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WO2024238969A1 - Trans-cyclooctene-modified targeted protein degrader conjugates - Google Patents

Trans-cyclooctene-modified targeted protein degrader conjugates Download PDF

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Publication number
WO2024238969A1
WO2024238969A1 PCT/US2024/030043 US2024030043W WO2024238969A1 WO 2024238969 A1 WO2024238969 A1 WO 2024238969A1 US 2024030043 W US2024030043 W US 2024030043W WO 2024238969 A1 WO2024238969 A1 WO 2024238969A1
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Prior art keywords
alkyl
trans
cyclooctene
aryl
occurrence
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French (fr)
Inventor
Jesse M. McFARLAND
José Manuel Mejía ONETO
Jaime R. CABRERA-PARDO
Maksim Royzen
Muhammad SAADIQ
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Tambo Inc
Research Foundation of the State University of New York
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Tambo Inc
Research Foundation of the State University of New York
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Publication of WO2024238969A1 publication Critical patent/WO2024238969A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • TPD trans-cyclooctene-modified targeted protein degrader
  • TPD targeted protein degrader
  • linker a linker
  • TPDs targeted protein degrader
  • PROTACs proteolysis-targeting chimeras
  • a bifunctional TPD molecule is typically comprised of two covalently-linked ligands, and functions to recruit target protein and E3 ubiquitin ligase together to trigger proteasomal degradation of target protein by the ubiquitin- proteasome system (UPS).
  • UPS ubiquitin- proteasome system
  • a TPD needs to enter cells to mobilize intracellular UPS, so its membrane permeability is the key to TPDs function.
  • large molecular weight and large exposed polar surface area of TPDs can result in low cell/tissue permeability.
  • some E3 ligands exhibit low specificity which results in such TPDs having off-target effects.
  • trans-cyclooctene-modified targeted protein degrader (TPD) (or PROTAC) conjugates, which comprise a targeted protein degrader (TPD) covalently bonded to at least one trans- cyclooctene moiety, optionally via a linker.
  • TPD targeted protein degrader
  • the trans-cyclooctene-modified TPD conjugates described herein are designed to be used with a tetrazine activator which then, when in contact with the trans- cyclooctene-modified TPD conjugate, liberates the TPD in vivo.
  • trans-cyclooctene-modified TPDconjugates described herein may have enhanced benefits, such as, but not limited to, improving the permeability of a TPD, masking the activity of the TPDin order to enhance the safety profile, and the like. It is further contemplated that cell permeability of the TPDcould be increased by the use of linkers between the TPDand trans-cyclooctene moiety or derivatizing one or more trans-cyclooctene moieties in order to form intramolecular hydrogen bonds that partially reduce polarity, or attaching cell-permeable peptides thereto, such as a poly-D- arginine sequence.
  • a method for treating cancer comprising administering to a subject in need thereof, a conjugate as described herein to a subject in need thereof, and administering to the subject a tetrazine activator, as described herein.
  • compounds which include a precursor to a TPD that upon administration individually, react to generate a TPDin vivo.
  • a system comprising a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine- modified target protein ligand.
  • a system comprising a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand.
  • 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 trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand, or a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand.
  • the cancer is metastatic.
  • 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, 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 Wilm’s tumor/neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma,
  • 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.
  • the cancer is a solid tumor.
  • the cancer is a lymphoma or leukemia.
  • the cancer is a hematologic malignancy. DETAILED DESCRIPTION [0016] 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. 1.
  • 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.
  • 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.
  • “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.
  • the conjunctive term “or” includes any and all combinations of one or more listed elements associated by the conjunctive term.
  • 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, ...
  • alkyl as used herein, means a straight or branched, saturated hydrocarbon chain containing from 1 to 30 carbon atoms.
  • lower alkyl or “C 1- C 6 -alkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms.
  • C 1 -C 3 - alkyl means a straight or branched chain hydrocarbon containing from 1 to 3 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert- butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, and n-decyl.
  • alkoxy 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.
  • 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.
  • alkynyl 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.
  • 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.
  • alkynyl groups include, but are not limited to acetylenyl (-C ⁇ CH), and propargyl (-CH 2 C ⁇ CH), and cycloalkynyl moieties, such as, but not limited to, substituted or unsubstituted cyclooctyne moieties.
  • alkoxyalkyl refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • alkylene 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, -CH 2 -, -CH(CH3)-, -C(CH3)2-, -CH 2 CH 2 -, -CH(CH3)CH 2 -, -C(CH3)2CH 2 -, -CH 2 CH 2 CH 2 -, -CH(CH3)CH 2 CH 2 -, -C(CH3)2CH 2 CH 2 -, -CH 2 C(CH 3 ) 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, and –CH 2 CH 2 CH 2 CH 2 CH 2 -.
  • 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 ⁇ - 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, and 4-(3-methyl-(1,2,4,5-tetrazine))-phenylalanine.
  • aryl refers to an aromatic carbocyclic group having a single ring (e.g. monocyclic) or multiple rings (e.g. bicyclic or tricyclic) including fused systems.
  • Representative examples of 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 the rings, and can be unsubstituted or substituted.
  • the aromatic bicyclic ring system or aromatic tricyclic ring system does not contain non-aromatic rings.
  • 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.
  • 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 the 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 are 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 the parent molecular moiety through any carbon atom contained within the rings, and can be unsubstituted or substituted.
  • the term “azide” as used herein, refers to the functional group –N3.
  • cycloalkyl refers to a non-aromatic carbocyclic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10, carbon atoms and zero heteroatoms. 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 the point of attachment to the remainder of the molecule.
  • 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.
  • 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 or cycloheptenyl.
  • cyclooctene refers to a substituted or unsubstituted non-aromatic cyclic alkyl group of 8 carbon atoms, having a single ring with a double bond.
  • cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).
  • TCO trans-cyclooctene
  • fluoroalkyl 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.
  • alkoxyfluoroalkyl refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.
  • fluoroalkoxy 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.
  • halogen or “halo” as used herein, means Cl, Br, I, or F.
  • 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.
  • haloalkoxy as used herein, means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.
  • 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.
  • heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, and alkyl sulfides.
  • heteroaryl 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.
  • heteroaryl 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 at least one heteroatom independently selected from the group consisting of N, O and S (e.g.1, 2, 3, or 4 heteroatoms independently selected from O, S, and N).
  • the five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds.
  • 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-1,2- dihydropyridinyl.
  • bicyclic heteroaryl include, but are not limited to, chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine, benzooxadiazolyl, and benzopyrazolyl.
  • 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.
  • the aromatic bicyclic ring system or aromatic tricyclic ring system does not contain non-aromatic rings.
  • 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.
  • the five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds.
  • 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.
  • 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.
  • heterocyclyl 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 (e.g., 1-5, 1-4, 1-3, 1-2, or 1) heteroatom, and optionally one or more oxo and/or double bonds.
  • heterocyclyl 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, morpholin
  • 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.
  • 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-1H-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.
  • tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane).
  • 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.
  • hydroxyl as used herein, means an –OH group.
  • 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.
  • substituted refers to a group that may be further substituted with one or more non- hydrogen substituent groups.
  • 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.
  • tetrazine groups include 1,2,3,4-tetrazine and 1,2,4,5-tetrazine.
  • 1,2,4,5-tetrazine is referred to as a “Tz” group.
  • tetrazine-based generally refers to a tetrazine as defined herein or a moiety which can be converted to a tetrazine, such as an optionally substituted dihydrotetrazine.
  • the term “selectively delivering” refers to delivering an agent (e.g., a TPD) to an organ or tissue (or portion thereof) in need of treatment or diagnosis, without significant binding to other non-target organs or tissues (or portions thereof).
  • an agent e.g., a TPD
  • the trans-cyclooctene-modified TPDconjugates described herein do not themselves have a therapeutic effect, but rather are designed to allow the selective or targeted delivery of a TPD. However, it may be that the tetrazine-based targeting agent does have a therapeutic effect, and thus, such constructs are not excluded by the present disclosure.
  • diagnosis agent refers to agents that assist in diagnosing conditions or diseases.
  • 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, 3 H, 11 C, 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 89 Zr, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In, 123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu, and 241 Am.
  • the term “ligand” as used herein to define a binding partner for a biological target molecule in vivo for example, an enzyme or receptor).
  • the ligands for use according to the invention are trypically small molecules that can be functionalized by the addition of a TCO moiety, optionally via a linker.
  • 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.
  • stable association means that a compound is bound to, e.g., covalently or non- covalently, or otherwise associated with, another moiety or structure under standard physiological conditions.
  • Stable associations, or binding 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), dipole-dipole interactions, and the like.
  • Targeting agents include ligands that specifically bind (or substantially specifically bind) a particular clinically-relevant target receptor or cell surface target. Specific targeting agents are described herein.
  • targeted organ or tissue refers to an organ or tissue that is being targeted for delivery of the TPD. 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.
  • the term “implanting” refers to surgical implantation into a subject’s body.
  • 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 the added reagents or moieties, which can be produced in the contacting mixture.
  • 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) (e.g., 1,2,4,5- tetrazine), and others.
  • TCO trans-cyclooctene
  • Tz tetrazine
  • the binding agents useful in the present disclosure may have a high reactivity with the corresponding binding agent so that the reaction is rapid.
  • 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.
  • 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.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • a pharmaceutically or therapeutically effective amount refers 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.
  • 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.
  • subject or patient 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.
  • 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.
  • livestock such as cattle, sheep, goats, cows, swine, and the like
  • poultry such as chickens, ducks, geese, turkeys, and the like
  • domesticated animals particularly pets such as dogs and cats.
  • suitable subjects may include mammals, such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.
  • treating 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 the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Stereoisomers include enantiomers and diastereomers and mixtures of enantiomers or diastereomers.
  • Individual stereoisomers of the 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.
  • 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.
  • 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, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl, respectively.
  • the compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors.
  • positron-emitting isotopes that can be incorporated are 11 C, 13 N, 15 O, and 18 F.
  • 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. Trans-Cyclooctene-Modified TPD Conjugates [0071] The trans-cyclooctene-modified TPD conjugates described herein are designed to, once administered to a subject, localize at a target site within the subject. The trans-cyclooctene-modified TPD conjugates can be administered locally or systemically.
  • a tetrazine activator can be administered, which when in contact with the trans-cyclooctene-modified TPD conjugate in vivo, allows for targeted drug delivery of a TPD.
  • a trans-cyclooctene-modified TPD conjugate comprising a targeted protein degrader (TPD) covalently bonded to at least one trans-cyclooctene moiety, optionally via a linker.
  • TPD targeted protein degrader
  • the trans-cyclooctene-modified TPD conjugate is of the formula X-L- TPD; wherein X is a trans-cyclooctene moiety; L is a linker; and TPD is a targeted protein degrader.
  • the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of a Von Hippel–Lindau (VHL) ligand, a cereblon (CRBN) ligand, a mouse double minute 2 homolog (MDM2) ligand, and a beta-transducin repeat-containing protein ( ⁇ -TrCP) ligand.
  • VHL Von Hippel–Lindau
  • CRBN cereblon
  • MDM2 mouse double minute 2 homolog
  • ⁇ -TrCP beta-transducin repeat-containing protein
  • the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of thalidomide, lenalidomide, pomalidomide, MLN4924 (Pevonedistat), Nutlin-3, and curcumin.
  • the TPD comprises an E3 ubiquitin ligase ligand selected from Table A, wherein the wavy line indicates the point of attachment to the remainder of the TPD.
  • the TPD comprises a target protein ligand selected from the group consisting of a receptor tyrosine kinase (RAF) kinase ligand (e.g., a BRAF kinase ligand), a steroid receptor ligand (e.g., an androgen receptor (AR) ligand, an estrogen receptor (ER) ligand, or a progesterone receptor (PR) ligand), a cyclin- dependent kinase (CDK) ligand, a BTK ligand, a BET ligand, and an Inhibitor of Apoptosis Protein (IAP) ligand.
  • RAF receptor tyrosine kinase
  • BRAF tyrosine kinase
  • a steroid receptor ligand e.g., an androgen receptor (AR) ligand, an estrogen receptor (ER) ligand, or a progesterone receptor (PR) ligand
  • the TPD comprises a target protein ligand selected from Table B, wherein the wavy line indicates the point of attachment to the remainder of the TPD.
  • the TPD is selected from Table C: Table C [0080]
  • the trans-cyclooctene moiety is of Formula X: wherein: R 1A , at each occurrence, is independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, and C1-4alkoxy; q is 0, 1, or 2; q1 is 0 or 1; R 1B , at each occurrence, is independently selected from the group consisting of G 1 , OH, –NR 1c –C 1-4 alkylene–G 1 , –NR 1c –C 1-4 alkylene–N(R 1d ) 2 , –NR 1c –C 1-6 alkylene–N(C 1-4 alkyl) 3 + , –N(R 1c )CHR 1e CO
  • the trans-cyclooctene moiety is of Formula XI: wherein: R 2 is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine; wherein R 2 may be optionally further substituted with a polyether.
  • the trans-cyclooctene moiety is of Formula: , o [0083] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, at least one trans-cyclooctene moiety is .
  • At least one trans-cyclooctene moiety is ; wherein A is an amino acid side chain (e.g., C1-4 alkyl, aryl (such as phenyl), etc., wherein each are optionally substituted with -OH, -SH, -SCH 3 , -C(O)OH, -NH 2 , -NHNH 2 NH, -C(O)NH 2 , etc..
  • A is an amino acid side chain (e.g., C1-4 alkyl, aryl (such as phenyl), etc., wherein each are optionally substituted with -OH, -SH, -SCH 3 , -C(O)OH, -NH 2 , -NHNH 2 NH, -C(O)NH 2 , etc..
  • trans-cyclooctene-modified TPD conjugate at least one trans-cyclooctene moiety is [0087] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the trans-cyclooctene moiety is covalently bonded to the TPD via a linker. Suitable linkers are described below. [0088] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the linker is a cleavable linker.
  • the linker comprises one or more of a carbamate (-NHC(O)O-), an ester, and amide, 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.
  • the linker 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.
  • the linker 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, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl.
  • the linker is a non-cleavable linker.
  • the linker is a cleavable linker.
  • the linker comprises one or more amino acids.
  • the linker comprises a polypeptide.
  • the linker is an alkylene linker optionally comprising one or more -O-, -S- , amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.
  • each R 110 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R 120 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl.
  • the linker is not a bond.
  • the linker 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-1,2-diylbis(methylcarbamate, an arylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.
  • polyethylene glycol e.g., PEG having an average molecular weight of from 300 g/mol to 10,000 g/mol
  • ethylene-1,2-diylbis(methylcarbamate e.e., an arylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.
  • the linker comprises one or more of: [0102] In some embodiments, the linker comprises one or more of:
  • the linker comprises one or more of: , [0104] In some embodiments, the linker comprises one or more . In some embodiments, the linker comprises one or more . [0105] In some embodiments, the linker is, or comprises one or more: [0106] In some embodiments, the linker is, or comprises one or more: [0107] In some embodiments, the linker is, or 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.
  • the linker is, or comprises a peptide containing linker represented by “A—Y—Z—X 2 —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 2 ” 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.
  • one or more of the amino acids in the peptide linker is N-methylated.
  • 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.
  • 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.
  • Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.
  • Exemplary Y-Z combinations include Valine-Citrulline; Valine-Alanine; and Alanine-Alanine.
  • A is -OC(O)-.
  • X 2 is -OC(O)-.
  • W is -OC(O)-.
  • X 2 is absent and W is -OC(O)-. 2
  • the moiety —X —W comprises .
  • the moiety —X 2 is .
  • 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.
  • 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.
  • the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe- Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-N 9 -tosyl-Arg, or Phe-N 9 -Nitro-Arg.
  • 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. [0121] In some embodiments, L, or the linker, is or comprises one or more of: ,
  • the linker comprises one or more of: ,
  • the linker comprises one or more of:
  • linkers may bond to an amino acid side chain present on X, such as a lysine or [0125]
  • the linker is –C(O)L 4 – or –C(O)C1-6alkyleneC(O)L 4 –;
  • L 4 is a bond, –N(R 12 )–C 2-3 alkylene–N(R 13 )C(O)–, -CH(NHC(O)R 14 )C 1-4 alkylene–S–S–C 1-4 alkylene– OC(O)–, –NHNHC(O)CH(NHC(O)R 15 )CH 2 C(O)–, –C1-6alkylene–CH(G x )OC(O)–,
  • R 12 , R 13 , R 14 , R 15 , and R 19 are each independently hydrogen or C1-4alkyl;
  • R 16 is hydrogen, C 1-4 alkyl, –C 1-4 alkylene–OH, –C 1-4
  • the linker comprises a carbonyl moiety for conjugating at least one trans- cyclooctene moiety to a TPD.
  • 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 the carbonyl moiety.
  • PPM polypeptide moiety
  • the linker may comprise [0127]
  • the linker is or comprises one or more of:
  • the linker is: , [0129] In some embodiments, the linker, is or comprises one or more , , or . [0130] In some embodiments, the linker, is or comprises: [0131] In some embodiments, provided is a trans-cyclooctene-modified TPD conjugate selected from Table 1.
  • a system comprising the trans-cyclooctene-modified TPD conjugate as described herein and a tetrazine activator.
  • the tetrazine activator may be formulated for systemic administration as a free tetrazine compound or as a tetrazine-containing group covalently bonded to an antibody or antibody fragment moiety, optionally via a linker, or is administered via a therapeutic support composition, where the therapeutic support composition comprises a biocompatible support and a tetrazine-containing group.
  • the trans-cyclooctene-modified TPD conjugate is capable of targeting a specific protein at a treatment site or on a cell in a subject.
  • the trans-cyclooctene moiety substantially blocks or inhibits the a portion of the TPD from effectively binding to its target.
  • contact with a tetrazine activator can release the trans-cyclooctene moiety, thus exposing the TPD and allowing it to bind its target.
  • the tetrazine activator is formulated for systemic administration.
  • each R 20 is independently 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; wherein each is independently optionally substituted with one to three Z 1 .
  • the tetrazine activator is: , [0138] In certain embodiments, the tetrazine activator is: , [0139] In certain embodiments, the tetrazine activator comprises 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.
  • R 22 is independently a linker of 1 to 100 linking atoms, and can include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups.
  • the tetrazine activator is of Formula IIA: wherein L, p, X, and R 20 are each independently as defined herein.
  • the tetrazine activator is of Formula IIB: wherein L, p, and X are each independently as defined herein.
  • the tetrazine activator is of Formula IIC:
  • the tetrazine activator is represented by Formula IID: wherein X and R 20 are each independently as defined herein. In some embodiments, R 20 is methyl. In some embodiments, X is an antigen-binding protein. In some embodiments, X is an antigen- binding protein which targets HER2. [0149] In some embodiments, the tetrazine activator is of Formula IIE: wherein p and X are each independently as defined herein. [0150] In some embodiments, the tetrazine activator is of Formula IIF: wherein p and X are each independently as defined herein.
  • the tetrazine activator is of Formula IIG: wherein p and X are each independently as defined herein. [0152] In some embodiments of Formula IIA, at least one of: , , , . [0153] In some embodiments of Formula IIA, at least one of: defined herein. [0154] In some embodiments of Formula IIA, at least one of: [0155] In some embodiments of Formula IIA, at least one of:
  • p is 1-12.
  • X is an antibody.
  • p is 1-6, or 5-6. 2.
  • 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.
  • X is an antibody fragment moiety (e.g., Fab).
  • ring A is cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R 3 and R 4 are both absent, or a dihydrotetrazine when R 3 and R 4 are both present;
  • X is a biocompatible support, antibody, or antibody fragment moiety;
  • p is 1-150;
  • L at each occurrence, is independently a linker;
  • the tetrazine activator is of Formula VI: 1 2 3 4 wherein each of R , R , R , R , ring A, L, p, t, and X are independently as defined herein.
  • R 4 is hydrogen.
  • R 3 is a group capable of being removed after a triggering event. In some embodiments, the triggering event occurs in vivo.
  • the dihydrotetrazine-containing group is oxidized to provide a tetrazine as in Formula VII: wherein each of R 1 , R 2 , ring A, L, p, t, and X are independently as defined herein.
  • the triggering event is initiated after administration of the tetrazine activator to the subject, and can be initiated by any means, such as internal means (e.g., via enzymatic cleavage of a functional group, optionally followed by a decomposition) or by external means (e.g., photocleavable linkers).
  • R 3 comprises a targeting moiety, such as an antibody or antibody fragment as described herein.
  • R 3 comprises an amino acid sequence specific for cleavage by a protease or esterase. [0165] In some embodiments, R 3 comprises an amino acid sequence specific for cleavage by a protease as shown in Table 1A. Table 1A
  • R 3 comprises an amino acid sequence specific for cleavage by a cathepsin, matrix metalloprotease (MMP), or PSMA.
  • MMP matrix metalloprotease
  • R 3 comprises Val-Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly for cleavage by cathepsins.
  • R 3 comprises Ac- ⁇ E-PLG–S(OBn)YL, or Ac-PLG–HofOrnL, where Hof is homophenylalanine and Orn is ornithine for cleavage by MMPs.
  • R 3 comprises an amino acid sequence as shown Table 1B. Table 1B
  • R 3 is photolabile.
  • the photolabile group is labile, or decomposes, with exposure to light at a wavelength matched to the absorbance profile of the photolabile group.
  • R 3 is L 5 is a direct bond or linker; and X 1 is -NO2, an optionally substituted sugar moiety, or an optionally substituted peptide unit comprising one or more natural or unnatural amino acids.
  • at least one of the moiety: is represented by a formula selected from:
  • R 1 , 2 3 4 R , R , and R are independently as defined herein, and optionally the ring A portion may be substituted with one or more R 2 moieties.
  • at least one of the moiety: is represented by a formula selected from:
  • X 2 is alkyl (e.g., methyl) optionally su bstituted with a PEG, an amino acid, ester, amide, amine, -C(O)OH, -SO2, -SO3, -PO3, -PO4, or other solubility enhancing substituent; and each of L, ring A, R 1 , R 2 , t, p, and X are independently as defined herein.
  • ring A is cycloalkyl.
  • ring A is heterocyclyl.
  • ring A is heteroaryl.
  • ring A is aryl.
  • 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.
  • ring A is phenyl.
  • R 1 at each occurrence, is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z 1 .
  • R 1 at each occurrence, is independently hydrogen or alkyl optionally substituted with one to three Z 1 .
  • R 2 at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl.
  • R 2 at each occurrence, is independently halo, alkyl, or haloalkyl.
  • R 2 at each occurrence, is independently halo or alkyl.
  • t at each occurrence is 0.
  • the tetrazine activator is of Formula: wherein p and X are each independently as defined herein. In some embodiments, the tetrazine activator is of Formula: wherein p and X are each independently as defined herein.
  • X is a biocompatible support.
  • ring A is other than pyridyl. In some embodiments, ring A is other than aryl. In some embodiments, ring A is other than phenyl.
  • 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.
  • 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).
  • a biocompatible support which comprises a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid
  • 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. [0185] In some embodiments, X is an antibody or antibody fragment moiety.
  • the 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 3559), CEA (NCBI Gene ID 634), CEACAM5 (
  • the targeting agent or X is an antibody or antibody fragment moiety which targets, HER2 (NCBI Gene ID 2064), CLDN4 (NCBI Gene ID 1364), TNC (NCBI Gene ID 3371), FN1 (NCBI Gene ID 2335), ITGAV (NCBI Gene ID 3685), TACSTD2 (NCBI Gene ID 4070), CD174 (NCBI Gene ID 2525), GPNMB (NCBI Gene ID 10457), GPC1 (NCBI Gene ID 2817), ITGB6 (NCBI Gene ID 3694), SEZ6 (NCBI Gene ID 124925), SLITRK6 (NCBI Gene ID 84189), NaPi-2b (NCBI Gene ID 20531), ZIP6 (NCBI Gene ID 25800), ROR1 (NCBI Gene ID 4919), ROR2 (NCBI Gene ID 4920), ANTXR1 (NCBI Gene ID 84168), or FAP (NCBI Gene ID 2191).
  • the targeting agent or 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.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody or antibody fragment moiety which targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, or CD25.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CEA, such as labetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CEACAM5, such as Tusamitiamab or CC4, or an antibody fragment moiety derived therefrom.
  • X is an antibody, or antibody fragment moiety, that targets ASPH, such as PAN-622, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • EGFR such as cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, duligotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, or lapri
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets VEGFR, such as ramucizumab, ramucirumab, or vulinacimab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets TROP2, such as Sacituzumab or Pr1E11, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets Nectin4, such as enfortumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets PSMA, such as J591 or MLN591, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets BCMA, such as belantamab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or 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.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD79b, such as polatuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD45, such as I-131-BC8, or Iomab-B, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets endoglin, such as carotuximab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets FGFR2, such as bemarituzumab or aprutumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets C4.4A, such as lupartumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets MMP9, such as andecaliximab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets DLL3, such as rovalpituzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD138, such as indatuximab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD74, such as milatuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets IL-6R, such as tocilizumab or sarilumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets SLAMF7, such as elotuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets BAFF, such as belimumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD30, such as brentuximab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets GPNMB, such as glembatumumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD142, such as tisotumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets NaPi2B, such as lifastuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets GCC, such as indusatumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets STEAP1, such as vandortuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets MUC16, such as sofituzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD70, such as vorsetuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CD44, such as bivatuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets vWF, such as caplacizumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets IL-6R, such as vobarilizumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets BCMA, such as LCAR-B38M, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets ADAMTS5, such as M6495, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets CX3CR1, such as BI 655088, or an antibody fragment moiety derived therefrom.
  • the targeting agent or 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.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets TfR1, such as TXB4, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets VEGFR, such as CDP791, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets PSMA, such as GY1, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets FN1, such as L19 or NJB2, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets FAP, such as F19, OMTX005 or sibrotuzumab, or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody, or antibody fragment moiety, that targets TNC, such as F16 or R6N or an antibody fragment moiety derived therefrom.
  • the targeting agent or X is an antibody.
  • the antibody is daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitiamab, 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
  • the targeting agent or 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.
  • the targeting agent or X is an antibody fragment moiety.
  • the targeting agent or X, or the antibody fragment moiety is 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 single-domain antibody (sdAb), 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,
  • the targeting agent or 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 divalent (or bivalent) single-domain antibody (sdAb).
  • scFv single-chain variable fragment
  • di-scFvs, bi-scFvs divalent (or bivalent) single-chain variable fragment
  • Fab antigen-binding fragment
  • sdAb single-domain antibody
  • sdAb divalent (or bivalent) single-domain antibody
  • 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.
  • the targeting agent or 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.
  • the Fab comprises four domains: VH, CH1, VL and CL1.
  • the Fab comprises 400-500 amino acids, or 440-480 amino acids.
  • the Fab has a molecular weight of about 50 kDa, or 40-55 kDa, or 45-50 kDa, or 45-55 kDa.
  • the antibody fragment moiety comprises one or more PEG units, which may enhance circulation life.
  • the antibody fragment moiety is an antigen-binding protein.
  • Antigen- binding proteins are proteins which are designed to be antibody-mimetics, exhibiting a high affinity and specificity for a given target.
  • the antigen-binding protein is a single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light- 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 the amino terminus of the VH sequence.
  • VL variable light- chain amino acid sequence
  • VH variable heavy-chain sequence
  • 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.
  • 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.
  • the antibody fragment moiety is an antigen-binding protein that targets TNC, FN1, CLDN4, MMP9, EpCAM, ITGAV, CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, or CD25.
  • 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).
  • the antibody fragment moiety is derived from daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitiamab, CC4, PAN-622, cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, duligotuzumab, futuximab, gc1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, laprituximab, oportuzumab, citat
  • X is an antibody fragment moiety derived from atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, or trastuzumab.
  • X is an antibody, or antibody fragment moiety, that targets vWF, such as Caplacizumab.
  • X is an antibody, or antibody fragment moiety, that targets TNF, such as Ozoralizumab, V565, or PF-05230905.
  • X is an antibody, or antibody fragment moiety, that targets IL-6R, such as Vobarilizumab.
  • X is an antibody, or antibody fragment moiety, that targets BCMA, such as LCAR-B38M.
  • X is an antibody, or antibody fragment moiety, that targets ADAMTS5, such as M6495.
  • X is an antibody, or antibody fragment moiety, that targets CX3CR1, such as BI 655088.
  • X is an antibody, or antibody fragment moiety, that targets CXCR4, such as AD-214 or ALX-0651.
  • X is an antibody, or antibody fragment moiety, that targets TfR1, such as TXB4.
  • X is an antibody, or antibody fragment moiety, that targets VEGFR, such as CDP791.
  • X is an antibody, or antibody fragment moiety, that targets PSMA, such as GY1.
  • the antibody fragment moiety is caplacizumab, ozoralizumab, V565, PF- 05230905, vobarilizumab, LCAR-B38M, M6495, BI 655088, AD-214, ALX-0651, TXB4, CDP791, or GY1.
  • X further comprises an imaging contrast agent.
  • the imaging contrast agent is a protein.
  • X is a support composition.
  • the support composition comprises a support. Supports may be biocompatible supports compositions, i.e., compatible with the subject’s body.
  • a support is non-toxic to the subject and does not substantially react with tissue or biological compounds in the subject.
  • 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 TPD 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.
  • 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, polyanhydrides, polyacetals, poly(ortho esters), polyphosphoesters, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, and blends/composites/co-polymers thereof.
  • polyethers include, but are not limited to, poly(ethylene glycol) (PEG), polypropylene glycol) (PPG), triblock Pluronic ([PEG] n -[PPG] m -[PEG] n ), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA), where n and m are each independently an integer from 1-100.
  • 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.
  • 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. In some embodiments, the support is chitosan. [0283] 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 ⁇ m or less).
  • average is meant the arithmetic mean.
  • the nanoparticles have a diameter ranging from 1 nm to 1 ⁇ m, such as from 10 nm to 1 ⁇ m, or 25 nm to 1 ⁇ m, or 50 nm to 1 ⁇ m, or 75 nm to 1 ⁇ m, or 100 nm to 1 ⁇ m, or 150 nm to 1 ⁇ m, or 200 nm to 1 ⁇ m, or 250 nm to 1 ⁇ m, or 300 nm to 1 ⁇ m, or 350 nm to 1 ⁇ m, or 400 nm to 1 ⁇ m, or 450 nm to 1 ⁇ m, or 500 nm to 1 ⁇ m.
  • 1 nm to 1 ⁇ m such as from 10 nm to 1 ⁇ m, or 25 nm to 1 ⁇ m, or 50 nm to 1 ⁇ m, or 75 nm to 1 ⁇ m, or 100 nm to 1 ⁇ m, or 150 nm to 1 ⁇ m, or 200 nm to 1
  • the microparticles have a diameter ranging from 1 ⁇ m to 1 mm, such as from 10 ⁇ m to 1 mm, or 25 ⁇ m to 1 mm, or 50 ⁇ m to 1 mm, or 75 ⁇ m to 1 mm, or 100 ⁇ m to 1 mm, or 150 ⁇ m to 1 mm, or 200 ⁇ m to 1 mm, or 250 ⁇ m to 1 mm, or 300 ⁇ m to 1 mm, or 350 ⁇ m to 1 mm, or 400 ⁇ m to 1 mm, or 450 ⁇ m to 1 mm, or 500 ⁇ m to 1 mm.
  • 1 ⁇ m to 1 mm such as from 10 ⁇ m to 1 mm, or 25 ⁇ m to 1 mm, or 50 ⁇ m to 1 mm, or 75 ⁇ m to 1 mm, or 100 ⁇ m to 1 mm, or 150 ⁇ m to 1 mm, or 200 ⁇ m to 1 mm, or 250 ⁇ m to 1 mm, or 300 ⁇ m to 1
  • 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 ⁇ m.
  • 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.
  • 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.
  • the particles may be made of an inert material, such as alginate or iron oxide.
  • 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.
  • 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.
  • the particle itself may be a virus or a phage with an inherent affinity for certain substrates.
  • 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 that may be used for the “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), poly(alkylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers, pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers or graft copolymers of any of the above.
  • 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)).
  • a targeting agent e.g., a ligand or antibody
  • 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.
  • the receptor or cell surface target is PD-1, CTLA-4, HER2/neu, HER1/EGFR, VEGFR, 4-1BB, GITR, CEACAM5 or other cellular receptors or cell surface targets.
  • the targeting agent is a monoclonal antibody.
  • a monoclonal antibody can be an entire monoclonal antibody, or a fragment thereof (e.g., antigen-binding fragment (Fab)).
  • the targeting agent is an antibody, or antibody fragment, 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
  • CD25
  • the targeting agent is a monoclonal antibody.
  • a monoclonal antibody can be an entire monoclonal antibody, or a fragment thereof (e.g., antigen-binding fragment (Fab)).
  • the targeting agent is an antibody, or antibody fragment, that targets one or more of, HER2 (NCBI Gene ID 2064), CLDN4 (NCBI Gene ID 1364), TNC (NCBI Gene ID 3371), FN1 (NCBI Gene ID 2335), ITGAV (NCBI Gene ID 3685), TACSTD2 (NCBI Gene ID 4070), CD174 (NCBI Gene ID 2525), GPNMB (NCBI Gene ID 10457), GPC1 (NCBI Gene ID 2817), ITGB6 (NCBI Gene ID 3694), SEZ6 (NCBI Gene ID 124925), SLITRK6 (NCBI Gene ID 84189), NaPi-2b (NCBI Gene ID 20531), ZIP6 (NCBI Gene ID 25800), ROR1 (NCBI Gene ID 4919), ROR2
  • the targeting agent is an antibody, or antibody fragment, that targets CEA, such as Labetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CEACAM5, such as tusamitamab or CC4, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets ASPH, such as PAN-622, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, 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 derived therefrom.
  • EGFR such as Cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, duligotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, alutumumab, tomuzotuximab, or laprituximab, or
  • the targeting agent is an antibody, or antibody fragment, that targets EPCAM, such as oportuzumab, citatuzumab, tucotuzumab, catumaxomab, edrecolomab, or adecatumumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets VEGFR, such as ramucizumab, ramucirumab, or vulinacimab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets TROP2, such as sacituzumab or Pr1E11, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets Nectin4, such as enfortumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets PSMA, such as J591 or MLN591, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets BCMA, such as belantamab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD22, such as moxetumomab, inotuzumab, epratuzumab, or pinatuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD20, such as ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, or ibritumomab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD19, such as loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, or MDX-1342, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD79b, such as polatuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD38, such as isatuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD45, such as I-131-BC8, or Iomab-B, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets endoglin, such as carotuximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets FGFR2, such as bemarituzumab or aprutumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets C4.4A, such as lupartumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets Claudin-18.2, such as zolbetuximab, or claudiximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets MMP9, such as andecaliximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets folate receptor, such as mirvetuximab, farletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets DLL3, such as rovalpituzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD138, such as indatuximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD37, such as BI 836826, otlertuzumab, or naratuximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD74, such as milatuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets mesothelin, such as anetumab, amatuximab, or MMOT-0530A, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets IL- 6R, such as tocilizumab or sarilumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets SLAMF7, such as elotuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets BAFF, such as belimumab, or an antibody fragment therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, or cantuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets GPC3, such as codrituzumab, ECT204, or MDX-1414, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets HER3, such as patritumab, seribantumab, lumretuzumab, elgemtumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD30, such as brentuximab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD33, such as gemtuzumab, BI 835858, vadastuximab, or lintuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets GPNMB, such as glembatumumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD142, such as tisotumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets NaPi2B, such as lifastuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets GCC, such as indusatumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets STEAP1, such as vandortuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets MUC16, such as sofituzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD70, such as vorsetuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CD44, such as bivatuzumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets vWF, such as caplacizumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets TNF, such as ozoralizumab, V565, or PF-05230905, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets IL- 6R, such as vobarilizumab, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets BCMA, such as LCAR-B38M, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets ADAMTS5, such as M6495, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CX3CR1, such as BI 655088, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets CXCR4, such as AD-214 or ALX-0651, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets TfR1, such as TXB4, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets VEGFR, such as CDP791, or an antibody fragment derived therefrom.
  • the targeting agent is an antibody, or antibody fragment, that targets PSMA, such as GY1, or an antibody fragment derived therefrom.
  • 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.
  • 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.
  • the bone graft substitute material is osteoinductive, such that it facilitates the formation of new bone through active recruitment of mesenchymal stem cells from the surrounding tissue.
  • growth factors such as bone morphogenetic proteins
  • 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.
  • tetrazine activators of the present disclosure comprise a support and a tetrazine-containing group covalently linked to the support.
  • the tetrazine-containing group may be attached to the support on a surface of the support, such as a solvent-accessible surface of the support (e.g., a surface of the support that is in contact with the surrounding solvent).
  • the tetrazine-containing group is attached directly to the support.
  • the tetrazine-containing group may be covalently attached to the surface of the support, e.g., through a covalent bond, such as an amide, amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, thiourea, etc.
  • the tetrazine-containing group is covalently attached to the support through an amide bond.
  • the tetrazine-containing group may be linked to the support via a linker.
  • Any suitable linker can be used to link the tetrazine-containing group to the support.
  • Representative linkers can have from 1 to 100 linking atoms, and can include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups.
  • linkers may have from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms.
  • Representative linkers include, but are not limited to, those shown below:
  • linkers may have from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms.
  • the tetrazine activators comprises units of formula: [0355] In some embodiments, the tetrazine activators comprise units of formula: . [0356] In some embodiments, the tetrazine activators comprise units of formula: [0357] In some embodiments, the tetrazine activators comprise substituted hyaluronic acid having units of formula: wherein linker of 1 to 1 20 00 linking atoms; and R is as defined herein. [0358] In further embodiments, still further embodiments, G 2 is and R 20 is hydrogen or C 1-4 alkyl.
  • R 20 is hydrogen or C1-4alkyl.
  • the tetrazine activators comprise units of formula: ; [0361] Additional tetrazine activators are exemplified in WO2017/044983, WO/2015/139025A1, and WO/2014/205126A1, the entire contents of each of which is incorporated herein by reference in their entirety. [0362]
  • 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.
  • the N-acetylglucosamine units of the hyaluronic acid are not linked or conjugated to the tetrazine-containing group.
  • 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 the 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 the tetrazine-containing group.
  • the linkers can be linear or branched (including, but not limited to, bivalent, trivalent, tetravalent branching).
  • the linker in one tetrazine-based targeting agent, there may be one or more targeting moiety covalently bonded to one or more tetrazine-containing group.
  • the “L” may be inside or outside of the “p” designation, however, it is intended that the linker can be linear with one bond to the tetrazine-containing group and one bond to the targeting moiety, wherein there are more than one [tertrazine-L]- moieties bonded to a single targeting moiety; or branched such that the linker has a bond to two tetrazine moieties and one bond to the targeting moiety.
  • 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.
  • SPP N-succinimidyl-4-(2-pyridyldithio)pentanoate
  • SPDB N-succinimidyl-4-(2- pyridyldithio)butyrate
  • AcBut 4-(4’-acetyl
  • 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.
  • 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, C 1-4 alkyl, C 1-4 alkoxy, and C 1-4 haloalkyl.
  • L, or the linker is bonded to X via a cysteine or lysine residue on X.
  • L, or the linker is a non-cleavable linker.
  • L, or the linker is a cleavable linker.
  • L, or the linker comprises one or more amino acids.
  • L, or the linker comprises a polypeptide.
  • L is an alkylene linker optionally comprising one or more - O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.
  • each R 110 is independently hydrogen, C 1-4 alkyl, C 1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R 120 is independently hydrogen, C 1-4 alkyl, C 1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl.
  • the linker is not a bond.
  • the L, or the linker 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-1,2-diylbis(methylcarbamate, an arylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.
  • the linker comprises one or more of: [0379] In some embodiments, the linker comprises one or more of: [0380] In some embodiments, the linker comprises one or more of: , [0 381] In some embodiments, the linker comprises one or more .
  • the linker comprises one or more . [0382] In some embodiments, the linker is, or comprises one or more: [0383] In some embodiments, the linker is, or comprises one or more: [0384] In some embodiments, the linker is, or 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.
  • the linker is, or comprises a peptide containing linker represented by “A—Y—Z—X 2 —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 2 ” 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. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated.
  • 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.
  • 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.
  • Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.
  • Exemplary Y-Z combinations include Valine-Citrulline; Valine-Alanine; and Alanine-Alanine.
  • A is -OC(O)-.
  • X 2 is -OC(O)-.
  • W is -OC(O)-.
  • X 2 is absent and W is -OC(O)-.
  • the moiety —X —W comprises .
  • the moiety —X 2 is .
  • 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.
  • 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.
  • the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe- Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-N 9 -tosyl-Arg, or Phe-N 9 -Nitro-Arg.
  • 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. [0398] In some embodiments, L, or the linker, is or comprises one or more of: , , , ,
  • the linker comprises one or more of: , ,
  • the linker comprises one or more of: .
  • the foregoing linkers may bond to an amino acid side chain present on X, such as a lysine or cysteine (e.g., )
  • the linker is –C(O)L 4 – or –C(O)C1-6alkyleneC(O)L 4 –;
  • L 4 is a bond, –N(R 12 )–C 2-3 alkylene–N(R 13 )C(O)–, -CH(NHC(O)R 14 )C 1-4 alkylene–S–S–C 1-4 alkylene– OC(O)–, —NHNHC(O)CH(NHC(O)R 15 )CH 2 C(O)–, –C 1-6 alkylene–CH(G x )OC(O)–,
  • R 12 , R 13 , R 14 , R 15 , and R 19 are each independently hydrogen or C1-4alkyl;
  • R 16 is hydrogen, C 1-4 alkyl, –C 1-4 alkylene–OH, –C 1-4 alkylene–OC 1-4 alkyl, –C 1-4 alkylene–CO 2 H, or –C1-4alkylene–CONH 2 ;
  • G x is phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, C 1-4 alkyl, C 1-4 haloalkyl, C 1-4 alkoxy, cyano, and nitro.
  • the linker comprises a carbonyl moiety for conjugating the tetrazine- containing group to the linker or X.
  • 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 the carbonyl moiety.
  • the linker may c .
  • the linker is or comprises one or more of:
  • the linker is: , [0406] In some embodiments, the linker, is or comprises one or more of: , D. TPD Precursors [0407]
  • compounds which include a precursor to a TPD that upon administration individually, react in vivo to generate a TPD. Accordingly, in certain embodiments, provided is a system comprising a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine- modified target protein ligand.
  • a system comprising a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand.
  • a method for making a TPD at a target location in a subject comprising administering to the subject, a therapeutically effective amount of a trans-cyclooctene- modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand.
  • a method for making a TPD at a target location in a subject comprising administering to the subject, a therapeutically effective amount of a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand.
  • a method of delivering a TPD to a target location in a subject comprising administering to the subject, a therapeutically effective amount of a trans- cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand.
  • a method of delivering a TPD to a target location in a subject comprising administering to the subject, a therapeutically effective amount of a tetrazine- modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand.
  • a compound of Table 2 In certain embodiments, provided is a kit comprising two or more compounds of Table 2. Table 2
  • aspects of the present disclosure include methods for delivering a TPD to a target location in a subject.
  • the method includes selectively delivering a TPD to the target location in a subject.
  • Selective delivery of the TPD may be achieved through use of a tetrazine activator as described herein.
  • a tetrazine activator of the present disclosure may be localized to a desired target location in a subject.
  • methods of the present disclosure may include administering to a subject a tetrazine activator as described herein. The tetrazine activator may be administered to the subject at a desired target location in the subject.
  • the tetrazine activator may be injected locally into the subject at the desired target location in the subject. In some embodiments, the tetrazine activator is administered systemically. In these embodiments, the tetrazine activator may localize at a desired target location in the subject through specific binding of the tetrazine activator to its target (e.g., antibody-antigen interaction, and the like), or may localize on the surface of a desired target (e.g., a cell surface) through specific binding of the tetrazine activator to its target (e.g., antibody-antigen interaction, and the like).
  • target e.g., antibody-antigen interaction, and the like
  • bioorthogonal binding partners e.g., between a tetrazine of the tetrazine activator and its complementary trans-cyclooctene of the trans-cyclooctene- modified TPD conjugate may occur. Due to the administration of the tetrazine activator and resulting localization to a desired location in the subject, the selective binding between the tetrazine and its complementary binding agent of the trans-cyclooctene on the trans-cyclooctene-modified TPD conjugate will localize the TPD to the desired target location.
  • a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a tetrazine activator as described herein, or a pharmaceutically acceptable salt thereof, and a trans-cyclooctene-modified TPD conjugate as described herein.
  • the cancer is metastatic.
  • 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, 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 Wilm’s tumor/neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.
  • 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.
  • the cancer is a solid tumor.
  • the cancer is a soft tissue sarcoma.
  • the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing’s sarcoma.
  • the method also comprises enhancing or eliciting an immune response.
  • the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.
  • 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.
  • 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.
  • Indications for this approach include cancer, both hematological and solid cancers.
  • the approach can be used for the treatment and/or diagnosis of soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing’s sarcoma, and all the different subtypes of soft tissue sarcoma as well as osteosarcoma.
  • the compositions can be for the treatment and/or diagnosis of pigmented vilonodular synovitis.
  • the 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 Ho
  • 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.
  • amelioration also includes situations where the 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 the condition, or at least the symptoms that characterize the condition.
  • Treatment may include inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease.
  • Treatment may include relief, that is, causing the regression of clinical symptoms.
  • the 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.
  • 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).
  • 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
  • non-human primates e.g., chimpanzees, and monkeys.
  • 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 breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, squamous cell carcinoma of head/neck, anal/vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma,
  • melanoma e
  • the disclosed approach lends itself well as an adjuvant / neoadjuvant system.
  • 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.
  • the surgeon could administer additional tetrazine activator 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.
  • a tetrazine activator and trans-cyclooctene-modified TPD conjugate as disclosed herein could be administered and 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 tetrazine activator or trans-cyclooctene-modified TPD conjugate 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.
  • further doses of treatment e.g. chemotherapy through the disclosed approach
  • the disclosed methods provide the ability to place particles as disclosed herein at the time of the biopsy. When the results return, the practitioner can deliver through to the biopsy site a TPD.
  • the disclosed methods provide the ability for a practitioner to deliver TPDs to enhance the immune system with fewer side effects. This approach would be beneficial to patients.
  • the TPD would treat the solid tumor or specific location.
  • Cancer 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 growth suppressors, resistance to cell death, enablement of replicative immortality, induction of angiogenesis, and the activation of invasion and metastasis.
  • 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
  • the 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.
  • the cancer is a solid tumor.
  • the cancer is a soft tissue carcinoma.
  • the cancer is afibrosarcoma.
  • the cancer is diffuse intrinsic pontine glioma.
  • the cancer is a metastatic cancer.
  • the 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
  • the tetrazine-based targeting agent 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.
  • the disclosed approach lends itself well as an adjuvant / neoadjuvant system.
  • tetrazine activators 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 the biopsy may indicate the amount and type of treatment to deliver to the site of a tumor.
  • chemokines agents that attract cancerous cells and/or immune cells
  • adjuvants to enhance the immune system with fewer side effects as well as the chemotherapeutics agents
  • the disclosed methods may include systemic or local administration.
  • the tetrazine-based targeting agent is delivered systemically.
  • the tetrazine activator and the trans-cyclooctene-modified TPD conjugate are both delivered systemically. In some embodiments, the tetrazine activator is delivered locally.
  • the disclosed compounds and compositions may be administered prior to surgical resection. The disclosed methods may minimize the size of the 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 tetrazine- based targeting agent 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.
  • the disclosed methods may include multiple systemic doses of trans-cyclooctene-modified TPD conjugate that focus at one location.
  • 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 Diffuse intrinsic pontine gliomas
  • 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 and 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 the triad of multiple cranial neuropathies, long tract signs such as hyperreflexia and clonus, as well as ataxia. Expansion of the pons section of the brainstem may cause obstructive hydrocephalus and increased intracranial pressure.
  • 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.
  • the disclosed methods may include multiple systemic doses of trans-cyclooctene-modified TPD conjugate or tetrazine activator that focus at one location.
  • Modes of Administration [0446] 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.
  • the conjugate, compound or compositions disclosed herein may also be dispersed in a microparticle, e.g. a nanoparticulate composition.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a kit comprising a tetrazine-based targeting agent, or a pharmaceutically acceptable salt thereof, as described herein, or the pharmaceutical composition comprising the same, and instructions for use thereof.
  • the kit further comprising a prodrug.
  • 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, state 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.
  • 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.
  • 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.
  • 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 (qid), or three times a day (tid).
  • the compositions of the present disclosure can be administered at any suitable frequency, interval and duration.
  • 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.
  • 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.
  • composition of the present 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.
  • 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.
  • Co- administration 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.
  • 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.
  • Co-administration can be accomplished by coimplantation or coinjection.
  • 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.
  • the composition of the present disclosure and the active agent can be formulated separately and co-administered to the subject.
  • 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).
  • composition of the present disclosure and the other active agent can be present in any suitable weight ratio, such as 1:100 (w/w), 1:75, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 (w/w).
  • suitable weight ratio such as 1:100 (w/w), 1:75, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 (w/w).
  • Other dosages and dosage ratios of the composition of the present disclosure and the active agent are suitable in the formulations and methods described herein.
  • 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 tetrazine-based targeting agent 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.
  • the disclosure also provides a pharmaceutical combination comprising a tetrazine-based targeting agent 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.
  • a pharmaceutical combination comprising a tetrazine-based targeting agent 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.
  • the disclosure also provides the use of a pharmaceutical combination comprising a tetrazine- based targeting agent 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 trans-cyclooctene prodrug thereof for the treatment or prevention of a cancer or for use in enhancing or eliciting an immune response.
  • 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.
  • the delay of progression or treatment of diseases 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 the 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 the term "administering" is to be interpreted accordingly.
  • a pharmaceutical combination 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.
  • the tetrazine-based targeting agent (or therapeutic tetrazine-based targeting agent) and prodrug may be administered/used simultaneously (e.g., through coinjection or coimplantation), separately, or sequentially, followed by administration of the additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.
  • the methods and uses in treating cancer include administering/localizing the tetrazine-based targeting agent at a tumor.
  • the administration of the prodrug, or a pharmaceutically acceptable salt, or composition thereof; the tetrazine-based targeting agent; and optionally an additional therapeutic agent may inhibit the growth of the tumor.
  • 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.
  • the additional therapeutic agent there may be an interval of time between administration of the additional therapeutic agent and the disclosed conjugates or compositions.
  • administration of an additional therapeutic agent with a disclosed conjugate or composition may allow lower doses of the other therapeutic agents and/or administration at less frequent intervals.
  • the conjugates or compositions of the present disclosure and the other active ingredients may be used in lower doses than when each is used singly.
  • the pharmaceutical compositions of the present disclosure include those that contain one or more other active ingredients, in addition to a conjugates of the present disclosure.
  • Anticancer agents include, but are not limited to, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidron), Abidron, ABVE,
  • 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
  • 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 [0468] 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.
  • Suitable 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.
  • Compounds, conjugates, and activators provided herein can be prepared by methods adapted from the literature (see, e.g., WO2020/077140, WO2018/187740, WO2017/044983, WO2015/139025, WO2014/205126, WO2022/032191, WO2021/007160, WO2020/077140, WO2018/187740, WO2017/044983, WO2015/139025, and WO2014/205126, which methods are incorporated herein in their entirety).
  • trans-cyclooctene-modified TPD conjugates (P1) as described herein can be prepared according to Scheme I below, where A is a TPD, linker is as defined herein, TCO is at least one trans-cyclooctene moiety, and Xx is a leaving group and Yy is a nucleophilic moiety, or Xx is a nucleophilic moiety and Yy is a leaving group, under standard coupling conditions.

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Abstract

The present disclosure relates generally to trans-cyclooctene-modified TPD conjugate comprising a targeted protein degrader (TPD) covalently bonded to at least one trans-cyclooctene moiety, optionally via a linker, which conjugates have applications, e.g., in the treatment of cancer, tumor growth, and immunotherapy.

Description

TRANS-CYCLOOCTENE-MODIFIED TARGETED PROTEIN DEGRADER CONJUGATES CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application 63/503,126, filed May 18, 2023, which is hereby incorporated by reference in its entirety. FIELD [0002] The present disclosure relates generally to trans-cyclooctene-modified targeted protein degrader (TPD) conjugates comprising a targeted protein degrader (TPD) covalently bonded to at least one trans- cyclooctene moiety, optionally via a linker, which conjugates have applications, e.g., in the treatment of cancer, tumor growth, and immunotherapy. BACKGROUND [0003] Targeted protein degraders (TPDs), sometimes referred to as proteolysis-targeting chimeras (PROTACs) are engineered techniques for targeted protein degradation, and have emerged as a promising approach for targeted therapy in various diseases, particularly in cancers. A bifunctional TPD molecule is typically comprised of two covalently-linked ligands, and functions to recruit target protein and E3 ubiquitin ligase together to trigger proteasomal degradation of target protein by the ubiquitin- proteasome system (UPS).. [0004] For proper function, a TPD needs to enter cells to mobilize intracellular UPS, so its membrane permeability is the key to TPDs function. However, large molecular weight and large exposed polar surface area of TPDs can result in low cell/tissue permeability. Moreover, some E3 ligands exhibit low specificity which results in such TPDs having off-target effects. [0005] Accordingly, a need exists to provide modified TPDs for effective and safe delivery of the TPD in vivo. SUMMARY [0006] 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. [0007] Provided herein are trans-cyclooctene-modified targeted protein degrader (TPD) (or PROTAC) conjugates, which comprise a targeted protein degrader (TPD) covalently bonded to at least one trans- cyclooctene moiety, optionally via a linker. The trans-cyclooctene-modified TPD conjugates described herein are designed to be used with a tetrazine activator which then, when in contact with the trans- cyclooctene-modified TPD conjugate, liberates the TPD in vivo. [0008] It is contemplated that the trans-cyclooctene-modified TPDconjugates described herein may have enhanced benefits, such as, but not limited to, improving the permeability of a TPD, masking the activity of the TPDin order to enhance the safety profile, and the like. It is further contemplated that cell permeability of the TPDcould be increased by the use of linkers between the TPDand trans-cyclooctene moiety or derivatizing one or more trans-cyclooctene moieties in order to form intramolecular hydrogen bonds that partially reduce polarity, or attaching cell-permeable peptides thereto, such as a poly-D- arginine sequence. [0009] In certain embodiments, provided is a conjugate of Table 1. [0010] In some embodiments, provided is a method for treating cancer, comprising administering to a subject in need thereof, a conjugate as described herein to a subject in need thereof, and administering to the subject a tetrazine activator, as described herein. [0011] In addition, provided herein are compounds which include a precursor to a TPD, that upon administration individually, react to generate a TPDin vivo. Accordingly, in certain embodiments, provided is a system comprising a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine- modified target protein ligand. Also provided is a system comprising a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand. [0012] In certain embodiments, provided is 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 a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand, or a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand. [0013] In certain embodiments, provided is a compound of Table 2. In certain embodiments, provided is a kit comprising two or more compounds of Table 2. [0014] 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, 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 Wilm’s tumor/neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma. [0015] 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 [0016] 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. 1. Definitions [0017] 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 [0018] 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 the 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. [0019] 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 plural 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. [0020] 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. [0021] 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. [0022] 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, 75th Ed., 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, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference. [0023] 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 “C1-C6-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, tert- butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl, and n-decyl. [0024] 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. [0025] 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. [0026] 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. [0027] 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. [0028] 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-. [0029] 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 α- 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, and 4-(3-methyl-(1,2,4,5-tetrazine))-phenylalanine. [0030] The term “aryl” as used herein, refers to an aromatic carbocyclic group having a single ring (e.g. monocyclic) or multiple rings (e.g. bicyclic or tricyclic) including fused systems. Representative 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 the 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, 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. [0031] 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 the 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 are 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 the parent molecular moiety through any carbon atom contained within the rings, and can be unsubstituted or substituted. [0032] The term “azide” as used herein, refers to the functional group –N3. [0033] 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 the point of attachment to the remainder of the molecule. [0034] 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. [0035] 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 or cycloheptenyl. [0036] 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). [0037] 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. [0038] 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. [0039] 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. [0040] The term “halogen” or “halo” as used herein, means Cl, Br, I, or F. [0041] 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. [0042] 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. [0043] 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. [0044] The term “heteroaryl” 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 at least one heteroatom independently selected from the group consisting of N, O and S (e.g.1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). 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-1,2- 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, 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. [0045] 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. [0046] 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 (e.g., 1-5, 1-4, 1-3, 1-2, or 1) heteroatom, 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, 1,3-dimethylpyrimidine-2,4(1H,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, 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-1H-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-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-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. [0047] The term “hydroxyl” as used herein, means an –OH group. [0048] 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. [0049] The term “substituted” refers to a group that may be further substituted with one or more non- hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =O, =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. [0050] 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. The term “tetrazine-based” generally refers to a tetrazine as defined herein or a moiety which can be converted to a tetrazine, such as an optionally substituted dihydrotetrazine. [0051] The term “selectively delivering” refers to delivering an agent (e.g., a TPD) to an organ or tissue (or portion thereof) in need of treatment or diagnosis, without significant binding to other non-target organs or tissues (or portions thereof). In some embodiments, the trans-cyclooctene-modified TPDconjugates described herein do not themselves have a therapeutic effect, but rather are designed to allow the selective or targeted delivery of a TPD. However, it may be that the tetrazine-based targeting agent does have a therapeutic effect, and thus, such constructs are not excluded by the present disclosure. [0052] The term “diagnostic agent” refers to agents that 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, 11C, 13N, 18F, 19F, 60Co, 64Cu, 67Cu, 68Ga, 82Rb, 89Zr, 90Sr, 90Y, 99Tc, 99mTc, 111In, 123I, 124I, 125I, 129I, 131I, 137Cs, 177Lu, 186Re, 188Re, 211At, Rn, Ra, Th, U, Pu, and 241Am. [0053] The term “ligand” as used herein to define a binding partner for a biological target molecule in vivo (for example, an enzyme or receptor). The ligands for use according to the invention are trypically small molecules that can be functionalized by the addition of a TCO moiety, optionally via a linker. [0054] 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. “Stable association” means that a compound is bound to, e.g., covalently or non- covalently, or otherwise associated with, another moiety or structure under standard physiological conditions. Stable associations, or binding, 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), dipole-dipole interactions, and the like. Targeting agents include ligands that specifically bind (or substantially specifically bind) a particular clinically-relevant target receptor or cell surface target. Specific targeting agents are described herein. [0055] The term “targeted organ or tissue” refers to an organ or tissue that is being targeted for delivery of the TPD. 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. [0056] The term “implanting” refers to surgical implantation into a subject’s body. [0057] 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 the added reagents or moieties, which can be produced in the contacting mixture. [0058] 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) (e.g., 1,2,4,5- 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. [0059] 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. [0060] 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. [0061] The term “parenteral” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. [0062] The term “therapeutically effective amount” refers 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. [0063] As used herein, the term “subject” or “patient” 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. [0064] 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 the 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. [0065] 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. [0066] 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. [0067] 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. [0068] 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. [0069] 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 the 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,” 5th edition (1989), Longman Scientific & Technical, Essex CM202JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods. [0070] 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, 17O, 31P, 32P, 35S, 18F, and 36Cl, 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 are 11C, 13N, 15O, and 18F. 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. Trans-Cyclooctene-Modified TPD Conjugates [0071] The trans-cyclooctene-modified TPD conjugates described herein are designed to, once administered to a subject, localize at a target site within the subject. The trans-cyclooctene-modified TPD conjugates can be administered locally or systemically. Once administered, a tetrazine activator can be administered, which when in contact with the trans-cyclooctene-modified TPD conjugate in vivo, allows for targeted drug delivery of a TPD. [0072] Provided herein is a trans-cyclooctene-modified TPD conjugate comprising a targeted protein degrader (TPD) covalently bonded to at least one trans-cyclooctene moiety, optionally via a linker. [0073] In some embodiments, the trans-cyclooctene-modified TPD conjugate is of the formula X-L- TPD; wherein X is a trans-cyclooctene moiety; L is a linker; and TPD is a targeted protein degrader. [0074] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of a Von Hippel–Lindau (VHL) ligand, a cereblon (CRBN) ligand, a mouse double minute 2 homolog (MDM2) ligand, and a beta-transducin repeat-containing protein (β-TrCP) ligand. [0075] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of thalidomide, lenalidomide, pomalidomide, MLN4924 (Pevonedistat), Nutlin-3, and curcumin. [0076] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the TPD comprises an E3 ubiquitin ligase ligand selected from Table A, wherein the wavy line indicates the point of attachment to the remainder of the TPD. Table A
Figure imgf000015_0001
[0077] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the TPD comprises a target protein ligand selected from the group consisting of a receptor tyrosine kinase (RAF) kinase ligand (e.g., a BRAF kinase ligand), a steroid receptor ligand (e.g., an androgen receptor (AR) ligand, an estrogen receptor (ER) ligand, or a progesterone receptor (PR) ligand), a cyclin- dependent kinase (CDK) ligand, a BTK ligand, a BET ligand, and an Inhibitor of Apoptosis Protein (IAP) ligand. [0078] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the TPD comprises a target protein ligand selected from Table B, wherein the wavy line indicates the point of attachment to the remainder of the TPD. Table B
Figure imgf000016_0001
Figure imgf000017_0002
[0079] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the TPD is selected from Table C: Table C
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0002
[0080] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the trans-cyclooctene moiety is of Formula X:
Figure imgf000020_0001
wherein: 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; q1 is 0 or 1; R1B, at each occurrence, is independently selected from the group consisting of G1, OH, –NR1c–C1-4alkylene–G1, –NR1c–C1-4alkylene–N(R1d)2, –NR1c–C1-6alkylene–N(C1-4alkyl)3 +, –N(R1c)CHR1eCO2H, –N(R1c)–C1-6alkylene–CO2H, –N(R1f)–C2-4alkylene–(N(C1-4alkylene–CO2H)– C2-4alkylene)n–N(C1-4alkylene–CO2H)2, –N(R1c)CHR1eC(O)OC1-6alkyl, –N(R1c)–C1-6alkylene– C(O)OC1-6alkyl, –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; R1e, at each occurrence, is independently –C1-4alkylene–CO2H, –C1-4alkylene–CONH2, or –C1-4alkylene–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; and G1, at each occurrence, is independently an optionally substituted heterocyclyl. [0081] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the trans-cyclooctene moiety is of Formula XI:
Figure imgf000021_0001
wherein: R2 is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine; wherein R2 may be optionally further substituted with a polyether. [0082] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the trans-cyclooctene moiety is of Formula: , o
Figure imgf000021_0002
[0083] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, at least one trans-cyclooctene moiety is
Figure imgf000021_0003
. [0084] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, at least one trans-cyclooctene moiety i
Figure imgf000022_0001
[0085] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, at least one trans-cyclooctene moiety is
Figure imgf000022_0002
; wherein A is an amino acid side chain (e.g., C1-4 alkyl, aryl (such as phenyl), etc., wherein each are optionally substituted with -OH, -SH, -SCH3, -C(O)OH, -NH2, -NHNH2NH, -C(O)NH2, etc.. [0086] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, at least one trans-cyclooctene moiety is
Figure imgf000022_0003
[0087] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the trans-cyclooctene moiety is covalently bonded to the TPD via a linker. Suitable linkers are described below. [0088] In some embodiments of the trans-cyclooctene-modified TPD conjugate disclosed herein, the linker is a cleavable linker. [0089] In some embodiments, the linker comprises one or more of a carbamate (-NHC(O)O-), an ester, and amide, 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. [0090] In some embodiments, the linker 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. [0091] In some embodiments, the linker 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, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl. [0092] In some embodiments, the linker is a non-cleavable linker. [0093] In some embodiments the linker is a cleavable linker. [0094] In some embodiments, the linker comprises one or more amino acids. [0095] In some embodiments, the linker comprises a polypeptide. [0096] In some embodiments, the linker is an alkylene linker optionally comprising one or more -O-, -S- , amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups. [0097] In some embodiments, the linker is of the formula: -Y10-(CHR130)n’-Y20-(CHR140)n''-Y30-(CHR150)m''-Y40- wherein: each of Y10, Y20, Y30, and Y40 are 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-, -(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, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl; each R110 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R120 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R130 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain; each R140 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; each R150 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain; and n', n'', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. [0098] In some embodiments, the linker is of the formula: -Y10-(CH2)n’-Y20-(CH2)m''-Y30- wherein: each of Y10, Y20, and Y30 are 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, 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, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl; each R110 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; each R120 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. [0099] In certain embodiments, each R110 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R120 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. In certain embodiments, the linker is not a bond. [0100] The linker, 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-1,2-diylbis(methylcarbamate, an arylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. [0101] In some embodiments, the linker comprises one or more of:
Figure imgf000024_0001
[0102] In some embodiments, the linker comprises one or more of:
Figure imgf000025_0001
[0103] In some embodiments, the linker comprises one or more of: ,
Figure imgf000025_0002
[0104] In some embodiments, the linker comprises one or more . In some embodiments, the linker comprises one or more
Figure imgf000025_0003
. [0105] In some embodiments, the linker is, or comprises one or more:
Figure imgf000025_0004
Figure imgf000026_0001
[0106] In some embodiments, the linker is, or comprises one or more:
Figure imgf000026_0002
[0107] In some embodiments, the linker is, or 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. [0108] In some embodiments, the linker is, or comprises 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. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated. [0109] 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. [0110] 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. [0111] Exemplary Y-Z combinations include Valine-Citrulline; Valine-Alanine; and Alanine-Alanine. [0112] In some embodiments, A is -OC(O)-. [0113] In some embodiments, X2 is -OC(O)-. [0114] In some embodiments, W is -OC(O)-. In some embodiments, X2 is absent and W is -OC(O)-. 2
Figure imgf000027_0001
[0115] In some embodiments, the moiety —X —W comprises . In some
Figure imgf000027_0002
embodiments, the moiety —X2 is . [0116] In some embodiments,
Figure imgf000027_0003
[0117] In some embodiments,
Figure imgf000027_0004
[0118] In some 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. [0119] 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. [0120] In some embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe- Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-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. [0121] In some embodiments, L, or the linker, is or comprises one or more of:
Figure imgf000027_0005
,
, ,
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
[0122] In some embodiments, the linker comprises one or more of:
Figure imgf000030_0002
,
Figure imgf000031_0001
[0123] In some embodiments, the linker comprises one or more of:
Figure imgf000031_0002
Figure imgf000032_0001
[0124] The foregoing linkers may bond to an amino acid side chain present on X, such as a lysine or
Figure imgf000032_0002
[0125] In some embodiments, the linker, is –C(O)L4– or –C(O)C1-6alkyleneC(O)L4–; L4 is a bond, –N(R12)–C2-3alkylene–N(R13)C(O)–, -CH(NHC(O)R14)C1-4alkylene–S–S–C1-4alkylene– OC(O)–, –NHNHC(O)CH(NHC(O)R15)CH2C(O)–, –C1-6alkylene–CH(Gx)OC(O)–,
Figure imgf000033_0001
R12, R13, R14, R15, and R19 are each independently hydrogen or C1-4alkyl; R16 is hydrogen, C1-4alkyl, –C1-4alkylene–OH, –C1-4alkylene–OC1-4alkyl, –C1-4alkylene–CO2H, or –C1-4alkylene–CONH2; and Gx is phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, cyano, and nitro. [0126] In some embodiments, the linker, comprises a carbonyl moiety for conjugating at least one trans- cyclooctene moiety to a TPD. 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 the carbonyl moiety. In some embodiments, the linker may comprise
Figure imgf000033_0003
[0127] In some embodiments, the linker, is or comprises one or more of:
Figure imgf000033_0002
,
Figure imgf000034_0001
Figure imgf000035_0001
[0128] In some embodiments, the linker, is: ,
Figure imgf000035_0002
[0129] In some embodiments, the linker, is or comprises one or more
Figure imgf000036_0001
, , or . [0130] In some embodiments, the linker, is or comprises:
Figure imgf000036_0002
[0131] In some embodiments, provided is a trans-cyclooctene-modified TPD conjugate selected from Table 1.
Table 1
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
C. Tetrazine Activators [0132] In certain embodiments, provided is a system comprising the trans-cyclooctene-modified TPD conjugate as described herein and a tetrazine activator. The tetrazine activator may be formulated for systemic administration as a free tetrazine compound or as a tetrazine-containing group covalently bonded to an antibody or antibody fragment moiety, optionally via a linker, or is administered via a therapeutic support composition, where the therapeutic support composition comprises a biocompatible support and a tetrazine-containing group. [0133] In certain embodiments, the trans-cyclooctene-modified TPD conjugate is capable of targeting a specific protein at a treatment site or on a cell in a subject. In certain embodiments, the trans-cyclooctene moiety substantially blocks or inhibits the a portion of the TPD from effectively binding to its target. Once the trans-cyclooctene-modified TPD conjugate binds to the treatment site, contact with a tetrazine activator can release the trans-cyclooctene moiety, thus exposing the TPD and allowing it to bind its target. [0134] In certain embodiments, the tetrazine activator is formulated for systemic administration. [0135] In certain embodiments, the tetrazine activator is of Formula IV:
Figure imgf000042_0001
wherein: each R20 is independently selected from the group consisting of hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R", C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1 is 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(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; and R''', at each occurrence, is independently selected from aryl and alkyl. [0136] In certain embodiments, the each R20 is independently 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; wherein each is independently optionally substituted with one to three Z1. [0137] In certain embodiments, the tetrazine activator is: ,
Figure imgf000043_0001
[0138] In certain embodiments, the tetrazine activator is: ,
Figure imgf000043_0002
[0139] In certain embodiments, the tetrazine activator comprises 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. [0140] In certain embodiments, the tetrazine activator is of Formula I, Formula II, or Formula V:
Figure imgf000044_0001
wherein: ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3 and R4 are both absent, or a dihydrotetrazine when R3 and R4 are both present; provided that when ring A is aryl, then R3 and R4 are both present; X is a biocompatible support, antibody, or antibody fragment moiety; p is 1-150; L, at each occurrence, is independently a linker; R1, at each occurrence, is independently selected from the group consisting of hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R", C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; 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; R3 and R4 are both absent; or R3 and R4 are each independently hydrogen or a group capable of being removed after a triggering event; R20, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S) R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; 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 R31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl; each Z1 is 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(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; R''', at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently is 0, 1, 2, 3, or 4. [0141] In certain embodiments, the tetrazine activator is of Formula I:
Figure imgf000045_0001
wherein: X is an antibody or antibody fragment moiety; p is 1-16; L, at each occurrence, is independently a linker; R20, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; 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; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; and R''' at each occurrence is independently selected from aryl and alkyl. [0142] In one embodiment, the tetrazine activator is of Formula II:
Figure imgf000046_0001
wherein: X is an antibody or antibody fragment moiety; p is 1-16; L, at each occurrence, is independently a linker; R20, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; 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 R31b are 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; and t is independently is 0, 1, 2, 3, or 4. [0143] In one embodiment, R22, at each occurrence, is independently a linker of 1 to 100 linking atoms, and can include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. [0144] In one embodiment, the tetrazine activator is of Formula IIA:
Figure imgf000047_0001
wherein L, p, X, and R20 are each independently as defined herein. [0145] In one embodiment, the tetrazine activator is of Formula IIB:
Figure imgf000047_0002
wherein L, p, and X are each independently as defined herein. [0146] In one embodiment, the tetrazine activator is of Formula IIC:
Figure imgf000048_0001
wherein L, p, and X are each independently as defined herein. [0147] In one embodiment, the tetrazine activator is of Formula III:
Figure imgf000048_0002
wherein: X is an antibody or antibody fragment moiety; p is 1-16; L, at each occurrence, is independently a linker; R20, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; 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; and t is independently is 0, 1, 2, 3, or 4. [0148] In some embodiments, the tetrazine activator is represented by Formula IID:
Figure imgf000049_0001
wherein X and R20 are each independently as defined herein. In some embodiments, R20 is methyl. In some embodiments, X is an antigen-binding protein. In some embodiments, X is an antigen- binding protein which targets HER2. [0149] In some embodiments, the tetrazine activator is of Formula IIE:
Figure imgf000049_0002
wherein p and X are each independently as defined herein. [0150] In some embodiments, the tetrazine activator is of Formula IIF:
Figure imgf000049_0003
wherein p and X are each independently as defined herein. [0151] In some embodiments, the tetrazine activator is of Formula IIG:
Figure imgf000049_0004
wherein p and X are each independently as defined herein. [0152] In some embodiments of Formula IIA, at least one of: , ,
Figure imgf000050_0001
,
Figure imgf000051_0001
. [0153] In some embodiments of Formula IIA, at least one of:
Figure imgf000051_0002
defined herein. [0154] In some embodiments of Formula IIA, at least one of:
Figure imgf000051_0003
[0155] In some embodiments of Formula IIA, at least one of:
Figure imgf000052_0001
defined herein. [0156] In some embodiments of Formula IIA, at least one of:
Figure imgf000052_0002
[0157] In some embodiments, p is 1-12. In some embodiments, X is an antibody. In some embodiments, p is 1-6, or 5-6. 2. In some embodiments, p is 1-16, or 1-8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2. In some embodiments, X is an antibody fragment moiety (e.g., Fab). [0158] In one embodiment, the tetrazine activator is of Formula V:
Figure imgf000052_0003
wherein: ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3 and R4 are both absent, or a dihydrotetrazine when R3 and R4 are both present; provided that when ring A is aryl, then R3 and R4 are both present; X is a biocompatible support, antibody, or antibody fragment moiety; p is 1-150; L, at each occurrence, is independently a linker; R1, at each occurrence, is independently selected from the group consisting of hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R", C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; 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; R3 and R4 are both absent; or R3 and R4 are each independently hydrogen or a group capable of being removed after a triggering event; each Z1 is 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(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; R''', at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently is 0, 1, 2, 3, or 4. [0159] In some embodiments, the tetrazine activator is of Formula V:
Figure imgf000054_0001
wherein: ring A is cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3 and R4 are both absent, or a dihydrotetrazine when R3 and R4 are both present; X is a biocompatible support, antibody, or antibody fragment moiety; p is 1-150; L, at each occurrence, is independently a linker; R1, at each occurrence, is independently selected from the group consisting of hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R", C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; 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; R3 and R4 are both absent; or R3 is a group capable of being removed after a triggering event; R4 is hydrogen or R3; each Z1 is 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(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; R''', at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently is 0, 1, 2, 3, or 4. [0160] In some embodiments, the tetrazine activator is of Formula VI: 1 2 3 4
Figure imgf000055_0001
wherein each of R , R , R , R , ring A, L, p, t, and X are independently as defined herein. [0161] In some embodiments, R4 is hydrogen. [0162] In some embodiments, R3 is a group capable of being removed after a triggering event. In some embodiments, the triggering event occurs in vivo. Once the triggering event occurs and R3 is removed, the dihydrotetrazine-containing group is oxidized to provide a tetrazine as in Formula VII:
Figure imgf000055_0002
wherein each of R1, R2, ring A, L, p, t, and X are independently as defined herein. [0163] The triggering event is initiated after administration of the tetrazine activator to the subject, and can be initiated by any means, such as internal means (e.g., via enzymatic cleavage of a functional group, optionally followed by a decomposition) or by external means (e.g., photocleavable linkers). In some embodiments, R3 comprises a targeting moiety, such as an antibody or antibody fragment as described herein. [0164] In some embodiments, R3 comprises an amino acid sequence specific for cleavage by a protease or esterase. [0165] In some embodiments, R3 comprises an amino acid sequence specific for cleavage by a protease as shown in Table 1A. Table 1A
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
[0166] In some embodiments, R3 comprises an amino acid sequence specific for cleavage by a cathepsin, matrix metalloprotease (MMP), or PSMA. For example, in some embodiments, R3 comprises Val-Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly for cleavage by cathepsins. In some embodiments, R3 comprises Ac-γE-PLG–S(OBn)YL, or Ac-PLG–HofOrnL, where Hof is homophenylalanine and Orn is ornithine for cleavage by MMPs. In some embodiments, R3 comprises an amino acid sequence as shown Table 1B. Table 1B
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
↓indicates cleavage site Special amino acid abbreviation: Cit: Citrulline; Cha: β-cyclohexylalanine; Hof: homophenylalanine; Nva: aminosuberic acid; Dpa: D- phenylalanine; Nle: Norleucine; Smc: S-methylcysteine * the listing of multiple amino acids before, between, or after a slash indicate alternative amino acids that can be substituted at the position; “-“ indicates that any amino acid may be substituted for the corresponding amino acid indicated in the middle column ** x is any L-amino acid other than proline Hy is any hydrophobic L-amino acid γ indicates that bond is a gamma carboxy linkage [0167] Additional cleavable groups are described in Choi, et al., Theranostics.2012; 2(2): 156–178, in which Table 2 is hereby incorporated by reference. [0168] In some embodiments, R3 is photolabile. In some embodiments, the photolabile group is labile, or decomposes, with exposure to light at a wavelength matched to the absorbance profile of the photolabile group. [0169] In some embodiments, R3 is
Figure imgf000065_0003
L5 is a direct bond or linker; and X1 is -NO2, an optionally substituted sugar moiety, or an optionally substituted peptide unit comprising one or more natural or unnatural amino acids. [0170] In some embodiments, at least one of the moiety:
Figure imgf000065_0001
is represented by a formula selected from:
Figure imgf000065_0002
Figure imgf000066_0001
Figure imgf000067_0001
, , , , , 1 ; wherein each of R1, 2 3 4
Figure imgf000067_0002
R , R , and R are independently as defined herein, and optionally the ring A portion may be substituted with one or more R2 moieties. [0171] In some embodiments, at least one of the moiety:
Figure imgf000067_0003
is represented by a formula selected from:
, and
Figure imgf000068_0001
; wherein X2 is alkyl (e.g., methyl) optionally su
Figure imgf000068_0002
bstituted with a PEG, an amino acid, ester, amide, amine, -C(O)OH, -SO2, -SO3, -PO3, -PO4, or other solubility enhancing substituent; and each of L, ring A, R1, R2, t, p, and X are independently as defined herein. [0172] In some embodiments, ring A is cycloalkyl. In some embodiments, ring A is heterocyclyl. In some embodiments, ring A is heteroaryl. In some embodiments, ring A is aryl. [0173] 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. [0174] In some embodiments, ring A is phenyl. [0175] In some embodiments, R1, at each occurrence, is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1. [0176] In some embodiments, R1, at each occurrence, is independently hydrogen or alkyl optionally substituted with one to three Z1. [0177] In some embodiments, Z1, at each occurrence, is independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'. [0178] 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. [0179] In some embodiments, t at each occurrence, is 0. [0180] In some embodiments, the tetrazine activator is of Formula:
Figure imgf000069_0001
wherein p and X are each independently as defined herein. In some embodiments, the tetrazine activator is of Formula:
Figure imgf000069_0002
wherein p and X are each independently as defined herein. [0181] In some embodiments, X is a biocompatible support. [0182] 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. [0183] 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. [0184] 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. [0185] In some embodiments, X is an antibody or antibody fragment moiety. Antibody and Antibody Fragment Moieties [0186] In certain embodiments, the 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 (NCBI Gene ID 972), mesothelin (NCBI Gene ID 10232), IL-6R (NCBI Gene ID 3570), SLAMF7 (NCBI Gene ID 57823), BAFF (NCBI Gene ID 10673), MUC1 (NCBI Gene ID 4582), GPC3 (NCBI Gene ID 2719), HER2 (NCBI Gene ID 2064), HER3 (NCBI Gene ID 2065), CD30 (NCBI Gene ID 943), CD33 (NCBI Gene ID 945), CD123 (NCBI Gene ID 3563), GPNMB (NCBI Gene ID 10457), cMET (NCBI Gene ID 4233), CD142 (NCBI Gene ID 2152), NaPi2B (NCBI Gene ID 10568), GCC (NCBI Gene ID 2984), STEAP1 (NCBI Gene ID 26872), MUC16 (NCBI Gene ID 94025), CD70 (NCBI Gene ID 970), CD44 (NCBI Gene ID 960), 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), or TfR1 (NCBI Gene ID 7037). [0187] In some embodiments, the targeting agent or X, is an antibody or antibody fragment moiety which targets, HER2 (NCBI Gene ID 2064), CLDN4 (NCBI Gene ID 1364), TNC (NCBI Gene ID 3371), FN1 (NCBI Gene ID 2335), ITGAV (NCBI Gene ID 3685), TACSTD2 (NCBI Gene ID 4070), CD174 (NCBI Gene ID 2525), GPNMB (NCBI Gene ID 10457), GPC1 (NCBI Gene ID 2817), ITGB6 (NCBI Gene ID 3694), SEZ6 (NCBI Gene ID 124925), SLITRK6 (NCBI Gene ID 84189), NaPi-2b (NCBI Gene ID 20531), ZIP6 (NCBI Gene ID 25800), ROR1 (NCBI Gene ID 4919), ROR2 (NCBI Gene ID 4920), ANTXR1 (NCBI Gene ID 84168), or FAP (NCBI Gene ID 2191). [0188] In some embodiments, the targeting agent or 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. [0189] In some embodiments, the targeting agent or 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. [0190] In some embodiments, the targeting agent or X, is an antibody or antibody fragment moiety which targets CEA, CEACAM5, ASPH, EGFR, EPCAM, VEGFR, PDGFR, TROP2, Nectin4, PSMA, BCMA, HER2, or CD25. [0191] In certain embodiments, the targeting agent or 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. [0192] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CEA, such as labetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment moiety derived therefrom. [0193] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CEACAM5, such as Tusamitiamab or CC4, or an antibody fragment moiety derived therefrom. [0194] 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. [0195] In certain embodiments, the targeting agent or 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. [0196] In certain embodiments, the targeting agent or 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. [0197] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets VEGFR, such as ramucizumab, ramucirumab, or vulinacimab, or an antibody fragment moiety derived therefrom. [0198] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment moiety derived therefrom. [0199] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets TROP2, such as Sacituzumab or Pr1E11, or an antibody fragment moiety derived therefrom. [0200] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets Nectin4, such as enfortumab, or an antibody fragment moiety derived therefrom. [0201] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets PSMA, such as J591 or MLN591, or an antibody fragment moiety derived therefrom. [0202] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets BCMA, such as belantamab, or an antibody fragment moiety derived therefrom. [0203] In certain embodiments, the targeting agent or 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. [0204] In certain embodiments, the targeting agent or 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. [0205] In certain embodiments, the targeting agent or 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. [0206] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD79b, such as polatuzumab, or an antibody fragment moiety derived therefrom. [0207] In certain embodiments, the targeting agent or 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. [0208] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD45, such as I-131-BC8, or Iomab-B, or an antibody fragment moiety derived therefrom. [0209] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets endoglin, such as carotuximab, or an antibody fragment moiety derived therefrom. [0210] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets FGFR2, such as bemarituzumab or aprutumab, or an antibody fragment moiety derived therefrom. [0211] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets C4.4A, such as lupartumab, or an antibody fragment moiety derived therefrom. [0212] In certain embodiments, the targeting agent or 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. [0213] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets MMP9, such as andecaliximab, or an antibody fragment moiety derived therefrom. [0214] In certain embodiments, the targeting agent or 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. [0215] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets DLL3, such as rovalpituzumab, or an antibody fragment moiety derived therefrom. [0216] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD138, such as indatuximab, or an antibody fragment moiety derived therefrom. [0217] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment moiety derived therefrom. [0218] In certain embodiments, the targeting agent or 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. [0219] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD74, such as milatuzumab, or an antibody fragment moiety derived therefrom. [0220] In certain embodiments, the targeting agent or 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. [0221] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets IL-6R, such as tocilizumab or sarilumab, or an antibody fragment moiety derived therefrom. [0222] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets SLAMF7, such as elotuzumab, or an antibody fragment moiety derived therefrom. [0223] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets BAFF, such as belimumab, or an antibody fragment moiety derived therefrom. [0224] In certain embodiments, the targeting agent or 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. [0225] In certain embodiments, the targeting agent or 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. [0226] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment moiety derived therefrom. [0227] In certain embodiments, the targeting agent or 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. [0228] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD30, such as brentuximab, or an antibody fragment moiety derived therefrom. [0229] In certain embodiments, the targeting agent or 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. [0230] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment moiety derived therefrom. [0231] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets GPNMB, such as glembatumumab, or an antibody fragment moiety derived therefrom. [0232] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment moiety derived therefrom. [0233] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD142, such as tisotumab, or an antibody fragment moiety derived therefrom. [0234] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets NaPi2B, such as lifastuzumab, or an antibody fragment moiety derived therefrom. [0235] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets GCC, such as indusatumab, or an antibody fragment moiety derived therefrom. [0236] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets STEAP1, such as vandortuzumab, or an antibody fragment moiety derived therefrom. [0237] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets MUC16, such as sofituzumab, or an antibody fragment moiety derived therefrom. [0238] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD70, such as vorsetuzumab, or an antibody fragment moiety derived therefrom. [0239] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CD44, such as bivatuzumab, or an antibody fragment moiety derived therefrom. [0240] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets vWF, such as caplacizumab, or an antibody fragment moiety derived therefrom. [0241] In certain embodiments, the targeting agent or 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. [0242] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets IL-6R, such as vobarilizumab, or an antibody fragment moiety derived therefrom. [0243] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets BCMA, such as LCAR-B38M, or an antibody fragment moiety derived therefrom. [0244] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets ADAMTS5, such as M6495, or an antibody fragment moiety derived therefrom. [0245] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets CX3CR1, such as BI 655088, or an antibody fragment moiety derived therefrom. [0246] In certain embodiments, the targeting agent or 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. [0247] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets TfR1, such as TXB4, or an antibody fragment moiety derived therefrom. [0248] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets VEGFR, such as CDP791, or an antibody fragment moiety derived therefrom. [0249] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets PSMA, such as GY1, or an antibody fragment moiety derived therefrom. [0250] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets FN1, such as L19 or NJB2, or an antibody fragment moiety derived therefrom. [0251] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets FAP, such as F19, OMTX005 or sibrotuzumab, or an antibody fragment moiety derived therefrom. [0252] In certain embodiments, the targeting agent or X, is an antibody, or antibody fragment moiety, that targets TNC, such as F16 or R6N or an antibody fragment moiety derived therefrom. [0253] In some embodiments, the targeting agent or X, is an antibody. [0254] In some embodiments, the antibody is daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitiamab, 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, Iomab-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, F16, or R6N. [0255] In some embodiments, the targeting agent or 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. [0256] In some embodiments, the targeting agent or X, is an antibody fragment moiety. [0257] In some embodiments, the targeting agent or X, or the antibody fragment moiety is 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 single-domain antibody (sdAb), 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. [0258] In some embodiments, the targeting agent or 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 divalent (or bivalent) single-domain antibody (sdAb). [0259] 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. [0260] In some embodiments, the targeting agent or 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, CH1, 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. [0261] In some embodiments, the Fab of trastuzumab, enfortumab, brentuximab, sacituzumab, L19 - binding to FN-1 (Gene ID 2335); F16 - binding to TNC (Gene ID 3371), or NJB2 (ECM-targeting sequences). [0262] In some embodiments, the antibody fragment moiety comprises one or more PEG units, which may enhance circulation life. [0263] In some embodiments, the antibody fragment moiety is an antigen-binding protein. Antigen- binding proteins are proteins which are designed to be antibody-mimetics, exhibiting a high affinity and specificity for a given target. In some embodiments, the antigen-binding protein is a single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light- 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 the amino terminus of the VH sequence. [0264] 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. [0265] 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, 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). [0266] In some embodiments, the antibody fragment moiety is derived from daclizumab, RG6292, basiliximab, HuMax-TAC, labetuzumab, 15-1-32, PR1A3, cT84.66, tusamitiamab, 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, Iomab-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, F16 or R6N. [0267] In some embodiments, X is an antibody fragment moiety derived from atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, mogamulizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, or trastuzumab. [0268] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets vWF, such as Caplacizumab. [0269] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TNF, such as Ozoralizumab, V565, or PF-05230905. [0270] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets IL-6R, such as Vobarilizumab. [0271] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets BCMA, such as LCAR-B38M. [0272] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets ADAMTS5, such as M6495. [0273] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CX3CR1, such as BI 655088. [0274] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets CXCR4, such as AD-214 or ALX-0651. [0275] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets TfR1, such as TXB4. [0276] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets VEGFR, such as CDP791. [0277] In certain embodiments, X is an antibody, or antibody fragment moiety, that targets PSMA, such as GY1. [0278] 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. [0279] In some embodiments, X further comprises an imaging contrast agent. In some embodiments, the imaging contrast agent is a protein. [0280] In some embodiments, X is a support composition. The support composition comprises a support. 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 TPD 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. [0281] 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, polyanhydrides, 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), polypropylene glycol) (PPG), triblock Pluronic ([PEG]n-[PPG]m-[PEG]n), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA), where n and m are each independently an integer from 1-100. 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. [0282] 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. In some embodiments, the support is chitosan. [0283] 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 μm or less). By “average” is meant the arithmetic mean. In some embodiments, the nanoparticles have a diameter ranging from 1 nm to 1 μm, such as from 10 nm to 1 μm, or 25 nm to 1 μm, or 50 nm to 1 μm, or 75 nm to 1 μm, or 100 nm to 1 μm, or 150 nm to 1 μm, or 200 nm to 1 μm, or 250 nm to 1 μm, or 300 nm to 1 μm, or 350 nm to 1 μm, or 400 nm to 1 μm, or 450 nm to 1 μm, or 500 nm to 1 μm. In other embodiments, the microparticles have a diameter ranging from 1 μm to 1 mm, such as from 10 μm to 1 mm, or 25 μm to 1 mm, or 50 μm to 1 mm, or 75 μm to 1 mm, or 100 μm to 1 mm, or 150 μm to 1 mm, or 200 μm to 1 mm, or 250 μm to 1 mm, or 300 μm to 1 mm, or 350 μm to 1 mm, or 400 μm to 1 mm, or 450 μm to 1 mm, or 500 μm to 1 mm. In further embodiments, small particles 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 μm. 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. [0284] 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 that may be used for the “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), poly(alkylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers, pluronic polyols, polyoxamers, poly(uronic 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. [0285] 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, CEACAM5 or other cellular receptors or cell surface targets. [0286] In some embodiments, the targeting agent is a monoclonal antibody. A monoclonal antibody can be an entire monoclonal antibody, or a fragment thereof (e.g., antigen-binding fragment (Fab)). In certain embodiments, the targeting agent is an antibody, or antibody fragment, 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 (NCBI Gene ID 972), mesothelin (NCBI Gene ID 10232), IL-6R (NCBI Gene ID 3570), SLAMF7 (NCBI Gene ID 57823), BAFF (NCBI Gene ID 10673), MUC1 (NCBI Gene ID 4582), GPC3 (NCBI Gene ID 2719), HER2 (NCBI Gene ID 2064), HER3 (NCBI Gene ID 2065), CD30 (NCBI Gene ID 943), CD33 (NCBI Gene ID 945), CD123 (NCBI Gene ID 3563), GPNMB (NCBI Gene ID 10457), cMET (NCBI Gene ID 4233), CD142 (NCBI Gene ID 2152), NaPi2B (NCBI Gene ID 10568), GCC (NCBI Gene ID 2984), STEAP1 (NCBI Gene ID 26872), MUC16 (NCBI Gene ID 94025), CD70 (NCBI Gene ID 970), CD44 (NCBI Gene ID 960), 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), or TfR1 (NCBI Gene ID 7037). [0287] In some embodiments, the targeting agent is a monoclonal antibody. A monoclonal antibody can be an entire monoclonal antibody, or a fragment thereof (e.g., antigen-binding fragment (Fab)). In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets one or more of, HER2 (NCBI Gene ID 2064), CLDN4 (NCBI Gene ID 1364), TNC (NCBI Gene ID 3371), FN1 (NCBI Gene ID 2335), ITGAV (NCBI Gene ID 3685), TACSTD2 (NCBI Gene ID 4070), CD174 (NCBI Gene ID 2525), GPNMB (NCBI Gene ID 10457), GPC1 (NCBI Gene ID 2817), ITGB6 (NCBI Gene ID 3694), SEZ6 (NCBI Gene ID 124925), SLITRK6 (NCBI Gene ID 84189), NaPi-2b (NCBI Gene ID 20531), ZIP6 (NCBI Gene ID 25800), ROR1 (NCBI Gene ID 4919), ROR2 (NCBI Gene ID 4920), ANTXR1 (NCBI Gene ID 84168), or FAP (NCBI Gene ID 2191) [0288] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD25, such as Daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom. [0289] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CEA, such as Labetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment derived therefrom. [0290] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CEACAM5, such as tusamitamab or CC4, or an antibody fragment derived therefrom. [0291] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets ASPH, such as PAN-622, or an antibody fragment derived therefrom. [0292] In certain embodiments, the targeting agent is an antibody, or antibody fragment, 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 derived therefrom. [0293] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets EPCAM, such as oportuzumab, citatuzumab, tucotuzumab, catumaxomab, edrecolomab, or adecatumumab, or an antibody fragment derived therefrom. [0294] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets VEGFR, such as ramucizumab, ramucirumab, or vulinacimab, or an antibody fragment derived therefrom. [0295] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment derived therefrom. [0296] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets TROP2, such as sacituzumab or Pr1E11, or an antibody fragment derived therefrom. [0297] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets Nectin4, such as enfortumab, or an antibody fragment derived therefrom. [0298] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets PSMA, such as J591 or MLN591, or an antibody fragment derived therefrom. [0299] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets BCMA, such as belantamab, or an antibody fragment derived therefrom. [0300] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD22, such as moxetumomab, inotuzumab, epratuzumab, or pinatuzumab, or an antibody fragment derived therefrom. [0301] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD20, such as ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, or ibritumomab, or an antibody fragment derived therefrom. [0302] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD19, such as loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, or MDX-1342, or an antibody fragment derived therefrom. [0303] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD79b, such as polatuzumab, or an antibody fragment derived therefrom. [0304] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD38, such as isatuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment derived therefrom. [0305] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD45, such as I-131-BC8, or Iomab-B, or an antibody fragment derived therefrom. [0306] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets endoglin, such as carotuximab, or an antibody fragment derived therefrom. [0307] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets FGFR2, such as bemarituzumab or aprutumab, or an antibody fragment derived therefrom. [0308] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets C4.4A, such as lupartumab, or an antibody fragment derived therefrom. [0309] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets Claudin-18.2, such as zolbetuximab, or claudiximab, or an antibody fragment derived therefrom. [0310] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets MMP9, such as andecaliximab, or an antibody fragment derived therefrom. [0311] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets folate receptor, such as mirvetuximab, farletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment derived therefrom. [0312] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets DLL3, such as rovalpituzumab, or an antibody fragment derived therefrom. [0313] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD138, such as indatuximab, or an antibody fragment derived therefrom. [0314] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment derived therefrom. [0315] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD37, such as BI 836826, otlertuzumab, or naratuximab, or an antibody fragment derived therefrom. [0316] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD74, such as milatuzumab, or an antibody fragment derived therefrom. [0317] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets mesothelin, such as anetumab, amatuximab, or MMOT-0530A, or an antibody fragment derived therefrom. [0318] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets IL- 6R, such as tocilizumab or sarilumab, or an antibody fragment derived therefrom. [0319] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets SLAMF7, such as elotuzumab, or an antibody fragment derived therefrom. [0320] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets BAFF, such as belimumab, or an antibody fragment therefrom. [0321] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, or cantuzumab, or an antibody fragment derived therefrom. [0322] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets GPC3, such as codrituzumab, ECT204, or MDX-1414, or an antibody fragment derived therefrom. [0323] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment derived therefrom. [0324] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets HER3, such as patritumab, seribantumab, lumretuzumab, elgemtumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment derived therefrom. [0325] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD30, such as brentuximab, or an antibody fragment derived therefrom. [0326] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD33, such as gemtuzumab, BI 835858, vadastuximab, or lintuzumab, or an antibody fragment derived therefrom. [0327] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment derived therefrom. [0328] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets GPNMB, such as glembatumumab, or an antibody fragment derived therefrom. [0329] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment derived therefrom. [0330] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD142, such as tisotumab, or an antibody fragment derived therefrom. [0331] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets NaPi2B, such as lifastuzumab, or an antibody fragment derived therefrom. [0332] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets GCC, such as indusatumab, or an antibody fragment derived therefrom. [0333] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets STEAP1, such as vandortuzumab, or an antibody fragment derived therefrom. [0334] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets MUC16, such as sofituzumab, or an antibody fragment derived therefrom. [0335] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD70, such as vorsetuzumab, or an antibody fragment derived therefrom. [0336] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CD44, such as bivatuzumab, or an antibody fragment derived therefrom. [0337] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets vWF, such as caplacizumab, or an antibody fragment derived therefrom. [0338] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets TNF, such as ozoralizumab, V565, or PF-05230905, or an antibody fragment derived therefrom. [0339] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets IL- 6R, such as vobarilizumab, or an antibody fragment derived therefrom. [0340] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets BCMA, such as LCAR-B38M, or an antibody fragment derived therefrom. [0341] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets ADAMTS5, such as M6495, or an antibody fragment derived therefrom. [0342] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CX3CR1, such as BI 655088, or an antibody fragment derived therefrom. [0343] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets CXCR4, such as AD-214 or ALX-0651, or an antibody fragment derived therefrom. [0344] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets TfR1, such as TXB4, or an antibody fragment derived therefrom. [0345] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets VEGFR, such as CDP791, or an antibody fragment derived therefrom. [0346] In certain embodiments, the targeting agent is an antibody, or antibody fragment, that targets PSMA, such as GY1, or an antibody fragment derived therefrom. [0347] 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 the 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. [0348] 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 it 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. [0349] In some embodiments, tetrazine activators of the present disclosure comprise a support and a tetrazine-containing group covalently linked to the support. The tetrazine-containing group may be attached to the support on a surface of the support, such as a solvent-accessible surface of the support (e.g., a surface of the support that is in contact with the surrounding solvent). In some cases, the tetrazine-containing group is attached directly to the support. For example, the tetrazine-containing group may be covalently attached to the surface of the support, e.g., through a covalent bond, such as an amide, amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, thiourea, etc. In some instances, the tetrazine-containing group is covalently attached to the support through an amide bond. In other instances, the tetrazine-containing group may be linked to the support via a linker. Any suitable linker can be used to link the tetrazine-containing group to the support. Representative linkers can have from 1 to 100 linking atoms, and can 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. Representative linkers include, but are not limited to, those shown below:
Figure imgf000087_0001
[0350] In certain embodiments, the tetrazine activators comprise a support and a tetrazine-containing group of formula:
Figure imgf000087_0002
wherein R20 is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S) R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R" at each occurrence are independently selected from hydrogen, aryl and alkyl; and R''' at each occurrence is independently selected from aryl and alkyl; R30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; Ra, R31a and R31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl; and t is 0, 1, 2, 3, or 4. [0351] In certain embodiments, the tetrazine activators have formula:
Figure imgf000087_0003
wherein R20 is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S) R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R" at each occurrence are independently selected from hydrogen, aryl and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; and R22 is a linker of 1 to 100 linking atoms, and can 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. [0352] In certain embodiments, the tetrazine activators have formula: wherein
Figure imgf000088_0001
R20 is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S) R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R" at each occurrence are independently selected from hydrogen, aryl and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; R30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; Ra, R31a and R31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl; and t is 0, 1, 2, 3, or 4. [0353] In certain embodiments, the tetrazine activators comprise substituted alginate having units of formula:
Figure imgf000089_0001
salt thereof, wherein R20 is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S) R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R" at each occurrence are independently selected from hydrogen, aryl and alkyl; and R''' at each occurrence is independently selected from aryl and alkyl. [0354] In certain embodiments, the tetrazine activators comprises units of formula:
Figure imgf000089_0002
[0355] In some embodiments, the tetrazine activators comprise units of formula:
Figure imgf000090_0001
. [0356] In some embodiments, the tetrazine activators comprise units of formula:
Figure imgf000090_0002
[0357] In some embodiments, the tetrazine activators comprise substituted hyaluronic acid having units of formula:
Figure imgf000090_0003
wherein linker of 1 to 1 20
Figure imgf000090_0004
00 linking atoms; and R is as defined herein. [0358] In further embodiments,
Figure imgf000091_0001
still further embodiments, G2 is
Figure imgf000091_0003
and R20 is hydrogen or C1-4alkyl. [0359] In some embodiments, the tetrazine activators comprise units of formula:
Figure imgf000091_0002
wherein R20 is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S) R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R" at each occurrence are independently selected from hydrogen, aryl and alkyl; and R''' at each occurrence is independently selected from aryl and alkyl. In some embodiments, R20 is hydrogen or C1-4alkyl. [0360] In some embodiments, the tetrazine activators comprise units of formula: ;
Figure imgf000092_0001
[0361] Additional tetrazine activators are exemplified in WO2017/044983, WO/2015/139025A1, and WO/2014/205126A1, the entire contents of each of which is incorporated herein by reference in their entirety. [0362] 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. [0363] 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 the 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 the tetrazine-containing group. [0364] In the tetrazine activators disclosed herein, the linkers can be linear or branched (including, but not limited to, bivalent, trivalent, tetravalent branching). Thus, in one tetrazine-based targeting agent, there may be one or more targeting moiety covalently bonded to one or more tetrazine-containing group. In the Formulas disclosed herein, the “L” may be inside or outside of the “p” designation, however, it is intended that the linker can be linear with one bond to the tetrazine-containing group and one bond to the targeting moiety, wherein there are more than one [tertrazine-L]- moieties bonded to a single targeting moiety; or branched such that the linker has a bond to two tetrazine moieties and one bond to the targeting moiety. [0365] In some embodiments, L, or the linker, 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. [0366] In some embodiments, L, or the linker, 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. [0367] In some embodiments, L, or the linker, 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, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl. [0368] In some embodiments, L, or the linker, is bonded to X via a cysteine or lysine residue on X. [0369] In some embodiments, L, or the linker, is a non-cleavable linker. [0370] In some embodiments, L, or the linker, is a cleavable linker. [0371] In some embodiments, L, or the linker, comprises one or more amino acids. [0372] In some embodiments, L, or the linker, comprises a polypeptide. [0373] In some embodiments, L, or the linker, is an alkylene linker optionally comprising one or more - O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups. [0374] In some embodiments, L, or the linker, is of the formula: -Y10-(CHR130)n’-Y20-(CHR140)n''-Y30-(CHR150)m''-Y40- wherein: each of Y10, Y20, Y30, and Y40 are 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-, -(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, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl; each R110 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R120 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R130 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain; each R140 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; each R150 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain; and n', n'', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. [0375] In some embodiments, L, or the linker, is of the formula: -Y10-(CH2)n’-Y20-(CH2)m''-Y30- wherein: each of Y10, Y20, and Y30 are 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, 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, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl; each R110 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; each R120 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. [0376] In certain embodiments, each R110 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R120 is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. In certain embodiments, the linker is not a bond. [0377] The L, or the linker, 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-1,2-diylbis(methylcarbamate, an arylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. [0378] In some embodiments, the linker comprises one or more of:
Figure imgf000095_0001
[0379] In some embodiments, the linker comprises one or more of:
Figure imgf000095_0002
[0380] In some embodiments, the linker comprises one or more of: ,
Figure imgf000095_0003
[0
Figure imgf000096_0001
381] In some embodiments, the linker comprises one or more . In some embodiments, the linker comprises one or more
Figure imgf000096_0002
. [0382] In some embodiments, the linker is, or comprises one or more:
Figure imgf000096_0003
[0383] In some embodiments, the linker is, or comprises one or more:
Figure imgf000096_0004
[0384] In some embodiments, the linker is, or 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. [0385] In some embodiments, the linker is, or comprises 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. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated. [0386] 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. [0387] 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. [0388] Exemplary Y-Z combinations include Valine-Citrulline; Valine-Alanine; and Alanine-Alanine. [0389] In some embodiments, A is -OC(O)-. [0390] In some embodiments, X2 is -OC(O)-. [0391] In some embodiments, W is -OC(O)-. In some embodiments, X2 is absent and W is -OC(O)-. [0392] In some embodiments 2
Figure imgf000097_0001
, the moiety —X —W comprises . In some
Figure imgf000097_0002
embodiments, the moiety —X2 is . [0393] In some embodiments,
Figure imgf000097_0003
[0394] In some embodiments,
Figure imgf000097_0004
[0395] In some 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. [0396] 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. [0397] In some embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe- Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-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. [0398] In some embodiments, L, or the linker, is or comprises one or more of: ,
Figure imgf000098_0001
, ,
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
[0399] In some embodiments, the linker comprises one or more of: ,
Figure imgf000101_0002
,
Figure imgf000102_0001
[0400] In some embodiments, the linker comprises one or more of:
Figure imgf000102_0002
Figure imgf000103_0001
. [0401] The foregoing linkers may bond to an amino acid side chain present on X, such as a lysine or cysteine (e.g.,
Figure imgf000103_0002
)
Figure imgf000103_0003
[0402] In some embodiments, the linker, is –C(O)L4– or –C(O)C1-6alkyleneC(O)L4–; L4 is a bond, –N(R12)–C2-3alkylene–N(R13)C(O)–, -CH(NHC(O)R14)C1-4alkylene–S–S–C1-4alkylene– OC(O)–, –NHNHC(O)CH(NHC(O)R15)CH2C(O)–, –C1-6alkylene–CH(Gx)OC(O)–,
Figure imgf000103_0004
Figure imgf000104_0001
R12, R13, R14, R15, and R19 are each independently hydrogen or C1-4alkyl; R16 is hydrogen, C1-4alkyl, –C1-4alkylene–OH, –C1-4alkylene–OC1-4alkyl, –C1-4alkylene–CO2H, or –C1-4alkylene–CONH2; and Gx is phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, cyano, and nitro. [0403] In some embodiments, the linker, comprises a carbonyl moiety for conjugating the tetrazine- containing group 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 the carbonyl moiety. In some embodiments, the linker may c
Figure imgf000104_0002
. [0404] In some embodiments, the linker, is or comprises one or more of:
Figure imgf000104_0003
,
Figure imgf000105_0001
Figure imgf000106_0001
. [0405] In some embodiments, the linker, is: ,
Figure imgf000106_0002
[0406] In some embodiments, the linker, is or comprises one or more of:
Figure imgf000106_0003
,
Figure imgf000107_0001
D. TPD Precursors [0407] In certain embodiments, provided herein are compounds which include a precursor to a TPD, that upon administration individually, react in vivo to generate a TPD. Accordingly, in certain embodiments, provided is a system comprising a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine- modified target protein ligand. Also provided is a system comprising a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand. [0408] In certain embodiments, provided is a method for making a TPD at a target location in a subject, comprising administering to the subject, a therapeutically effective amount of a trans-cyclooctene- modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand. [0409] In certain embodiments, provided is a method for making a TPD at a target location in a subject, comprising administering to the subject, a therapeutically effective amount of a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand. [0410] In certain embodiments, provided is a method of delivering a TPD to a target location in a subject, comprising administering to the subject, a therapeutically effective amount of a trans- cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand. [0411] In certain embodiments, provided is a method of delivering a TPD to a target location in a subject, comprising administering to the subject, a therapeutically effective amount of a tetrazine- modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand. [0412] In certain embodiments, provided is 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 a trans-cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand, or a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand. [0413] In certain embodiments, provided is a compound of Table 2. In certain embodiments, provided is a kit comprising two or more compounds of Table 2. Table 2
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
E. Methods of Treatment [0414] Aspects of the present disclosure include methods for delivering a TPD to a target location in a subject. In certain embodiments, the method includes selectively delivering a TPD to the target location in a subject. Selective delivery of the TPD may be achieved through use of a tetrazine activator as described herein. [0415] In some instances, a tetrazine activator of the 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 tetrazine activator as described herein. The tetrazine activator may be administered to the subject at a desired target location in the subject. In some instances, the tetrazine activator may be injected locally into the subject at the desired target location in the subject. In some embodiments, the tetrazine activator is administered systemically. In these embodiments, the tetrazine activator may localize at a desired target location in the subject through specific binding of the tetrazine activator to its target (e.g., antibody-antigen interaction, and the like), or may localize on the surface of a desired target (e.g., a cell surface) through specific binding of the tetrazine activator to its target (e.g., antibody-antigen interaction, and the like). [0416] As described herein, selective binding between bioorthogonal binding partners (e.g., between a tetrazine of the tetrazine activator and its complementary trans-cyclooctene of the trans-cyclooctene- modified TPD conjugate may occur. Due to the administration of the tetrazine activator and resulting localization to a desired location in the subject, the selective binding between the tetrazine and its complementary binding agent of the trans-cyclooctene on the trans-cyclooctene-modified TPD conjugate will localize the TPD to the desired target location. [0417] Provided herein is a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a tetrazine activator as described herein, or a pharmaceutically acceptable salt thereof, and a trans-cyclooctene-modified TPD conjugate as described herein. [0418] 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, 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 Wilm’s tumor/neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma. [0419] 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. [0420] In some embodiments, the cancer is a solid tumor. [0421] In some embodiments, the cancer is a soft tissue sarcoma. [0422] In some embodiments, the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing’s sarcoma. [0423] 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. [0424] 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. [0425] Indications for this approach include cancer, both hematological and solid cancers. In certain embodiments, the approach can be used for the treatment and/or diagnosis of soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing’s sarcoma, and all the different subtypes of soft tissue sarcoma as well as osteosarcoma. The compositions can be for the treatment and/or diagnosis of pigmented vilonodular synovitis. [0426] In certain embodiments, the 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. [0427] 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 the 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 the condition, or at least the symptoms that characterize the condition. Treatment may include inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease. Treatment may include relief, that is, causing the regression of clinical symptoms. For example, in the context of cancer, the 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. [0428] 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). [0429] 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 breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric 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 Wilm's 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 tetrazine activator 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. [0430] In certain embodiments, a tetrazine activator and trans-cyclooctene-modified TPD conjugate as disclosed herein could be administered and 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 tetrazine activator or trans-cyclooctene-modified TPD conjugate 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. [0431] In certain embodiments, the disclosed methods provide the ability to place particles as disclosed herein at the time of the biopsy. When the results return, the practitioner can deliver through to the biopsy site a TPD. [0432] In certain embodiments, the disclosed methods provide the ability for a practitioner to deliver TPDs to enhance the immune system with fewer side effects. This approach would be beneficial to patients. The TPD would treat the solid tumor or specific location. Cancer [0433] 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 growth suppressors, resistance to cell death, enablement of replicative immortality, induction of angiogenesis, and the 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. [0434] 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, macroglobulinemia, melanoma, mesothelioma, mouth cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non- Hodgkin’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). [0435] In some embodiments, the 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, the cancer is a solid tumor. In some embodiments, the cancer is a soft tissue carcinoma. In some embodiments, the cancer is afibrosarcoma. In some embodiments, the cancer is diffuse intrinsic pontine glioma. In some embodiments, the cancer is a metastatic cancer. [0436] In some embodiments, the 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. [0437] Thus, local release of ICD inducers using the compounds and methods of the disclosure may be beneficially combined with one or more immunomodulatory agents. [0438] In certain embodiments, the tetrazine-based targeting agent 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. [0439] The disclosed approach lends itself well as an adjuvant / neoadjuvant system. For example, tetrazine activators 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 the 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 enhance the immune system with fewer side effects as well as the chemotherapeutics agents could be delivered and combined with immunotherapy agents. [0440] The disclosed methods may include systemic or local administration. In some embodiments, the tetrazine-based targeting agent is delivered systemically. In some embodiments, the tetrazine activator and the trans-cyclooctene-modified TPD conjugate are both delivered systemically. In some embodiments, the tetrazine activator is delivered locally. [0441] The disclosed compounds and compositions may be administered prior to surgical resection. The disclosed methods may minimize the size of the 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 tetrazine- based targeting agent 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. [0442] The disclosed methods may include multiple systemic doses of trans-cyclooctene-modified TPD conjugate that focus at one location. [0443] 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 and 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 the triad of multiple cranial neuropathies, long tract signs such as hyperreflexia and clonus, as well as ataxia. Expansion of the pons section of the brainstem may cause obstructive hydrocephalus and increased intracranial pressure. [0444] 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. [0445] The disclosed methods may include multiple systemic doses of trans-cyclooctene-modified TPD conjugate or tetrazine activator that focus at one location. Modes of Administration [0446] 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. [0447] 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. [0448] 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. [0449] 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. [0450] In some embodiments, provided is a kit comprising a tetrazine-based targeting agent, or a pharmaceutically acceptable salt thereof, as described herein, or the pharmaceutical composition comprising the same, and instructions for use thereof. [0451] In some embodiments, the kit further comprising a prodrug. [0452] 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, state 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. [0453] 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 (qid), or three times a day (tid). [0454] 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 present 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. [0455] 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. Co- administration 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. [0456] Co-administration can be accomplished by coimplantation or coinjection. [0457] 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. [0458] 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 1:100 (w/w), 1:75, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 (w/w). Other dosages and dosage ratios of the composition of the present disclosure and the active agent are suitable in the formulations and methods described herein. Combination Therapies [0459] 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 tetrazine-based targeting agent 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. [0460] The disclosure also provides a pharmaceutical combination comprising a tetrazine-based targeting agent 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. [0461] The disclosure also provides the use of a pharmaceutical combination comprising a tetrazine- based targeting agent 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 trans-cyclooctene prodrug thereof for the treatment or prevention of a cancer or for use in enhancing or eliciting an immune response. [0462] 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 the 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 the 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 tetrazine-based targeting agent (or therapeutic tetrazine-based targeting agent) and prodrug may be administered/used simultaneously (e.g., through coinjection or coimplantation), separately, or sequentially, followed by administration of the additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof. [0463] The methods and uses in treating cancer include administering/localizing the tetrazine-based targeting agent at a tumor. In the methods and uses disclosed herein, the administration of the prodrug, or a pharmaceutically acceptable salt, or composition thereof; the tetrazine-based targeting agent; and optionally an additional therapeutic agent may inhibit the growth of the tumor. [0464] 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 additional therapeutic agent with a disclosed conjugate or composition may allow lower doses of the 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 the 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 the present disclosure. Anticancer agents [0465] 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, Casodex (Bicalutamide), CeeNU (Lomustine), Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, Chlorambucil, Chlorambucil-Prednisone, CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cometriq (Cabozantinib-S-Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine, Cytarabine 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), 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), Ifex (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, 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 1131 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). [0466] 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. [0467] 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 [0468] 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, e.g., from Sigma Aldrich or other chemical suppliers. [0469] 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. [0470] Compounds, conjugates, and activators provided herein can be prepared by methods adapted from the literature (see, e.g., WO2020/077140, WO2018/187740, WO2017/044983, WO2015/139025, WO2014/205126, WO2022/032191, WO2021/007160, WO2020/077140, WO2018/187740, WO2017/044983, WO2015/139025, and WO2014/205126, which methods are incorporated herein in their entirety). [0471] For example, the trans-cyclooctene-modified TPD conjugates (P1) as described herein can be prepared according to Scheme I below, where A is a TPD, linker is as defined herein, TCO is at least one trans-cyclooctene moiety, and Xx is a leaving group and Yy is a nucleophilic moiety, or Xx is a nucleophilic moiety and Yy is a leaving group, under standard coupling conditions.
Figure imgf000123_0001
SM3 SM4 P1 Example 1: Synthesis of Compound 16
Figure imgf000123_0002
Figure imgf000124_0002
[0472] Intermediate 1a (0.50 g, 2.34 mmol), Intermediate 2a (1.00 g, 2.34 mmol) and HATU (1.34 g, 3.51 mmol) were combined and dissolved in anhydrous CH2Cl2 (20mL). Anhydrous DIPEA (1.63 mL, 9.36 mmol) was added and mixture was stirred at rt under N2 atmosphere for 18 h. The reaction mixture was directly loaded onto silica column and compound 3a was purified by flash chromatography using a gradient of MeOH in CH2Cl2 (0 to 6%). [0473] HRMS (ESI) Calcd for C32H44N5O6S [M+1] 626.3007; found 626.3549 [0474] 1H NMR (500 MHz, MeOD) δ 8.89 (s, 1H), 7.47 (t, J = 7.2 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 4.67 (d, J = 9.2 Hz, 1H), 4.61 (d, J = 8.4 Hz, 1H), 4.58 – 4.56 (m, 1H), 4.52 (d, J = 6.4 Hz, 2H), 4.39 (dd, J = 15.5, 4.5 Hz, 1H), 4.28 (dd, J = 12.2, 7.3 Hz, 1H), 3.87 (d, J = 11.1 Hz, 1H), 3.82 (dd, J = 11.0, 3.8 Hz, 1H), 2.68 – 2.55 (m, 2H), 2.49 (s, 3H), 2.36 (s, 1H), 2.23 (dd, J = 13.1, 7.7 Hz, 1H), 2.10 (ddd, J = 12.9, 8.9, 4.3 Hz, 1H), 1.46 (s, 9H), 1.05 (s, 9H). [0475] 13C NMR (126 MHz, MeOD) δ 172.99, 171.28, 170.37, 151.77, 147.71, 138.85, 132.15, 130.15, 128.82, 127.66, 79.73, 70.73, 69.50, 59.32, 57.31, 42.59, 37.71, 35.89, 27.55, 25.54, 14.53.
Figure imgf000124_0001
[0476] A solution of HCl (4N) in dioxane (6 mL) was cooled in an ice water-bath under Nitrogen atmosphere. A solution of compound 3a in CH2Cl2 (6 mL) was added and the reaction mixture was gradually warmed up to rt and stirred for 1 h. The solvents were evaporated under reduced pressure. The crude product was carried out for further synthesis without any purification. [0477] HRMS (ESI) Calcd for C27H38ClN5O4S [M+2]2+ 281.6161; found 282.3049 [0478] 1H NMR (500 MHz, MeOD) δ 9.89 (s, 1H), 7.60 – 7.57 (m, 2H), 7.54 (d, J = 8.0 Hz, 2H), 4.67 (s, 1H), 4.60 (d, J = 8.7 Hz, 1H), 4.57 (d, J = 8.0 Hz, 1H), 4.53 (s, 1H), 4.43 (d, J = 15.7 Hz, 1H), 4.22 – 4.18 (m, 1H), 3.89 (d, J = 10.8 Hz, 1H), 3.83 (dd, J = 10.8, 3.7 Hz, 1H), 3.76 (dd, J = 11.4, 5.5 Hz, 2H), 3.69 (d, J = 7.3 Hz, 8H), 3.62 – 3.59 (m, 1H), 3.51 (q, J = 7.0 Hz, 1H), 3.25 (q, J = 7.4 Hz, 1H), 2.90 – 2.84 (m, 1H), 2.79 (dd, J = 10.1, 7.5 Hz, 1H), 2.66 (s, 1H), 2.62 (s, 3H), 2.26 (dd, J = 12.9, 7.9 Hz, 1H), 2.11 (ddd, J = 13.2, 9.2, 4.3 Hz, 1H), 1.40 (t, J = 7.1 Hz, 7H), 1.20 (t, J = 7.0 Hz, 2H), 1.09 (s, 9H). [0479] 13C NMR (126 MHz, MeOD) δ 173.14, 170.20, 167.13, 154.87, 141.79, 140.88, 135.64, 128.98, 127.94, 75.96, 73.79, 72.17, 71.05, 69.67, 66.79, 65.51, 60.80, 59.45, 58.25, 56.59, 54.46, 51.29, 42.38, 42.24, 37.61, 35.19, 25.65, 21.07, 17.36, 15.92, 14.06, 11.97, 11.80.
Figure imgf000125_0001
[0480] Intermediate 4a (200 mg, 0.38 mmol), compound 5a (100.05 mg, 0.38 mmol) and HATU (217.24 mg, 0.571 mmol) were combined in anhydrous CH2Cl2 (20 mL). DIPEA (0.264 ml, 1.52 mmol) was added dropwise and the reaction mixture was stirred for 18 h at rt. The reaction mixture was loaded onto silica column and Intermediate 6a was purified by flash chromatography using a gradient of MeOH in CH2Cl2 (0 to 8%). [0481] HRMS (ESI) Calcd for C38H55N6O9S [M+1] 771.3746; found 771.4397 [0482] 1H NMR (500 MHz, MeOD) δ 8.90 (s, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 4.69 (t, J = 6.7 Hz, 1H), 4.66 (s, 1H), 4.60 – 4.55 (m, 2H), 4.52 (s, 1H), 4.38 (d, J = 15.5 Hz, 1H), 4.08 (d, J = 2.2 Hz, 2H), 3.87 (d, J = 10.8 Hz, 1H), 3.82 (dd, J = 10.9, 3.8 Hz, 1H), 3.74 (dd, J = 5.1, 3.2 Hz, 3H), 3.69 – 3.66 (m, 2H), 3.56 (t, J = 5.7 Hz, 2H), 3.37 (s, 2H), 3.25 (dd, J = 11.9, 6.3 Hz, 3H), 2.71 (dt, J = 14.5, 5.0 Hz, 2H), 2.50 (s, 4H), 2.41 (s, 1H), 2.22 (d, J = 7.8 Hz, 1H), 2.10 (ddd, J = 13.3, 9.0, 4.5 Hz, 1H), 1.45 (s, 9H), 1.38 (t, J = 6.0 Hz, 6H), 1.05 (s, 9H). [0483] 13C NMR (126 MHz, MeOD) δ 172.93, 171.51, 170.58, 151.11, 148.14, 138.43, 132.19, 130.14, 129.01, 127.52, 78.51, 71.13, 70.57, 69.37, 59.30, 57.53, 51.37, 42.23, 37.48, 35.15, 27.71, 25.09, 21.27, 14.18.
Figure imgf000125_0002
[0484] Intermediate 6a (0.150 g, 0.38 mmol) was dissolved in CH2Cl2 (5 mL) and TFA (5 mL). The reaction mixture was stirred for 2 h at rt under N2 atmosphere. The solvents were removed under reduced pressure and the product was azeotroped with CH2Cl2 (5 mL) to remove any residual TFA. [0485] 1H NMR (500 MHz, MeOD) δ 9.58 (s, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.1 Hz, 2H), 4.83 (t, J = 6.1 Hz, 1H), 4.66 – 4.61 (m, 2H), 4.58 – 4.53 (m, 2H), 4.44 – 4.39 (m, 1H), 4.09 (s, 2H), 3.92 (d, J = 11.0 Hz, 1H), 3.82 (dd, J = 11.0, 3.4 Hz, 1H), 3.78 – 3.70 (m, 8H), 3.25 – 3.19 (m, 2H), 2.75 – 2.62 (m, 2H), 2.56 (s, 3H), 2.40 (s, 1H), 2.29 (dd, J = 12.6, 7.4 Hz, 1H), 2.16 – 2.08 (m, 1H), 1.38 (t, J = 6.2 Hz, 5H), 1.06 (s, 9H).
Figure imgf000126_0001
[0486] Intermediate 8a (0.060 g, 0.15 mmol) and PyBOP (0.117 g, 0.225 mmol) were combined in anhydrous DMF (6 mL). DIPEA (0.104 ml, 0.6 mmol) was added dropwise and the reaction was stirred for 15 min at rt under a N2 atmosphere. Intermediate 7a (0.10 g, 0.15 mmol) was added and the reaction mixture was stirred for 18 h. The reaction mixture was diluted with 1:1 DMSO:H2O and purified by preparative HPLC using a gradient of CH3CN (0.1% formic acid) in H2O (0.1% formic acid) 30-95%. [0487] HRMS (ESI) Calcd for C52H62ClN10O8S2 [M+1] 1053.3877; found 1053.2644 [0488] 1H NMR (500 MHz, MeOD) δ 8.90 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.47 (d, J = 7.9 Hz, 4H), 7.42 (d, J = 8.4 Hz, 4H), 4.71 (t, J = 6.7 Hz, 1H), 4.66 (t, J = 6.4 Hz, 2H), 4.59 (t, J = 8.2 Hz, 1H), 4.53 (d, J = 15.3 Hz, 1H), 4.47 (s, 1H), 4.37 (d, J = 15.4 Hz, 1H), 4.07 (d, J = 3.5 Hz, 2H), 3.87 (d, J = 10.8 Hz, 1H), 3.80 (dd, J = 10.9, 3.8 Hz, 1H), 3.75 (d, J = 5.4 Hz, 2H), 3.71 (d, J = 5.1 Hz, 2H), 3.66 (t, J = 5.2 Hz, 2H), 3.52 – 3.46 (m, 3H), 3.37 (dd, J = 15.2, 5.3 Hz, 1H), 2.71 (s, 5H), 2.48 (s, 3H), 2.46 (s, 3H), 2.23 – 2.17 (m, 1H), 2.11 – 2.06 (m, 1H), 2.05 (s, 1H), 1.71 (s, 3H), 1.41 – 1.37 (m, 2H), 1.04 (s, 9H). [0489] 13C NMR (126 MHz, DMSO) δ 172.32, 170.15, 169.80, 169.74, 169.55, 163.57, 163.51, 151.98, 148.18, 139.94, 137.16, 135.74, 132.72, 131.26, 130.64, 130.34, 130.13, 130.07, 129.14, 128.94, 127.93, 80.97, 73.45, 70.72, 70.25, 69.85, 69.71, 69.32, 59.21, 57.13, 56.82, 54.26, 51.19, 42.14, 40.90, 38.34, 37.91, 36.09, 26.72, 22.17, 16.39, 14.52, 13.16, 11.78.
Figure imgf000127_0001
[0490] Intermediate 9a (0.050 g, 0.047 mmol), compound 10a (9.4 uL, 0.047 mmol), sodium ascorbate (0.002 g, 0.0094 mmol, 20 mol%) and Cu(II)sulfate (0.002 g, 0.0094 mmol, 20 mol%) were combined in THF (1.5 mL).5-6 drops of H2O was added and the reaction mixture was stirred for 16 h. The solvents were evaporated under reduced pressure and the mixture was redissolved in 1:1 DMSO:H2O (1.5 mL each). Intermediate 11a was purified by preparative HPLC using a gradient of CH3CN (0.1% formic acid) in H2O (0.1% formic acid) 30-95%. [0491] HRMS (ESI) Calcd for C60H80ClN14O11S2 [M+1] 1271.5255; found 1271.2644 [0492] 1H NMR (500 MHz, MeOD) δ 8.89 (s, 1H), 8.19 (s, 3H), 7.75 (s, 1H), 7.46 (d, J = 2.6 Hz, 4H), 7.43 (d, J = 3.7 Hz, 4H), 4.86 – 4.82 (m, 1H), 4.66 (d, J = 3.3 Hz, 1H), 4.62 (s, 1H), 4.53 (d, J = 5.5 Hz, 3H), 4.49 (s, 1H), 4.41 – 4.36 (m, 1H), 4.02 (d, J = 5.3 Hz, 2H), 3.91 (t, J = 9.1 Hz, 1H), 3.85 (d, J = 4.1 Hz, 2H), 3.80 (dd, J = 10.8, 3.5 Hz, 1H), 3.68 (t, J = 6.1 Hz, 7H), 3.66 – 3.61 (m, 6H), 3.58 (d, J = 7.1 Hz, 6H), 3.49 (dd, J = 12.9, 7.1 Hz, 3H), 3.37 (dd, J = 15.2, 5.2 Hz, 1H), 3.24 (dd, J = 14.9, 6.0 Hz, 1H), 3.14 (d, J = 5.7 Hz, 3H), 2.70 (s, 3H), 2.68 (s, 1H), 2.48 (s, 3H), 2.46 (s, 3H), 2.26 (dd, J = 12.8, 7.3 Hz, 1H), 2.12 – 2.06 (m, 1H), 2.05 (s, 1H), 1.71 (s, 3H), 1.02 (s, 9H). [0493] 13C NMR (126 MHz, MeOD) δ 173.01, 171.59, 171.22, 170.84, 170.45, 164.78, 164.22, 155.64, 151.48, 150.76, 147.65, 142.67, 138.89, 136.74, 136.56, 132.15, 131.98, 131.81, 130.63, 130.56, 130.12, 129.97, 128.97, 128.39, 127.90, 127.64, 127.58, 123.75, 70.77, 70.05, 69.90, 69.83, 69.67, 69.46, 69.01, 66.50, 59.47, 57.75, 56.76, 53.84, 52.31, 49.88, 42.32, 39.28, 39.16, 39.03, 37.61, 37.42, 35.35, 27.74, 25.61, 14.46, 13.04, 11.57, 10.20.
Figure imgf000128_0001
[0494] Intermediate 11a (0.090 g, 0.071 mmol), TCO-bis-NHS (0.036 g, 0.085 mmol), and DIPEA (37 mL, 0.21 mmol) were combined in DMF (5 mL). The reaction mixture was stirred for 16 h. The solvents were evaporated under reduced pressure and the mixture was redissolved in CH2Cl2 (3 mL). Intermediate 12a was purified by preparative TLC using 15% MeOH in CH2Cl2 as mobile phase. [0495] HRMS (ESI) Calcd for C75H97ClN15O17S2 [M+1] 1578.6311; observed: 1578.3682 [0496] 1H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.08 – 7.98 (m, 1H), 7.89 – 7.85 (m, 1H), 7.67 – 7.59 (m, 1H), 7.34 (s, 1H), 6.09 – 5.76 (m, 1H), 5.71 – 5.47 (m, 1H), 5.19 – 5.13 (m, 1H), 4.94 – 4.80 (m, 1H), 4.75 – 4.62 (m, 1H), 4.61 – 4.42 (m, 1H), 4.40 – 4.31 (m, 1H), 4.07 – 3.98 (m, 1H), 3.84 – 3.75 (m, 1H), 3.67 (s, 1H), 3.56 (s, 1H), 3.45 (s, 1H), 3.23 – 3.01 (m, 1H), 2.82 (s, 1H), 2.50 (s, 1H), 2.41 (s, 1H), 2.34 – 2.20 (m, 1H), 2.20 – 2.13 (m, 1H), 2.12 – 1.79 (m, 1H), 1.66 (s, 1H), 1.25 (s, 1H), 1.07 – 1.02 (m, 1H), 0.94 (s, 1H).
Figure imgf000129_0001
[0497] Intermediate 12a (0.050 g, 0.031 mmol), aspartic acid (0.021 g, 0.15 mmol), DIPEA (108 mL, 0.63 mmol) and BSA (0.063 g, 0.31 mmol) were combined in CH2Cl2 (5 mL). The reaction mixture was stirred for 16 h. The solvents were evaporated under reduced pressure and the mixture was redissolved in a 1:1 solution of CH3CN (4 mL) and 5% aqueous citric acid (4 mL, pH 3.3). The mixture was stirred at rt for 1 h. Compound 16 was purified by preparative HPLC using a gradient of CH3CN (0.1% formic acid) in H2O (0.1% formic acid) 30-95%. [0498] HRMS (ESI) Calcd for C75H99ClN15O18S2 [M+1] 1596.6417; observed: 1596.3116 [0499] 1H NMR (500 MHz, MeOD) δ 8.88 (s, 1H), 8.64 – 8.59 (m, 1H), 8.51 – 8.47 (m, 1H), 8.16 – 7.95 (m, 2H), 7.74 (s, 1H), 7.51 – 7.36 (m, 7H), 5.95 – 5.86 (m, 1H), 5.68 – 5.61 (m, 1H), 5.08 (s, 1H), 4.69 – 4.59 (m, 4H), 4.58 – 4.44 (m, 4H), 4.42 – 4.34 (m, 1H), 4.06 – 3.98 (m, 2H), 3.93 (d, 1H), 3.89 – 3.76 (m, 3H), 3.73 – 3.61 (m, 6H), 3.58 (s, 8H), 3.54 – 3.46 (m, 4H), 3.40 – 3.35 (m, 2H), 3.30 – 3.10 (m, 4H), 2.84 (t, 1H), 2.73 – 2.65 (m, 3H), 2.49 (d, 5H), 2.30 – 2.22 (m, 2H), 2.16 – 2.05 (m, 2H), 2.02 – 1.94 (m, 2H), 1.90 – 1.86 (m, 1H), 1.84 – 1.77 (m, 1H), 1.72 – 1.68 (m, 3H), 1.66 – 1.57 (m, 1H), 1.12 (s, 2H), 1.02 (s, 8H). [0500] 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. [0501] 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 the claims. [0502] 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. [0503] 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

What is claimed is: 1. A trans-cyclooctene-modified TPD conjugate comprising a targeted protein degrader (TPD) covalently bonded to at least one trans-cyclooctene moiety, optionally via a linker. 2. The trans-cyclooctene-modified TPD conjugate of claim 1, wherein the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of a Von Hippel–Lindau (VHL) ligand, a cereblon (CRBN) ligand, a mouse double minute 2 homolog (MDM2) ligand, and a beta-transducin repeat-containing protein (β-TrCP) ligand. 3. The trans-cyclooctene-modified TPD conjugate of claim 1 or 2, wherein the TPD comprises an E3 ubiquitin ligase ligand selected from the group consisting of thalidomide, lenalidomide, pomalidomide, MLN4924 (Pevonedistat), Nutlin-3, and curcumin. 4. The trans-cyclooctene-modified TPD conjugate of claim 1 or 2, wherein the TPD comprises an E3 ubiquitin ligase ligand selected from Table A, wherein the wavy line indicates the point of attachment to the remainder of the TPD. 5. The trans-cyclooctene-modified TPD conjugate of claim 1, wherein the TPD comprises a target protein ligand selected from the group consisting of a receptor tyrosine kinase (RAF) kinase ligand (e.g., a BRAF kinase ligand), a steroid receptor ligand (e.g., an androgen receptor (AR) ligand, an estrogen receptor (ER) ligand, or a progesterone receptor (PR) ligand), a cyclin-dependent kinase (CDK) ligand, a BTK ligand, a BET ligand, and an Inhibitor of Apoptosis Protein (IAP) ligand. 6. The trans-cyclooctene-modified TPD conjugate of claim 1, wherein the TPD comprises a target protein ligand selected from Table B. 7. The trans-cyclooctene-modified TPD conjugate of claim 1, wherein the TPD is selected from Table C. 8. The trans-cyclooctene-modified bispecific antibody of any preceding claim, wherein the trans- cyclooctene moiety is of Formula X:
Figure imgf000131_0001
wherein: 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; q1 is 0 or 1; R1B, at each occurrence, is independently selected from the group consisting of G1, OH, –NR1c–C1-4alkylene–G1, –NR1c–C1-4alkylene–N(R1d)2, –NR1c–C1-6alkylene–N(C1-4alkyl)3+, –N(R1c)CHR1eCO2H, –N(R1c)–C1-6alkylene–CO2H, –N(R1f)–C2-4alkylene–(N(C1-4alkylene–CO2H)– C2-4alkylene)n–N(C1-4alkylene–CO2H)2, –N(R1c)CHR1eC(O)OC1-6alkyl, –N(R1c)–C1-6alkylene– C(O)OC1-6alkyl, –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; R1e, at each occurrence, is independently –C1-4alkylene–CO2H, –C1-4alkylene–CONH2, or –C1-4alkylene–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; and G1, at each occurrence, is independently an optionally substituted heterocyclyl. 9. The trans-cyclooctene-modified bispecific antibody of any preceding claim, wherein the trans- cyclooctene moiety is of Formula XI:
Figure imgf000132_0001
wherein: R2 is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine; wherein R2 may be optionally further substituted with a polyether. 10. The trans-cyclooctene-modified bispecific antibody of any preceding claim, wherein the trans- cyclooctene moiety is of Formula: H
Figure imgf000132_0002
, , o
Figure imgf000133_0001
11. The trans-cyclooctene-modified TPD conjugate of any preceding claim, wherein at least one trans-cyclooctene moiety is
Figure imgf000133_0002
. 12. The trans-cyclooctene-modified TPD conjugate of any preceding claim, wherein at least one trans-cyclooctene moiety is
Figure imgf000133_0004
13. The trans-cyclooctene-modified TPD conjugate of any preceding claim, wherein the trans- cyclooctene moiety is covalently bonded to the TPD via a linker. 14. The trans-cyclooctene-modified TPD conjugate of any preceding claim, wherein the linker is a cleavable linker. 15. A trans-cyclooctene-modified TPD conjugate selected from Table 1. 16. A system comprising the trans-cyclooctene-modified TPD conjugate of any preceding claim and a tetrazine activator. 17. The system of claim 16, wherein the tetrazine activator is formulated for systemic administration. 18. The system of claim 16 or 17, wherein the tetrazine activator is of Formula IV:
Figure imgf000133_0003
wherein: each R20 is independently selected from the group consisting of hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R", C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1 is 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(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; and R''', at each occurrence, is independently selected from aryl and alkyl. 19. The system of claim 18, wherein each R20 is independently 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; wherein each is independently optionally substituted with one to three Z1. 20. The system of claim 16, wherein the tetrazine activator comprises a support composition comprising a biocompatible support and a tetrazine-containing group. 21. The system of claim 20, wherein the tetrazine activator comprises a biocompatible support and a tetrazine-containing group of Formula IVA, IVB, or IVC:
Figure imgf000134_0001
wherein: R20 is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S) R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R" at each occurrence are independently selected from hydrogen, aryl and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; R30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; Ra, R31a and R31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl; and t is 0, 1, 2, 3, or 4. 22. The system of claim 16, wherein the tetrazine activator is of Formula V: wherein:
Figure imgf000135_0001
ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3 and R4 are both absent, or a dihydrotetrazine when R3 and R4 are both present; provided that when ring A is aryl, then R3 and R4 are both present; X is a biocompatible support, antibody, or antibody fragment moiety; p is 1-150; L, at each occurrence, is independently a linker; R1, at each occurrence, is independently selected from the group consisting of hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR'R", C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; 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; R3 and R4 are both absent; or R3 and R4 are each independently hydrogen or a group capable of being removed after a triggering event; each Z1 is 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(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; R''', at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently is 0, 1, 2, 3, or 4. 23. The system of any one of claims 20-22, wherein the biocompatible support is a polymer, viscous or non-viscous liquid material, gel, hydrogel, polysaccharide hydrogel, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, particle, nanoparticle, microparticle, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, or chondroitin sulfate. 24. The system of claim 16, wherein the tetrazine activator is of Formula I:
Figure imgf000136_0001
wherein: X is an antibody or antibody fragment moiety; p is 1-16; L, at each occurrence, is independently a linker; R20, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; 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; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; and R''' at each occurrence is independently selected from aryl and alkyl. 25. The system of claim 16, wherein the tetrazine activator is of Formula II:
Figure imgf000137_0001
wherein: X is an antibody or antibody fragment moiety; p is 1-16; L, at each occurrence, is independently a linker; R20, at each occurrence, is independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=O)R'", OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)2R"', S(=O)2NR' R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR' R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R''; R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;
Figure imgf000137_0002
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; and t is independently is 0, 1, 2, 3, or 4. 26. 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 trans- cyclooctene-modified TPD conjugate of any one of claims 1-15, or the system of any one of claims 16-25. 27. 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 a trans- cyclooctene-modified E3 ubiquitin ligase ligand and a tetrazine-modified target protein ligand, or a tetrazine-modified E3 ubiquitin ligase ligand and a trans-cyclooctene-modified target protein ligand, wherein each is independently selected from Table 2. 28. The method of claim 26 or 27, wherein the cancer is a melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer , glioblastoma, 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, Wilm’s tumor/neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, Prostatic adenocarcinoma, Nasopharyngeal carcinoma, or cutaneous T-cell lymphoma. 29. The method of claim 26 or 27, wherein the cancer is a solid tumor. 30. The method of claim 26 or 27, wherein the cancer is a soft tissue sarcoma. 31. The method of claim 26 or 27, wherein the cancer is a hematological malignancy. 32. The method of claim 31, wherein the hematological malignancy is myelodysplastic syndrome, acute myeloid leukemia, myelodysplastic 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 lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy-cell leukemia, precursor T-lymphoblastic leukemia, or nodular lymphocyte predominant Hodgkin lymphoma. 33. The method of claim 32, wherein the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.
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