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WO2024229427A1 - Agents de ciblage à base de tétrazine pour la distribution in vivo d'une charge utile - Google Patents

Agents de ciblage à base de tétrazine pour la distribution in vivo d'une charge utile Download PDF

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Publication number
WO2024229427A1
WO2024229427A1 PCT/US2024/027840 US2024027840W WO2024229427A1 WO 2024229427 A1 WO2024229427 A1 WO 2024229427A1 US 2024027840 W US2024027840 W US 2024027840W WO 2024229427 A1 WO2024229427 A1 WO 2024229427A1
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targeting agent
alkyl
tetrazine
independently
occurrence
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Jesse M. McFARLAND
Jaime R. CABRERA-PARDO
José Manuel Mejía ONETO
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Tambo Inc
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Tambo Inc
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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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/54—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 organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • A61K47/54—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 organic compound
    • A61K47/555—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 organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/08—Six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg

Definitions

  • the present disclosure relates generally to tetrazine-based targeting agents for bioorthogonal delivery of a payload to a targeted location in a subject, which tetrazine-based targeting agents have applications, e.g., in the treatment of cancer, tumor growth, and immunotherapy.
  • Bioorthogonal conjugation or click reactions are selective and orthogonal (non-interacting with) functionalities found in biological systems, and have found use in various applications in the fields of chemistry, chemical biology, molecular diagnostics, and medicine, where they can be used to facilitate the selective manipulation of molecules, cells, particles and surfaces, and the tagging and tracking of biomolecules in vitro and in vivo.
  • These reactions include the Staudinger ligation, the azide-cyclooctyne cycloaddition, and the inverse-electron-demand Diels-Alder reaction.
  • tetrazine-based targeting agents comprising one or more targeting moieties covalently conjugated to one or more tetrazine moieties optionally via a linker, provided that at least one targeting moiety is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent.
  • a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L
  • the tetrazine-based targeting agents described herein are designed to, once administered to a subject, localize at a target site within the subject.
  • the tetrazine-based targeting agent can be administered locally or systemically.
  • a prodrug comprising a payload or therapeutic agent and one or more complimentary bioorthogonal components (i.e., a trans-cyclooctene moiety) can be administered, which when in contact with the tetrazine -based targeting agent in vivo, allows for targeted delivery of the payload or therapeutic agent.
  • the tetrazine-based targeting agent comprises is a therapeutic targeting agent.
  • the tetrazine-based targeting agents described herein comprise a diagnostic agent such that the tetrazine-based targeting agents described herein can be used in diagnosing conditions or diseases, with or without administering a payload or therapeutic agent.
  • a method for treating cancer comprising administering to a subject in need thereof, a tetrazine-based targeting agent as described herein to a subject in need thereof, and administering to the subject a conjugate, or the pharmaceutically acceptable salt or composition thereof, 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 lymphoma or leukemia.
  • the cancer is a hematologic malignancy.
  • Figure 1A and Figure IB show Compound 111 binds to both human (Fig. 1 A) and mouse (Fig. IB) isoforms of recombinant Nectin-4 in a ligand binding assay, while Compound 111-C does not bind in either case.
  • Figure ! shows Compounds B and F convert to MMAE and exatecan, respectively, after contact with Compound 111.
  • Figure 3 shows the tumor response of Compound 111 + Compound B in NCI-H292 tumors in BALB/c nude mice.
  • 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 C1-C6-alkyl means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms.
  • C1-C3- 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 (-CH2C ⁇ 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(CH 3 )-, -C(CH 3 ) 2 -, -CH 2 CH 2 -, -CH(CH 3 )CH 2 -, -C(CH 3 ) 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -C(CH 3 ) 2 CH 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 a- carbon attached to a hydrogen, carboxyl group, amino group, and R group.
  • Such analogs can have a modified R group (e.g., norleucine as an example) or retain a modified peptide backbone while retaining the same basic chemical structure as a natural amino acid.
  • Non-limiting examples of non-natural amino acids or amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, homophenylalanine, ornithine, formyl glycine, phenyl glycine, para-azidophenyl glycine, para-azidophenylalanine, para-acetophenylalanine, 4-(3-methyl-(l,2,4,5-tetrazine))- phenylglyine, and 4-(3-methyl-(l,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.
  • 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.
  • aryl 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.
  • 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.
  • azide refers to the functional group -N 3 .
  • 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 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.
  • 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).
  • 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, trifluoromcthyl, difluoromcthyl, pcntafluorocthyl, 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.
  • 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 arc replaced by a halogen.
  • haloalkoxy means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.
  • heteroalkyl 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.
  • 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-l,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, azctidinyl, azcpanyl, aziridinyl, diazcpanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, l,3-dimethylpyrimidine-2,4(lH,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, o
  • 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-l W-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-2//-2,5-methanocyclopenta[b]furan, hexahydro- l//-l,4-methanocyclopenta[c]furan, aza-adamantane (l-azatricyclo[3.3.1.1 3,7 ]decane), and oxa- adamantane (2-oxatricyclo[3.3.1.1 3,7 ]dccanc).
  • 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 nonhydrogen 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 dihydro tetrazine.
  • the term “selectively delivering” refers to delivering an agent (e.g., a payload) to an organ or tissue (or portion thereof) in need of treatment or diagnosis, without significant binding to other nontarget organs or tissues (or portions thereof).
  • an agent e.g., a payload
  • the tetrazine-based targeting agents described herein do not themselves have a therapeutic effect, but rather are designed to allow the selective or targeted delivery of a therapeutic agent. However, it may be that the tetrazine-based targeting agent does have a therapeutic effect, and thus, such constructs are not excluded by the present disclosure.
  • payload refers to an agent for delivery to a target site in a subject. Tn some embodiments, payloads include therapeutic or diagnostic agents.
  • therapeutic agent refers to an agent capable of treating and/or ameliorating a condition or disease, or one or more symptoms thereof, in a subject.
  • Therapeutic agents of the present disclosure also include prodrug forms of therapeutic agents.
  • 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 (l,r-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, H C, 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 58 Ga, 82 Rb, 89 Zr, 90 Sr, 90 Y, "Tc, 99m Tc, n i 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.
  • 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 payload.
  • Representative organs and tissues for targeting include those that can be targeted by chemical or biological targeting agents, as well as those organs and tissues that cannot be targeted by chemical or biological targeting agents.
  • implanting refers to surgical implantation into a subject’s body.
  • contacting 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 moictics, 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.
  • 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.
  • a slow-release device e.g., a mini-osmotic pump
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • leaving group refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons.
  • suitable leaving groups include halides (e.g., Br, Cl, I), sulfonate esters (e.g., triflate, mesylate, tosylate, and brosylate), and nitrophenols.
  • 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.
  • the term “subject,” “patient,” or “organism” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which an agent(s) of the present disclosure may be administered may include mammals, particularly primates, especially humans.
  • 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.
  • 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.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • 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. 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,” 5 th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.
  • 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 C1, 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 n 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.
  • the tetrazine-based targeting agents described herein are designed to, once administered to a subject, localize at a target site within the subject.
  • the tetrazine-based targeting agents can be administered locally or systemically.
  • a prodrug comprising a complimentary bioorthogonal component (e.g., a payload or therapeutic agent with a trans-cyclooctene moiety) can be administered, which when in contact with the tetrazine-based targeting agent in vivo, allows for targeted drug delivery of a payload or therapeutic agent.
  • the tetrazine -based targeting agents described herein comprise a diagnostic agent such that the tetrazine-based targeting agent as described herein can be used in diagnosing conditions or diseases, with or without administering a payload or therapeutic agent.
  • tetrazine-based targeting agents which comprise one or more targeting moieties covalently conjugated to one or more tetrazine moieties optionally via a linker, provided that at least one targeting moiety is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent.
  • at least one targeting moiety is selected from Table 1.
  • each tetrazine moiety is independently of Formula I:
  • R 3 and R 4 are both absent; or R 3 and R 4 are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double 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;
  • Y 1 at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NR a , or CR 31a R 31b NR a ;
  • Y 2 at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or hetero arylene, is independently optionally substituted with one to four R 21 ;
  • R a , R 31a , and R 31b are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;
  • R' and R" at each occurrence, are independently selected from hydrogen, alkyl, and aryl;
  • R"' at each occurrence, is independently selected from alkyl and aryl.
  • R 3 and R 4 are both absent, and the dotted lines are optional double bonds to form a tetrazine.
  • R 3 and R 4 are each independently hydrogen or a group capable of being removed after a triggering event, and the dotted lines are optional double bonds to form a dihydrotetrazine.
  • R 2 at each occurrence, is independently cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to three Z 1 .
  • each tetrazine moiety is independently of Formula II: wherein:
  • R' and R" at each occurrence, are independently selected from hydrogen, alkyl, and aryl;
  • R T " at each occurrence, is independently selected from alkyl and aryl.
  • the linker is branched.
  • the tetrazine-based targeting agent comprises more than one targeting moieties.
  • the tetrazine-based targeting agent comprises more than one tetrazine moiety.
  • the linker is linear.
  • linker can be branched or linear and independently one or more (e.g., 1-16) linkers covalently attached to a targeting moiety. Further, more than one targeting moiety can be covalently attached via a branched linker.
  • tetrazine -based targeting agent of Formula IX is provided.
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; each L is independently a linker; each p is independently 1-8;
  • R 3 and R 4 are both absent; or R 3 and R 4 are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double 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;
  • Y 1 at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NR a , or CR 31a R 31b NR a ;
  • Y 2 at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to four R 21 ; R l , R 31a , and R 31b , at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;
  • R" and R" at each occurrence, are independently selected from hydrogen, alkyl, and aryl;
  • R"' at each occurrence, is independently selected from alkyl and aryl.
  • a tetrazine-based targeting agent of Formula IX wherein X is a targeting agent selected from Table 1.
  • tetrazine-based targeting agent of Formula III wherein:
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent: L is a linker: p is 1-16;
  • R 3 and R 4 are both absent; or R 3 and R 4 are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double 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;
  • Y 1 at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NR a , or CR 31a R 31b NR a ;
  • Y 2 at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or hetero arylene, is independently optionally substituted with one to four R 21 ;
  • R a , R 31a , and R 31b are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;
  • R' and R at each occurrence, are independently selected from hydrogen, alkyl, and aryl; and R'", at each occurrence, is independently selected from alkyl and aryl.
  • tetrazine-based targeting agent of Formula III wherein:
  • X is a targeting agent selected from Table 1;
  • R 3 and R 4 are both absent; or R 3 and R 4 are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double 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;
  • Y 1 at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NR a , or CR 31a R 31b NR a ;
  • Y 2 at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to four R 21 ; R l , R 31a , and R 31b , at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;
  • R" and R" at each occurrence, are independently selected from hydrogen, alkyl, and aryl;
  • R"' at each occurrence, is independently selected from alkyl and aryl.
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an intcgrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;
  • p is 1-16;
  • L at each occurrence, is independently a linker
  • R 3 and R 4 are both absent; or R 3 and R 4 are each independently hydrogen or a group capable of being removed after a triggering event;
  • R 22 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 30 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, at each occurrence, is independently 0, 1, 2, 3, or 4.
  • tetrazine moiety represented by:
  • 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 21 at each occurrence, is independently halo, alkyl, or haloalkyl.
  • p is 1-5, or 1-4, or 1-3, or 1-2, or 1.
  • t at each occurrence, is 0.
  • R 2 or Ring A is pyridyl or phenyl.
  • R 2 or Ring A is other than pyridyl or phenyl.
  • R 2 or Ring A is other than pyridyl or phenyl.
  • a tetrazine-based targeting agent of Formula IID wherein X is a targeting agent as disclosed herein. In some embodiments, X is a targeting agent selected from Table 1.
  • a tetrazine-based targeting agent of Formula HE wherein X is a targeting agent as disclosed herein.
  • X is a targeting agent selected from Table 1.
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;
  • p is 1-16;
  • L at each occurrence, is independently a linker
  • R 3 and R 1 are both absent; or R 3 and R 1 are each independently hydrogen or a group capable of being removed after a triggering event;
  • R 22 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 30 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, at each occurrence, is independently 0, 1, 2, 3, or 4.
  • R 3 and R 4 are both present.
  • a tetrazine-based targeting agent of Formula I-A wherein:
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent: p is 1-16;
  • L at each occurrence, is independently a linker
  • R 22 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 T and R are independently selected from hydrogen, aryl, and alkyl
  • R'" at each occurrence is independently selected from aryl and alkyl.
  • tetrazine-based targeting agent of Formula IIA-a wherein:
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;
  • p is 1-16;
  • L at each occurrence, is independently a linker
  • R 30 is independently halogen, cyano, nitro, hydroxy, alkyl, haloalky 1, alkenyl, alkynyl, alkoxy, haloalkoxy, hctcroalkyl, aryl, hctcroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;
  • R a , R 31a and R 31b are each independently hydrogen, Ci-Ce-alkyl, or Ci-Ce-haloalkyl;
  • R T and R 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.
  • roio6i In one embodiment, R 22 , 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.
  • tetrazine-based targeting agent of Formula IIB-a wherein L, p, and X are each independently as defined herein.
  • a tetrazine-based targeting agent of Formula IlC-a wherein L, p, and X are each independently as defined herein.
  • tetrazine-based targeting agent of Formula Ill-a is provided.
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;
  • p is 1-16;
  • L at each occurrence, is independently a linker
  • R 30 is independently halogen, cyano, nitro, hydroxy, alkyl, haloalky 1, alkenyl, alkynyl, alkoxy, haloalkoxy, hctcroalkyl, aryl, hctcroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;
  • R' and R" at each occurrence, are independently selected from hydrogen, aryl, and alkyl;
  • R T " at each occurrence, is independently selected from aryl and alkyl; and t is independently is 0, 1, 2, 3, or 4.
  • a tetrazine-based targeting agent represented by Formula IID-a: wherein X and R 20 arc each independently as defined herein. In some embodiments, R 20 is methyl.
  • tetrazine-based targeting agent of Formula IIE-a wherein p and X are each independently as defined herein.
  • L0113J Also provided is a tetrazine-based targeting agent of Formula HF-a: wherein p and X are each independently as defined herein.
  • tetrazine -based targeting agent of Formula IIG-a wherein p and X are each independently as defined herein.
  • a tetrazine-based targeting agent of Formula IIH wherein X is as defined herein.
  • a tetrazine-based targeting agent of Formula IIH-a wherein X is a targeting agent selected from Table 1.
  • X is a nectin-4 targeting agent.
  • a tetrazine-based targeting agent of Formula V-A ring A is aryl, 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; provided that when ring A is aryl, then R 3 and R 4 are both present;
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;
  • p is 1-16;
  • L at each occurrence, is independently a linker
  • R' and R" at each occurrence, are independently selected from hydrogen, aryl, and alkyl;
  • R T " at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently 0, 1, 2, 3, or 4.
  • a tetrazine-based targeting agent of Formula V-A 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 non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent: p is 1-16;
  • L at each occurrence, is independently a linker
  • R 3 and R 4 are both absent;
  • R 3 is a group capable of being removed after a triggering event
  • 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 0, 1, 2, 3, or 4.
  • t at each occurrence, is independently 0, 1, 2, 3, or 4.
  • R 4 is hydrogen
  • R 3 is a group capable of being removed after a triggering event.
  • the triggering event occurs in vivo. Once the triggering event occurs and R 3 is removed, the dihydrotetrazine moiety is oxidized to provide a tetrazine as in Formula VII-A: 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-based targeting agent 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).
  • internal means e.g., via enzymatic cleavage of a functional group, optionally followed by a decomposition
  • external means e.g., photocleavable linkers
  • R 3 comprises an amino acid sequence specific for cleavage by a protease or esterase.
  • 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 Vai- Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly for cleavage by cathepsins.
  • R 3 comprises Ac-yE-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 IB.
  • Cit Citrulline; Cha: P-cyclohexylalanine; Hof: homophenylalanine; Nva: aminosuberic acid; Dpa: D- phenylalanine; Nle: Norleucine; Smc: S-methylcysteine
  • Hy is any hydrophobic L-amino acid y indicates that bond is a gamma carboxy linkage
  • 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.
  • 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:
  • 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:
  • 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.
  • 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
  • At least one of the moiety is represented by a formula selected from:
  • 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. In some embodiments, R 2 , at each occurrence, is independently halo, alkyl, or haloalkyl. In some embodiments, R 2 , at each occurrence, is independently halo or alkyl.
  • t at each occurrence is 0.
  • a tetrazine-based targeting agent of Formula VA-a wherein p and X are each independently as defined herein.
  • a tetrazine -based targeting agent of Formula VB-a wherein p and X are each independently as defined herein.
  • ring A is other than pyridyl. In some embodiments, ring A is other than aryl. In some embodiments, ring A is other than phenyl.
  • tetrazine-based targeting agents which comprise one or more targeting moieties covalently bonded to one or more tetrazine moieties.
  • the targeting moieties described herein are designed to, once administered to a subject, localize at a target site within the subject.
  • a prodrug comprising a complimentary bioorthogonal component (i.e., a trans-cyclooctene moiety) can be administered, which when in contact with the tetrazine-based targeting agent in vivo, allows for targeted drug delivery of a payload or therapeutic agent.
  • the targeting moiety, or X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent.
  • the targeting moieties as used herein are derived from known compounds (i.e., targeting agents) which target (or bind) to a desired target.
  • the parent or known targeting agent e.g., compounds or peptides
  • the targeting moieties are modified to be conjugated to a tetrazine moiety, optionally via a linking moiety as defined herein.
  • the targeting moieties even after modification to arrive at the compounds described herein, maintain biological activity, which is comparable to that observed in the original, unmodified targeting agent.
  • the targeting moieties exhibit a binding activity which is at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of that observed in the original, unmodified targeting agent.
  • the terms “modified” and “derived from” as used in reference to a targeting agent means that one or more atoms of the original, unmodified targeting agent (i.e., a known targeting agent) is replaced by a covalent bond to the tetrazine moiety, optionally via a linking moiety.
  • one hydrogen atom bound to a heteroatom e.g., N, O, or S
  • one reactive functional group e.g., -OH
  • the targeting agent comprises amino acids, including an additional amino acid or a conservative substitution provides a synthetic handle (e.g., introducing a lysine or cysteine),
  • the targeting moiety, or X is a non-antibody or non-antibody fragment NECTIN-4 peptide targeting agent. See, e.g., Table 1, entry 1.
  • tetrazine-based targeting agents disclosed herein can be tested for NECTIN-4-binding using assays known in the art (e.g., Babeker, et al., Journal of Nuclear Medicine Jun 2022, 63 (supplement 2) 2865).
  • the targeting moiety, or X is a non-antibody or non-antibody fragment PSMA peptide targeting agent.
  • tetrazine-based targeting agents disclosed herein can be tested for PSMA-binding using assays known in the art (e.g., Rogers, et al. Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors. Sci Rep. 2021 Jul 21 ; 11 (1): 14925. doi: 10.1038/s41598-021-94534-5).
  • the targeting moiety, or X is a non-antibody or non-antibody fragment cholecystokinin-2 receptor (CCK2R) peptide targeting agent.
  • CCK2R cholecystokinin-2 receptor
  • tetrazine-based targeting agents disclosed herein can be tested for CCK2R-binding using assays known in the art (e.g., Hbrmann et al., Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177. Molecules. 2020 Oct 8;25(19):4585. doi:
  • tetrazine-based targeting agents disclosed herein can be used in methods for treating cancer or gastrointestinal disorders.
  • the targeting moiety, or X is a non-antibody or non-antibody fragment PD-L1 peptide targeting agent.
  • tetrazine-based targeting agents disclosed herein can be tested for PD-L1 -binding using assays known in the art (e.g., Liu, et al. Clinical applications of PD-L1 bioassays for cancer immunotherapy. J Hematol Oncol 10, 110 (2017). https://doi.org/10.1186/sl3045-017-0479-y).
  • the targeting moiety, or X is a non-antibody or non-antibody fragment integrin alpha V beta 3 targeting agent.
  • tetrazine-based targeting agents disclosed herein can be tested for integrin alpha V beta 3- binding using assays known in the art (e.g., Danhier et al., RGD-Based Strategies To Target Alpha(v) Beta(3) Integrin in Cancer Therapy and Diagnosis, Mol. Pharmaceutics 2012, 9, 11, 2961-2973).
  • the targeting moiety, or X is a non-antibody or non-antibody fragment integrin alpha V beta 6 targeting agent.
  • the tetrazine-based targeting agents disclosed herein can be tested for integrin alpha V beta 6- binding using assays known in the art (e.g., Monieri et aL, A stapled chromogranin A-derived peptide homes in on tumors that express av06 or av[38 integrins, Int. J. Biol. Sci., 2023; 19(1): 156-166. doi: 10.7150/ijbs.76148; and Nardelli, M., et al. (2019). Switching on phosphorescence in [Ir(C A N)2(N A N)]+ complexes by trifluoromethylation of the N A N ligand. Chemical Communications, 55(98), 14777-14780. doi: 10.1039/C9CC07666E).
  • assays known in the art e.g., Monieri et aL, A stapled chromogranin A-derived peptide homes in on tumors that express
  • the targeting moiety, or X is a non-antibody or non-antibody fragment integrin alpha V beta 8 targeting agent.
  • the tetrazine -based targeting agents disclosed herein can be tested for integrin alpha V beta 8- binding using assays known in the art (e.g., Monieri et aL, A stapled chromogranin A-derived peptide homes in on tumors that express av06 or av08 integrins, Int. J. Biol. Sci., 2023; 19(1): 156-166. doi: 10.7150/ijbs.76148; and Nardelli, M., et al. (2019). Switching on phosphorescence in [Ir(C A N)2(N A N)]+ complexes by trifluoromethylation of the N A N ligand. Chemical Communications, 55(98), 14777-14780. doi: 10.1039/C9CC07666E).
  • assays known in the art e.g., Monieri et aL, A stapled chromogranin A-derived peptide homes in on tumors that express
  • the targeting moiety, or X is a non-antibody or non-antibody fragment FAP targeting agent.
  • the tetrazine-based targeting agents disclosed herein can be tested for FAP-binding using assays known in the art (e.g., Zhang, et al. (2020). A cell-based fluorescent assay for FAP inhibitor discovery. Bioorganic & Medicinal Chemistry Letters. 30. 127253. 10.1016/j.bmcl.2020.127253.).
  • the targeting moiety, or X is a non-antibody or non-antibody fragment carbonic anhydrase IX (CAIX) targeting agent.
  • non-antibody or non-antibody fragment is intended to refer to targeting agents that are generally considered antibodies or antibody fragments, such as compounds, including peptides, less than 15 kDa, or less that 10k Da, or less than 9 kDa, or less that 8 kDa, or less that 7 kDa, or less that 6 kDa, or less that 5 kDa.
  • the targeting moiety, or X is selected from Table 1.
  • Table 1 For representative procedures for the preparation of various Targeting moieties, see, e.g., Cazzamalli, et al., Clin Cancer Res; 24(15) August 1, 2018; Wayua, et al., Mol. Pharmaceutics 2015, 12, 2477-2483; Pastorino, et al., Current Radiopharmaceuticals, 2020, Vol. 13, No. 1, 63-79; Lerchen, et al., Cancers 2022, 14, 391;
  • the linkers can be linear or branched (including, but not limited to, bivalent, trivalent, tetravalent branching).
  • the linkers in one tetrazine-based targeting agent, there may be one or more targeting moiety covalently bonded to one or more tetrazine moiety.
  • 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 moiety and one bond to the targeting moiety, wherein there are more than one [tetrazine-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, CM alkyl, CM alkoxy, and CM 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.
  • L is of the formula:
  • L is of the formula:
  • each R 110 is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R 120 is independently hydrogen, CM alkyl, CM 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/rnol), ethylene- l,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/rnol
  • ethylene- l,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:
  • the linker comprises one or more of:
  • the linker comprises one or more of:
  • the linker comprises one or more H . In some embodiments, the linker comprises one or more
  • the linker is, or comprises one or more:
  • the linker is, or comprises one or more:
  • the linker comprises one or more natural or unnatural amino acids, which may be referred to as a peptide linker.
  • the linker may be a peptide linker made up of a carboxylic acyl unit, and one or more amino acids making up a protein or peptide sequence.
  • the linker may also contain a self-immolating spacer which spaces the drug and the protein peptide sequence.
  • the linker may be 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 which spaces the peptide and the payload, D, or the bioorthogonal moiety.
  • 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. In some embodiments 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 2 — W comprises H . In some embodiments, the moiety — X 2 is H
  • 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, Vai- Ala, Ala- Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe- Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-N 9 -tosyl-Arg, or Phe-N 9 -Nitro-Arg.
  • the peptide linker is Phe-Lys, Val-Lys, Vai-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Vai-Ala, or Ala-Ala.
  • L or the linker, is or comprises one or more of:
  • the linker L comprises one or more of:
  • the linker L comprises one or more of: [0210]
  • the foregoing linkers may bond to an amino acid side chain present on X, such as a lysine or
  • L or the linker, is -C(O)L 4 - or -C(O)Ci4>alkyleneC(O)L 4 -;
  • R 12 , R 13 , R 14 , R 15 , and R 19 are each independently hydrogen or Cmalkyl;
  • R 16 is hydrogen, Cwalkyl, -Ci-4alkylene-OH, -CMalkylene-OCwalkyl, -Ci-.alkylcnc-CXhH, or -Ci-4alkylene-CONH2;
  • G x is phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, Cwalkyl, Cwhaloalkyl, Cwalkoxy, cyano, and nitro.
  • L comprises a carbonyl moiety for conjugating the tetrazine moiety 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 L may comprise polypeptide lysine side chain
  • L, or the linker is or comprises one or more of: [0214] In some embodiments, L, or the linker, is or comprises one or more of:
  • L or the linker, is or comprises one or more of:
  • L or the linker, is or comprises one or more of:
  • L is:
  • L is:
  • L is or comprises one or more
  • L is or comprises one or more
  • L is or comprises one or more
  • L is or comprises one or more
  • L, or the linker is or comprises one or more L, or the linker, is or comprises one or more
  • L is or comprises one or more
  • L is or comprises one or more
  • the linker L is, or comprises one or more
  • a tetrazine-based targeting agent of Formula I wherein: p is 1-16;
  • R 22 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;
  • R" at each occurrence is independently selected from aryl and alkyl
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;
  • L at each occurrence, is independently a linker selected from the group consisting of:
  • tetrazine-based targeting agent of Formula II wherein:
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;
  • p is 1-16;
  • R 1 , R 31a and R 31b are each independently hydrogen, Ci-Cs-alkyl, or Ci-Ce-haloalkyl;
  • R' and R" at each occurrence, are independently selected from hydrogen, aryl, and alkyl;
  • R' at each occurrence, is independently selected from aryl and alkyl; t is independently is 0, 1, 2, 3, or 4;
  • L at each occurrence, is independently a linker selected from the group consisting of:
  • a tetrazine-based targeting agent of Formula V wherein: ring A is aryl, 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; provided that when ring A is aryl, then R 3 and R 4 are both present;
  • X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CA1X) targeting agent; p is 1-16;
  • 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; t, at each occurrence, is independently 0, 1, 2, 3, or 4;
  • L at each occurrence, is independently a linker selected from the group consisting of:
  • ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl; wherein each may be optionally substituted.
  • p is 1-5, or 1-4, or 1-3, or 1-2.
  • p is dependent on the size and/or number of available binding sites on X for forming a covalent bond to L.
  • the tetrazine-based targeting agent is of Formula: wherein n is 1-10.
  • the tetrazine -based targeting agent is as shown in Table 2.
  • Table 2
  • the support composition comprises a support.
  • the support composition is a therapeutic support composition.
  • 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., a TCO-containing group), as well as payloads after the binding agents conjugate.
  • Representative supports include, but are not limited to polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and the like. Supports also include particles, such as nanoparticles, microparticles, and the like.
  • 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).
  • 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.
  • 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.
  • 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.
  • the particles can be nanoparticles or microparticles.
  • Nanoparticles include particles having average dimensions in the nanometer scale (e.g., 1000 nm or less).
  • Microparticles are particles having average dimensions in the micrometer scale (e.g., 1000 pm or less). By “average” is meant the arithmetic mean.
  • the nanoparticles have a diameter ranging from 1 nm to 1 pm, such as from 10 nm to 1 pm, or 25 nm to 1 pm, or 50 nm to 1 pm, or 75 nm to 1 pm, or 100 nm to 1 pm, or 150 nm to 1 pm, or 200 nm to 1 pm, or 250 nm to 1 pm, or 300 nm to 1 pm, or 350 nm to 1 pm, or 400 nm to 1 pm, or 450 nm to 1 pm, or 500 nm to 1 pm.
  • the microparticles have a diameter ranging from 1 pm to 1 mm, such as from 10 pm to 1 mm, or 25 pm to 1 mm, or 50 pm to 1 mm, or 75 pm to 1 mm, or 100 pm to 1 mm, or 150 pm to 1 mm, or 200 pm to 1 mm, or 250 pm to 1 mm, or 300 pm to 1 mm, or 350 pm to 1 mm, or 400 pm to 1 mm, or 450 pm to 1 mm, or 500 pm to 1 mm.
  • small particles on the order of 10-100 nm in diameter may be assembled to form larger complexes, such as clusters or assemblies on the order of 1-10 pm.
  • Particles of the present disclosure may be substantially spherical, such that the particles have a substantially circular cross-section.
  • Other particle shapes may also be used, such as, but not limited to, ellipsoid, cubic, cylindrical, conical, needle, or other irregular shapes.
  • 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(alkylcnc oxides), poly(allylamincs)(PAM), poly(acrylatcs), 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.
  • the particles, or a group of several particles in a complex may be functionalized with a tetrazine -based 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 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 tctrazinc-bascd targeting agent may be attached directly to the particle itself.
  • the tetrazine-based 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, or other cellular receptors or cell surface targets.
  • Other compounds or molecules such as fluorophores or autofluorescent or luminescent markers, which may assist in detecting the particles (e.g., in vivo detection), may also be attached to 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.
  • 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 facilitates the formation of new bone through active recruitment of mesenchymal stem cells from the surrounding tissue.
  • growth factors such as bone morphogenetic proteins, may be included in the bone graft substitute
  • 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.
  • the support compositions comprise substituted alginate having units of formula: wherein the dashed line represents a bond to L.
  • the support compositions comprise substituted hyaluronic acid having units of formula: wherein the dashed line represents a bond to L.
  • 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 L of the tetrazine-containing group.
  • Trans-cyclooctene functionalized prodrugs are known in the art, including prodrugs of anticancer agents, as described in WO2018/187740, WO2014/205126, WO2015/139025, and WO2017/044983, which are incorporated herein by reference. Further embodiments using trans-cyclooctene functionalized prodrugs follow.
  • the trans-cyclooctene functionalized prodrugs is a conjugate comprised of a payload linked to one or more trans-cyclooctene moieties.
  • the conjugate (or trans-cyclooctene functionalized prodrug) comprises an immunomodulatory agent payload, such as for example, an immunomodulatory agent payload selected from the group consisting of a cytokine, chemokine, chemokine antagonist, therapeutic monoclonal antibody, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.
  • an immunomodulatory agent payload selected from the group consisting of a cytokine, chemokine, chemokine antagonist, therapeutic monoclonal antibody, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.
  • the immunomodulatory agent payload is an inhibitor of a cytokine payload, or a pharmaceutically acceptable salt thereof.
  • the inhibitor of a cytokine payload is an inhibitor of TNF-a, infliximab, certolizumab, TGF-0, galunisertib, fresolimumab, M7824, CSF-1, pexidartinib, or cabiralizumab.
  • the conjugate comprises a monoclonal antibody, or a pharmaceutically acceptable salt thereof.
  • the conjugate comprises a therapeutic protein payload, or a pharmaceutically acceptable salt thereof.
  • the therapeutic protein payload is an antibody -based drug, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin, or thrombolytic.
  • the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen.
  • the therapeutic protein payload is a payload of erythropoietin (EPO, e.g., native EPO or synthetic EPO (see, e.g., US 2003/0191291), such as, but not limited to, e.g., PROCRIT®, EPREX®, or EPOGEN® (epoetin-a), ARANESP® (darbepoietin-a), NEORECORMON®, EPOGIN® (epoetin-P), and the like); a growth hormone (e.g., a somatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®, SEROSTIM®, HUMATROPE®, etc.); theraputic monoclonal antibody (e.g Atezolizumab, Avelumab, Bevacizumab, Cemiplimab, Cetuximab
  • EPO erythrop
  • the conjugate is of Formula X, or a pharmaceutically acceptable salt thereof: wherein m is an integer from 1-150;
  • G at each occurrence, is independently an optionally substituted trans-cyclooctene moiety
  • D is a payload
  • L 1 at each occurrence, is independently a linker.
  • each trans-cyclooctene moiety is independently: wherein:
  • R IA at each occurrence, is independently selected from the group consisting of Cwalkyl, Ci- dialoalkyl, and Ci.4alkoxy; q is 0, 1, or 2; ql is 0 or 1;
  • R IB is independently selected from the group consisting of G 1 , -OH, -NR 1L -C walkylene-G 1 , -NR 1C -C i. 4 alkylene-N(R ld ) 2 , -NR 1C -C ⁇ alkylene-NIC i _ 4 alky 1) 3 + , -N(R 1C )CHR 1C CO 2 H, -N(R 1C )-Ci.
  • R lc and R ld are independently hydrogen or Ci ⁇ alkyl
  • R le at each occurrence, is independently -Ci-4alkylene-CO 2 H, -Ci-4alkylene-CONH 2 , or -C i-4alkylene-OH ;
  • R lf at each occurrence, is independently hydrogen, Ci-ealkyl, or Ci-4alkylene-CO 2 H; n, at each occurrence, is independently 0, 1, 2, or 3;
  • L 2 at each occurrence, is independently selected from the group consisting of -C(O)- and Ci. 3 alkylene;
  • G 1 is independently an optionally substituted heterocyclyl.
  • the conjugate is of Formula X, or a pharmaceutically acceptable salt thereof, wherein
  • G is the trans-cyclooctene moiety, and G, at each occurrence, is independently
  • L 1 at each occurrence, is independently a linker; m is an integer from 1-150;
  • D is a payload
  • R IA at each occurrence, is independently selected from the group consisting of Ci ⁇ alkyl, Ci-Jialoalkyl, and Ci-4alkoxy; q is 0, 1 or 2; ql is 0 or 1;
  • R IB is independently selected from the group consisting of G 1 , OH, -NR lc -Ci. 4 alkylene-G 1 , -NR lc -Ci- 4 alkylene-N(R ld ) 2 , -NR lc -Ci- 6 alkylene-N(Ci-4alkyl)j + , -N(R lc )CHR le CO 2 H, -N(R 1C )-Ci. 6 alkylene-CO 2 H, -N(R lc )CHR le C(O)OCi.
  • R lc and R ld are independently hydrogen or Ci- 4 alkyl
  • R le at each occurrence, is independently -Ci- 4 alkylene-CO 2 H, -Ci- 4 alkylene-CONH 2 , or -C i . 4 alkylene-OH ;
  • R lf at each occurrence, is independently hydrogen, Ci-ealkyl, or Ci. 4 alkylene-CO 2 H; n, at each occurrence, is independently 0, 1, 2, or 3;
  • L 2 at each occurrence, is independently selected from the group consisting of -C(O)- and Ci. 3 alkylene;
  • G 1 is independently an optionally substituted heterocyclyl.
  • ql is 1.
  • the payload is an immunomodulatory agent payload.
  • the payload is a therapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.
  • the payload is selected from a therapeutic agent for treating cancer (e.g., doxorubicin, daunorubicin, PNU-159682, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, baccatin III, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, exatecan, auristatin (dolastatin 10, MMAE, MMAD, MMAF), duocarmycin, pyrrolobenzodiazapene dimer, mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., Amphotericin, and the like), an antibiotic (e.g., van
  • the payload is selected from a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, exatecan, auristatin (dolastatin 10, MMAE, MMAD, MMAF) mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, and the like), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., Amphotericin, and the like), an antibiotic (e.g., vancomycin, daptomycin, doxycycline, ceftriax
  • Reference to a payload means that one or more atoms, including hydrogen or non-hydrogen atoms, of the original, unmodified payload is replaced by a covalent bond to one or more linker.
  • the payloads are derived from the known nuclear payload and are modified to be covalently bonded to at least one optionally substituted trans-cyclooctene via a linker. The payloads, even after modification to arrive at the compounds described herein, maintain biological activity which is comparable to that observed in the original, unmodified payload.
  • the payloads exhibit a binding activity or inhibition which is at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of that observed in the original, unmodified payload.
  • a hydrogen atom bound to a heteroatom (e.g., N, O, or S) of the original, unmodified payload is replaced by a covalent bond to a linker.
  • a halogen atom on a payload is replaced for attachment to the remainder of the compound.
  • a hydrogen atom on a payload is replaced for attachment to the remainder of the compound.
  • the hydrogen atom is on a heteroatom.
  • the hydrogen atom is on a nitrogen.
  • the hydrogen atom is on an oxygen.
  • the hydrogen atom is on a carbon.
  • G at each occurrence, is independently
  • G at each occurrence, is independently
  • the payload is a monoclonal antibody payload.
  • a monoclonal antibody for use herein as a payload can be an entire monoclonal antibody, or a fragment thereof (c.g., antigenbinding fragment (Fab)).
  • the antibody is an immune cell engager, and as such would induce or elicit an immune response.
  • the monoclonal antibody, or fragment thereof targets one or more of CD3 (NCBI Gene ID 916), CD28 (NCBI Gene ID 940), CD137 (4-1BB) (NCBI Gene ID 3604), CD16 (NCBI Gene ID 2214), NKG2D (NCBI Gene ID 22914), CD64 (NCBI Gene ID 2209), GITR/TNFRSF18 (NCBI Gene ID 8487), CD25 (NCBI Gene ID 3559), CD40 (NCBI Gene ID 958), CD4 (NCBI Gene ID 920), CXCR4 (NCBI Gene ID 7852), G-CSFR (NCBI Gene ID 1441), GM-CSFR (NCBI Gene ID 1438), CD122 (NCBI Gene ID 3560), PD1 (NCBI Gene ID 5133), CTLA4 (NCBI Gene ID 1493), LAG3 (NCBI Gene ID 3902), TIGIT (NCBI Gene ID 201633), NCR1 (NCBI Gene ID 9437), TIM3 (NCBI Gene ID 84868), VISTA (
  • the payload is an antibody or antibody fragment which targets CD3, such as OKT3, SP34, UCHT1, Teplizumab, Otelixizumab, Visilizumab, or Foralumab, or an antibody fragment derived therefrom.
  • CD3 such as OKT3, SP34, UCHT1, Teplizumab, Otelixizumab, Visilizumab, or Foralumab, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD28, such as Thcralizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD 137 (4-1BB), such as Utomilumab, Urelumab, LVGN6051, or AGEN2373, or an antibody fragment derived therefrom.
  • CD 137 4-1BB
  • Utomilumab, Urelumab, LVGN6051, or AGEN2373 an antibody fragment derived therefrom.
  • the pay load is an antibody or antibody fragment which targets CD 16, such as AFM13, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets NKG2D, such as NNC0152-0002 orJNJ-64304500, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD64, such as H22, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets GITR/TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD25, such as Daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD40, such as Iscalimab, ABBV-323, bleselumab (ASKP-1240), BI-655064, FFP-104, BMS986090, Dacetuzumab, or Lucatumumab, or an antibody fragment derived therefrom.
  • CD40 such as Iscalimab, ABBV-323, bleselumab (ASKP-1240), BI-655064, FFP-104, BMS986090, Dacetuzumab, or Lucatumumab, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD4, such as MAX.16H5, IT1208, Zanolimumab (HuMax-CD4), UB-421, or MTRX1011A, or an antibody fragment derived therefrom.
  • CD4 such as MAX.16H5, IT1208, Zanolimumab (HuMax-CD4), UB-421, or MTRX1011A, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CXCR4, such as F50067, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets G-CSFR, such as CSL324, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets GM- CSFR, such as Methosimumab, or an antibody fragment derived therefrom.
  • the pay load is an antibody or antibody fragment which targets CD 122, such as Hu-Mik(beta)!, or an antibody fragment derived therefrom.
  • the pay load is an antibody or antibody fragment which targets PD-1, such as CC-90006, Ccmiplimab, Camrelizumab, or TSR-042, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CTLA4, such as Tremelimumab or ipilimumab, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets LAG3, such as Relatlimab (BMS-986016), GSK2831781, Cemiplimab (REGN3767), Favezelimab, leramilimab, or Mavezelimab, or an antibody fragment derived therefrom.
  • LAG3 Relatlimab
  • GSK2831781 GSK2831781
  • Cemiplimab REGN3767
  • Favezelimab Favezelimab
  • leramilimab or Mavezelimab
  • an antibody fragment derived therefrom such as Relatlimab (BMS-986016), GSK2831781, Cemiplimab (REGN3767), Favezelimab, leramilimab, or Mavezelimab, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets TIGIT, such as BMS-986207, Tiragolumab, Vibostolimab, Etigilimab, Domvanalimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.
  • TIGIT such as BMS-986207, Tiragolumab, Vibostolimab, Etigilimab, Domvanalimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.
  • NCR1 such as hNKp46.02
  • the payload is an antibody or antibody fragment which targets TIM3, such as Cobolimab, Sym023, LY3321367, BMS-986258, SHR-1702, Sabatolimab, or INCAGN02390, or an antibody fragment derived therefrom.
  • TIM3 such as Cobolimab, Sym023, LY3321367, BMS-986258, SHR-1702, Sabatolimab, or INCAGN02390, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets VISTA, such as SG7, K01401-020, CI-8993, or JNJ-61610588, or an antibody fragment derived therefrom.
  • the pay load is an antibody or antibody fragment which targets CD 134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD27, such as Varlilumab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets CD40L, such as Dapirolizumab, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets ICOS, such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.
  • ICOS such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.
  • the payload is an antibody or antibody fragment which targets BAFFR, such as lanalumab, or an antibody fragment derived therefrom.
  • the pay load is an antibody or antibody fragment which targets LFA-1, such as Efalizumab, or an antibody fragment therefrom.
  • the payload is an antibody or antibody fragment which targets BTLA, such as Icatolimab, or an antibody fragment derived therefrom.
  • the payload is an anti-CD3 (aCD3) monoclonal antibody, or a derivative, or analog thereof.
  • the anti-CD3 (aCD3) monoclonal antibody is SP34, UCHT1, or OKT3, or a derivative, or analog thereof.
  • At least one payload is selected from an inhibitor of poly (ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, an anti- CD3 (aCD3) monoclonal antibody, lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, Paclitaxel, Gemcitabine, Mitomycin C, Etoposide, exatecan, and MMAE, or a derivative, or analog thereof.
  • PARP poly (ADP-ribose) polymerase
  • PPD pyrrolobenzodiazepine
  • HTI-286 hemiasterlin
  • HTI-286 an anti- CD3 (aCD3) monoclonal antibody
  • lurbinectedin MSA-2, gardiquimod, ciprofloxacin
  • Paclitaxel Gemcitabine
  • Mitomycin C Etoposide
  • exatecan exatecan
  • D is a payload selected from an inhibitor of poly (ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, and an anti- CD3 (aCD3) monoclonal antibody, or a derivative, or analog thereof.
  • PARP poly (ADP-ribose) polymerase
  • PPD pyrrolobenzodiazepine
  • hemiasterlin hemiasterlin
  • HTI-286 hemiasterlin
  • aCD3 anti- CD3
  • At least one payload is selected from lurbinectedin, MSA- 2, gardiquimod, ciprofloxacin, Paclitaxel, Gemcitabine, Mitomycin C, Etoposide, exatecan, Seco-Duocarmycin SA, and MMAE, or a derivative, or analog thereof.
  • a payload is an inhibitor of poly (ADP-ribose) polymerase (PARP), or a derivative, or analog thereof.
  • the inhibitor of poly (ADP-ribose) polymerase is niraparib, talazoparib, olaparib, pamiparib, rucaparib, veliparib, iniparib, 3- aminobenzamide, CEP-9722, E7016, or a derivative, or analog thereof.
  • a payload is:
  • a payload is a duocarmycin, or a derivative, or analog thereof.
  • the duocarmycin is Duocarmycin A, Duocarmycin Bl, Duocarmycin B2, Duocarmycin Cl, Duocarmycin C2, Duocarmycin D, Duocarmycin SA, CC-1065, adozelesin, carzelesin, bizelesin, or a derivative, or analog thereof.
  • a pay load comprises:
  • a payload comprises a pyrrolobenzodiazepine (PBD), or a derivative, or analog thereof.
  • the pyrrolobenzodiazepine (PBD) is [l,2]diazepino[3,4-e]indole, or a derivative, or analog thereof.
  • a payload comprises:
  • a payload is an inhibitor of tubulin polymerization.
  • a payload is hemiasterlin, HTI-286, or a derivative, or analog thereof.
  • a payload comprises:
  • a payload comprises: [0314] In some embodiments, the payload is a topoisomerase inhibitor. In some embodiments, the payload comprises camptothecin, or a derivative, or analog thereof. In some embodiments, the payload comprises topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, or rubitecan.
  • the pay load comprises:
  • the payload comprises:
  • the payload comprises:
  • the pay load comprises:
  • the pay load comprises:
  • the pay load comprises:
  • the payload comprises a polypeptide.
  • the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.
  • the linker L 1 is covalently bonded to a lysine, serine, threonine, or tyrosine residue present on the payload.
  • the polypeptide comprises one or more lysine residues.
  • the linker L 1 is covalently bonded to a lysine residue present on the payload.
  • the payload comprises an N-terminal amino acid, wherein the linker L 1 is covalently bonded to a N-terminal amino acid.
  • n is 1-20. In some embodiments, m is 1-10. In some embodiments, m is 1-5.
  • the payload is an immunomodulatory agent payload.
  • the immunomodulatory agent payload is an antibody payload.
  • the immunomodulatory agent payload is the immune checkpoint inhibitor payload.
  • the immune checkpoint inhibitor payload is a payload of pidilizumab, sintilimab, AMP-224, atezolizumab, durvalumab, BMS-936559, tremelimumab, indoximod, epacadostat, a TIGIT inhibitor (e.g., LAG-3, such as an anti-LAG-3 antibody; TIM-3, such as an anti-TIM-3 antibody), a B7 molecule, or a BTLA pathway antagonist.
  • LAG-3 such as an anti-LAG-3 antibody
  • TIM-3 such as an anti-TIM-3 antibody
  • B7 molecule e.g., BTLA pathway antagonist.
  • the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload.
  • the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload.
  • the PD-1 inhibitor payload is a nivolumab, pembrolizumab, pidilizumab, sintilimab, or AMP-224 payload.
  • the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload.
  • the PD-L1 inhibitor payload is an atezolizumab, avelumab, durvalumab, or BMS-936559 payload.
  • the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload.
  • the CTLA4 inhibitor payload is an ipilimumab or tremelimumab payload.
  • the immune checkpoint inhibitor payload is an indoleamine 2,3- dioxygenase (IDO) inhibitor payload.
  • IDO indoleamine 2,3- dioxygenase
  • the IDO inhibitor payload is an indoximod or epacadostat payload.
  • the immunomodulatory agent payload is a cytokine payload.
  • the cytokine payload is an interferon, interleukin, tumor necrosis factor, erythropoietin, MIP3a, ICAM, macrophage colony stimulating factor, Erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), or granulocyte-macrophage colony stimulating factor payload.
  • the interleukin pay load is chosen from IL-1 to IL-40. In some embodiments, the interleukin payload is IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.
  • the immunomodulatory agent payload is a type 1 cytokine (IL-2, IL- 12, TNF-B, IFN-g).
  • the cytokine payload is selected from the group consisting of IFN-alpha, IFN-beta, IFN-gamma, pegylated IFN-a, and apolipoprotein A-I fusion protein with IFN-a, interleukin, IL-2, IL-2 covalently bound to immunoglobulins (e.g., cergutuzumab amunaleukin, RO6874281), IL-2 covalently bound to PEG molecules (e.g., NKTR-214), IL-10, PEGylated IL-10 (e.g., pegilodecakin), IL- 7, IL- 12, IL-15, recombinant aglycosylated IL- 15, fusion protein of IL- 15 with the binding domain of IL- 15Ra (e.g., RLI), triple fusion protein comprising human IL-15, the binding domain of IL-15Ra and apolipoprotein A-
  • immunoglobulins
  • the immunomodulatory agent payload is the chemokine payload.
  • the chemokine payload is a CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CXCL14 payload.
  • the immunomodulatory agent payload is the chemokine antagonist payload. In some embodiments, the chemokine antagonist payload is a plerixafor payload. [0340] In some embodiments, the immunomodulatory agent is a monoclonal antibody specific to a cytokine or a cytokine receptor.
  • the immunomodulatory agent payload comprises a polypeptide.
  • the polypeptide comprises one or more lysine residues.
  • the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.
  • the trans-cyclooctene is linked to one of the one or more lysine residues.
  • the trans-cyclooctene is independently linked to one or more lysine, serine, threonine, or tyrosine residues.
  • the polypeptide comprises an N-terminal amino acid, wherein an occurrence of the bioorthogonal moiety is linked to the N-terminal amino acid.
  • m is 1-20. In some embodiments, m is 1-10. In some embodiments, m is 1-5. In some embodiments, m is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, m is 1.
  • trans-cyclooctene functionalized prodrug of Formula XI or a pharmaceutically acceptable salt thereof, wherein:
  • R la at each occurrence, is independently selected from the group consisting of hydrogen, Ci- 4alkyl, and Ci-ihaloalkyl;
  • R lb at each occurrence, is independently selected from the group consisting of hydrogen, Ci- 4 alkyl, CiJialoalkyl, C(O)OH, C(O)OC M alkyl, C(O)N(R lc )CHR le CO 2 H, C(O)N(R lc )CHR le C(O)OCi. 4 alkyl, C(O)N(R 1C )-Ci.6alkylene-CO 2 H, and C(O)N(R 1C )-Ci. 6 alky lene-C(O)OC w alkyl;
  • R lc at each occurrence, is independently hydrogen or C i al ky I :
  • R le at each occurrence, is independently -Ci. 4 alkylene-CO 2 H, -Ci. 4 alkylene-CONH 2 , or -C i . 4 alkylene-OH ;
  • D at each occurrence, is independently a payload
  • L 1 at each occurrence, is independently a linker; p', at each occurrence, is independently 0, 1, or 2; and p'', at each occurrence, is independently 1, 2, or 3.
  • D at each occurrence, is independently selected from the group consisting of an anticancer agent payload, a toll-like receptor (TLR) agonist payload and a stimulator of interferon genes (STING) agonist payload.
  • R 1a is hydrogen.
  • R 1a is C1-4alkyl.
  • R 1a is CH3.
  • R 1b is selected from the group consisting of C(O)OH, C(O)OC1-4alkyl, C(O)N(R 1c )CHR 1e CO2H, C(O)N(R 1c )CHR 1e C(O)OC1-4alkyl, C(O)N(R 1c )–C1-6alkylene–CO2H, and C(O)N(R 1c )–C 1-6 alkylene–C(O)OC 1-4 alkyl.
  • R 1b is selected from the group consisting of C(O)OH, C(O)N(R 1c )CHR 1e CO2H, and C(O)N(R 1c )CH2CO2H.
  • R 1b is selected from the group consisting of –NR 1c –CH 2 CH 2 –N(CH 3 ) 3 + , –N(R 1c )–CH 2 CH 2 –SO 3 H, –N(R 1c )–(CH 2 CH 2 O) 3 –CH 2 CH 2 N((CH 2 CH 2 O) 3 –CH 2 CH 2 –CO 2 H) 2 , and – N(R 1c )–CH(CH2O–CH2CH2–CO2H)2.
  • the trans-cyclooctene moiety (G) is: OH
  • the trans-cyclooctene moiety is N-cyclooctene
  • the trans-cyclooctene moiety is the trans-cyclooctene moiety
  • the trans-cyclooctene moiety is N-cyclooctene
  • the trans-cyclooctene moiety is the trans-cyclooctene moiety
  • the trans-cyclooctene moiety is the trans-cyclooctene moiety
  • the trans-cyclooctene moiety is the trans-cyclooctene moiety
  • the trans-cyclooctene moiety is N-cyclooctene
  • the trans-cyclooctene moiety is [0366] In some embodiments, the trans-cyclooctene moiety
  • R 2 may be optionally further substituted with a polyether.
  • the trans-cyclooctene moiety comprises
  • the trans-cyclooctene moiety comprises
  • the trans-cyclooctene moiety of comprises
  • the trans-cyclooctene moiety of comprises
  • R le is -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH 3 )OH.
  • R le is -Chalky lene-CO 2 H.
  • R le is -CH2CO2H.
  • R lb is -C(O)N(R lc )-Ci-6alkylene-CO2H.
  • R lb is -C(O)N(R 1C )CH 2 CO 2 H.
  • R lc is hydrogen
  • R lb is hydrogen.
  • R 1b is C(O)OH.
  • linker L 1 may have 1 to 100 linking atoms, and may 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, 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.
  • linker L 1 may comprise one or more (e.g., 1-10 or 1-5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1-10 or 1-5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moieties; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, C 1-4 alkyl, C 1-4 alkoxy, and C 1-4 haloalkyl.
  • chain heteroatoms e.g., O, N, S
  • alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moieties wherein each alkylene, alkenylene,
  • 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.
  • Representative linkers include, but are not limited to, those shown below: [0385] Representative link n below: [0386] In some embodim , yethylene 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. In some embodiments, linker L 1 may comprise . [0387] In some embodiments, linker L 1 may comprise one or more natural or unnatural amino acids, which may be referred to as a peptide linker.
  • linker L 1 may comprise one or more natural or unnatural amino acids, which may be referred to as a peptide linker.
  • linker may be bound thereto using a peptide linker made up of a carboxylic acyl unit, and one or more amino acids making up a protein or peptide sequence.
  • linker L 1 may also contain a self- immolating spacer which spaces the drug and the protein peptide sequence.
  • linker L 1 may be a peptide linker represented by “A—Y—Z—Q—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 “Q” and “W” are optional additional linkers having from 1 to 50 linking atoms, or from 5 to 10 linking atoms, or from 1 to 10 linking atoms which spaces the peptide and the drug, D, or the bioorthogonal moiety.
  • 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)-.
  • Q is -OC(O)-.
  • W is -OC(O)-.
  • Q is absent and W is -OC(O)-.
  • 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: ], Ala-Leu-Ala-Leu [SEQ ID NO: ], 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.
  • the peptide linker is Val-Cit, Val-Ala, or Ala-Ala. , h , ).
  • the payload may be an amine-containing payload for attachment of the payload to a carbonyl group of the linker, or, in other cases, the payload may be a carboxyl-containing payload for attachment of the payload to an amine group of the linker.
  • the payload and linker together form a carbamate group; e.g., the payload may be an amine-containing payload for attachment of the payload to an acyloxy group of the linker.
  • the payload and linker together form a carbonate group; e.g., the payload may be a hydroxy-containing payload for attachment of the payload to an acyloxy group of the linker.
  • L 3a is a bond or C1-6al L 4a is a bond, —NHN:, –N(R 10 )–C 2-6 alkylene–N(R 11 )–, –N(R 12 )–C 2-3 alkylene–N(R 13 )C(O)–, –N(R 10 )–C 1-6 alkylene–C(O)NHN: —NHNHC(O)C 1-6 alkylene–C(O)NHN: , R , R , R , R , R , R , and R 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 –C 1-4 alkylene–CONH 2 ; R 17 , at each occurrence, is independently hydrogen or
  • linker L 1 is -OC(O)-. O O 1 L 3a L 4a
  • L is ; L 3a is a bond; and [0406]
  • p is 1.
  • p’ is 1. , , , at eac occurrence, s n epenenty y rogen or – 2 ( )N ; R D is hydrogen or C 1-4 alkyl on a nitrogen atom of a payload; and D and D’ are independently a payload moiety.
  • D or D’ is a cyclic dinucleotide payload moiety, imidazo[4,5-c]quinolin- 4-amine payload moiety, TLR agonist payload moiety, STING agonist payload moiety, or anticancer agent payload moiety. or D and D’ are independently a payload moiety (e.g., anticancer agent payload moiety).
  • p’ is 0.
  • p is 2 or 3.
  • [0412] In some embodiments .
  • a payload (D or D’) may be an anticancer agent payload of any of the anticancer agents described herein.
  • the payload comprises a TLR7/8 agonist.
  • the payload comprises gardiquimod.
  • the payload comprises camptothecin, or derivative thereof.
  • the payload comprises exatecan.
  • the payload comprises MMAE, or derivative thereof.
  • the payload comprises paclitaxel, or derivative thereof.
  • the payload comprises docetaxel, or derivative thereof.
  • the payload has the structure: , , , ,
  • the method includes selectively delivering a payload to the target location in a subject.
  • Selective delivery of the payload includes delivering the payload to the target location (e.g., an organ or tissue, or portion thereof), without targeting other locations in the subject (e.g., other organs or tissues, or portions thereof) that do not need administration of the payload.
  • Selective delivery of the payload may be achieved through use of the tetrazine-based targeting agent and the functionalized payloads described herein.
  • a tetrazine-based targeting agent 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-based targeting agent as described herein.
  • the tetrazine-based targeting agent may be administered to the subject at a desired target location in the subject.
  • the tetrazine-based targeting agent may be injected locally into the subject at the desired target location in the subject.
  • the tetrazine-based targeting agent is administered systemically.
  • the tetrazine-based targeting agent may localize at a desired target location in the subject through specific binding of the tetrazine-based targeting agent 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-based targeting agent to its target (e.g., antibody- antigen interaction, and the like).
  • target e.g., antibody-antigen interaction, and the like
  • a desired target e.g., a cell surface
  • selective binding between bioorthogonal binding partners e.g., between a tetrazine of the tetrazine-based targeting agent and its complementary trans-cyclooctene of a prodrug may occur.
  • a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a tetrazine-based targeting agent as described herein, or a pharmaceutically acceptable salt thereof, and a trans-cyclooctene prodrug.
  • 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
  • compositions of the present disclosure find use in treatment and/or diagnosis of a condition or disease in a subject that is amenable to treatment or diagnosis by administration of the payload (e.g., the parent drug (i.e., the drug prior to conjugation to the composition)).
  • 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.
  • 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.
  • 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
  • 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-based targeting agent 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 -based targeting agent 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 -based targeting agent 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.
  • the practitioner can deliver through to the biopsy site immunomodulatory agents.
  • the disclosed methods provide the ability for a practitioner to deliver immunomodulatory agents, such as TLR agonists, STING agonists, chemokines (agents that attract cancerous cells and/or immune cells) and adjuvants to enhance the immune system with fewer side effects as well as the chemotherapeutics agents combined with immunotherapy agents.
  • immunomodulatory agents such as TLR agonists, STING agonists, chemokines (agents that attract cancerous cells and/or immune cells) and adjuvants to enhance the immune system with fewer side effects as well as the chemotherapeutics agents combined with immunotherapy agents.
  • This combination approach would be beneficial to patients.
  • the chemotherapy agent would treat the solid tumor or specific location, while the enhanced response of the immunotherapy would help with distant metastatic sites.
  • compositions and methods could employ or be used with anthracyclines, taxanes, gemcitabine and other agents to enhance the efficacy of one or more immunomodulatory agents such as ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).
  • anthracyclines such as ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).
  • 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.
  • 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
  • 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 lymph
  • ICD immunogenic cell death
  • Calreticulin one of the DAMP molecules, which is normally in the lumen of endoplasmic reticulum (ER), is translocated after the induction of immunogenic apoptosis to the surface of dying cell where it functions as an "eat me” signal for professional phagocytes.
  • Other important surface exposed DAMPs are heat-shock proteins (HSPs), namely HSP70 and HSP90, which are under stress condition also translocated to the plasma membrane.
  • HMGB1 antigen-presenting cell
  • TLR Toll-like receptor
  • 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.
  • melanoma e.g. , unresectable, metastatic melanoma
  • renal cancer e.g., renal cell carcinoma
  • prostate cancer e.g., metastatic
  • the disclosed approach lends itself well as an adjuvant / neoadjuvant system.
  • targeting moieties as disclosed herein could be placed during the biopsy, once the results from the study come back, the practitioner could administer the appropriate cocktail to deliver treatment to the desired site in the body (compounds as disclosed herein and optional additional therapeutic agent(s)).
  • the results of 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 could be delivered and combined with immunotherapy agents.
  • the disclosed methods may include one or multiple systemic doses of targeting moieties that focus at one location or more locations.
  • the disclosed methods may be used to deliver a functionalized payload to these location through systemic or local administration.
  • the tetrazinebased targeting agent is delivered systemically.
  • the tetrazine-based targeting agent and the payload i.e., a TCO-labeled payload
  • the payload i.e., a TCO-labeled payload
  • the tetrazine-based targeting agent 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 tetrazinebased 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 functionalized payload that focus at one location.
  • the disclosed methods may be used to deliver a second payload.
  • the disclosed methods may be used to administer a second functionalized payload if the tumor is resistant to the first payload.
  • a second payload may be a TCO-labeled payload of gemcitabine or docetaxel.
  • the TCO-labeled payload of gemcitabine, paclitaxel, or docetaxel may be administered in combination with doxorubicin.
  • the second functionalized payload may be activated by the tetrazine-based targeting agent used for the first prodrug.
  • the functionalized payloads disclosed herein may function as adjuvants. This combination approach would be beneficial to patients.
  • the chemotherapy agent would treat the solid tumor or specific location and may enhance or elicit an immune response, while the enhanced response of the immunotherapy of the functionalized payload and/or separate agent may help with distant metastatic sites.
  • the disclosed compositions and methods could employ or be used with anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, camptothecin analogues and other agents to enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).
  • the disclosed methods may be used to treat diffuse intrinsic pontine gliomas.
  • Diffuse intrinsic pontine gliomas 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.
  • the disclosed methods may include multiple systemic doses of functionalized payload that focus at one location.
  • the disclosed methods may be used to deliver a second payload.
  • the disclosed methods may be used to administer a second functionalized payload if the tumor is resistant to the first payload.
  • a second payload may be a TCO-labeled payload of gemcitabine or docetaxel.
  • the TCO-labeled payload of gemcitabine or docetaxel may be administered in combination with doxorubicin.
  • the second functionalized payload may be activated by the tetrazine-based targeting agent used for the first prodrug.
  • 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.
  • parenterally refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • compositions 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.
  • 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.
  • kits 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).
  • 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.
  • compositions of the present disclosure can be co- administered with another active agent.
  • Co-administration includes administering the composition of the present disclosure and active agent within 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, or 24 hours of each other.
  • Coadministration also includes administering the composition of the present disclosure and active agent simultaneously or approximately simultaneously (e.g., within about 1 min, 5 min, 10 min, 15 min, 20 min, or 30 minutes of each other), or sequentially in any order.
  • 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.
  • composition of the present disclosure and the active agent can be present in a formulation in any suitable weight ratio, such as from 1: 100 to 100:1 (w/w), or 1 :50 to 50: 1, or 1:25 to 25: 1, or 1 : 10 to 10:1, or 1:5 to 5: 1 (w/w).
  • the composition of the present disclosure and the other active agent can be present in any suitable weight ratio, such as l :100 (w/w), 1 :75, 1 :50, 1:25, 1: 10, 1:5, 1 :4, 1:3, 1 :2, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 10: 1, 25: 1, 50: 1, 75: 1, or 100: 1 (w/w).
  • Other dosages and dosage ratios of the composition of the present disclosure and the active agent are suitable in the formulations and methods described herein.
  • 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 tetrazinebased 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.
  • a pharmaceutical combination comprising a tetrazinebased 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 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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-
  • 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.
  • 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.
  • 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, NJ., Wiley- Interscience, and references cited therein.
  • Compounds provided herein can be prepared by methods described in the Examples below or adapted from the literature (see, e.g., W02020/077140, WO2018/187740, WO2017/044983, WO2015/139025, and WO2014/205126).
  • Exemplary payloads can be prepared can be prepared according to methods adapted from the literature (see, e.g., WO2022/032191, WO2021/007160, W02020/077140, WO2018/187740, WO2017/044983, WO2015/139025, and WO2014/205126, which methods are incorporated herein in their entirety).
  • Targeting agent in PBS is mixed with of tetrazine-NHS (e.g., 20 equivalents).
  • the reaction is mixed thoroughly and aged at room temperature for 1 hour, at which time the reaction is quenched by the addition of 1 volume of 0.1 M Tris buffer.
  • the resulting solution is buffer exchanged to 0.01 M PBS to remove excess reagent and buffer salts.
  • the resulting solution of targeting agent is analyzed by SDS- Page and LCMS confirming the formation of the tetrazine-based targeting agent.
  • the peptide was synthesized using standard Fmoc chemistry.
  • cleavage buffer (5% DTT I 2.5% TIS / 2.5% H2O / 90% TFA) to the flask containing the side chain protected peptide at room temperature and stir for 1 hr.
  • the peptide was synthesized using standard Fmoc chemistry.
  • cleavage buffer (90% TFA /2.5% TIS 72.5% H 2 O 75.0% DTT) to the flask containing the side chain protected peptide at room temperature and stir for 2 hrs.
  • the peptide is precipitated with cold isopropyl ether and centrifuged (3 mins at 3000 rpms).
  • TFA condition The reaction mixture was dissolved in DMF, purified by prep-HPLC (Gilson GX-281; Gemini® 250*30 mm, C18, 5 um, 110A; Eluent of 28%-48% of solution (A: 0.1% TFA in H2O, B: MeCN) for 40 mins at room temperature; observed under wavelength (220/254 nm), flow rate was 20 mL/Min, retention time was 23 mins).
  • HC1 condition The TFA salt was dissolved in ACN/H2O, eluent of 10% of solution (A: 0.5 % NH4CI in H2O, B: MeCN) for 20 mins at room temperature, then eluent of 10% of solution (A: 0.05 % HC1 in H2O, B: MeCN) for 10 mins at room temperature, followed by prep-HPLC (AUNO LC2000; Gemini-C18, 150*30 mm, 5 um, 110A; Eluent of 20%-40% of solution (A: 0.05 % HC1 in H 2 O, B: MeCN) for 40 mins at room temperature; observed under wavelength (220/254 nm), flow rate was 20 mL/Min, retention time was 27 mins).
  • Peptide Synthesis The peptide was synthesized using standard Fmoc chemistry.
  • Resin preparation Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol/g) and swell for 2 hrs.
  • Deprotection 20% piperidine in DMF (30.0 mL) was added and agitated the resin with Ni at 22 °C for 30 mins. The resin was washed with DMF (30.0 mL x 5) and filtered to get the resin.
  • Resin preparation Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol/g) and swell for 2 hrs.
  • reaction mixture was purified by prep-HPLC (TFA condition) to give Target 254 (43.0 mg, 12.8 pmol, 23.6% yield, 96.6% purity, HOAC) as a pink solid which was confirmed via LCMS and HPLC.
  • Resin preparation Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol/g) and swell for 2 hrs. 2) Deprotection: 20% piperidine in DMF (30.0 mL) was added and agitated the resin with Ni at 22 °C for 30 mins. The resin was washed with DMF (30.0 mL x 5) and filtered to get the resin.
  • Resin preparation Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol/g) and swell for 2 hrs.
  • mice Female Balb/c nude mice are implanted with HCC1954 grown in exponential phase in right flank (5e6 cells + Matrigel) in 0.2 mL PBS. The animals are randomized when the tumor volume reached -200 mm 3 . Animals are dosed IV with saline (days 1-4), the tetrazine -based targeting agent (day 0, 5 mg/kg) + saline (days 1-4), or tetrazine-based targeting agent (day 0, 5 mg/kg) + TCO prodrug (days 1, 2, 3 & 4, 120 mg/kg).
  • Tumor volume is measured twice weekly in two dimensions using a caliper, and the volume is expressed in mm 3 using the formula: V - 0.5 a x b 2 where a and b are the long and short diameters of the tumor, respectively.
  • a decrease in tumor growth with administration of the prodrug shows efficacy of the system described therein.
  • Suitable prodrugs for use in the methods disclosed herein can be prepared and administered as described in W02020/077140, WO2018/187740, WO2017/044983, WO2015/139025, and WO2014/205126.
  • Example 6 General procedure for preparation of Compound B O O NH 2 O NO 2 O HCl O O Cl O O H [0602] To a solution o compoun ( . g, 5. mmo) n ( m ) was added DIEA (2.10 g, 16.3 mmol), EDCI (2.08 g, 10.9 mmol) and DMAP (1.33 g, 10.9 mmol) and compound 3 (1.61 g, 8.14 mmol). The mixture was stirred at 25 °C for 16 hrs. TLC indicated compound 2 was consumed completely and one new spot formed. The reaction mixture was partitioned between DCM (20 mL) and H2O (10 mL). The organic phase was separated, washed with sat.
  • Example 7 Alternative route to Compound B and Synthesis of Compound C.
  • the aqueous layer was extracted with MTBE (3 ⁇ 400 mL).
  • the combined MTBE layers were was dried with Na 2 SO 4 , filtered and concentrated in vacuo to provide the compound 2 (5.50 g, 35.9% yield).
  • the crude product was used into the next step without further purification.
  • Example 9 General procedure for preparation of Compound E [0643] To a solution of compound 13 (350 mg, 0.30 ⁇ mol) and DMAP (221 mg, 1.81 mmol) and compound 14 (249 mg, 0.39 mmol, HCl) in DMF (0.3 mL). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed compound 13 was consumed completely and one main peak with desired mass was detected. The residue was purified by prep-HPLC (Water (0.1% TFA)-ACN) to give Compound E (205 mg, 41.3% yield). [0644] LCMS (m/z): 1646.5 (M+H) + .
  • Example 10 3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-tetrazine [ c-2- cyano-5-aminomethyl-pyrimidine (1) in dry acetonitrile is added hydrazine and nickel (II) triflate. The reaction mixture is then heated overnight; the starting material is consumed by TLC. To the reaction mixture is added sodium nitrite (dissolved in water), followed by 1 M hydrochloric acid. The reaction mixture is then stirred at ambient temperature until the reaction is determined to be complete by HPLC. The reaction mixture is then partitioned between ethyl acetate and water. The organic layer is washed with water (3x), followed by brine (1x), and then is dried over sodium sulfate.
  • 6-(6-Methyl-1,2,4,5-tetrazin-3-yl)-3-pyridinemethanamine can also be utilized in the compounds and methods described herein, which compound can be prepared according to the art or purchased from a commercial source (e.g., Enamine US Inc., New Jersey, USA).
  • Example 12 Dihydrotetrazine (Target 5) [0651] To a solution of N2H4.H2O (9.74 g, 190 mmol, 9.44 mL, 98% purity, 7.08 eq) in EtOH (35.0 mL) was added compound 1 (5.00 g, 26.9 mmol, 1.00 eq, HCl) and compound 2 (2.55 g, 26.9 mmol, 1.00 eq, HCl) at 20 °C. The mixture was stirred at 78 °C for 3 hrs. LCMS analysis of the reaction mixture showed compound 1 was consumed completely.
  • Example 15 3-methyl-6-(1H-pyrazol-1-yl)-1,2,4,5-tetrazine (Target 13) [0661] methyl (E)-hydrazinecarbohydrazonothioate (2): To a mixture of 1,3-diaminothiourea (100 g, 942.06 mmol) in MeOH (500 mL) was added MeI (160.46 g, 1.13 mol) at 25 o C and the mixture was stirred at 80 o C for 1.5 h under N2. H NMR showed the reaction was completed. The resulting pale yellow solution was cooled to room temperature until solid precipitated. And then it was diluted with MTBE (500 mL).
  • MeI 160.46 g, 1.13 mol
  • Example 16 (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 15) [0667] tert-butyl (4-cyano-2-(trifluoromethyl)benzyl)carbamate (2): To a solution of 4- (aminomethyl)-3-(trifluoromethyl)benzonitrile (300 mg, 1.50 mmol) in DCM (10 mL) was added Boc 2 O (360 mg, 1.65 mmol) and TEA (227 mg, 2.25 mmol) at 20 °C under N2. The mixture was stirred at 20 °C for 2 h.
  • Example 17 (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 17) [0674] 3-((1,3-dioxoisoindolin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile (2): To a solution of 3- (hydroxymethyl)-4-(trifluoromethyl)benzonitrile (4.5 g, 22.38 mmol) in THF (50 mL) was added isoindoline-1,3-dione (3.3 g, 22.38 mmol), PPh3 (11.7 g, 44.76 mmol) and DIAD (6.79 g, 33.57 mmol) at 0 °C under N2.
  • the mixture was stirred at 45 °C for 16 h. Then the mixture was cooled to 20 o C and added with a solution of NaNO2 in H2O (40 mL) dropwise at 20 o C. The mixture was stirred at 20 o C for 1 h. Under ice-cooling, the pH was adjusted to 3 with 1 M aqueous hydrochloride and then extracted with DCM (3 ⁇ 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • tert-butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate To a mixture of 1-[2-[6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl]ethyl]-3-methyl-urea (1 g, 2.60 mmol) and (tert- butoxycarbonylamino)methyl-trifluoro-boron;potassium hydride (926 mg, 3.90 mmol) in 2-METHYL-2- BUTANOL (4 mL) and H2O (1 mL) was added Cs2CO3 (1.70 g, 5.21 mmol) and ditert- butyl(cyclopentyl)phosphane;dichloropalladium;iron (170 mg, 0.26 mmol) at 25 °C under N2.
  • Example 19 4-((S)-2-((S)-2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl-6- methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (Target 1b) [0 696] 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (2): To a solution of benzonitrile (10 g, 96.97 mmol), ACN (31.85 g, 775.79 mmol), 3-mercaptopropanoic acid (10.29 g, 96.97 mmol) in EtOH (100 mL) was added dropwise NH2NH2.H2O (79.26 g, 1.55 mol) at 0 °C under N2.
  • the mixture was stirred for 16 h at 40 °C.
  • the combined organic layers were washed with brine (100 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the mixture was slurried with MTBE:EtOAc (10:1, 100 mL), filtered and the solid was desired.
  • the solid was purified by p-HPLC (FA) under the following condition: column: Phenomenex luna C18 (250 ⁇ 70 mm, 15 um); mobile phase: [water(FA)-ACN]; B%: 8%-35%, 22 min to give 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (10 g, 29%).
  • Example 20 Synthesis of Exatecan Prodrug Compound F [ ] roceure or prepara on o ompoun [0709]
  • methyl compound 1 (25.0 g, 104 mmol, 1.0 eq.) in MeOH (125 mL)
  • NaOMe 5.40 M, 105 mL, 5.5 eq.
  • H2O 125 mL
  • the reaction mixture was diluted with H2O (50 mL) and extracted with MTBE (4 ⁇ 500 mL).
  • the aqueous layer was extracted with MTBE (5 ⁇ 500 mL).
  • the combined MTBE layers were dried with Na 2 SO 4 , filtered and concentrated under reduced pressure to give residue and evaporated with MeCN three times to give compound 2 (9.1 g, 49.3 mmol, 47.4% yield).
  • Example 22 Synthesis of Exatecan Prodrug Compound H [ ] o a u e o p osge e g, . o was a e , , -6- hydroxy-1-methylcyclooct-4-ene-1-carboxylic acid (220 mg, 1.20 mmol) and DMAP (292 mg, 2.40 mmol). The mixture was stirred at room temperature for 30 min.
  • Mobile phase A was 0.01% HCOOH in water
  • mobile phase B was 0.01% HCOOH in HPLC grade CH 3 CN.
  • the column used for the chromatography is a 4.6 x 50 mm XBridge C18 column (3.5 ⁇ m particles).
  • Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive/negative electrospray ionization.
  • Example 23 Synthesis of Exatecan Prodrug Compound J [0736]
  • DMF 5 mL
  • DIEA 38 mg, 300 ⁇ mol
  • glycine 16 mg, 200 ⁇ mol
  • NaHCO3 17 mg, 200 ⁇ mol
  • the mixture was stirred at 25°C for 5 hr.
  • the mixture was concentrated and purified by Prep-HPLC (CH3CN/H2O(FA)) 0% to 70%) to give Compound J (32 mg, 45.2 %).
  • Mobile phase A was 0.01% HCOOH in water
  • mobile phase B was 0.01% HCOOH in HPLC grade CH3CN.
  • the column used for the chromatography is a 4.6 x 50 mm XBridge C18 column (3.5 ⁇ m particles).
  • Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive/negative electrospray ionization.
  • Example 24 Synthesis of Compound K and Compound L
  • Resin preparation To the vessel containing 2-CTC resin (1.50 mmol, Sub: 0.81 mmol/g, 1.85 g) and Fmoc-7-Ahp-OH (1.50 mmol, 0.55 g, 1.00 eq.), in DCM (30.0 mL) and then DIEA (4.00 eq.) was added with N 2 bubbling for 2 hrs at 15 °C. Add MeOH (15.0 mL) and mix for 30 mins. Then 20% piperidine in DMF (50.0 mL) was added and the mixture was bubbled with N 2 for 30 mins at 15 °C. Then the mixture was filtered to obtain the resin.
  • Peptide Cleavage [0768] Add cleavage buffer (HFIP/DCM, 2/8, v/v, 100 ml) to the flask containing the side chain protected peptide at room temperature and stir for 0.5 h for 2 times. [0769] Filter and collect the filtrate. The mixture was concentrated under reduced pressure. [0770] The residue was dissolved in ACN (100 ml) and H2O (150 ml), then lyophilized to give the compound 16 (520 mg, 93.2% purity, 27.3% yield) which was confirmed via LCMS and HPLC.
  • cleavage buffer HFIP/DCM, 2/8, v/v, 100 ml

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Abstract

La présente invention concerne de manière générale des agents de ciblage à base de tétrazine pour l'administration bio-orthogonale d'une charge utile à un emplacement ciblé chez un sujet, lesdits agents de ciblage à base de tétrazine ayant des applications, par exemple, dans le traitement du cancer, de la croissance tumorale et de l'immunothérapie.
PCT/US2024/027840 2023-05-04 2024-05-03 Agents de ciblage à base de tétrazine pour la distribution in vivo d'une charge utile Pending WO2024229427A1 (fr)

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