 (d, J = 8.4 Hz, 2 H), 8.33 (br s, 3 H), 7.73 (d, J = 8.4 Hz, 2 H), 6.64 (t, J = 4.3Atty Docket No. 63XT-401927-WO Hz, 1 H), 4.24 - 4.31 (m, 2 H), 4.19 (br d, J = 3.0 Hz, 2 H), 2.35 - 2.41 (m, 2 H), 1.96 (quin, / = 5.6 Hz, 2 H).

[0531] General procedure for preparation of compound 20

[0532] To a solution of compound 19 (165.1 mg, 544 pmol. 1.5 eq) in DMF (3 mL) was added TBTU (174.8 mg, 544 pmol, 1.5 eq) and DIPEA (140.7 mg, 1.09 mmol. 190 pL. 3 eq). The mixture was stirred at 0-5 °C for 0.5 hr, then compound 18 (150 mg, 363 pmol, 1 eq, TFA) was added. The mixture was stirred at 0-5 °C for 0.5 hr. LCMS analysis showed the starting material was consumed completely, and one main peak with desired mass-to-charge ratio. 1’he reaction mixture was filtered and the filtrate was purified by prep-HPLC (FA condition) to give compound 20 (160 mg, 288 pmol, 79.5% yield, 100% purity) as a purple solid, confirmed by 1H NMR, LCMS and HPLC. LCMS: Rt = 0.606 min. MS cal.: 554.6, MS observed: [M+H]+= 555.3. HPLC: Rt= 4.635 min, purity: 100%. ¹H NMR: 400 MHz, CDCl₃ δ: 8.57 (d, J = 8.4 Hz, 2 H), 7.49 (d, J = 8.4 Hz, 2 H), 6.81 (br s, 1 H), 6.77 (t, J = 4.4 Hz, 1 H), 5.29 -5.37 (m, 1 H), 4.58 (br t, J = 6.3 Hz, 2 H), 4.36 - 4.41 (m, 2 H), 4.11 - 4.23 (m, 1 H), 2.41 - 2.50 (m, 3 H), 2.31 - 2.38 (m, 1 H), 2.04 - 2.15 (m, 3 H), 1.97 (dt, J= 14.6, 7.3 Hz, 1 H), 1.46 (s, 9 H), 1.44 (s, 9 H).

[0533] General procedure for preparation of compound 21

[0534] A mixture of compound 20 (160 mg, 288 pmol, 1 eq), TFA (492.8 mg, 4.322 mmol, 320 pL, 15 eq) in DCM (1.28 mL) was stirred at 25 °C for 11 hrs. LC-MS analysis showed compound 20 was consumed completely and the desired mass-to-charge ratio (Rt= 0.340 min) was detected. The reaction mixture was slowly added to 160 mL cool isopropyl ether (100 V ) and the precipitate was pelleted by centrifugation. The supernatant was discarded to give a residue. Then the residue was concentrated under reduced pressure at 30 °C. The residue was purified by prep-HPLC (AcOH condition). Compound 21 (39 mg. 97 pmol, 33.6% yield, 98.9% purity) was obtained, confirmed by LCMS and HPLC. LCMS: Rt= 0.346 min, MS cal.: 398.17, MS observed: [M+H]+= 399.1. HPLC: Rt= 3.105 min. purity: 98.9%

[0535] General procedure for preparation of Target 388Atty Docket No. 63XT-401927-WOTarget 388

[0536] To a solution of compound 21 (34 mg, 84 pmol, 1 eq) in DMF (300 pL) was added DIPEA (32.7 mg, 253 pmol, 44 μL, 3 eq). Then the above mixture was added to a solution of compound 7 (67.4 mg, 253 pmol, 3 eq) in DMF (300 pL). The mixture was stirred at 25 °C for 20 min. LC-MS analysis showed compound 21 was consumed completely and the desired mass (Rt= 1.391 min) was detected. The reaction mixture was combined with a second batch and purified by prep-HPLC (AcOH condition) directly. The desired product, Mal-Glu-Tz-DHP (Target 388) (50 mg, 91 pmol, 93.6% yield for two batches, 99.6% purity) was obtained. LCMS: Rt= 0.423 min, MS cal.: 549.20, MS observed: [M+H]+= 550.2. HPLC: Rt= 2.811 min, purity: 99.6%. ¹H NMR: 400 MHz, DMSO-d₆ δ: 8.55 (t, J = 6.13 Hz, 1 H), 8.42 (d, J = 8.25 Hz, 2 H), 8.31 (brd, 7= 7.13 Hz, 1 H), 7.51 (d, J = 8.38 Hz, 2 H), 6.99 (s, 2 H), 6.61 (t, J = 4.25 Hz. 1 H), 4.40 (br d, J = 6.00 Hz, 2 H), 4.21 - 4.28 (m, 3 H), 3.63 (t, J = 7.32 Hz, 2 H), 2.36 -2.45 (m, 4 H), 2.17 - 2.26 (m, 2 H). 1.89 - 1.99 (m, 3 H). 1.68 - 1.83 (m, 1 H).Example 3: Conjugation procedureT84 Fab-TzlO conjugate:

[0537] T84 Fab (4.0 mg) (Neumaier. et al. (1990) Cancer Res.50(T): 2128-34) was buffer exchanged to 25 mM HEPES, pH 8.0 overnight. The NHS-PEG9-TzlO was dissolved in DMSO to make a 10 mM stock solution. For conjugation, the two components were reacted at 3.3:1 (NHS-PEG9-TzlO to Fab) molar ratio at 25 °C for 2 hours. The reaction was quenched by addition of 1.0 mM hydroxylamine and the resulting mixture was aged at ambient temperature for 30 min. Then the solution was buffer exchanged to PBS pH 7.4 to remove excess tetrazine and buffer salts. The total amount of conjugate T84 Fab-TzlO recovered was 3.7 mg. The average degree of labeling, as determined by mass spectrometry, was 2.36. The conjugate was >99% monomeric by SEC.Atty Docket No. 63XT-401927-WO T84 Fah-MeTz conjugate:

[0538] T84 Fab (350.0 mg) was buffer exchanged to 25 mM HEPES, pH 8.0 overnight. The NHS-PEG9-MeTz was dissolved in DMSO to make a 10 mM stock solution. For conjugation, the two components were reacted at 4: 1 (NHS-PEG9-MeTz to Fab) molar ratio at 25 °C for 2 hours. The reaction was quenched by addition of 1.0 m hydroxylamine and the resulting mixture was aged at ambient temperature for 60 min. Then the solution was buffer exchanged to PBS pH 7.4 to remove excess tetrazine and buffer salts. The total amount of conjugate T84 Fab-MeTz recovered was 315 mg. The average degree of labeling, as determined by mass spectrometry, was 2.60. The conjugate was >99% monomeric by SEC.Example 4: Analysis of Conjugates

[0539] The following protodrugs were tested and the following conditions employed.:InstrumentationVanquish UHPLC System (Thermo Fisher Scientific, USA)Orbitrap Exploris 240 (Thermo Fisher Scientific, USA)LC conditionsColumn: Agilent PLRP-S 1000 A 8pm 50*2.1mmColumn Temperature: 70 °CSolvents: A, 0.1% FA and 10% IPA in Water; B, 0.1% FA and 10% IPA in ACNFlow rate: 300 pL / minAtty Docket No. 63XT-401927-WO Program: 0-20 min, 15%-38%B, 20-22 min, 38%-90%B, 22-22.5 min, 90%-99%B, 22.5-24min, 99%B, 24-25min, 99-15%B;Injection Volume: 12 pLMS conditionsIonization mode: Positive ion modeSpray Voltage: StaticAux gas flow rate: 10Vaporizer Temp (°C): 350 °CScan type: Full MSResolution: 15,000AGC Target: Standard

[0540] A mixture containing the tetrazine activator (Fab-MeTz or Fab-TzlO, 100 pg / mL) and protodrug (200 pg / mL) in PBS was incubated at 37 °C for 4 hours. The resulting product was analyzed by LCMS, the charge ladder reconstructed, and the resulting peaks were quantified by area percent. The peaks were assigned according to the mass shift from the unconjugated protein peak and the percent of desired product vs dead-end product was calculated. The release efficiency was calculated as the area percent of the desired product divided by the sum of area percents of both products multiplied by 100. The results are provided below.

[0541] As can be seen from the above-table, the payload release efficiency is superior in the tetrazine comprising the dihydropyran.Atty Docket No. 63XT-401927-WO

[0542] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0543] The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of tire claims.

[0544] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0545] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Claims

Atty Docket No. 63XT-401927-WO What is claimed is:A targeting moiety of Formula I or Formula II or Formula III:wherein:X is a biocompatible support, antibody, antibody fragment moiety;p is 1-150;L, at each occurrence, is independently a linker;R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Ra, R31a, and R31bare each independently hydrogen, Ci-Ce-alkyl, or Ci-Ce-haloalkyl;ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl;R2, at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl. alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1is independently selected from halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR’, SR', C(=O)R’, C(=S)R’, OC(=O)R"’, SC(=O)R’", OC(=S)R"’, SC(=S)R"', S(=O)R', S(=O)2R"’, S(=O)2NR' R", C(=O)O-R’, C(=O)S-R’, C(=S)O-R', C(=S)S-R’, C(=0)NR’R", C(=S)NR'R", NR’R", NR'C(=0)R", NR'C(=S)R", NR’C(=0)0R", NR'C(=S)OR", NR’C(=0)SR", NR'C(=S)SR", 0C(=0)NR’R", SC(=O)NR’R", OC(=S)R’R’", SC(=S)R’R", NR’C(=0)NR" R", and NR'C(=S)NR" R”;R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;Atty Docket No. 63XT-401927-WO R'", at each occurrence, is independently selected from aryl and alkyl; andt, at each occurrence, is independently 0, 1, 2, 3, or 4.

2. The targeting moiety of claim 1, wherein p is 1-16, or 4-12, or 2-10, or 5-10, or 1-8, or 3-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 0-4.The targeting moiety of claim 1, wherein the targeting moiety is of Formula I or II:wherein:X is an antibody or antibody fragment moiety;p is 1-16;L, at each occurrence, is independently a linker;R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group; R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Ra, R31aand R31bare each independently hydrogen, Ci-Cs -alkyl, or Ci-Ce-haloalkyl;R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; R'" at each occurrence is independently selected from aryl and alkyl; andt, at each occurrence, is independently 0, 1, 2, 3, or 4.Atty Docket No. 63XT-401927-WO 4. The targeting moiety of claim 3, represented by Formula IIA:

5. The targeting moiety of claim 1, represented by Formula III:

6. The targeting moiety of claim 5, wherein ring A is phenyl, pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido [4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl.

7. The targeting moiety of claim 6, wherein at least one of the moiety:is represented by a formula selected from:Atty Docket No. 63XT-401927-WOwherein R1is dihydropyran.

8. The targeting moiety of any one of claims 1-2 or 5-7, wherein R2, at each occurrence, is independently halo, alkyl, or haloalkyl.

9. The targeting moiety of any one of claims 1-3 or 5-8, wherein t, at each occurrence, is 0.

10. A targeting moiety of formula:Atty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOwhereinp is 1-10; andX is an antibody or antibody fragment moiety.

11. The targeting moiety of any one of claims 1-10, wherein X is a biocompatible support.

12. The targeting moiety of any one of claims 1-11 wherein X is a biocompatible support which comprises a particle, polymer, viscous or non- viscous liquid material, gel, hydrogel, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, or a protein.

13. The targeting moiety of any one of claims 1-10, wherein X is an antibody or antibody fragment moiety.

14. The targeting moiety of any one of claims 1-10 or 13, wherein X is an antibody or antibody fragment moiety which targets TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, CD25, ANTXR1, CSPG4, or FAP.

15. The targeting moiety of any one of claims 1-10 or 13-14, wherein X is an antibody selected from daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, duligotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, laprituximab, oportuzumab, citatuzumab, tucotuzumab, catumaxomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, vulinacimab, olaratumab, ramucirumab, sacituzumab, Pr1E11, enfortumab,J591, MLN591, belantamab, moxetumomab, inotuzumab,Atty Docket No. 63XT-401927-WO epratuzumab, pinatuzumab, ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, ibritumomab, loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, MDX-1342, polatuzumab, isatuximab, daratumumab, MOR202, TAK-079, 1-131-BC8, lomab-B, carotuximab, bemarituzumab, aprutumab, lupartumab, zolbetuximab, claudiximab, andecaliximab, mirvetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, rovalpituzumab, indatuximab, lorvotuzumab, promiximab, BI 836826, otlertuzumab, naratuximab, milatuzumab, anetumab, amatuximab, MMOT-0530A, sarilumab, elotuzumab, belimumab, KL-6, MY.1E12, hMUCl-lH7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, cantuzumab, codrituzumab, ECT204, MDX-1414, pertuzumab, trastuzumab, margetuximab, patritumab, seribantumab, lumretuzumab, elgemtumab, AV-203, CDX-3379, GSK284933, brentuximab, gemtuzumab, BI 835858, vadastuximab, lintuzumab, KHK2823, taclotuzumab, G4723A. glembatumumab, telisotuzumab, onartuzumab, SAIT301, tisotumab, lifastuzumab, indusatumab, vandortuzumab, sofituzumab, vorsetuzumab, bivatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, vobarilizumab, LCAR-B38M, BI 655088, AD-214. ALX-0651, TXB4, CDP791, GY1. L19, NJB2, E19, OMTX005, sibrotuzumab, C17, C44, F16 or R6N.

16. The targeting moiety of claim any one of claims 1-10 or 13-15, wherein X is an antibody fragment moiety selected from the group consisting of a single-chain variable fragment (scFv), a divalent (or bivalent) single-chain variable fragment (di-scFvs, bi-scFvs), an antigen-binding fragment (Fab), a single-domain antibody (sdAb), a VHH, an antigen-binding protein, a DotBody, an affibody, a DARPin, a DART, a TandAb, a diabody. a ribobody, a centyrin. a knottin, an affilin, an affimer, an alphabody, an anticalin, an atrimer. an avimer, a fynomer, a kunitz domain, an obody. a pronectin, a repebody, and a bicyclic peptide or a Humabody.

17. The targeting moiety of any one of claims 1-10, 13-14, or 16, wherein X is an antibody fragment moiety derived from daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595. depatuxizumab, dapatuxizumab, duligotuzumab, futuximab, GC1118, imgatuzumab, panitumumab. alutumumab, tomuzotuximab, laprituximab, oportuzumab. citatuzumab, tucotuzumab, catumaxomab, edrecolomab, adecatumumab, ramucizumab, ramucirumab, vulinacimab. olaratumab, ramucirumab, sacituzumab, Pr1E11, Enfortumab, J591, MLN591, belantamab. moxetumomab, inotuzumab, epratuzumab, pinatuzumab, ublituximab. ofatumumab, rituximab, obinutuzumab. tositumomab, ibritumomab, loncastuximab. XMAB-5574. MOR208. coltuximab, denintuzumab. taplitumomab. MDX-1342, polatuzumab. isatuximab, daratumumab. MOR202, TAK-079. 1-131-BC8. lomab-B. carotuximab, bemarituzumab, aprutumab. lupartumab. zolbetuximab. claudiximab, andecaliximab, mirvetuximab, farletuzumab, MORAb-202, MORAb-003, SP8166, rovalpituzumab, indatuximab, lorvotuzumab, promiximab, BI 836826, otlertuzumab, naratuximab, milatuzumab, anetumab, amatuximab, MMOT-0530A, sarilumab, elotuzumab, belimumab, KL-6, MY.1E12, hMUCl-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, cantuzumab, codrituzumab, ECT204, MDX-1414, pertuzumab, trastuzumab, margetuximab, patritumab, seribantumab,Atty Docket No. 63XT-401927-WO lumretuzumab, elgemtumab, AV-203, CDX-3379, GSK284933, brentuximab, gemtuzumab, BI 835858, vadastuximab, lintuzumab, KHK2823, taclotuzumab, G4723A, glembatumumab, telisotuzumab, onartuzumab, SAIT301, tisotumab, lifastuzumab, indusatumab, vandortuzumab, sofituzumab, vorsetuzumab, bivatuzumab, caplacizumab, ozoralizumab, V565, PF-05230905, vobarilizumab, LCAR-B38M, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, C17, C44, GMTX005, sibrotuzumab, F16 or R6N.

18. The targeting moiety of any one of claims 1-10 or 13-17, wherein the antibody or antibody fragment moiety is caplacizumab, ozoralizumab, V565, PF-05230905, vobarilizumab, LCAR-B38M, T84.66, M6495, BI 655088, AD-214, ALX-0651, TXB4, CDP791, GY1, L19, NJB2, F19, GMTX005, sibrotuzumab, F16, C17, C44, or R6N.

19. The targeting moiety of any one of claims 1-10 or 13-18, wherein the antibody or antibody fragment moiety targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNG, or ECM, EPCAM, CSPG4, CEA, or CEACAM5.

20. The targeting moiety of any one of claims 1-19, wherein X further comprises an imaging contrast agent.

21. The targeting moiety of any one of claims 1-20, wherein linker L is bonded to X via a cysteine or lysine residue on X.

22. The targeting moiety of any one of claims 1-21, wherein L is a non-cleavable linker.

23. The targeting moiety of any one of claims 1-22, wherein L is a cleavable linker.

24. The targeting moiety of any of claims 1-23, wherein L comprises one or more amino acids.

25. The targeting moiety of any of claims 1-24, wherein L comprises one or more of a hydrazone, a hydrazide, a disulfide, a N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), a N-succinimidyl-4-(2-pyridyldithio)butyratc (SPDB), a 4-(4’-acctylphcnoxy)butanoic acid (AcBut), one or more linear or branched, natural or unnatural amino acid, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety.

26. The targeting moiety of any one of claims 1-25, wherein L comprises 1 to 100 linking atoms, from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms, or from 1 to 40 linking atoms, or from 1 to 30 linking atoms, or from 1 to 20 linking atoms, or from 1 to 10 linking atoms, or from 1 to 5 linking atoms, or from 5 to 30 linking atoms, or from 10 to 30 linking atoms, or from 5 to 40 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms.

27. The targeting moiety of any one of claims 1-26, wherein L comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene,Atty Docket No. 63XT-401927-WO cycloalkylene, or heterocycloalkylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, CM alkoxy, and CM haloalkyl.

28. The targeting moiety of any one of claims 1-27, wherein L is an alkylene linker optionally comprising one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thio- succinimide, or ketone functional groups.

29. The targeting moiety of any one of claims 1-28, wherein L is of the formula:-Y10-(CHR130)n'-Y20-(CHR140)n''-Y30-(CHR150)m''-Y40- wherein:each of Y10, Y20, Y30, and Y40are independently a bond, -NR110-, -O-, -S(O)0.2-, -NR110C(C))-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-. -NR110-N=CR120-, -C(O)-, -OC(O)-, -OC(O)O-, -(CH2CH2O)1-5-. -C(O)O-. alkylene, alkenylene, alkynylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, arylene, or heteroarylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, C alkoxy, and C1.4 haloalkyl;each R110is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;each R120is independently hydrogen, C alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl;each R130is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain;each R140is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain;each R150is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; andn', n", and m" are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

30. The targeting moiety of any one of claims 1-29, wherein L is of the formula:-Y10-(CH2)„’-Y20-(CH2)m"-Y30-wherein:each of Y10, Y20, and Y30are independently a bond, -NR110-, -O-, -S(O)0-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, hetero arylene, cycloalkylene, or heterocycloalkylene; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, orAtty Docket No. 63XT-401927-WO heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, C1-4 alkoxy, and C1.4 haloalkyl;each R110is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;each R120is independently hydrogen, CM alkyl, CM haloalkyl. aryl, heteroaryl, cycloalkyl or heterocyclyl; andn' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

31. The targeting moiety of any one of claims 1-21, wherein L is:Atty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WO33. The targeting moiety of any one of claims 1-21, wherein X is an antibody or antibody fragment moiety targets HER2, TROP2, Nectin-4, Claudin-18.2, MMP9, mesothelin, FN1, FAP, TNC, or ECM, EPCAM, CSPG4, CEA, or CEACAM5; andL, at each occurrence, is independently a linker selected from the group consisting of:Atty Docket No. 63XT-401927-WO34. A pharmaceutical composition comprising the targeting moiety of any of claims 1-33, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

35. A method of treating cancer or enhancing or eliciting an immune response, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the targeting moiety of any of claims 1-33, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 34, and a conjugate comprising payload linked to one or more trans-cyclooctene moieties, or a pharmaceutically acceptable salt thereof.

36. The method of claim 35, wherein the conjugate is of Formula X, or a pharmaceutically acceptable salt thereof:wherein:G, at each occurrence, is independentlyL1, at each occurrence, is independently a linker;m is an integer from 1-150;D is a payload;R1A, at each occurrence, is independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, and C1-4alkoxy;q is 0, 1, or 2;ql is 0 or 1;R1B, at each occurrence, is independently selected from the group consisting of G1, OH,C1-6alkyl, –N(R1f)–C2-4alkylene–(N(C1-4alkylene–C(O)OC1-6alkyl)–C2-4alkylene)n–N(C1-4alkylene–C(O)OC1-6alkyl)2, –N(R1c)–C1-6alkylene–SO3H, –N(R1c)–(CH2CH2O)1-3–CH2CH2N((CH2CH2O)1-3–C1-6alkylene–CO2H)2, and –N(R1c)–CH(CH2O–(CH2CH2O)0-2–C1-6alkylene–CO2H)2;R1c and R1d, at each occurrence, are independently hydrogen or C1-4alkyl;Atty Docket No. 63XT-401927-WO Rle, at each occurrence, is independently -C1-4alkylene-CO2H, -Ci-4alkylene-CONH2, or -Ci-alkylene-OH;R1f, at each occurrence, is independently hydrogen, C1-6alkyl, or C1-4alkylene–CO2H;n, at each occurrence, is independently 0, 1, 2, or 3;L2, at each occurrence, is independently selected from the group consisting of –C(O)– and C1-3alkylene; andG1, at each occurrence, is independently an optionally substituted heterocyclyl.

37. The method of claim 35 or 36, wherein the payload is an immunomodulatory agent payload.

38. The method of any one of claims 35-37, wherein the payload is a dierapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.

39. The method of any one of claims 35-37, wherein the payload is selected from a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, camptothccin, cxatccan, auristatin (dolastatin 10, MMAE, MMAD, MMAF) mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, duocarmycin, PBD, and die like), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., amphotericin, and die like), an antibiotic (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimedioprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, and the like), lurbinectedin, gardiquimod, a matrix metalloproteinase (MMP) inhibitor, L-dopa, oseltamivir, cefalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic-adenosine monophosphatidyl (c-AMP).

40. The method of claim 35, wherein die conjugate is selected from:Atty Docket No. 63XT-401927-WOAtty Docket No. 63XT-401927-WO41. The method of any of claims 35-40, wherein the method is a method of treating cancer.

42. The method of claim 41, wherein the cancer is a melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, colorectal cancer, squamous cell carcinoma of head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi’s sarcoma, Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma Wilms’ tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, Prostatic adenocarcinoma, Nasopharyngeal carcinoma, or Cutaneous T-cell lymphoma.

43. The method of claim 41, wherein the cancer is a hematological malignancy such as myelodysplastic syndrome, acute myeloid leukemia, myelodisplastic syndromes, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin’s lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B -lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy-cell leukemia, precursor T-lymphoblastic leukemia, or nodular lymphocyte predominant Hodgkin’s lymphoma.

44. A kit comprising the targeting moiety of any of claims 1-33, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 34, and instructions for use thereof.

45. The kit of claim 44, further comprising and a conjugate comprising payload linked to one or more trans-cyclooctene moieties, or a pharmaceutically acceptable salt thereof.

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