EP4661914A1 - Antibody-drug conjugates via inverse electron demand diels-alder reactions - Google Patents

Antibody-drug conjugates via inverse electron demand diels-alder reactions

Info

Publication number
EP4661914A1
EP4661914A1 EP24714268.0A EP24714268A EP4661914A1 EP 4661914 A1 EP4661914 A1 EP 4661914A1 EP 24714268 A EP24714268 A EP 24714268A EP 4661914 A1 EP4661914 A1 EP 4661914A1
Authority
EP
European Patent Office
Prior art keywords
alkylene
compound
antibody
pegn
certain embodiments
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24714268.0A
Other languages
German (de)
French (fr)
Inventor
Amy Han
Christopher D'SOUZA
Ning Zou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regeneron Pharmaceuticals Inc
Original Assignee
Regeneron Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Publication of EP4661914A1 publication Critical patent/EP4661914A1/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • ADCs Antibody-drug conjugates
  • ADCETRIS ® Bossetimab vedotin
  • KADCYLA ® ado-trastuzumab emtansine
  • Linkers covalently link the payload portion, for example, small molecule therapeutic agent of an ADC to its antibody.
  • a significant challenge in linker design is in finding moieities that keep the payload stably attached to the antibody during storage, formulation, administration, and plasma circulation in the patient, yet allow efficient release upon the antibody binding its target, that allow facile conjugation to the payload under synthesis conditions, and that allow release of the intended payload without alteration in structure.
  • Chemical site-selective protein modification has become increasingly popular for antibody-based bio-conjugates.
  • tetrazine-linkers were designed to have two functions: (1) a tetrazine-linker as a handle that includes an additional chemical moiety (e.g., an amine) to be attached to an antibody while the tetrazine-moiety can react with a linker-payload to generate an ADC; and (2) a tetrazine-linker as a linker of a linker-payload that can be attached with an antibody-handle (FIG.13).
  • an additional chemical moiety e.g., an amine
  • R 1 )(R 2 )N-W-X-Y-Z wherein W is C 1 -C 10 alkylene or C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene;
  • X is -N(R 3 )C(O)-C 1 -C 10 alkylene, -C(O)N(R 3 )-C 1 -C 10 alkylene, -C(O)N(R 3 )-C 1 -C 10 alkylene-C(O)N(R 3 )-C 1 -C 10 alkylene-C(O)-(AA)p-N(R 3 )-, -C(O)-(AA)p-N(R 3 )-, -C(O)N(R 3 )-, -N(R 5 )2, -C(O)
  • R 1 )(R 2 )N-W-X-Y-Z wherein W is C 1 -C 10 alkylene or C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -N(R 3 )C(O)-C 1 -C 10 alkylene, -C(O)N(R 3 )-, -N(R 5 ) 2 , or -N(R 5 )(R 6 ); Y when present is C 1 -C 10 alkylene; Z is selected from the group consisting of , , , , , and ; R 1 and R 2 are independently hydrogen, C 1 -C 10 alkyl, or -C(O)-C 1 -C 10 alkyl-COOH; R 3 and R 4 are independently hydrogen or C 1 -C 10 alkyl; R 5 is -Y-C(O)N(R 3 )-Y-Z;
  • a compound having the following chemical structure L10aE [0012] In one embodiment, provided is a compound selected from the group consisting of LP1; LP7; LP15; LP16; and LP17. In one embodiment, the compound is LP1. In one embodiment, the compound is LP7. In one embodiment, the compound is LP15. In one embodiment, the compound is LP16. In one embodiment, the compound is LP17.
  • a compound having the following structure (I) or (II) or a pharmaceutically acceptable salt thereof wherein L is hydrogen or a linker comprising a moiety reactive with a binding agent; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent; SP is a spacer group; R 1a and R 1b are, independently, hydrogen or alkyl; R 2 is hydrogen or an amino acid side chain; R 3 , R 4 , and R 6 is hydrogen or alkyl; R 5 is oxygen, NR 6 , or sulfur; R 7 is an O-amino acid residue; R 8 is C 1 -C 10 alkylene or C 1 -C 10 heteroalkylene; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein
  • a compound having the following structure (I) or (II) or a pharmaceutically acceptable salt thereof wherein L is a linker comprising a moiety reactive with a binding agent; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent; SP is a spacer group; R 1a and R 1b are, independently, hydrogen or alkyl; R 2 is hydrogen or an amino acid side chain; R 3 , R 4 , and R 6 is hydrogen or alkyl; R 5 is oxygen, NR 6 , or sulfur; R 7 is an O-amino acid residue; R 8 is C 1 -C 10 alkylene or C 1 -C 10 heteroalkylene; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein when X is or , wherein T is part
  • D* is a residue of a therapeutic moiety, wherein the therapeutic moiety is a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, or an analogue or derivative thereof, or an imaging agent moiety.
  • D* is a residue of a maytansinoid.
  • D* is a residue of a tubulysin.
  • D* is a residue of an auristatin.
  • D* is a residue of a dolastatin. In one embodiment, D* is a residue of a camptothesin. In one embodiment, D* is a residue of a pyrrolobenzodiazepine. In one embodiment, D* is a residue of an antibiotic. In one embodiment, D* is a residue of an antiviral agent. In one embodiment, D* is a residue of an anti-inflammatory agent. In one embodiment, D* is a residue of an immunomodulator. In one embodiment, D* is a residue of an antifungal agent. In one embodiment, D* is a residue of a steroid. In one embodiment, D* is a residue of an imaging agent.
  • D* is a residue of a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, or an imaging agent moiety, or an analogue or derivative thereof.
  • a conjugate selected from the group consisting of and
  • FIGS.1-10 show synthetic chemistry schemes for linkers including AL4, AL10, and L5; branched AL11, AL12, AL13, AL14, AL15, AL16, AL17, L10a, and L10aE; linear vcPABC linker-payloads LP4 and LP12-LP15; GGFG linker-paylaods LP2 and LP8; branched GGFG linker-payloads LP5 and LP10; and branched EvcPABC linker-payloads LP6 and LP11.
  • FIG.11 shows 4DAR ADC conjugations.
  • FIG.12 shows exemplary 4DAR, 8DAR, and 16DAR ADC conjugations.
  • FIG.13 shows ADC generation via metal-free 9enzotriazol inverse electron demand Diels-Alder reactions
  • FIG. 14 shows Her2-MMAE ADCs conjugated with amino-dienophile linkers via AL2 or AL3 versus Mc-vcPAB-MMAE that potently kill Her2 positive cells
  • FIG. 15 shows the potency of 4DAR ADCs 14, 20-23, 32, and 33 conjugated via AL6 and AL7 Amino-tetrazine linkers or azido-PEGn-N3.
  • FIG.16 shows the potency of 8DAR ADCs conjugated with branched linker-payload LP6 via AL6 and AL7 amino-tetrazine linkers or azido-PEGn-N 3 .
  • FIG.17 shows the potency of 8DAR ADCs conjugated with branched linker-payload LP5 via AL6 and AL7 amino-tetrazine linker or azido-PEGn-N3.
  • FIG. 18 shows 8DAR ADC potency via amino-tetrazine linker AL11 compared to AL6, AL7, and azido-PEGn-N3.
  • FIG. 19 shows the potency of 15DAR ADC via amino-tetrazine linker AL11 compared to AL6, AL7, and azido-PEGn-N 3 .
  • FIG.20 shows ADC synthetic Approaches I and II.
  • FIG.21 shows stability data for Tz handles.
  • FIG.22 shows aHer2-(AL11)4.
  • FIG.23 shows ES-MS ADC characterization data for aHer2-(AL11)4, aHer2-(AL11- LP4)n, and FelD1-(AL11-LP4)n.
  • FIG. 24 shows ES-MS ADC characterization data for aHer2-(AL11-LP6)4 and FelD1-(AL11-LP6)4.
  • FIG.25 shows that both aHer2-(AL7-LP4)n and aHer2-(AL7-LP6)n are stable in all plasma for fourteen days.
  • DESCRIPTION OF EXEMPLARY EMBODIMENTS Definitions [0031] When referring to the compounds provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term provided herein, these Definitions prevail unless stated otherwise. [0032] As used herein, “alkyl” refers to a monovalent and saturated hydrocarbon radical moiety.
  • Alkyl is optionally substituted and can be linear, branched, or cyclic, (i.e., cycloalkyl).
  • Alkyl includes, but is not limited to, those radicals having one to twenty carbon atoms, for example, C 1-20 alkyl; one to twelve carbon atoms, for example, C 1-12 alkyl; one to eight carbon atoms, for example, C 1-8 alkyl; one to six carbon atoms, for example, C 1-6 alkyl; and one to three carbon atoms for example, C 1 -3 alkyl.
  • alkyl moieties include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, i-butyl, a pentyl moiety, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • a pentyl moiety includes, but is not limited to, n-pentyl and i-pentyl.
  • a hexyl moiety includes, but is not limited to, n-hexyl.
  • alkylene refers to a divalent alkyl group. Unless specified otherwise, alkylene includes, but is not limited to, one to twenty carbon atoms. The alkylene group is optionally substituted as described herein for alkyl. In some embodiments, alkylene is unsubstituted. Examples of alkylene moieties include -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, and the like. [0034] As used herein, “heteroalkylene” refers to a divalent alkyl group wherein one or more carbon atoms is replaced with a heteroatom.
  • heteroalkylene includes, but is not limited to, one to twenty total atoms (i.e., carbons and heteroatoms).
  • the heteroalkylene group is optionally substituted as described herein for alkyl.
  • heteroalkylene is unsubstituted.
  • heteroatoms contemplated within heteroalkylene moieties include oxygen, nitrogen, sulfur (i.e., including sulfoxide, sulphite, sulfate, and sulfone), silicon, and phosphorous (i.e., including phosphite and phosphate), and/or combinations thereof.
  • Nonlimiting exemplary embodiments of heteroalkylene moieties include -CH 2 O-, -CH 2 OCH 2 -, -CH 2 OCH 2 CH 2 -, -CH 2 CH 2 CH 2 OCH 2 -, and the like; -CH 2 NR-, -CH 2 NRCH 2 -, -CH 2 NRCH 2 CH 2 -, -CH 2 CH 2 CH 2 NRCH 2 -, and the like; and -CH 2 S-, -CH 2 SCH 2 -, -CH 2 SCH 2 CH 2 -, -CH 2 CH 2 CH 2 SCH 2 -, and the like wherein R includes, but is not limited to, hydrogen or alkyl.
  • haloalkyl refers to alkyl, as defined above, wherein the alkyl includes at least one substituent selected from a halogen, for example, fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).
  • haloalkyl include, but are not limited to, –CF3, –CH 2 CF 3 , –CCl 2 F, –CHF 2 , and –CCl 3 .
  • alkenyl refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more non-aromatic carbon-carbon double bonds or olefins. Alkenyl is optionally substituted and can be linear, branched, or cyclic. Alkenyl includes, but is not limited to, those radicals having two to twenty carbon atoms, for example, C 2-20 alkenyl; two to twelve carbon atoms, for example, C 2-12 alkenyl; two to eight carbon atoms, for example, C 2-8 alkenyl; two to six carbon atoms, for example, C 2-6 alkenyl; and two to four carbon atoms for example, C 2-4 alkenyl.
  • alkenyl moieties include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl.
  • alkynyl refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl is optionally substituted and can be linear, branched, or cyclic.
  • Alkynyl includes, but is not limited to, those radicals having two to twenty carbon atoms, for example, C 2-20 alkynyl; two to twelve carbon atoms, for example, C 2-12 alkynyl; two to eight carbon atoms, for example, C 2-8 alkynyl; two to six carbon atoms, for example, C 2-6 alkynyl; and two to four carbon atoms, for example, C 2-4 alkynyl.
  • alkynyl moieties include, but are not limited to ethynyl, propynyl, propargyl, and butynyl.
  • alkoxy refers to a monovalent and saturated hydrocarbon radical moiety wherein the hydrocarbon includes a single bond to an oxygen atom and wherein the radical is localized on the oxygen atom, for example, CH 3 CH 2 -O ⁇ for ethoxy.
  • Alkoxy substituents bond to the compound which they substitute through this oxygen atom of the alkoxy substituent.
  • Alkoxy is optionally substituted and can be linear, branched, or cyclic, for example, cycloalkoxy.
  • Alkoxy includes, but is not limited to, those radicals having one to twenty carbon atoms, for example, C 1-20 alkoxy; one to twelve carbon atoms, for example, C 1-12 alkoxy; one to eight carbon atoms, for example, C 1-8 alkoxy; one to six carbon atoms, for example, C 1-6 alkoxy; and one to three carbon atoms, for example, C 1-3 alkoxy.
  • alkoxy moieties include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, i- butoxy, a pentoxy moiety, a hexoxy moiety, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.
  • haloalkoxy refers to alkoxy, as defined above, wherein the alkoxy includes at least one substituent selected from a halogen, for example, F, Cl, Br, or I.
  • aryl refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms are carbon atoms.
  • Aryl is optionally substituted and can be monocyclic or polycyclic, for example, bicyclic or tricyclic.
  • aryl moieties include, but are not limited to, those having six to twenty ring carbon atoms, for example, C 6-20 aryl; six to fifteen ring carbon atoms, for example, C 6-15 aryl, and six to ten ring carbon atoms, for example, C 6-10 aryl.
  • aryl moieties include, but are limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, and pyrenyl.
  • arylalkyl refers to a monovalent moiety that is a radical of an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, for example, the aromatic compound includes a single bond to an alkyl group and wherein the radical is localized on the alkyl group.
  • An arylalkyl group bonds to the illustrated chemical structure via the alkyl group.
  • An arylalkyl can be represented by a structure, for example, , , , , or , wherein B is an aromatic moiety, for example, aryl or phenyl.
  • Arylalkyl is optionally substituted, for example, the aryl group and/or the alkyl group can be substituted as disclosed herein. Examples of arylalkyl include, but are not limited to, benzyl.
  • alkylaryl refers to a monovalent moiety that is a radical of an aryl compound, wherein the aryl compound is substituted with an alkyl substituent, for example, the aryl compound includes a single bond to an alkyl group and wherein the radical is localized on the aryl group.
  • An alkylaryl group bonds to the illustrated chemical structure via the aryl group.
  • An alkylaryl can be represented by a structure, for example, , , , or , wherein B is an aromatic moiety, for example, phenyl.
  • Alkylaryl is optionally substituted, for example, the aryl group and/or the alkyl group can be substituted as disclosed herein.
  • alkylaryl examples include, but are not limited to, toluyl.
  • aryloxy refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms are carbon atoms and wherein the ring is substituted with an oxygen radical, for example, the aromatic compound includes a single bond to an oxygen atom and wherein the radical is localized on the oxygen atom, for example, for phenoxy.
  • Aryloxy substituents bond to the compound that they substitute through this oxygen atom. Aryloxy is optionally substituted.
  • Aryloxy includes, but is not limited to, those radicals having six to twenty ring carbon atoms, for example, C 6-20 aryloxy; six to fifteen ring carbon atoms, for example, C6-15 aryloxy, and six to ten ring carbon atoms, for example, C6-10 aryloxy.
  • aryloxy moieties include, but are not limited to phenoxy, naphthoxy, and anthroxy.
  • arylene refers to a divalent moiety of an aromatic compound wherein the ring atoms are only carbon atoms.
  • Arylene is optionally substituted and can be monocyclic or polycyclic, for example, bicyclic or tricyclic.
  • arylene moieties include, but are not limited to those having six to twenty ring carbon atoms, for example, C 6-20 arylene; six to fifteen ring carbon atoms, for example, C 6-15 arylene, and six to ten ring carbon atoms, for example, C 6-10 arylene.
  • heteroalkyl refers to an alkyl in which one or more carbon atoms are replaced by heteroatoms.
  • heteroalkenyl refers to an alkenyl in which one or more carbon atoms are replaced by heteroatoms.
  • heteroalkynyl refers to an alkynyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.
  • heteroaryl refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms contain carbon atoms and at least one oxygen, sulfur, nitrogen, or phosphorus atom.
  • heteroaryl moieties include, but are not limited to those having five to twenty ring atoms; five to fifteen ring atoms; and five to ten ring atoms. Heteroaryl is optionally substituted.
  • heteroarylene refers to a divalent heteroaryl in which one or more ring atoms of the aromatic ring are replaced with an oxygen, sulfur, nitrogen, or phosphorus atom. Heteroarylene is optionally substituted.
  • heterocycloalkyl or “heterocyclyl” refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms (i.e., including sulfoxide and sulfone). Heterocycloalkyl or heterocyclyl is optionally substituted.
  • heterocycloalkyl and heterocyclyl moieties include, but are not limited to, morpholinyl, piperidinyl, tetrahydropyranyl, pyrrolidinyl, aziridnyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, dioxolanyl, dithiolanyl, oxanyl, and thianyl.
  • Lewis acid refers to a molecule or ion that accepts an electron lone pair.
  • the Lewis acids used in the methods described herein are those other than protons.
  • Lewis acids include, but are not limited to, non-metal acids, metal acids, hard Lewis acids, and soft Lewis acids.
  • Lewis acids include, but are not limited to, Lewis acids of aluminum, boron, iron, tin, titanium, magnesium, copper, antimony, phosphorus, silver, ytterbium, scandium, nickel, and zinc.
  • Illustrative Lewis acids include, but are not limited to, AlBr 3 , AlCl 3 , BCl 3 , boron trichloride methyl sulfide, BF3, boron trifluoride methyl etherate, boron trifluoride methyl sulfide, boron trifluoride tetrahydrofuran, dicyclohexylboron trifluoromethanesulfonate, iron (III) bromide, iron (III) chloride, tin (IV) chloride, titanium (IV) chloride, titanium (IV) isopropoxide, Cu(Otf) 2 , CuCl 2 , CuBr 2 , zinc chloride, alkylaluminum halides (RnAlX 3- n, wherein R is hydrocarbyl), Zn(Otf)2, ZnCl2, Yb(Otf) 3 , Sc(Otf) 3 , MgBr2, NiCl2, Sn(
  • N-containing heterocycloalkyl refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms and wherein at least one replacing heteroatom is a nitrogen atom. Suitable heteroatoms in addition to nitrogen include, but are not limited to, oxygen and sulfur atoms. N-containing heterocycloalkyl is optionally substituted. Examples of N-containing heterocycloalkyl moieties include, but are not limited to, morpholinyl, piperidinyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, or thiazolidinyl.
  • optionally substituted when used to describe a radical moiety, for example, optionally substituted alkyl, means that such moiety is optionally bonded to one or more substituents.
  • substituents include, but are not limited to, halo, cyano, nitro, amino, hydroxyl, optionally substituted haloalkyl, aminoalkyl, hydroxyalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, , , , , , , , , , , or , wherein R A , R B , and R C are, independently at each occurrence, hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heteroaryl, or heterocycloalkyl, or R A and R B together with the atoms to which they are bonded, form a saturated
  • a radical moiety is optionally substituted with an optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring
  • the substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring, if they are substituted, are not substituted with substituents which are further optionally substituted with additional substituents.
  • the substituent bonded to the group is unsubstituted unless otherwise specified.
  • binding agent refers to any molecule, for example, protein, antibody, or antigen binding fragment thereof, capable of binding with specificity to a given binding partner, for example, an antigen.
  • linker refers to a divalent, trivalent, or multivalent moiety that covalently links, or is capable of covalently linking (e.g., via a reactive group), the binding agent to one or more compounds described herein, for instance, payload compounds and enhancement agents.
  • amide synthesis conditions refers to reaction conditions suitable to effect the formation of an amide, for example, by the reaction of a carboxylic acid, activated carboxylic acid, or acyl halide with an amine.
  • amide synthesis conditions refer to reaction conditions suitable to effect the formation of an amide bond between a carboxylic acid and an amine.
  • the carboxylic acid is first converted to an activated carboxylic acid before the activated carboxylic acid reacts with an amine to form an amide.
  • Suitable conditions to effect the formation of an amide include, but are not limited to, those utilizing reagents to effect the reaction between a carboxylic acid and an amine including, but not limited to, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), (16enzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (16enzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7- azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O-(16enzotriazole-1-yl)- N,N,N’,N’-tetramethyluronium hex
  • a carboxylic acid is first converted to an activated carboxylic ester before treating the activated carboxylic ester with an amine to form an amide bond.
  • the carboxylic acid is treated with a reagent.
  • the reagent activates the carboxylic acid by deprotonating the carboxylic acid and then forming a product complex with the deprotonated carboxylic acid as a result of nucleophilic attack by the deprotonated carboxylic acid onto the protonated reagent.
  • the activated carboxylic esters for certain carboxylic acids are subsequently more susceptible to nucleophilic attack by an amine than the carboxylic acid is before activation. This results in amide bond formation.
  • regioisomer refers to the product(s) of 1,3-cycloadditions or strain-promoted alkyne-azide cycloadditions (SPAACs)—otherwise known as click reactions—that derive from suitable azides (e.g.,-N 3 , or –PEG-N 3 derivitized antibodies) treated with suitable alkynes; or refers to the product(s) of inverse electron demand Diels-Alder reactions that derive from suitable dienophiles (e.g., alkenes or alkynes) treated with suitable dienes (e.g., tetrazines), or suitable dienes (e.g., tetrazines) treated with suitable dienophiles.
  • suitable dienophiles e.g., alkenes or alkynes
  • suitable dienes e.g., tetrazines
  • suitable dienes e.g., tetrazines
  • regioisomers and mixtures of regioisomers are characterized by the click reaction products shown below: wherein represents attachment to a binding agent as described elsewhere herein.
  • more than one suitable azide and more than one suitable alkyne can be utilized within a synthetic scheme en route to a product, where each pair of azide-alkyne can participate in one or more independent click reactions to generate a mixture of regioisomeric click reaction products.
  • a first suitable azide may independently react with a first suitable alkyne
  • a second suitable azide may independently react with a second suitable alkyne, en route to a product, resulting in the generation of four possible click reaction regioisomers or a mixture of the four possible click reaction regioisomers.
  • regioisomers and mixtures of regioisomers are characterized by the inverse electron demand Diels-Alder reaction products below: or wherein represents attachment to a binding agent as described elsewhere herein.
  • more than one suitable dienophile and more than one suitable diene can be utilized within a synthetic scheme en route to a product, where each pair of dienophile-diene can participate in one or more independent inverse electron demand Diels-Alder reactions to generate a mixture of regioisomeric Diels-Alder reaction products.
  • a first suitable dienophile may independently react with a first suitable diene
  • a second suitable dienophile may independently react with a second suitable diene, en route to a product, resulting in the generation of four possible Diels-Alder reaction regioisomers or a mixture of the four possible Diels-Alder reaction regioisomers.
  • the term “residue” refers to the chemical moiety within a compound that remains after a chemical reaction.
  • amino acid residue refers to the product of an amide coupling or peptide coupling of an amino acid, O-amino acid, OH-amino acid, or a N-alkyl amino acid to a suitable coupling partner; wherein, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid, peptide, O- amino acid, OH-amino acid, or the N-alkylamino acid, resulting in the product having the amino acid residue, O-amino acid residue, OH-amino acid residue, or N-alkyl amino acid residue incorporated therein.
  • “therapeutically effective amount” refers to an amount (e.g., of a compound) that is sufficient to provide a therapeutic benefit to a patient in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder.
  • “constitutional isomers” refer to compounds that have the same molecular formula, but different chemical structures resulting from the way the atoms are arranged. Exemplary constitutional isomers include n-propyl and isopropyl; n-butyl, sec-butyl, and tert-butyl; and n-pentyl, isopentyl, and neopentyl, and the like.
  • Certain groups, moieties, substituents, and atoms are depicted with a wiggly line that intersects a bond or bonds to indicate the atom through which the groups, moieties, substituents, atoms are bonded.
  • cyclic group e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl
  • substituents bonded to a cyclic group are meant to indicate, unless specified otherwise, that the cyclic group may be substituted with that substituent at any ring position in the cyclic group or on any ring in the fused ring group, according to techniques set forth herein or which are known in the field to which the instant disclosure pertains.
  • the group, or , wherein subscript q is an integer from zero to four and in which the positions of substituent R 1 are described generically, for example, not directly attached to any vertex of the bond line structure, for example, a specific ring carbon atom includes the following, non-limiting examples of groups in which the substituent R 1 is bonded to a specific ring carbon atom: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
  • RL refers to a monovalent group that includes a reactive group (“RG”) and spacer group (“SP”), depicted for example as , wherein RG is the reactive group and SP is the spacer group.
  • RG reactive group
  • SP spacer group
  • a reactive linker may include more than one reactive group and more than one spacer group.
  • the spacer group is any divalent moiety that bridges the reactive group to another group, such as a payload (e.g., a biologically active compound).
  • the reactive linker includes a reactive group, which is a functional group or moiety that is capable of reacting with a reactive portion of another group, for instance, a binding agent, an antibody, modified antibody, or antigen binding fragment thereof, or an enhancement group.
  • the moiety resulting from the reaction of the reactive group with the binding agent, antibody, modified antibody, or antigen binding fragment thereof, together with the linking group include the “binding agent linker” (“BL”) portion of the conjugate, described herein.
  • the “reactive group” is a functional group or moiety (e.g., maleimide or N-hydroxysuccinimide (NHS) ester) that reacts with a cysteine or lysine residue of an antibody or antigen-binding fragment thereof.
  • the “reactive group” is a functional group or moiety that is capable of undergoing a click chemistry reaction (see, e.g., click chemistry, Huisgen Proc. Chem. Soc.1961, Wang et al. J. Am. Chem. Soc. 2003, and Agard et al. J. Am. Chem. Soc. 2004).
  • the reactive group is an alkyne that is capable of undergoing a 1,3- cycloaddition reaction with an azide.
  • suitable reactive groups include, but are not limited to, strained alkynes, for example, those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, for example, cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts.
  • Suitable alkynes also include, but are not limited to, (TMTH); (COMBO); (PYRROC); cyclooctyne (OCT); (SNO-OCTs); azacyclooctyne (DIMAC); dibenzoazacyclooctyne or (DIBAC); dibenzocyclooctyne or (DIBO); biarylazacyclooctynone or (BARAC); monofluorinated cyclooctyne (MOFO); difluorinated cyclooctyne or , or , or (DIFO), substituted, for example, fluorinated alkynes, aza-cycloalkynes; bicycle[6.1.0]nonyne or (BCN, where R is alkyl, alkoxy, or acyl); and derivatives thereof.
  • TMTH TMTH
  • COMP COMBO
  • PYRROC cyclooctyne
  • OCT
  • Particularly useful alkynes include and .
  • Linker-payloads including such reactive groups are useful for conjugating binding agents or antibodies that have been functionalized with azido groups.
  • Such functionalized binding agents or antibodies include antibodies functionalized with azido-polyethylene glycol groups.
  • such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an an amino group and an azide group, in the presence of the enzyme transglutaminase.
  • the “reactive group” is a functional group or moiety that is capable of undergoing an inverse electron demand Diels-Alder reaction (see, e.g., Chem. Rev.
  • the reactive group is a dienophile (e.g., alkenes or alkynes) that is capable of undergoing an inverse electron demand Diels-Alder reaction with a diene (e.g., a tetrazine).
  • a diene e.g., a tetrazine
  • suitable reactive groups include, but are not limited to, dienophiles or strained dienophiles suitable for the inverse electron demand Diels-Alder reaction, for example, or .
  • Linker-payloads including such reactive groups are useful for conjugating antibodies that have been functionalized with diene groups.
  • Such functionalized antibodies include antibodies functionalized with compounds described herein.
  • such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an an amino agroup and a diene group (e.g., a tetrazine), in the presence of the enzyme transglutaminase.
  • the “reactive group” is a functional group or moiety that is capable of undergoing an inverse electron demand Diels-Alder reaction (see, e.g., Chem. Rev. 2021, 121, 12, 6850–6914).
  • the reactive group is a diene (e.g., a tetrazine) that is capable of undergoing an inverse electron demand Diels-Alder reaction with a dieneophile (e.g., alkenes or alkynes).
  • a dieneophile e.g., alkenes or alkynes.
  • suitable reactive groups include, but are not limited to, dienes suitable for the inverse electron demand Diels-Alder reaction, for example, , , , and .
  • Linker-payloads including such reactive groups are useful for conjugating antibodies that have been functionalized with dieneophile groups.
  • Such functionalized antibodies include antibodies functionalized with compounds described herein.
  • such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an an amino agroup and a dieneophile group, in the presence of the enzyme transglutaminase.
  • the reactive group is an alkyne, for example, , which can react via click chemistry with an azide, for example, , to form a click chemistry product, for example, or .
  • the group reacts with an azide on a modified antibody or antigen binding fragment thereof.
  • the reactive group is an alkyne, for example, , which can react via click chemistry with an azide, for example, to form a click chemistry product, for example, .
  • the reactive group is a dienophile, for example, or , which can react via an inverse electron demand Diels-Adler reaction with a diene, for example, , to form a Diels-Alder product, for example, or .
  • the group reacts with a diene (e.g., a tetrazine) on a modified antibody or antigen binding fragment thereof.
  • the reactive group is a diene, for example, , which can react via an inverse electron demand Diels-Alder reaction with a dienophile, for example, to form a Diels-Alder product, for example, or .
  • the group reacts with a dienophile on a modified antibody or antigen binding fragment thereof.
  • the reactive group is an alkyne, for example, , which can react via click chemistry with an azide, for example, , to form a click chemistry product, for example, or .
  • the reactive group is a functional group, for example, , which reacts with a cysteine residue on an antibody or antigen-binding fragment thereof (i.e., Michael addition), to form a carbon-sulfur bond thereto, for example, , wherein Ab refers to an antibody or antigen-binding fragment thereof and sulphur S refers to the S atom on a cysteine residue through which the functional group bonds to the Ab.
  • the reactive group is a functional group, for example, ,which reacts with a lysine residue on an antibody or antigen-binding fragment thereof, to form an amide bond thereto, for example, , wherein Ab refers to an antibody or antigen-binding fragment thereof and NH refers to the NH moiety on a lysine side chain residue through which the functional group bonds to the Ab.
  • the phrase “biodegradable moiety” refers to a moiety that degrades in vivo to non-toxic, biocompatible components which can be cleared from the body by ordinary biological processes.
  • a biodegradable moiety completely or substantially degrades in vivo over the course of about ninety days or less, about sixty days or less, or about thirty days or less, where the extent of degradation is based on percent mass loss of the biodegradable moiety, and wherein complete degradation corresponds to 100% mass loss.
  • biodegradable moieties include, without limitation, aliphatic polyesters such as poly( ⁇ -caprolactone) (PCL), poly(3-hydroxybutyrate) (PHB), poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and its copolymers with glycolic acid (i.e., poly(D,L-lactide- coglycolide) (PLGA) (Vert M, Schwach G, Engel R and Coudane J (1998) J Control Release 53(1-3):85-92; Jain R A (2000) Biomaterials 21(23):2475-2490; Uhrich K E, Cannizzaro S M, Langer R S and Shakesheff K M (1999) Chemical Reviews 99(11): 3181-3198; and Park T G (1995) Biomaterials 16(15):1123-1130, each of which are incorporated herein by reference in their entirety).
  • PCL poly( ⁇ -caprolactone)
  • PHB poly(3-hydroxybutyrate)
  • binding agent linker refers to any divalent, trivalent, or multi-valent group or moiety that links, connects, or bonds a binding agent (e.g., an antibody or an antigen-binding fragment thereof) with a payload compound set forth herein and, optionally, with one or more side chain compounds.
  • suitable binding agent linkers for the antibody-drug conjugates described herein are those that are sufficiently stable to exploit the circulating half-life of the antibody-drug conjugates and, at the same time, capable of releasing a payload after antigen-mediated internalization of the conjugate. Linkers can be cleavable or non-cleavable.
  • Cleavable linkers are linkers that are cleaved by intracellular metabolism following internalization, for example, cleavage via hydrolysis, reduction, or enzymatic reaction.
  • Non-cleavable linkers are linkers that release an attached payload via lysosomal degradation of the antibody following internalization.
  • Suitable linkers include, but are not limited to, acid-labile linkers, hydrolytically-labile linkers, enzymatically cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers.
  • Suitable linkers also include, but are not limited to, those that are or comprise peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units (e.g., PEGn, (CH 2 CH 2 O)n, and (CH 2 OCH 2 )n, and used interchangeably herein), hydrazones, mal-caproyl units, dipeptide units, valine-citruline units, para-aminobenzyloxycarbonyl (PABC), and para-aminobenzyl (PAB) units.
  • PEG polyethylene glycol
  • PABC para-aminobenzyloxycarbonyl
  • PAB para-aminobenzyl
  • the binding agent linker includes a moiety that is formed by the reaction of the reactive group (RG) of a reactive linker (RL) and reactive portion of the binding agent, for example, antibody, modified antibody, or antigen binding fragment thereof.
  • the BL includes the following moiety: or , wherein is the bond to the binding agent.
  • the BL includes the following moiety: , wherein is the bond to the binding agent.
  • the BL includes the following moiety: or , wherein is the bond to the binding agent.
  • the BL includes the following moiety: or , wherein is the bond to the binding agent.
  • the BL includes the following moiety: , , , or , wherein is the bond to the binding agent.
  • the BL includes the following moiety: , wherein is the bond to the cysteine of the antibody or antigen-binding fragment thereof. In some examples, the BL includes the following moiety: , wherein is the bond to the lysine of the antibody or antigen-binding fragment thereof.
  • amino acid side chain refers to the additional chemical moiety on the same carbon that bears a primary or secondary amine and a carboxylic acid of an amino acid. As would be appreciated by a person of skill in the art, there are twenty-one “standard” amino acids. Exemplary “standard” amino acids include, without limitation, alanine, serine, proline, arginine, and aspartic acid.
  • amino acids include, cysteine, selenocysteine, and glycine (e.g., wherein the additional chemical moiety on the same carbon that bears the primary amine and carboxylic acid of glycine is hydrogen).
  • exemplary amino acid side chains include, without limitation, methyl (i.e., alanine), sec-buytl (i.e., isoleucine), iso-butyl (i.e., leucine), –CH 2 CH 2 SCH3 (i.e., methionine), –CH 2 Ph (i.e., phenylalanine), (i.e., tryptophan), (i.e., tyrosine), iso-propyl (i.e., valine), hydroxymethyl (i.e., serine), –CH(OH)CH 3 (i.e., threonine), –CH 2 C(O)NH 2 (i.e., asparagine), –CH 2 CH 2 C(
  • biologically active compound refers to a compound, prodrug, or payload that elicits a biological response when administered to a biological entity.
  • exemplary biological responses include, without limitation, increase or decrease in DNA or protein synthesis, up-regulation or down-regulation of signalling pathways, and increase or decrease in cell proliferation, and the like.
  • O-amino acid or “HO-amino acid” designates an amino acid wherein the native amino group at the N-terminus of an amino acid or an amino acid sequence has been replaced with an oxygen or hydroxyl group, respectively.
  • O- AAAA or “HO-AAAA” is intended to designate an amino acid sequence (AAAA) wherein the native amino group at the N-terminus has been replaced with an oxygen or hydroxyl group, respectively (e.g., , where each R is an amino acid side chain).
  • O-amino acid residue or “HO-amino acid residue” refers to the chemical moiety within a compound that remains after a chemical reaction.
  • O- amino acid residue or “HO-amino acid residue” refers to the product of an amide coupling or peptide coupling of an O-amino acid or a HO-amino acid to a suitable coupling partner; wherein, for example, a water molecule is expelled after the amide or peptide coupling of the O-amino acid or a HO-amino acid, resulting in the product having the O-amino acid residue or a HO- amino acid residue incorporated therein.
  • n sixteen.
  • n is twenty.
  • n is twenty-three.
  • n is twenty-four.
  • n is twenty- five.
  • n is twenty-nine.
  • n forty-three.
  • n forty-four.
  • n forty- seven.
  • n eighty- three.
  • R 1 and R 2 are hydrogen.
  • W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene
  • X is -N(R 3 )C(O)-C 1 -C 10 alkylene
  • Z is , then n is two, three, eight, or twelve.
  • the compound is selected from the group consisting of AL4c; AL4d; and AL4e. In one embodiment, the compound is . In one embodiment, the compound is AL4c. In one embodiment, the compound is AL4d. In one embodiment, the compound is AL4e. [0070] In certain embodiments, provided are compounds having the following formula (R 1 )(R 2 )N-W-X-Y-Z wherein when W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 3 )-; Y is C 1 -C 10 alkylene; and Z is , then n is one.
  • n twelve.
  • n sixteen.
  • n is twenty.
  • R 1 and R 2 are hydrogen.
  • n is an integer from one to three, five to seven, or nine to one hundred.
  • the compound is selected from the group consisting of ; ; ; ; ; ; ; and AL10c.
  • the compound is .
  • the compound is .
  • the compound is .
  • the compound is .
  • the compound is .
  • the compound is .
  • the compound is .
  • the compound is .
  • the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is AL10c. [0071] In one embodiment, W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -N(R 5 ) 2 or -N(R 5 )(R 6 ); R 5 is -Y-C(O)N(R 3 )-Y-Z; and R 6 is -Y-C(O)N(R 3 )-PEGn1-C 1 -C 10 alkylene-N 3 . In one embodiment, the compound is AL11. In one embodiment, the compound is AL12.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 ) 2 ; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is an integer from one to ten.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 ) 2 ; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is one.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 ) 2 ; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is two.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is three.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is four.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is five.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is six.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is seven.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is eight.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is nine.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene; X is -C(O)N(R 5 )2; R 5 is -Y-(R 3 )NC(O)-Y-Z; Z is ; and n is ten.
  • the compound is AL13.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene; X is -C(O)N(R 3 )-C 1 -C 10 alkylene-C(O)-(AA)p-N(R 3 )- or -C(O)-(AA)P-N(R 3 )-; and Z is .
  • the compound is selected from the group consisting of AL14a; AL14b; and AL14c. In one embodiment, the compound is AL14a. In one embodiment, the compound is AL14b. In one embodiment, the compound is AL14c.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene; X is -CH(R 5 )(R 6 ); R 5 is -(R 3 )NC(O)-Y-C(O)N(R 3 )-Y-Z; R 6 is -C(O)N(R 3 )-PEGn1-C 1 -C 10 alkylene-N 3 ; and Z is . In one embodiment, the compound is 513.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene; X is -C(O)-(AA)p-N(R 3 )-C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene-N(R 5 ) 2 ; R 5 is -Y-C(O)N(R 3 )-Y-Z; and Z is .
  • the compound is AL15.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene; X is -C(O)-(AA)p-N(R 3 )- ; and Z is .
  • the compound is 473.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene; X is -C(O)-(AA)p-N(R 5 ) 2 ; R 5 is -Y-C(O)N(R 3 )-Y-Z; and Z is .
  • the compound is AL16.
  • R 1 and R 2 are hydrogen; W is C 1 -C 10 alkylene; X is -C(O)-(AA)p-N(R 3 )-CH(R 5 )(R 6 ); and Z is .
  • the compound is AL17.
  • n is one.
  • n twelve.
  • n sixteen.
  • n is twenty.
  • n is twenty-three.
  • n is twenty-four.
  • n is twenty-six.
  • n forty-two.
  • n forty-three.
  • n forty-four.
  • n eighty-three.
  • R 1 is hydrogen and R 2 is -C(O)-C 1 -C 10 alkyl-COOH.
  • R 1 is hydrogen and R 2 is -C(O)CH 2 CH 2 COOH.
  • n is an integer from one to one hundred. In one embodiment, n is four, eight, or twelve.
  • the compound is selected from the group consisting of L5a; L5b; and L5c. In one embodiment, the compound is L5a. In one embodiment, the compound is L5b. In one embodiment, then compound is L5c. [0073] Provided herein are prodrugs or payloads. The payload can be any payload deemed suitable by the person of skill in the art.
  • the compounds or payloads include prodrugs thereof.
  • the terms or phrases “compounds,” “biologically active compounds,” “prodrugs,” and “payloads” are used interchangeably throughout this disclosure.
  • the biologically active compound (D*) or residue thereof includes hydroxyl functionality (e.g., D*–OH or D*–O–R).
  • R 5 represents the hydroxyl, amino, and thiol functional groups within the biologically active compounds described herein, as would be appreciated by a person of skill, or a portion thereof such as -O-, -NI-, or -S-.
  • R 5 may be part of the biologically active compounds described herein (e.g., D*), and may be used as a functional group for conjugation purposes.
  • the hydroxyl functionality is a primary hydroxyl moiety (e.g., D*–CH 2 OH or D*–CH 2 O–R; or D*–C(O)CH 2 OH or D*–C(O)CH 2 O–R).
  • the hydroxyl functionality is a secondary hydroxyl moiety (e.g., D*–CH(OH)R or D*–CH(O–R)R; or D*–C(O)CHI(OH) or D*–C(O)CHI(O–R)).
  • the hydroxyl functionality is a tertiary hydroxyl moiety (e.g., D*–C(R 1 )(R 2 )(OH) or D*– C(R 1 )(R 2 )(O–R); or D*–C(O)C(R 1 )(R 2 )(OH) or D*–C(O)C(R 1 )(R 2 )(O–R)).
  • a tertiary hydroxyl moiety e.g., D*–C(R 1 )(R 2 )(OH) or D*– C(R 1 )(R 2 )(O–R)
  • the D* including the hydroxyl functionality is an aryl hydroxyl or phenolic hydroxyl (e.g., D*–Ar–OH, D*–Ar–O–R.
  • the biologically active compound (D*) or residue thereof includes amino functionality (e.g., D*–NR 2 or D*–N(R)–R).
  • the amino functionality is a primary amino moiety (e.g., D*–CH 2 NR 2 or D*–CH 2 N(R)–R; or D*–C(O)CH 2 NR 2 or D*–C(O)CH 2 N(R)–R).
  • the amino functionality is a secondary amino moiety (e.g., D*–CH(NR 2 )R or D*–CH(NR–R)R; or D*–C(O)CH(R)(NR 2 ) or D*–C(O)CH(R)(NR–R)).
  • the amino functionality is a tertiary amino moiety (e.g., D*–C(R 1 )(R 2 )(NR 2 ) or D*–C(R 1 )(R 2 )(N(R)–R); or D*–C(O)C(R 1 )(R 2 )(NR 2 ) or D*–C(O)C(R 1 )(R2)(N(R)–R)).
  • the D* including the amino functionality is an aryl amine (e.g., D*–Ar–NR 2 , D*–Ar–N(R)–R.
  • the payload is a cytotoxin.
  • the payload is a calicheamicin.
  • the payload is an auristatin, for instance, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).
  • the payload is a maytansinoid, for instance, DM1 or DM4.
  • payload is a duocarmycin.
  • payload is an amanitin.
  • payload is a pyrrolobenzodiazepine.
  • payload is an exatecan.
  • the biologically active compound (D*) including amino functionality is exatecan, and the residue including the amino functionality is , wherein indicates attachment to a prodrug moiety (as shown in Formulae I-IV), a linker, and/or binding agent, as described herein.
  • the biologically active compound (D*) including amino functionality is MMAE, and the residue including the amino functionality is , wherein indicates attachment to a prodrug moiety (as shown in Formulae I-IV), a linker, and/or binding agent, as described herein.
  • the biologically active compound (D*) or residue thereof includes thiol functionality (e.g., D*–SH or D*–S–R).
  • the thiol functionality is a primary thiol moiety or sulfide (e.g., D*–CH 2 SH or D*–CH 2 S–R; or D*–C(O)CH2SH or D*–C(O)CH 2 S–R).
  • the thiol functionality is a secondary thiol moiety or sulfide (e.g., D*–CH(SH)R or D*–CH(S–R)R; or D*–C(O)CH(R)(SH) or D*–C(O)CH(R)(S–R)).
  • the thiol functionality is a tertiary thiol moiety or sulfide (e.g., D*–C(R 1 )(R 2 )(SH) or D*–C(R 1 )(R 2 )(S–R); or D*–C(O)C(R 1 )(R 2 )(SH) or D*– C(O)C(R 1 )(R 2 )(S–R)).
  • the D* including the thiol functionality is an aryl thiol or thiophenol or sulfide (e.g., D*–Ar–SH, D*–Ar–S–R.
  • the compounds can be delivered to cells as part of a conjugate.
  • the compounds are capable of carrying out any activity of exatecan or MMAE, or derivatives thereof at or in a target, for instance, a target cell.
  • Certain compounds can have one or more additional activities.
  • all diastereomers are contemplated.
  • the stereochemistry at the exatecan primary amine or the exatecan hydroxyl is undefined or racemic.
  • the stereochemistry at the exatexan primary amine is (R)-.
  • the stereochemistry at the exatecan primary amine is (S)-.
  • the stereochemistry at the exatexan primary amine is (R)- in excess of (S)-.
  • the stereochemistry at the exatecan primary amine is (S)- in excess of (R)-.
  • a compound having the following structure (I) or a pharmaceutically acceptable salt thereof wherein L is hydrogen; R 1a and R 1b are hydrogen; R 2 is hydrogen or an amino acid side chain; R 3 , R 4 , and R 6 is hydrogen or alkyl; R 5 is NR 6 ; R 7 is an O-amino acid residue; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; and m is zero, one, two, three, four, five, or six. In certain embodiments, m is zero. In certain embodiments, m is one. In certain embodiments, m is two. In certain embodiments, m is three. In certain embodiments, m is four. In certain embodiments, m is five. In certain embodiments, m is six. In one embodiment, the compound is selected from the group consisting of SerDXd; GlnDXd; GluDXd;
  • the compound is SerDXd. In one embodiment, the compound is GlnDXd. In one embodiment, the compound is GluDXd. In one embodiment, the compound is LysDXd. In one embodiment, the compound is PheDXd. In one embodiment, the compound is D PheDXd. In one embodiment, the compound is GlyNMeCH 2 DXd.
  • binding agents for any of the conjugates provided in the instant disclosure include, but are not limited to, antibodies, lymphokines (e.g., IL-2 or IL-3), hormones (e.g., insulin and glucocorticoids), growth factors (e.g., EGF, transferrin, and fibronectin type III), viral receptors, interleukins, or any other cell binding or peptide binding molecules or substances. Binding agents also include, but are not limited to, ankyrin repeat proteins and interferons. [0076] In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof. The antibody can be in any form known to those of skill in the art.
  • antibody refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen.
  • CDR complementarity determining region
  • antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, C H 1, C H 2, and C H 3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the antibodies (or antigen-binding portion thereof) suitable for the compounds herein may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • the term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules.
  • the terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable, standard technique(s) such as proteolytic digestion or recombinant genetic engineering technique(s) involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, for example, commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non- limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • an antibody e.g., an isolated CDR such as a CDR3 peptide
  • a constrained FR3-CDR3-FR4 peptide e.g., an isolated CDR such as a CDR3 peptide
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR- grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen- binding fragment,” as used herein.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the V H and V L domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric V H or V L domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of this disclosure include: (i) VH-CH1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H - CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL- CH2-CH3; and (xiv) VL-CL.
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • antigen- binding fragments may be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.
  • antibodies described herein are human antibodies.
  • the term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs and in particular CDR3.
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the term “human antibody” does not include naturally occurring molecules that normally exist without modification or human intervention/manipulation, in a naturally occurring, unmodified living organism.
  • the antibodies of this disclosure may, in some embodiments, be recombinant human antibodies.
  • recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • Human antibodies can exist in two forms that are associated with hinge heterogeneity.
  • an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond.
  • the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody).
  • These forms have been extremely difficult to separate, even after affinity purification.
  • the frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody.
  • a single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al.
  • the instant disclosure encompasses antibodies having one or more mutations in the hinge, C H 2, or C H 3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.
  • the antibodies described herein may be isolated antibodies.
  • An “isolated antibody,” as used herein, refers to an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody” for purposes of the instant disclosure.
  • An isolated antibody also includes an antibody in situ within a recombinant cell.
  • Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • the antibodies used herein can comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • This disclosure includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”).
  • Germline mutations such sequence changes are referred to herein collectively as “germline mutations”.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first eight amino acids of FR1 or within the last eight amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.
  • Antibodies useful for the compounds herein also include antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope.
  • different antibodies may bind to different areas on an antigen and may have different biological effects.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • the antibody comprises a light chain.
  • the light chain is a kappa light chain.
  • the light chain is a lambda light chain.
  • the antibody comprises a heavy chain.
  • the heavy chain is an IgA.
  • the heavy chain is an IgD.
  • the heavy chain is an IgE.
  • the heavy chain is an IgG.
  • the heavy chain is an IgM.
  • the heavy chain is an IgG1.
  • the heavy chain is an IgG2. In some embodiments, the heavy chain is an IgG3. In some embodiments, the heavy chain is an IgG4. In some embodiments, the heavy chain is an IgA1. In some embodiments, the heavy chain is an IgA2. [0078] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab′) 2 fragment. In some embodiments, the antibody fragment is a Fab′ fragment. In some embodiments, the antibody fragment is an scFv (sFv) fragment.
  • the antibody fragment is an scFv-Fc fragment.
  • the antibody is a monoclonal antibody.
  • the antibody is a polyclonal antibody.
  • the antibody is a bispecific antibody including a first antigen-binding domain (also referred to herein as “D1”), and a second antigen-binding domain (also referred to herein as “D2”).
  • the expression “antigen-binding domain” means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding a particular antigen of interest (e.g., PRLR, STEAP2, HER2, FelD1, and/or FGFR2).
  • a particular antigen of interest e.g., PRLR, STEAP2, HER2, FelD1, and/or FGFR2
  • the term “specifically binds” or the like, as used herein, means that the antigen-binding domain forms a complex with a particular antigen characterized by a dissociation constant (KD) of 1 ⁇ M or less, and does not bind other unrelated antigens under ordinary test conditions.
  • KD dissociation constant
  • “Unrelated antigens” are proteins, peptides, or polypeptides that have less than 95% amino acid identity to one another.
  • Exemplary categories of antigen-binding domains that can be used in the context of the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, e.g., Boersma and Pluckthun, 2011, Curr.
  • an antigen-binding domain includes polypeptides that bind a particular antigen (e.g., a target molecule [T] or an internalizing effector protein [E]) or a portion thereof with a KD of less than about 1 ⁇ M, less than about 500 nM, less than about 250 nM, less than about 125 nM, less than about 60 nM, less than about 30 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM,
  • a particular antigen e.g.,
  • the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. [0084] In some embodiments, the antibody is an anti-PSMA, anti-PRLR, anti-MUC16, anti- HER2, Her2, or anti-Her2, anti-EGFRvIII, anti-FelD1, anti-FGFR2, FGFR2, or anti-STEAP2 antibody. In some embodiments, the antibody is an anti-PRLR or anti HER2 antibody. In some embodiments, the antibody, or antigen-binding fragment thereof, is anti-STEAP2. In some embodiments, the antibody, or antigen-binding fragment thereof, is anti-PRLR.
  • the antibody, or antigen-binding fragment thereof is aHer2. In some embodiments, the antibody, or antigen-binding fragment thereof, is aFelD1. In some embodiments, the antibody, or antigen-binding fragment thereof, is aFGFR2. [0085]
  • the antibody can have binding specificity for any antigen deemed suitable to those of skill in the art.
  • the antigen is a transmembrane molecule (e.g., receptor). In one embodiment, the antigen is expressed on a tumor.
  • the binding agents interact with or bind to tumor antigens, including antigens specific for a type of tumor or antigens that are shared, overexpressed, or modified on a particular type of tumor.
  • the antigen is expressed on solid tumors.
  • Exemplary antigens include, but are not limited to, lipoproteins; alpha1-antitrypsin; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; fibroblast growth factor receptor 2 (FGFR2), EpCAM, GD3, FelD1, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD
  • the binding agent is adalimumab or infliximab. In some embodiments, the binding agent is alemtuzumab, muromonab, rituximab, tosituzumab, or agonistic antibodies (where immune stimulation might be part of the intended mechanism of action).
  • the antigen is PRLR or HER2. In some embodiments, the antigen is STEAP2. In some embodiments the antigen is human STEAP2. In some embodiments, the antigen is Her2. In some embodiments, the antigen is FelD1. In some embodiments, the antigen is FGFR2. In some examples, the MAGE proteins are selected from MAGE-1, -2, -3, -4, -6, and -12.
  • the GAGE proteins are selected from GAGE-1 and GAGE-2.
  • Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, GPNMB, MSR1, and UPK3A.
  • Exemplary antigens also include, but are not limited to, MUC16, STEAP2, and HER2.
  • the antigens include MUC16.
  • the antigens include STEAP2.
  • the antigens include PSMA.
  • the antigens include MSR1.
  • the antigens include HER2.
  • the antigen is prolactin receptor (PRLR) or prostate-specific membrane antigen (PSMA).
  • the antigen is MUC16. In some embodiments, the antigen is HER2. In some embodiments, the antigen is STEAP2. In some embodiments, the antigen is MSR1. [0088] In certain embodiments, the antibody comprises a glutamine residue at one or more heavy chain positions numbered 295 in the EU numbering system. In the present disclosure, this position is referred to as glutamine 295, or as Gln295, or as Q295. Those of skill will recognize that this is a conserved glutamine residue in the wild type sequence of many antibodies. In other useful embodiments, the antibody can be engineered to comprise a glutamine residue. In certain embodiments, the antibody is glycosylated, for instance at N297.
  • the antibody is deglycosylated. In certain embodiments, the antibody is aglycosylated. In certain embodiments, the antibody comprises one or more N297 mutations. In certain embodiments, the antibody comprises one or more N297Q mutations. Techniques for modifying an antibody sequence to include a glutamine residue are within the skill of those in the art (see, e.g., Ausubel et al. Current Protoc. Mol. Biol.). [0089] In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets STEAP2.
  • Sutiable anti-STEAP antibodies or antigen-binding fragments thereof include those, for example, in International Publication No. WO 2018/058001 A1, including those comprising amino acid sequences disclosed in Table 1, on page 75 therein.
  • an anti- STEAP2 antibody is H1H7814N of WO 2018/058001 A1, comprising the CDRs of H1M7814N in the same publication.
  • an anti-STEAP2 antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 2; an HCDR2 comprising SEQ ID NO: 3; an HCDR3 comprising SEQ ID NO: 4; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 6; an LCDR2 comprising SEQ ID NO: 7; and an LCDR3 comprising SEQ ID NO: 8.
  • HCDR heavy chain complementarity determining region
  • LCVR light chain variable region
  • the anti-STEAP2 antibody can be prepared by site-directed mutagenesis to insert a glutamine residue at a site without resulting in disabled antibody function or binding.
  • the anti-STEAP2 antibody can comprise an Asn297Gln (N297Q) mutation.
  • Such antibodies having an N297Q mutation can also contain one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to, for example, transglutaminase and therefore capable of conjugation to a linker compound, payload, or a linker-payload (Table A).
  • the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO:1; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) amino acid sequence of SEQ ID NO:5.
  • the antibody or antigen-binding fragment thereof comprises an HCVR amino acid sequence of SEQ ID NO:1; and an LCVR amino acid sequence of SEQ ID NO:5.
  • the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets human prolactin receptor (PRLR).
  • PRLR human prolactin receptor
  • Suitable anti-PRLR antibodies or antigen-binding fragments thereof include those, for example, in International Publication No. WO 2015/026907 A1, including those comprising amino acid sequences disclosed in Table 1, on page 36 therein.
  • an anti-PRLR antibody is H1H6958N2 of WO 2015/026907 A1, comprising the CDRs of H2M6958N2 in the same publication
  • PRLR includes both monomeric and multimeric PRLR molecules, such as those described in WO 2015/026907.
  • an anti-PRLR antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 10; an HCDR2 comprising SEQ ID NO: 11; an HCDR3 comprising SEQ ID NO: 12; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 14; an LCDR2 comprising SEQ ID NO: 15; and an LCDR3 comprising SEQ ID NO: 16.
  • an anti-PRLR antibody comprises a heavy chain variable region (HCVR) comprising SEQ ID NO: 9 and a light chain variable region (LCVR) comprising SEQ ID NO: 13.
  • the anti- PRLR antibody can be prepared by site-directed mutagenesis to insert a glutamine residue at a site without resulting in disabled antibody function or binding.
  • the anti-PRLR antibody can comprise an Asn297Gln (N297Q) mutation.
  • Such antibodies having an N297Q mutation can also contain one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to, for example, transglutaminase and therefore capable of conjugation to a linker compound, payload, or a linker-payload (Table A).
  • the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO:9; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) amino acid sequence of SEQ ID NO:13.
  • the antibody or antigen-binding fragment thereof comprises an HCVR amino acid sequence of SEQ ID NO:9; and an LCVR amino acid sequence of SEQ ID NO:13.
  • International Publication No. WO 2015/026907 A1 is hereby incorporated herein by reference in its entirety. Table A. Sequences of Exemplary Antibodies H1H7814N (anti-STEAP2) and H1H6958N (anti-PRLR)
  • the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets Her2. In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets FelD1. In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets FGFR2.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table A paired with any of the LCVR amino acid sequences listed in Table A.
  • this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-STEAP2 antibodies listed in Table A.
  • the HCVR/LCVR amino acid sequence pair is selected from the group consisting of: 250/258; as described in International Publication No.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCDR3 heavy chain CDR3
  • LCDR1 light chain CDR1
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • LCDR2 light chain CDR2
  • LCDR3 light chain CDR3
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table A paired with any of the LCDR3 amino acid sequences listed in Table A.
  • this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-STEAP2 antibodies listed in Table A.
  • the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of: 256/254; as described in International Publication No.
  • WO 2018/058001 A1 the contents of which are incorporated herein by reference in its entirety.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-STEAP2 antibodies listed in Table A.
  • the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of: 252-254-256-260-262-264; as described in International Publication No. WO 2018/058001 A1, the contents of which are incorporated herein by reference in its entirety.
  • this disclosure provides antibodies, or antigen-binding fragments thereof that specifically bind STEAP2, comprising a set of six CDRs (i.e., HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-STEAP2 antibodies listed in Table A.
  • this disclosure includes antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of: 250/258; as described in International Publication No.
  • WO 2018/058001 A1 the contents of which are incorporated herein by reference in its entirety.
  • Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
  • Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Kabat and Chothia approaches.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table A paired with any of the LCVR amino acid sequences listed in Table A.
  • this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-PRLR antibodies listed in Table A.
  • the HCVR/LCVR amino acid sequence pair is selected from the group consisting of: 18/26; 66/74; 274/282; 290/298; and 370/378; as described in International Publication No.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a heavy chain CDR1 (HCDR1 ) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCDR1 heavy chain CDR1
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a light chain CDR1 (LCDR1 ) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • LCDR1 light chain CDR1
  • LCDR2 light chain CDR2
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table A paired with any of the LCDR3 amino acid sequences listed in Table A.
  • this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-PRLR antibodies listed in Table A.
  • the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of: 24/32; 72/80; 280/288; 296/304; and 376/384; as described in International Publication No. WO 2015/026907 A1, the contents of which are incorporated herein by reference in its entirety.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3) contained within any of the exemplary anti-PRLR antibodies listed in Table A.
  • the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of: 20-22-24-28-30-32; 68-70-72-76- 78-80; 276-278-280-284-286-288; 292-294-296-300-302-304; and 372-374-376-380-382-384; as described in International Publication No.
  • this disclosure provides antibodies, or antigen-binding fragments thereof that specifically bind PRLR, comprising a set of six CDRs (i.e., HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-PRLR antibodies listed in Table A.
  • this disclosure includes antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of: 18/26; 66/74; 274/282; 290/298; and 370/378; as described in International Publication No. WO 2015/026907 A1, the contents of which are incorporated herein by reference in its entirety.
  • Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
  • Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol.273:927- 948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).
  • BA is an antibody, or antigen binding fragment thereof, that binds PRLR. In any of the compound or conjugate embodiments provided, BA is an antibody, or antigen binding fragment thereof, that binds Her. In any of the compound or conjugate embodiments provided, BA is an antibody, or antigen binding fragment thereof, that binds FelD1. In any of the compound or conjugate embodiments provided, BA is an antibody, or antigen binding fragment thereof, that binds FGFR2. In any of the compound or conjugate embodiments provided, BA is an antibody or antigen-binding fragment thereof, and conjugation is through at least one Q295 residue.
  • BA is an antibody or antigen-binding fragment thereof, and conjugation is through two Q295 residues.
  • BA is a N297Q antibody or antigen-binding fragment thereof.
  • BA is a N297Q antibody or antigen-binding fragment thereof, and conjugation is through at least one Q295 and at least one Q297 residue.
  • BA is a N297Q antibody or antigen-binding fragment thereof, and conjugation is through two Q295 residues and two Q297 residues.
  • numbering is according to the EU numbering system.
  • BA is an anti-MSR1 antibody.
  • BA is the anti-MSR1 antibody H1H21234N.
  • BA is the anti-MSR1 antibody H1H21234N N297Q.
  • BA is an anti-MSR1 antibody comprising an HCVR according to SEQ ID NO:19 and an LCVR according to SEQ ID NO: 27.
  • BA is an anti-MSR1 antibody comprising one, two, three, four, five, or six of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to SEQ ID NOS: 21, 23, 25, 29, 31, and 33, respectively.
  • the HCVR is encoded by SEQ ID NO:18. In certain embodiments, the LCVR is encoded by SEQ ID NO: 26. In certain embodiments, one, two, three, four, five, or six of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are encoded by the polynucleotide sequences SEQ ID NOS: 20, 22, 24, 28, 30, and 32, respectively.
  • N297Q indicates that one or more residues 297 are mutated from asparagine (N) to glutamine (Q). In certain embodiments, each residue 297 is mutated to Q. In certain embodiments, numbering is according to the EU numbering system.
  • the drug:antibody ratio is from one to twenty-four.
  • DAR is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, or twenty-four.
  • DAR is two.
  • DAR is four.
  • DAR is eight.
  • DAR is twelve.
  • DAR is sixteen.
  • DAR is twenty-four.
  • This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table B, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table B, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCDR3 heavy chain CDR3
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a light chain CDR1 (LCDR1 ) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • LCDR1 light chain CDR1
  • LCDR2 light chain CDR2
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table B paired with any of the LCDR3 amino acid sequences listed in Table B.
  • this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-MSR1 antibodies listed in Table B.
  • the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of: 8/16, 40/48, 56/64, 96/104, and 288/296; as described in International Publication No. WO 2019/217591 A1, the contents of which are incorporated herein by reference in its entirety.
  • This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3) contained within any of the exemplary anti-MSR1 antibodies listed in Table B.
  • the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of: 4-6-8-12-14-16; 36-38-40-44-46-48; 52- 54-56-60-62-64; 92-94-96-100-102-104, and 284-286-288-292-294-296; as described in International Publication No.
  • this disclosure provides antibodies, or antigen-binding fragments thereof that specifically bind MSR1, comprising a set of six CDRs (i.e., HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-MSR1 antibodies listed in Table B.
  • this disclosure includes antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of: 2/10, 23/42, 50/58, 90/98, and 282/290; as described in International Publication No. WO 2019/217591 A1, the contents of which are incorporated herein by reference in its entirety.
  • Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
  • Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol.273:927- 948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).
  • binding agent linkers can be bonded to the binding agent, for example, antibody or antigen-binding molecule, through an attachment at a particular amino acid within the antibody or antigen-binding molecule.
  • Exemplary amino acid attachments that can be used in the context of this embodiment of the disclosure include, for example, lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808; US 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al., Proc.
  • lysine see, e.g., US 5,208,020; US 2010/0129314; Hollander et al.,
  • Linkers including the linkers described herein, can also be conjugated to one or more glutamine residues via transglutaminase-based chemo-enzymatic conjugation (see, e.g., Dennler et al., Bioconjugate Chem. 2014, 25, 569-578).
  • transglutaminase one or more glutamine residues of an antibody can be coupled to a primary amine compound or a linker compound described herein.
  • Primary amine compounds include, for example, payloads, linkers, linkers described herein, or linker-payloads, which directly provide antibody drug conjugates via transglutaminase-mediated coupling.
  • Primary amine compounds also include linkers, including linkers described herein, and spacers that are functionalized with reactive groups that can be subsequently treated with further compounds towards the synthesis of antibody-drug conjugates (i.e., click chemistry and/or inverse electron deman Diels-Alder chemistry as described elsewhere herein).
  • Antibodies comprising glutamine residues can be isolated from natural sources or engineered to comprise one or more glutamine residues. Techniques for engineering glutamine residues into an antibody polypeptide chain (glutaminyl- modified antibodies or antigen binding molecules) are within the skill of the practitioners in the art. In certain embodiments, the antibody is aglycosylated.
  • the antibody or a glutaminyl-modified antibody or a transglutaminse-modified antibody or antigen binding molecule comprises at least one glutamine residue in at least one polypeptide chain sequence.
  • the antibody or a glutaminyl-modified antibody or a transglutaminase-modified antibody or antigen binding molecule comprises two heavy chain polypeptides, each with one Gln295 or Q295 residue.
  • the antibody or a glutaminyl-modified antibody or transglutaminase- modified antibody or antigen binding molecule comprises one or more glutamine residues at a site other than a heavy chain 295.
  • primary amine compounds useful for the transglutaminase- mediated coupling of an antibody (or antigen binding compound) comprising a glutamine can be any primary amine compound deemed useful by the practitioner of ordinary skill.
  • the primary amine compound has the formula H 2 N-R, where R can be any group compatible with the antibody and reaction conditions.
  • R is alkyl, substituted alkyl, heteroalkyl, or substituted heteroalkyl.
  • the primary amine compound comprises a reactive group or protected reactive group.
  • Useful reactive groups include azides, alkynes, dienophiles, dienes, cycloalkynes, thiols, alcohols, ketones, aldehydes, carboxylic acids, esters, amides, hydrazides, anilines, and amines.
  • the reactive group is selected from the group consisting of azide, alkyne, sulfhydryl, cycloalkyne, aldehyde, dienophile, diene, and carboxyl.
  • the primary amine compound is according to the formula H2N-LL-X, where LL is a divalent spacer and X is a reactive group or protected reactive group.
  • LL is a divalent polyethylene glycol (PEG) group.
  • X is selected from the group consisting of –SH, –N 3 , alkyne, aldehyde, and tetrazole. In particular embodiments, X is -N 3 .
  • the primary amine compound is according to the formula (R 1 )(R 2 )N-W-X-Y-Z as described elsewhere herein.
  • the primary amine compound is according to one of the following formulae: H2N-(CH2)n-X; H 2 N-(CH 2 CH 2 O)n-(CH 2 )p-X; H 2 N-(CH 2 )n-N(H)C(O)-(CH 2 )m-X; H 2 N-(CH 2 CH 2 O)n-N(H)C(O)-(CH 2 CH 2 O)m-(CH 2 )p-X; H 2 N-(CH 2 )n-C(O)N(H)-(CH 2 )m-X; H 2 N-(CH 2 CH 2 O)n-C(O)N(H)-(CH 2 CH 2 O)m-(CH 2 ) p -X; H 2 N-(CH 2 )n-N(H)C(O)-(CH 2 CH 2 O)m-(CH 2 )p-X; H 2 N-(CH 2 )n-N(H)C(O)-(CH 2 CH 2 O)m
  • any of the alkyl or alkylene (i.e., –CH 2 –) groups can optionally be substituted, for example with C 1-8 alkyl, methylformyl, or –SO 3 H.
  • the alkyl groups are unsubstituted.
  • the primary amine compound is selected from the group consisting of:
  • linker L portion of the conjugates described herein is a moiety, for instance a divalent moiety, that covalently links a binding agent to a payload compound described herein.
  • linker L is a trivalent or multivalent moiety that covalently links a binding agent to a payload compound described herein. Suitable linkers may be found, for example, in Antibody-Drug Conjugates and Immunotoxins; Phillips, G.
  • the linker L portion of the linker-payloads described herein is a moiety covalently linked to a payload compound described herein, capable of divalently and covalently linking a binding agent to a payload compound described herein.
  • the linker L portion of the linker-payloads described herein is a moiety covalently linked to a payload compound described herein, capable of covalently linking, as a trivalent or multivalent moiety, a binding agent to a payload compound described herein.
  • Payload compounds include MMAE, exatecan, DXd, and the like, and their residues following bonding or incorporation with linker L are linker-payload compounds.
  • the linker-payloads can be further bonded to binding agents such as antibodies or antigen binding fragments thereof to form antibody-drug conjugates. Those of skill in the art will recognize that certain functional groups of payload moieties are convenient for linking to linkers and/or binding agents.
  • the linker is absent and payloads are directly bonded to binding agents.
  • prodrugs or payloads include hydroxyl, amine, or thiol functionality capable of bonding with linkers and/or peptide residues within binding agents.
  • the linkers are stable in physiological conditions.
  • the linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value.
  • a linker comprises an enzyme-cleavable moiety.
  • Illustrative enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages.
  • the linker comprises a cathepsin-cleavable linker.
  • the linker comprises a moiety that is stable at certain pHs and cleavable to release the payload portion at other pHs. For instance, in certain embodiments, the linker is stable at physiological pH and capable of releasing the payload portion at a local pH in the vicinity of a target.
  • the linker comprises a non-cleavable moiety. In some embodiments, the non-cleavable linker is derived from maleimide.
  • the non-cleavable linkers are derived from an ester. In some embodiments, the non-cleavable linker is derived from an N-hydroxysuccinimide ester. In some embodiments, the non-cleavable linkers are derived from a dienophile (e.g., alkenes or alkynes). In some embodiments, the non-cleavable linkers are derived from a diene (e.g., a tetrazine). In some embodiments, the non-cleavable linker is derived from or a residue thereof. In some embodiments, the non-cleavable linker-payload residue is , or a regioisomer thereof.
  • the non-cleavable linker is derived from or a residue thereof. In some embodiments, the non-cleavable linker-payload residue is , or a regioisomer thereof.
  • the linker is maleimide cyclohexane carboxylate or 4-(N- maleimidomethyl)cyclohexanecarboxylic acid (MCC), where the payload can be added to either end of the MCC linker. In another embodiment, the linker is , where the payload can be added to either end of this linker. In certain embodiments, the linker is a self-stabilizing maleimide.
  • the self-stabilizing maleimide linker is , where the bond from the amide nitrogen to the payload can be a direct bond to the payload; or the bond from the amide nitrogen to the payload, as shown, contemplates the remainder of the linker.
  • the self-stabilizing linker manifests as , where the bond from the amide nitrogen to the payload can be a direct bond to the payload; or the bond from the amide nitrogen to the payload, as shown, contemplates the remainder of the linker.
  • the self-stabilizing linker includes moieties that stabilize the bond from the self-stabilizing linker to a binding agent.
  • the self-stabilizing linker when the bond from a binding agent to a self-stabilizing linker is a carbon-sulfur bond (e.g., following a Michael addition of a binding agent cysteine to the self-stabilizing maleimide linker), the self-stabilizing linker mitigates retro-Michael additions.
  • the aminomethyl functionality facilitates rapid hydrolysis of the succinimide Michael addition product to provide , where the bond from the amide nitrogen to the payload can be a direct bond to the payload; or the bond from the amide nitrogen to the payload, as shown, contemplates the remainder of the linker; thus, decreasing susceptibility to retro-Michael additions.
  • Moieties other than aminomethyl within self-stabilizing maleimide linkers that stablilize conjugates will be appreciated by those of skill in the art. In the structures, indicates a bond to a binding agent.
  • the structures in some examples, indicates a click chemistry residue which results from the reaction of, for example, a binding agent having an azide or alkyne functionality and a linker-payload having a complementary alkyne or azide functionality.
  • suitable linkers include, but are not limited to, those that are chemically bonded to two cysteine residues of a single binding agent, for example, an antibody or antigen binding fragment thereof.
  • linkers can serve to mimic the antibody’s disulfide bonds that are disrupted as a result of the conjugation process.
  • the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D- ⁇ -amino acids.
  • the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or any combination thereof (e.g., dipeptides, tripeptides, oligopeptides, polypeptides, and the like).
  • one or more side chains of the amino acids are linked to a side chain group, described below.
  • the linker is a peptide comprising or consisting of the amino acids valine and citrulline (e.g., divalent –Val-Cit– or divalent –VCit–). In some embodiments, the linker is a peptide comprising or consisting of the amino acids alanine and alanine, or divalent –AA–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid and alanine, or –EA–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid and glycine, or –EG–.
  • the linker is a peptide comprising or consisting of the amino acids glycine and glycine, or –GG–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamine, valine, and citrulline, or –Q-V- Cit– or –QVCit–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid, valine, and citrulline, or –E-V-Cit– or –EVCit–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –GGGGS–.
  • the linker is a peptide comprising or consisting of the amino acids –GGGGG–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –GGGGK–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –GFGG–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids lysine, valine, and citrulline, or –KVCit–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –KVA–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –VA–.
  • the linker is , , or wherein SP is a spacer SP 1 is a spacer; SP 2 is a spacer; SP 3 is a spacer; is one or more bonds to the binding agent; is one or more bonds to the payload; each AA is an amino acid residue; R 8 is alkylene or heteroalkylene; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent;and p is an integer from zero to ten. [00149] In one embodiment, SP is .
  • the SP 1 spacer is a moiety that connects the (AA) n moiety or residue to the binding agent (BA) or to a reactive group residue which is bonded to BA.
  • Suitable SP 1 spacers include, but are not limited to, those comprising alkylene or polyether, or both.
  • the ends of the spacers can be moieties derived from reactive moieties that are used for purposes of coupling the antibody or an AA to the spacer during chemical synthesis of the conjugate.
  • n is one, two, three, or four. In particular embodiments, n is two. In particular embodiments, n is three. In particular embodiments, n is four. In certain embodiments, when n is zero, then (AA) n is a bond.
  • p is one, two, three, or four. In particular embodiments, p is two.
  • the SP 1 spacer comprises an alkylene. In some embodiments, the SP 1 spacer comprises a C5-7 alkylene. In some embodiments, the SP 1 spacer comprises a polyether. In some embodiments, the SP 1 spacer comprises a polymer of ethylene oxide such as polyethylene glycol.
  • the SP 1 spacer is , , , or wherein RG' is a reactive group residue following reaction of a reactive group RG with a binding agent; is a bond to the binding agent; is a bond to (AA) n wherein n is an integer from zero to ten; and b is an integer from two to eight.
  • the reactive group RG can be any reactive group known to those of skill in the art to be capable of forming one or more bonds to the binding agent.
  • the reactive group RG is a moiety comprising a portion in its structure that is capable of reacting with the binding agent (e.g., reacting with an antibody at its cysteine or lysine residues; or at an azide moiety, for example, a PEG-N3 functionalized antibody at one or more glutamine residues; or at a dienophile moiety, for example, a dienophile functionalized antibody at one or more glutamine residues; or a diene moiety, for example, a diene functionalized antibody at one or more glutamine residues) to form a conjugate.
  • the reactive group becomes the reactive group residue (RG′).
  • Illustrative reactive groups include, but are not limited to, those that comprise haloacetyl, isothiocyanate, succinimide, N-hydroxysuccinimide, dienophile, diene, or maleimide portions that are capable of reacting with the binding agent or functionalized binding agent.
  • reactive groups include, but are not limited to, alkynes, dienophiles, and dienes.
  • the alkynes are alkynes capable of undergoing 1,3-cycloaddition reactions with azides in the absence of copper catalysts, such as strained alkynes.
  • the dienes are dienes (e.g., tetrazines) capable of undergoing inverse electron demand Diels- Alder reactions with dienophiles. Suitable dienophiles include, but are not limited to, , , and . Suitable dienes include, but are not limited to, and . [00154]
  • the binding agent is bonded directly to RG′. In certain embodiments, the binding agent is bonded to RG′ via a spacer, for instance SP 4 , located between and RG′. In particular embodiments, the binding agent is bonded indirectly to RG′ via SP 4 , for example, a PEG spacer.
  • the binding agent is prepared by functionalizing with one or more azido groups. Each azido group is capable of reacting with RG to form RG′.
  • the binding agent is derivatized with –PEG-N 3 linked to a glutamine residue.
  • Exemplary -N 3 derivatized binding agents, methods for their preparation, and methods for their use in reacting with RG are provided herein.
  • RG is an alkyne suitable for participation in 1,3-cycloadditions
  • RG′ is a regioisomeric 1,2,3-triazolyl moiety formed from the reaction of RG with an azido- functionalized binding agent.
  • RG′ is linked to the binding agent as shown in or , or a mixture of each regioisomer.
  • Each R and R′ is as described or exemplified herein.
  • the SP 2 spacer when present, is a moiety that connects the (AA) n or (AA) p moiety to the binding agent.
  • the SP 2 spacer when present, can be any spacer suitable for connection of the (AA) n or (AA) p moiety to the binding agent, for example, those comprising alkylene or polyether, or both.
  • the SP 2 spacer, when present, is where b is an integer from one to ten.
  • the SP 3 spacer when present, is a moiety that connects the (AA) n or (AA) p moiety to the payload.
  • Suitable spacers include, but are not limited to, those described above as SP 1 spacers.
  • Further suitable SP 3 spacers include, but are not limited to, those comprising alkylene or polyether, or both.
  • the ends of the SP 3 spacers for example, the portion of the spacer directly bonded to the payload or an AA, can be moieties derived from reactive moieties that are used for purposes of coupling the payload or AA to the SP 3 spacer during the chemical synthesis of the conjugate.
  • the ends of the SP 3 spacers for example, the portion of the SP 3 spacer directly bonded to the payload or an AA, can be residues of reactive moieties that are used for purposes of coupling the payload or an AA to the spacer during the chemical synthesis of the conjugate.
  • the SP 3 spacer when present, is selected from the group consisting of –NH-(p-C 6 H 4 )-CH 2 –, –NH-(p-C 6 H 4 )-CH 2 OC(O)–, an amino acid, a dipeptide, a tripeptide, an oligopeptide, –O–, –N(H)–, , , , , , , , , ,
  • each is a bond to the payload, and each is a bond to (AA) n or (AA) p [00158]
  • each (AA) n or (AA) p is an amino acid or, optionally, a p- aminobenzyloxycarbonyl residue (PABC). If PABC is present, in certain embodiments, then only one PABC is present. In certain embodiments, the PABC residue, if present, is bonded to a terminal AA in the (AA) n group, proximal to the payload.
  • Suitable amino acids for each AA include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D- ⁇ -amino acids.
  • the AA comprises alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or any combinations thereof (e.g., dipeptides, tripeptides, and oligopeptides, and the like).
  • one or more side chains of the amino acids is linked to a side chain group, described below.
  • n or p is two.
  • the (AA) n or (AA) p is valine-citrulline. In some embodiments, (AA) n or (AA) p is citrulline- valine. In some embodiments, (AA) n or (AA) p is valine-alanine. In some embodiments, (AA) n or (AA) p is alanine-valine. In some embodiments, (AA) n or (AA) p is valine-glycine. In some embodiments, (AA) n or (AA) p is glycine-valine. In some embodiments, n or p is three. In some embodiments, the (AA) n or (AA) p is valine-citrulline-PABC.
  • (AA) n or (AA) p is citrulline-valine-PABC. In some embodiments, (AA) n or (AA) p is glutamate-valine- citrulline. In some embodiments, (AA) n or (AA) p is glutamine-valine-citrulline. In some embodiments, (AA) n or (AA) p is lysine-valine-alanine. In some embodiments, (AA) n or (AA) p is lysine-valine-citrulline. In some embodiments, n or p is four. In some embodiments, (AA) n or (AA) p is glutamate-valine-citrulline-PABC.
  • (AA) n or (AA) p is glutamine- valine-citrulline-PABC.
  • PABC as a residue of p- aminobenzyloxycarbonyl with the following structure: .
  • the PABC residue has been shown to facilitate cleavage of certain linkers in vitro and in vivo.
  • PAB as a divalent residue of p-aminobenzyl or –NH-(p-C 6 H 4 )-CH 2 –.
  • linker-payloads include any specific compound, prodrug, or payload described herein, bonded to a linker, wherein the linker(s) described herein include a moiety that is reactive with an antibody or antigen binding fragment thereof described herein.
  • the compound or linker-payload is a structure of Formula III or IV (III) or (IV) or a pharmaceutically acceptable salt thereof, wherein X is or ; SP 1 , SP 2 , and SP 3 , when present, are in each instance independent spacer groups wherein SP 1 further comprises the moiety reactive with the binding agent; each AA is an amino acid; and p is an integer from zero to ten.
  • the linker further comprises .
  • p is zero. In one embodiment, p is one. In one embodiment, p is two. In one embodiment, p is three. In one embodiment, p is four. In one embodiment, p is five. In one embodiment, p is six. In one embodiment, p is seven. In one embodiment, p is eight. In one embodiment, p is nine. In one embodiment, p is ten.
  • SP 1 comprises a reactive group that comprises an alkene, alkyne, , and/or . In one embodiment, the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction.
  • the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels- Alder reaction to form regioisomeric Diels-Alder adduct moieties
  • the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent
  • SP 3 comprises .
  • the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction to form regioisomeric Diels- Alder adduct moieties
  • the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent
  • SP 3 comprises
  • R 2 is hydrogen or benzyl.
  • the binding agent is an antibody or antigen binding fragment thereof.
  • the compound or linker-payload is a structure of Formula III (III) wherein R 1a , R 1b , R 2 , and R 3 are hydrogen; and m is one.
  • the compound or linker-payload is a structure of Formula III (III) wherein R 1a , R 1b , and R 3 are hydrogen; R 2 is an amino acid side chain; and m is one.
  • the compound or linker- payload is a structure of Formula III (III) wherein R 1a , R 1b , R 2 , and R 3 are hydrogen; R 4 is alkyl; and m is one.
  • R 1a , R 1b , R 2 , and R 3 are hydrogen; and m is two or four. In one embodiment, m is two. In one embodiment, m is four.
  • D* is a residue of a maytansinoid. In one embodiment, D* is a residue of a tubulysin. In one embodiment, D* is a residue of an auristatin. In one embodiment, D* is a residue of a dolastatin. In one embodiment, D* is a residue of a camptothesin. In one embodiment, D* is a residue of a pyrrolobenzodiazepine. In one embodiment, D* is a residue of an antibiotic. In one embodiment, D* is a residue of an antiviral agent. In one embodiment, D* is a residue of an anti-inflammatory agent. In one embodiment, D* is a residue of an immunomodulator.
  • D* is a residue of an antifungal agent. In one embodiment, D* is a residue of a steroid. In one embodiment, D* is a residue of an imaging agent. In certain embodiments in this paragraph, D* is a residue of a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, or an imaging agent moiety, or an analogue or derivative thereof.
  • the binding agent is an antibody or antigen binding fragment thereof.
  • Conjugates/Antibody-Drug Conjugates (ADCs) [00161] Provided herein are binding agents and antibodies or antigen binding fragments thereof, wherein said binding agent or antibody is conjugated to one or more compounds described herein.
  • a compound wherein the compound is a binding agent or transglutaminase-modified binding agent comprising a compound of the formula (R 1 )(R 2 )N-W-X-Y-Z wherein W is C 1 -C 10 alkylene or C 1 -C 10 alkylene-PEGn-C 1 -C 10 alkylene;
  • X is -N(R 3 )C(O)-C 1 -C 10 alkylene, -C(O)N(R 3 )-C 1 -C 10 alkylene-C(O)-(AA) p -N(R 3 )-, -C(O)- (AA) p -N(R 3 )-, -C(O)- (AA) p -N(R 3 )-, -C(O)N(R 3 )-, -N(R 5 )2, -C(O)N(R 5 )2, -CH(R 5 )(R 6 ),
  • the binding agent further comprises a compound of (I) or (II) or a pharmaceutically acceptable salt thereof, wherein L is a linker comprising a moiety reactive with a binding agent; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent; SP is a spacer group; R 1a and R 1b are, independently, hydrogen or alkyl; R 2 is hydrogen or an amino acid side chain; R 3 , R 4 , and R 6 is hydrogen or alkyl; R 5 is oxygen, NR 6 , or sulfur; R 7 is an O-amino acid residue; R 8 is C 1 -C 10 alkylene or C 1 -C 10 heteroalkylene; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein when m is four,
  • the binding agent or transglutaminase-modified binding agent compound is , wherein BA is the binding agent.
  • the binding agent is an antibody or transglutaminase-modified antibody, or antigen binding fragments thereof.
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof are selected from an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRvIII antibody, an anti-MUC16 antibody, an anti-prolactin receptor (PRLR) antibody, an anti-prostate- specific membrane antigen (PSMA) antibody, an anti-FGFR2 antibody, an anti-FOLR1 antibody, an anti-HER2/HER2 bispecific antibody, an anti-MET/MET bispecific antibody, or an antigen-binding fragment thereof.
  • the antibody or transglutaminase- modified antibody, or antigen binding fragments thereof is an anti-HER2 antibody or an antigen- binding fragment thereof.
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-STEAP2 antibody or an antigen- binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MET antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-EGFRvIII antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MUC16 antibody or an antigen-binding fragment thereof.
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-prolactin receptor (PRLR) antibody or an antigen-binding fragment thereof.
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-prostate-specific membrane antigen (PSMA) antibody or an antigen-binding fragment thereof.
  • PSMA anti-prostate-specific membrane antigen
  • the antibody or transglutaminase- modified antibody, or antigen binding fragments thereof is an anti-FGFR2 antibody or an antigen-binding fragment thereof.
  • the antibody or transglutaminase- modified antibody, or antigen binding fragments thereof is an anti-FOLR1 antibody or an antigen-binding fragment thereof.
  • a conjugate comprising (R 1 )(R 2 )N-W-X-Y-Z wherein R 1 , R 2 , W, X, Y, and Z are as described elsewhere herein; LP1; LP7; (I); or (II) wherein L, X, T, SP, R 1a , R 1b , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , D*, m, and each n are as described elsewhere herein.
  • the conjugate is selected from the group consisting of ADC10; ADC11; ADC12; ADC13; ADC17; ADC22; ADC23;
  • the binding agent is an antibody or transglutaminase-modified antibody, or antigen binding fragments thereof.
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof are selected from an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti- EGFRvIII antibody, an anti-MUC16 antibody, an anti-prolactin receptor (PRLR) antibody, an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-FGFR2 antibody, an anti- FOLR1 antibody, an anti-HER2/HER2 bispecific antibody, an anti-MET/MET bispecific antibody, or an antigen-binding fragment thereof.
  • PRLR anti-prolactin receptor
  • PSMA anti-prostate-specific membrane antigen
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-HER2 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-STEAP2 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MET antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-EGFRvIII antibody or an antigen-binding fragment thereof.
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MUC16 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-prolactin receptor (PRLR) antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti- prostate-specific membrane antigen (PSMA) antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-FGFR2 antibody or an antigen-binding fragment thereof.
  • PRLR anti-prolactin receptor
  • PSMA prostate-specific membrane antigen
  • the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-HER2/HER2 bispecific antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MET/MET bispecific antibody or an antigen-binding fragment thereof.
  • the compound or linker-payload used in step (b) is LP7. In one embodiment, the compound or linker-payload used in step (b) is LP3. In one embodiment, the compound or linker-payload used in step (b) is LP9. In one embodiment, the compound or linker-payload used in step (b) is LP8. In one embodiment, the compound or linker-payload used in step (b) is LP10. In one embodiment, the compound or linker-payload used in step (b) is LP11.
  • Linker-payloads can be coupled to cysteine residues, for example, by subjecting the antibody to a reducing agent, for example, dithiotheritol, to cleave the disulfide bonds of the antibody, purifying the reduced antibody, for example, by gel filtration, and subsequently treating the antibody with a linker-payload containing a suitable reactive moiety, for example, a maleimido group (i.e., via Michael addition).
  • Suitable solvents include, but are not limited to water, DMA, DMF, and DMSO.
  • Linkers or Linker-payloads containing a reactive group for example, an activated ester or acid halide group, can be coupled to lysine residues of the antibody.
  • Suitable solvents include, but are not limited to, water, DMA, DMF, and DMSO.
  • Conjugates can be purified using known protein techniques, including, for example, size exclusion chromatography, dialysis, and ultrafiltration/diafiltration.
  • Binding agents for example antibodies, can also be conjugated via click chemistry reactions.
  • the linker-payload includes a reactive group, for example an alkyne, that is capable of undergoing a regioisomeric 1,3-cycloaddition reaction with an azide.
  • suitable reactive groups are described above.
  • the antibody includes one or more azide groups.
  • Such antibodies include antibodies functionalized with, for example, azido-polyethylene glycol groups.
  • such functionalized antibody is derived by treating an antibody having at least two glutamine residues, for example, heavy chain Gln295 and heavy chain Gln297, with a primary amine compound in the presence of the enzyme transglutaminase.
  • Such antibodies include Asn297Gln (N297Q) mutants.
  • the antibody has two heavy chains as described in this paragraph for a total of two or a total of four glutamine residues. Binding agents, for example antibodies, can also be conjugated via inverse electron demand Diels-Alder reactions.
  • the linker-payload includes a reactive group, for example an alkyne, that is capable of undergoing a regioisomeric Diels-Alder reaction with a tetrazine.
  • a reactive group for example an alkyne
  • suitable reactive groups are described above.
  • the antibody includes one or more dienophile or diene compounds.
  • Such antibodies include antibodies functionalized with, for example, (R 1 )(R 2 )N-W- X-Y-Z as described elsewhere herein.
  • such functionalized antibody is derived by treating an antibody having at least two glutamine residues, for example, heavy chain Gln295 and heavy chain Gln297, with a primary amine compound (e.g., (R 1 )(R 2 )N-W-X-Y-Z as described elsewhere herein) in the presence of the enzyme transglutaminase.
  • a primary amine compound e.g., (R 1 )(R 2 )N-W-X-Y-Z as described elsewhere herein
  • Such antibodies include Asn297Gln (N297Q) mutants.
  • the antibody has two heavy chains as described in this paragraph for a total of two or a total of four glutamine residues.
  • the antibody comprises two glutamine residues, one in each heavy chain.
  • the antibody comprises a Q295 residue in each heavy chain.
  • the antibody comprises one, two, three, four, five, six, seven, eight, or more glutamine residues. These glutamine residues can be in heavy chains, light chains, or in both heavy chains and light chains. These glutamine residues can be wild-type residues, or engineered residues, or the antibody can comprise both wild-type and engineered glutamine residues.
  • the antibody is glycosylated. In certain embodiments, the antibody is aglycosylated. In certain embodiments, the antibody is deglycosylated.
  • the antibodies can be prepared according to standard techniques. [00168] Those of skill will recognize that antibodies are often glycosylated at residue N297, near residue Q295 in a heavy chain sequence.
  • the antibody is not glycosylated.
  • the antibody is deglycoslated or aglycosylated.
  • an antibody heavy chain has an N297 mutation.
  • the antibody is mutated to no longer have an asparagine residue at position 297.
  • an antibody heavy chain has an N297Q mutation.
  • Such an antibody can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at a site apart from any interfering glycosylation site or any other interfering structure.
  • Such an antibody also can be isolated from natural or artificial sources.
  • the antibody without interfering glycosylation is then treated with a primary amine compound (e.g., (R 1 )(R 2 )N-W-X-Y-Z as described elsewhere herein).
  • a primary amine compound e.g., (R 1 )(R 2 )N-W-X-Y-Z as described elsewhere herein.
  • an aglycosylated antibody is reacted or treated with a primary amine compound to produce a glutaminyl-modified antibody.
  • a deglycosylated antibody is reacted or treated with a primary amine compound to produce a glutaminyl-modified antibody.
  • the primary amine can be any primary amine that is capable of forming a covalent bond with a glutamine residue in the presence of a transglutaminase.
  • Useful primary amines are described herein (e.g., (R 1 )(R 2 )N-W-X-Y-Z).
  • the transglutaminase can be any transglutaminase deemed suitable by those of skill in the art.
  • transglutaminases have been isolated from Streptomyces mobaraense, Streptomyces cinnamoneum, Streptomyces griseo-carneum, Streptomyces lavendulae, and Bacillus subtilis.
  • Non-microbial transglutaminases including mammalian transglutaminases, can also be used.
  • the transglutaminase can be produced by any technique or obtained from any source deemed suitable by the practitioner of skill. In particular embodiments, the transglutaminase is obtained from a commercial source.
  • the primary amine compound (e.g., (R 1 )(R 2 )N-W-X-Y- Z as described elsewhere herein) comprises a reactive group capable of further reaction after transglutamination.
  • the glutaminyl-modified antibody can be treated with a reactive payload compound or a reactive linker-payload compound to form an antibody- payload conjugate.
  • the primary amine compound comprises an azide.
  • the primary amine compound is (R 1 )(R 2 )N-W-X-Y-Z as described elsewhere herein.
  • the glutaminyl-modified antibody is treated with a reactive linker-payload to form an antibody-payload conjugate.
  • the reaction can proceed under conditions deemed suitable by those of skill in the art.
  • the glutaminyl- modified antibody is contacted with the reactive linker-payload compound under conditions suitable for forming a bond between the glutaminyl-modified antibody and the linker-payload compound. Suitable reaction conditions are well known to those in the art. Exemplary reactions are provided in the Examples below. Accordingly, provided herein is a method of preparing an antibody-drug conjugate comprising contacting a binding agent, as described herein, with a linker or linker-payload, also as described herein.
  • compositions and Methods of Treatment are methods of treating and preventing diseases, conditions, or disorders comprising administering a therapeutically or prophylactically effective amount or one or more of the compounds disclosed herein, for example, one or more of the compounds of a formula provided herein (e.g., (R 1 )(R 2 )N-W-X-Y-Z or Formula I-IV each as described elsewhere herein).
  • Diseases, disorders, and/or conditions include, but are not limited to, those associated with the antigens described herein.
  • the compounds described herein can be administered alone or together with one or more additional therapeutic agents.
  • the one or more additional therapeutic agents can be administered just prior to, concurrent with, or shortly after the administration of the compounds described herein.
  • the compounds described herein can also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and/or NSAIDs.
  • multiple doses of a compound described herein may be administered to a subject over a defined time course.
  • the methods according to this embodiment of the disclosure comprise sequentially administering to a subject multiple doses of a compound described herein.
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses can all include the same amount of a compound described herein, but generally can differ from one another in terms of frequency of administration. In certain embodiments, the amount of the compound included in the initial, secondary, and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • two or more (e.g., two, three, four, or five) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
  • each secondary and/or tertiary dose is administered one to twenty-six (e.g., 1, 11 ⁇ 2, 2, 21 ⁇ 2, 3, 31 ⁇ 2, 4, 41 ⁇ 2, 5, 51 ⁇ 2, 6, 61 ⁇ 2, 7, 71 ⁇ 2, 8, 81 ⁇ 2, 9, 91 ⁇ 2, 10, 101 ⁇ 2, 11, 111 ⁇ 2, 12, 121 ⁇ 2, 13, 131 ⁇ 2, 14, 141 ⁇ 2, 15, 151 ⁇ 2, 16, 161 ⁇ 2, 17, 171 ⁇ 2, 18, 181 ⁇ 2, 19, 191 ⁇ 2, 20, 201 ⁇ 2, 21, 211 ⁇ 2, 22, 221 ⁇ 2, 23, 231 ⁇ 2, 24, 241 ⁇ 2, 25, 251 ⁇ 2, 26, 261 ⁇ 2, or more) weeks after the immediately preceding dose.
  • twenty-six e.g., 1, 11 ⁇ 2, 2, 21 ⁇ 2, 3, 31 ⁇ 2, 4, 41 ⁇ 2, 5, 51 ⁇ 2, 6, 61 ⁇ 2, 7, 71 ⁇ 2, 8, 81 ⁇ 2, 9, 91 ⁇ 2, 10, 101 ⁇ 2, 11, 111 ⁇ 2, 12, 121 ⁇ 2, 13, 131 ⁇ 2, 14, 141 ⁇ 2, 15, 151 ⁇ 2, 16, 161 ⁇ 2, 17, 171 ⁇ 2, 18, 181
  • each secondary dose may be administered at the same frequency as the other secondary doses.
  • each secondary dose may be administered to the patient one to two weeks or one to two months after the immediately preceding dose.
  • each tertiary dose may be administered at the same frequency as the other tertiary doses.
  • each tertiary dose may be administered to the patient two to twelve weeks after the immediately preceding dose.
  • the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • the present disclosure includes administration regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by administration of two or more maintenance doses to the patient on a less frequent basis.
  • the maintenance doses may be administered to the patient once every six weeks, once every two months, once every three months, etc.
  • the present disclosure includes pharmaceutical compositions of the compounds and/or conjugates described herein, for example, the (R 1 )(R 2 )N-W-X-Y-Z compounds and/or compounds of Formulae I-IV, for example, compositions comprising a compound described herein, a salt, stereoisomer, regioisomer, polymorph thereof, and a pharmaceutically acceptable carrier, diluent, and/or excipient.
  • a pharmaceutical composition including the compounds of (R 1 )(R 2 )N-W-X-Y-Z and Formulae I-IV, and a pharmaceutically acceptable excipient, carrier, or diluent.
  • set forth herein is a method of treating a disease, disorder or condition including administering to a patient having said disorder a therapeutically effective amount of a compound set forth herein, or a pharmaceutical composition thereof.
  • set forth herein is a method of treating a disease, disorder or condition including administering to a patient having said disorder a therapeutically effective amount of (R 1 )(R 2 )N-W-X-Y-Z and/or Formulae I-IV, or a pharmaceutical composition thereof.
  • the present disclosure includes methods of preventing certain disorders or conditions comprising administering a therapeutically effective amount of one or more of the compounds disclosed herein (i.e., prophylactic uses). Examples include, but are not limited to preventing cytokine release syndrome for CD3 bispecifics, and adoptive cellular therapies such as CAR T cells, systemic IL-2 administration, graft-versus-host disease, and post-operative nausea and vomiting.
  • Examples also include, but are not limited to, therapeutic antibodies such as alemtuzumab, muromonab, rituximab, tosituzumab, and agonistic antiboides where immune stimulation might be part of the intended mechanism of action.
  • therapeutic antibodies such as alemtuzumab, muromonab, rituximab, tosituzumab, and agonistic antiboides where immune stimulation might be part of the intended mechanism of action.
  • set forth herein is a method for treating a disease, disorder, or condition selected from an autoimmune disease, an allergy, arthritis, asthma, a breathing disorder, a blood disorder, a cancer, a collagen disease, a connective tissue disorder, a dermatological disease, an eye disease, an endocrine problem, an immunological disease, an inflammatory disease, an intestinal disorder, a gastrointestinal disease, a neurological disorder, an organ transplant condition, a rheumatoid disorder, a skin disorder, a swelling condition, a wound healing condition, and combinations thereof, comprising administering a payload or conjugate thereof described herein.
  • a disease, disorder, or condition selected from an autoimmune disease, an allergy, arthritis, asthma, a breathing disorder, a blood disorder, a cancer, a collagen disease, a connective tissue disorder, a dermatological disease, an eye disease, an endocrine problem, an immunological disease, an inflammatory disease, an intestinal disorder, a gastrointestinal disease, a neurological disorder, an organ transplant condition, a r
  • the autoimmune disorder is selected from multiple sclerosis, autoimmune hepatitis, shingles, systemic lupus erythematosus (i.e., lupus), myasthenia gravis, Duchenne muscular dystrophy, and sarcoidosis.
  • the breathing disorder is selected from asthma, chronic respiratory disease, chronic obstructive pulmonary disease, bronchial inflammation, and acute bronchitis.
  • the cancer is selected from leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma (NHL), and multiple myeloma.
  • the collagen disease is systemic lupus erythematosus.
  • the eye disease is keratitis.
  • the endocrine problem is selected from Addison's Disease, adrenal insufficiency, adrenal cortical dysfunction, adrenocortical, and congenital adrenal hyperplasia.
  • the inflammatory disease is selected from inflammation after cataract surgery, joint inflammation, immune inflammation, tendon inflammation, bursitis, epicondylitis, Crohn's disease, inflammatory bowels disease, lipid pneumonitis thyroiditis, urticaria (hives), pericarditis, nephrotic syndrome, and uveitis.
  • the intestinal disorder is selected from ulcerative colitis, Crohn’s disease, and inflammatory bowel disease.
  • the rheumatoid disorder is selected from rheumatoid arthritis, polymyalgia rheumatic, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus.
  • the skin disorder is selected from psoriasis, eczema, and poison ivy.
  • the neurological disorder is chronic inflammatory demyelinating polyradiculoneuropathy.
  • the compounds described herein are administered to a patient to treat an acute inflammatory event including, but not limited to, shock, brain edema, and graft- vs-host disease.
  • the compounds described herein are administered to treat lympholytic effects including, but not limited to, those associated with hematological malignancies, for example, leukemias, lymphomas, and myelomas.
  • set forth herein is a method for reducing inflammation in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein.
  • set forth herein is a method for modulating the immune system in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a conpound or conjugate thereof described herein.
  • set forth herein is a method for modulating cortisol levels in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein.
  • set forth herein is a method of reducing lymphocyte migration in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein.
  • set forth herein is a method of treating hypercalcemia due to cancer, Meniere's disease, a migraine headache, a cluster headache, a severe aphthous ulcer, laryngitis, severe tuberculosis, a Herxheimer reaction to syphilis, a decompensated heart failure, allergic rhinitis or nasal polyps, comprising administering to a subject in need thereof a payload or conjugate thereof described herein.
  • the compounds disclosed herein can be used for treating inflammatory bowel disease, Crohn's disease, or ulcerative colitis.
  • the disease, disorder, or condition is a chronic inflammatory condition including, but not limited to, asthma, skin infections, and ocular infections.
  • compounds described herein are used for immunosuppression in patients undergoing organ transplantation.
  • the payloads and conjugates thereof described herein are administered to a patient to treat a nervous disorders associated with GR signalling including, but not limited to, psychiatric disorders such as schizophrenia, drug addiction, post-traumatic stress disorder (PTSD), and mood disorders, substance abuse, stress, and anxiety.
  • a nervous disorders associated with GR signalling including, but not limited to, psychiatric disorders such as schizophrenia, drug addiction, post-traumatic stress disorder (PTSD), and mood disorders, substance abuse, stress, and anxiety.
  • the payloads and conjugates thereof described herein are administered to a patient to treat a visual system disorder including, but not limited to, ocular inflammation (e.g., conjunctivitis, keratitis, uveitis), macular edema, and macular degeneration.
  • a visual system disorder including, but not limited to, ocular inflammation (e.g., conjunctivitis, keratitis, uveitis), macular edema, and macular degeneration.
  • the payloads and conjugates thereof described herein are administered to a patient to treat a cardiovascular disorder.
  • the payloads and conjugates thereof described herein are administered to a patient to treat a glucose and/or liver metabolism disorder.
  • the payloads and conjugates thereof described herein are administered to a patient to treat a musculoskeletal system disorder.
  • the side effect to be reduced or prevented is selected from elevation of blood pressure; sodium retention; water/fluid retention (edema, angioedema, pulmonary edema); increased excretion of potassium; reversible hypothalamic-pituitary adrenal (HPA) axis suppression; potential corticosteroid insufficiency after withdrawal of treatment; susceptibility to infecctions; exacerbation of systemic fungal infections; worsening of severity of chickenpox in pediatric and adult patients; worsening of severity of measles in pediatric and adult patients; posterior subcapsular cataracts; glaucoma with possible damage to the optic nerves; enhancement of the establishment of secondary ocular infections due to bacteria, fungi, or viruses; increase in new episodes of optic neuritis; Kaposi’s sarcoma; drug-induced secondary adrenocortical insufficiency; increased risk of a perforation when active or latent peptic ulcers, diverticulitis, fresh intestinal
  • the side effect to be reduced or prevented are those associated with drug-drug interactions.
  • the side effect to be reduced or prevented is associated with drug-drug interactions from the use of a corticosteroid with aminoglutethimide including diminishment of adrenal suppression by corticosteroids; amphotericin B injection and potassium-depleting agents, including development of hypokalemia, cardiac enlargement, and congestive heart failure; antibiotics including a significant decrease in corticosteroid clearance; anticholinesterases including producing severe weakness in patients with myasthenia gravis; oral anticoagulants including inhibition of response to warfarin; antidiabetics including increased blood glucose concentrations; antitubercular drugs including decreased serum concentrations of isoniazid; cholestyramine including increased clearance of corticosteroids; cyclosporine including increased activity of both cyclosporine and corticosteroids, and incidence of convulsions; dexamethasone suppression test (DST) interference including false-negative results in patients being treated with
  • glucocorticoid receptor comprising administering a conjugate of (R 1 )(R 2 )N-W-X-Y-Z and/or Formulae I-IV, to a patient having said disease, disorder, or condition, wherein the side effects associated with administration of the free steroid payload of said conjugate is reduced.
  • a compound of (R 1 )(R 2 )N-W-X-Y-Z and/or Formulae I-IV to a cell comprising contacting said cell with a protein conjugate the compound of (R 1 )(R 2 )N-W-X-Y-Z and/or Formulae I-IV, wherein the protein conjugate comprises an antibody or antigen binding fragment thereof that binds a surface antigen of said cell.
  • a disease, disorder or condition selected from the group consisting of an immunological disease, autoimmune disease, inflammation, asthma, or an inflammatory bowel disorder, Crohn's disease, ulcerative colitis.
  • the antigen is IL2R– ⁇ .
  • set forth herein is a method for treating a disease, disorder, or condition selected from an immunological disease, an autoimmune disease, an inflammatory disease, a dermatological disease, or a gastrointestinal disease.
  • the disease is Crohn’s disease, ulcerative colitis, Cushing's syndrome, adrenal insufficiency, or congenital adrenal hyperplasia.
  • the disease is inflammation, asthma, or an inflammatory bowel disorder.
  • the disease is an autoimmune diseases selected from multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, psoriasis, or eczema.
  • the disease is a cancer.
  • set forth herein is a method for reducing or ameliorating the side effects of chemotherapy, wherein the method includes administering to a patient having said disorder a therapeutically effective amount of a compound or a composition described herein.
  • set forth herein is a method for reducing or ameliorating the side effects of immunosuppressive therapy, wherein the method includes administering to a patient having said disorder a therapeutically effective amount of a compound or a composition described herein.
  • a method for treating cancer wherein the method includes administering to a patient having said disorder a therapeutically effective amount of a compound or a composition described herein.
  • the cancer is selected from acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin’s lymphoma, Non–Hodgkin’s lymphoma (NHL), or multiple myeloma, as well as others.
  • set forth herein are methods for treating or preventing any disease, disorder, or condition responsive to modulation of LXR signalling.
  • the disease or disorder is associated with LXR function, LXR polymorphisms, LXR agonist activity, or LXR antagonist activity.
  • set forth herein is a method of treating or preventing a disease, disorder, or condition selected from the group consisting of a proliferative disorder, a neurodegenerative disorder, an immunological disorder, an autoimmune disease, an inflammatory disorder, a dermatological disease, a metabolic disease, cardiovascular disease, and a gastrointestinal disease.
  • the proliferative disorder can be any proliferative disorder known to those of skill.
  • proliferative disorders include, without limitation, oncology disorders, where the oncology disorder can be any cancer disorder known to those of skill.
  • provided herein are methods of treating or preventing a melanoma.
  • methods of treating or preventing metastatic melanoma are methods of treating or preventing lung cancer.
  • methods of treating or preventing EGFR-tyrosine kinase inhibitor resistant lung cancer are provided herein.
  • provided herein are methods of treating or preventing oral cancer.
  • provided herein are methods of treating or preventing oral squamous cell carcinoma.
  • provided herein are methods of treating or preventing prostate cancer. In certain embodiments, provided herein are methods of treating or preventing Hodgkin’s lymphoma. In certain embodiments, provided herein are methods of treating or preventing breast cancer. In certain embodiments, provided herein are methods of treating or preventing gastric cancer.
  • the neurodegenerative disorder can be any neurodegenerative disorder known to those of skill. In certain embodiments, provided herein are methods of treating or preventing Alzheimer’s disease. In certain embodiments, provided herein are methods of treating or preventing Parkinson’s disease. In certain embodiments, provided herein are methods of treating or preventing Huntington’s disease. In certain embodiments, provided herein are methods of treating or preventing amyotrophic lateral sclerosis.
  • provided herein are methods of treating or preventing myelin gene expression. In certain embodiments, provided herein are methods of treating or preventing myelination and remyelination conditions, diseases, or disorders.
  • the immunological disorder can be any immunological disorder known to those of skill. In certain embodiments, provided herein are methods of treating or preventing imflammatory bowel disease. In certain embodiments, provided herein are methods of treating or preventing ulcerative colitis. In certain embodiments, provided herein are methods of treating or preventing Crohn’s disease.
  • the inflammatory disorder can be any inflammatory disorder known to those of skill. In certain embodiments, provided herein are methods of treating or preventing arthritis.
  • the metabolic disease can be any metabolic disease known to those of skill.
  • the metabolic disease is dyslipidemia.
  • Dyslipidemia can be any dyslipidemia known to those of skill.
  • provided herein are methods of treating or preventing endocarditis. In certain embodiments, provided herein are methods of treating or preventing peripheral artery disease. In certain embodiments, provided herein are methods of treating or preventing combinations of any of the diseases provided in this paragraph. [00210] In some examples, set forth herein is a method for modulating the function of a nuclear receptor.
  • the function may be selected from expression/secretion of inflammatory mediators (e.g.
  • Boc-AL10 as a red oil, which was dissolved in DCM (0.1 M). To the solution was added TFA (1/3 volume of DCM) and the reaction mixture was stirred at room temperature for an hour until Boc was completely removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.01%)) to give AL10 as a light-red oil.
  • N-Boc-Gly-OSu (0.62 g, ESI m/z: 295.0 (M + Na) + ) as a white solid.
  • N-Boc-Gly-OSu (0.62 g, ESI m/z: 295.0 (M + Na) + ) as a white solid.
  • Boc-AL15 38 mg, ESI m/z: 551.4 (M/2 + H) + ) as a red solid.
  • Boc-AL15 was dissolved in DCM (2 mL). To the solution was added TFA (0.4 mL) and the reaction mixture was stirred at room temperature for an hour. The reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by prep- HPLC (5-95% acetonitrile in aq. TFA (0.1%)) to give AL15 (10 mg, 11% yield) as a red solid.
  • the resulting solution was directly purified by reversed phase flash chromatography (0- 100% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give a yellow solid containing vcPABC-Exatecan and its E-ring-open product.
  • the mixture was suspended in water (5 mL) and acidified with aq. TFA (1 M) to pH 4.0.
  • the aqueous solution was then lyophilized to give pure vcPABC-Exatecan 6-1b (39 mg, 43% yield) as a pale yellow solid.
  • LP4 ⁇ 4-[(2S)-5-(carbamoylamino)-2-[(2S)-2- ⁇ 1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12- tetraoxapentadecan-15-amido ⁇ -3-methylbutanamido]pentanamido]phenyl ⁇ methyl N- ( ⁇ [( ⁇ [(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl ⁇ methoxy)methyl]carbamoyl ⁇ methyl)carbamate (LP4) [00
  • the reaction mixture was stirred at 25-30 °C for sixteen hours until most of compound 16-2 was consumed, as monitored by LCMS.
  • the resulting mixture was filtered through a short silica gel plug and the silica gel was washed with ethyl acetate (2x).
  • the combined filtrate was diluted with ethyl acetate and water.
  • the mixture was separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give a brown crude product.
  • the crude was dissolved into DCM (3 L).
  • the mixture was filtered through short silica gel plug eluting with DCM (3x) until all of compound 16-3 was collected.
  • the aqueous layer was then stirred at room temperature for sixteen hours until the ring-open form turned to the lactone form, as monitored by LCMS.
  • the resulting aqueous mixture was lyophilized to give crude ProDXds and purified by reversed phase flash chromatography (0- 100% acetonitrile in aq. TFA (0.03%)) to give pure ProDXds (TFA salt) as a solid; or prep-HPLC (5-95% acetonitrile in aq. formic acid (0.1%)) to give pure payloads (free base) as a solid.
  • the resulting mixture was directly purified by reserved phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to give a de-Fmoc product (68 mg, ESI m/z: 724 (M + H) + ) as a yellow solid).
  • the de-Fmoc product was dissolved in DMF (1 mL).
  • To the solution was added cesium fluoride (31 mg, 0.20 mmol) at 0 °C.
  • the mixture was then stirred at room temperature for an hour and monitored by LCMS.
  • the mixture was separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to give SerDXd (17 mg, 22% yield) as a white solid.
  • GlnDXd (2S)-2-amino-N-[( ⁇ [(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl ⁇ methoxy)methyl]pentanediamide (GlnDXd) [00393] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, GlnDXd (20 mg, 49% yield) was obtained as a light-yellow solid.
  • LysDXd [00398] (2S)-2,6-diamino-N-[( ⁇ [(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl ⁇ methoxy)methyl]hexanamide (LysDXd) [00399] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, LysDXd (13 mg, 43% yield) was obtained as a white solid.
  • Lys(N 3 )DXd [00401] (2S)-2-amino-6-azido-N-[( ⁇ [(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl- 5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl ⁇ methoxy)methyl]hexanamide (Lys(N 3 )DXd) [00402] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, Lys(N 3 )DXd (27 mg, 89%
  • GlyNMeCH 2 DXd [00413] 2-amino-N-[( ⁇ [(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8- oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)- heptaen-23-yl]carbamoyl ⁇ methoxy)methyl]-N-methylacetamide (GlyNMeCH2DXd) [00414] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, GlyNMeCH2DXd (22 mg, 60% yield) was obtained as
  • linker-payload LP13 (16 mg, 49% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)).
  • LP20 [00434] ⁇ 4-[(2S)-5-(carbamoylamino)-2-[(2S)-2- ⁇ 1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido ⁇ -3-methylbutanamido]pentanamido]phenyl ⁇ methyl N-[(1R)-1- ⁇ [( ⁇ [(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl ⁇ methoxy)methyl]carbamoyl ⁇
  • the reaction mixture was purified by prep-HPLC to give a white solid (0.13 g, ESI m/z: 587.3 (M + Na) + ), which was dissolved in dry DMF (3 mL). To the solution was added N-Boc-PEG 4 -acid (70 mg, 0.19 mmol), HATU (87 mg, 0.23 mmol), and DIPEA (99 mg, 0.23 mmol). The reaction mixture was stirred at room temperature for two hours and monitored by LCMS. The mixture was directly purified by prep-HPLC to give 17-2 (0.15 g, 43% yield) as a white solid. ESI m/z: 913.3 (M + H) + .
  • linker-payload LP5 (12 mg, 24% yield) as a white solid.
  • Step 1 Site-specific Conjugation of Handle-functionalized Amine With an Antibody Generated Drug Conjugate Containing Two, Four, or Eight Handles per Antibody
  • Aglycosylated human antibody IgG containing an N297Q mutation or an N297D mutation in BupH buffer (pH 7.4) was mixed with ⁇ 100 molar equivalents of non-branched Handle-amine or branched Handle-amine (AL).
  • the resulting solution was mixed with transglutaminase (350 U/mL; 1 U mTG per mg of antibody, SLCK1576, Sigma; or 25 U/mL; 1 U mTG per mg of antibody, Zedira, Darmstadt, Germany; or 10 U/mL; 0.06 mg mTG per mg of antibody, Modernist Pantry-ACTIVA TI contains Maltodextrin from Ajinomoto, Japan) resulting in a final concentration of the antibody at 0.5-20 mg/mL.
  • the reaction mixture was incubated at 25-37 °C for twenty-four hours while gently shaking and monitored by ESI-MS.
  • Step 2 Click Reactions Between Handle-functionalized Antibodies and a Linker- Payload in Table 2 to Generate the Site-specific ADCs
  • the Handle-functionalized antibody (Ab-(AL)n, 1-20 mg/mL) in PBS (pH 7.4) was incubated with ⁇ 2-10 molar equivalents of a linker-payload (LP) dissolved in an organic solvent such as DMSO or DMA (10 mg/mL) to have the overall reaction mixture containing 5-15% organic solvent (v/v), at 25-37 °C for 1-48 hours while gently shaking.
  • LP linker-payload
  • the reaction was monitored by ESI-MS. Upon reaction completion, the excess amount of LP and organic solvent were removed via a desalting column with BupH (pH 7.4), and protein aggregates (if any) were removed by size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • All ADCs were purified by SEC using an ⁇ KTA instrument from Cytiva, using a 16/600 Superdex® 200 column, eluting with DPBS, at a flow rate of 1.0 mL/min at pH 7.4.
  • the DAR values of the ADCs were measured by ESI-MS.
  • a mass increase of 4 x AL-LP (i.e., AL6- LP1) from Ab to Ab-[AL-LP] 4 was observed, correlating to 4DAR ADC. Shown in FIG.
  • the reaction mixture was incubated at 32 °C for one hour while gently shaking and was monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS.
  • the tetrazine-modified antibody resulted in a 745Da mass increase compared to mAb, indicating four AL7 were conjugated to the antibody (Ab- (AL7)4) with four tetrazine handles.
  • the resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 11.5 mg/mL.
  • the reaction mixture was incubated at 32 °C for one hour while gently shaking and monitored by ESI-MS.
  • ESI-MS size exclusion chromatography
  • the conjugate was characterized by UV-Vis, SEC, and ESI-MS.
  • the tetrazine-modified antibody resulted in a 745Da mass increase compared to mAb, indicating four AL7 were conjugated to the antibody (Ab-(AL7)4) with four tetrazine handles.
  • ADC resulted in a 12164 Da mass increase for the DAR7.5 conjugate. Conjugate monomer purity was >99% by SEC.
  • a representative 8DAR ADC from Approach I An aglycosylated anti-Her2 human IgG antibody containing an N297Q mutation was mixed with 100 molar equivalents of a TFA salt of 14-amino-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3-(2-((4-(6-methyl-1,2,4,5-tetrazin-3- yl)benzyl)amino)-2-oxoethyl)-6,9,12-trioxa-3-azatetradecanamide (AL11, MW 788.8 g/moL).
  • the resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 9.2 mg/mL.
  • the reaction mixture was incubated at 37 °C for seven hours while gently shaking and monitored by ESI-MS.
  • ESI-MS size exclusion chromatography
  • the conjugate was characterized by UV-Vis, SEC, and ESI-MS.
  • the tetrazine-modified antibody resulted in a 2626Da mass increase compared to mAb, indicating four AL11 were conjugated to the antibody (Ab-(AL11) 4 ) with eight tetrazine handles.
  • the site-specific antibody tetrazine conjugate (8.56 mg/mL) in PBS (pH 7.4) was mixed with twelve molar equivalents of linker-payload (LP6) in 7.2 mM of DMA resulting in the reaction mixture containing 8.2 % organic solvent (v/v), and the solution was set at 37 °C for twenty-one hours while gently shaking.
  • the reaction was monitored by ESI-MS.
  • the excess amount of linker-payload and protein aggregates were removed by size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • the purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. Conjugate monomer purity was 99.8% by SEC.
  • the resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 9.2 mg/mL.
  • the reaction mixture was incubated at 37 °C for seven hours while gently shaking and monitored by ESI-MS.
  • ESI-MS size exclusion chromatography
  • the conjugate was characterized by UV-Vis, SEC, and ESI-MS.
  • the tetrazine-modified antibody resulted in a 2626Da mass increase compared to mAb, indicating four AL11 were conjugated to the antibody (Ab-(AL11) 4 ) with eight tetrazine handles.
  • the site-specific antibody tetrazine conjugate (8.56 mg/mL) in PBS (pH 7.4) was mixed with twelve molar equivalents of linker- payload (LP4) in 7.2 mM of DMA resulting in the reaction mixture containing 8.2% organic solvent (v/v), and the solution was set at 37 °C for twenty-one hours while gently shaking.
  • the reaction was monitored by ESI-MS.
  • the excess amount of linker- payload and protein aggregates were removed by size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • the purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI- MS. Conjugate monomer purity was 99.8% by SEC.
  • An aglycosylated anti-Her2 human IgG antibody containing an N297Q mutation was mixed with 100 molar equivalents of 14-amino-N- (4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3-(2-((4-(6-methyl-1,2,4,5-tetrazin-3- yl)benzyl)amino)-2-oxoethyl)-6,9,12-trioxa-3-azatetradecanamide (AL11, MW 674.77 g/moL).
  • the resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 10 mg/mL.
  • the reaction mixture was incubated at 37 °C for seven hours while gently shaking and monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS.
  • the tetrazine-modified antibody resulted in a 2626Da mass increase compared to mAb, indicating four AL11 were conjugated to the antibody (Ab-(AL11) 4 ) with eight tetrazine handles.
  • the site-specific antibody tetrazine conjugate (8.56 mg/mL) in PBS (pH 7.4) was mixed with twelve molar equivalents of linker-payload (LP6) in 0.0065 mM of DMA resulting in the reaction mixture containing 8.6% organic solvent (v/v), and the solution was set at 37 °C for four hours while gently shaking.
  • the reaction was monitored by ESI-MS.
  • the excess amount of linker-payload and protein aggregates were removed by size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • the purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. Conjugate monomer purity was 99.8% by SEC.
  • SDS-PAGE for Analysis of ADC Integrity and Purity [00510]
  • SDS-PAGE conditions included non-reduced and reduced samples (1- 2 ⁇ g) along with Precision Plus Protein Dual Color Standards (Bio-Rad, 500 ⁇ L, Cat# 1610374) loaded per lane in (1.0 mm ⁇ 10 well) Novex 4-20% No Tris-Glycine Gel and and run at 180V, 300 mA, for eighty minutes.
  • a non-reduced sample was prepared using NuPAGE® LDS Sample Buffer (4X) (Thermo Fisher Scientific, Cat#1887691) and the reduced sample was prepared with SDS sample buffer (4X) containing 10% sample reducing agent (10X) (Thermo Fisher Scientific, Cat#1769410).
  • SDS sample buffer (4X) containing 10% sample reducing agent (10X) (Thermo Fisher Scientific, Cat#1769410).
  • Molecular weights of the antibodies and ADCs on SDS-PAGE were determined under non-reducing and reducing conditions. The mass shifts may not be obvious under non-reducing conditions due to relatively small percentages of mass changes. However, the masses of the heavy chains are increased from the naked antibodies to the azido-functionalized antibodies, and further to the ADC conjugates.
  • ADCs were purified by SEC and concentrated by using ultra centrifugation. To separate the antibody-drug conjugates from the reaction mixture, preparative SEC purifications were performed using the ⁇ KTA instrument from GE Healthcare, on a Superdex® 200 increase 10/300 GL (1.0 ⁇ 30cm) column, at a flow rate of 0.6 mL/min eluting with BupH at pH 7.4, and monitored at ⁇ 280 nm. To concentrate the product Amicon ® Ultra-4 Centrifugal Filters (Ultracel-10K) were used in an Allegra x-12r centrifuge, and the solution was stirred after each concentration to avoid high aggregation. [00515] Plasma Stability [00516] Both aHer2-(AL7-LP4)n and aHer2-(AL7-LP6)n are stable in all plasma for fourteen days.
  • Her2 is highly expressed in this cell line.
  • o Growth media McCoy’s, 10% FBS, 100 units/mL Penicillin, 100 ⁇ g/mL Streptomycin, 50 ⁇ g/mL glutamine. • NCI-N87, human gastric carcinoma cell line, ACL6581. Her2 is highly expressed in this cell line.
  • o Growth media RPMI, 10% FBS, 100 units/mL Penicillin, 100 ⁇ g/mL Streptomycin, 50 ⁇ g/mL glutamine. • Calu-3, human lung adenocarcinoma cell line, ACL15056. Her2 is highly expressed in this cell line.
  • o Growth media MEM, 10% FBS, 100 units/mL Penicillin, 100 ⁇ g/mL Streptomycin, 50 ⁇ g/mL glutamine, 1 mM sodium pyruvate, 100 ⁇ M non-essential amino acids. • JIMT-1, human breast ductal carcinoma, ACL14141. Her2 is moderately expressed in this cell line.
  • o Growth media DME, 100 units/mL Penicillin, 100 ⁇ g/mL Streptomycin, 50 ⁇ g/mL glutamine. • NCI-H1975, human lung adenocarcinoma, non-small cell lung cancer, ACL6573. This line expresses Her2 at very low level and serves as a negative cell line.
  • o Growth media RPMI, 10% FBS, 100 units/mL Penicillin, 100 ⁇ g/mL Streptomycin, 50 ⁇ g/mL glutamine.
  • An in vitro cytotoxicity assay was performed to test the potency of Her2-DXd ADCs, described herein and conjugated by an IEDDA method, in killing human cell lines.
  • In vitro cytotoxicity of the ADCs described herein as well as reference ADCs and compounds were evaluated using the CellTiter-Glo Assay Kit (Promega, Cat# G9243), in which the quantity of ATP present was used to determine the number of viable cells in culture.
  • the SK-BR-3 1000/well
  • NCI-N87 1000/well
  • Calu-3 1000/well
  • JIMT-1 1000/well
  • NCI-H1975 800/well
  • ADCs described herein and control ADCs were serially diluted at 1:4 starting from 400 nM in assay media (Opt-MEM+0.1% BSA) for ten points, leaving the last point as blank.
  • ADCs described herein with amino-dienophile linkers demonstrated potent killing of Her2 positive SK-BR-3, NCI-N87, and Calu-3 cells with IC50 values between 0.0015 nM and 0.013 nM.
  • the potency differences between ADCs and their corresponding isotype controls were >1000 fold, so the killing is highly target specific.
  • ADC (26 or 27) derived from the same payload MMAE and same linker and conjugated via cysteine is 7-25 fold less potent than the ADCs described herein with amino-dienophile linkers and conjugated to glutamine. All the ADCs described herein and the reference(s) weakly killed the moderate Her2 expression in JIMT-1 cells. Table 9.
  • ADCs described herein with amino tetrazine linkers in comparison with the azido PEG3-amine linker are summarized in Table 10. Both AL7 and AL6 amino-tetrazine linkers support single chain linkers and branched linkers. When AL7 and AL6 were conjugated with a single chain linker payload, the DAR values were comparable to those ADCs conjugated via the azido-PEG3-amine linker (e.g., see AL7-LP4, AL6-LP4).
  • the DAR values can get close to the maximum capacity of eight (e.g., see AL7- LP6, AL6-LP6, AL6-LP5).
  • the payloads were efficiently released from ADCs conjugated via amino-tetrazine linkers and their potencies were similar to those ADCs conjugated via the azido- PEG3-amine linkers via click chemistry (FIG.15, FIG.16, FIG.17).
  • a 15DAR ADC was made using the AL11 linker and aggregation was not observed. In the cell killing assay, the 15DAR ADC worked slightly better than DAR8 ADCs made from all amino-tetrazine linkers herein (Table 2 and FIG.19). [00524]
  • the embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of this patent application and are encompassed by the scope of the appended claims.

Landscapes

  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

Provided herein are compounds, compounds including novel linkers, protein conjugates thereof, and compositions thereof. Also provided herein are in vivo, in vitro, or ex vivo methods for the treatment of diseases, disorders, and conditions, and/or the management of the symptoms thereof, including administration of the compounds or payloads via novel linkers or novel linker-payloads, and protein conjugates thereof.

Description

ANTIBODY-DRUG CONJUGATES VIA INVERSE ELECTRON DEMAND DIELS-ALDER REACTIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This patent application claims the benefit of U.S. provisional patent application number 63/484,136, filed February 9, 2023, and the content thereof is incorporated herein by reference in its entirety. FIELD [0002] Provided herein are novel linkers, and protein conjugates thereof, and methods for treating a variety of diseases, disorders, and conditions including administering compounds or payloads via linker-payloads, and protein conjugates thereof. BACKGROUND [0003] Antibody-drug conjugates (ADCs) are antibodies that are covalently linked to biologically active small molecule drugs, often referred to as payloads, thus combining the targeting specificity of antibodies with the mode-of-action and potency of small molecule drugs. The therapeutic utility of ADC(s) has been validated in cancer treatment and is a major ongoing focus of study. ADCETRIS® (bentruximab vedotin) and KADCYLA® (ado-trastuzumab emtansine) are ADCs approved for the treatment of certain cancer types, and several other ADCs are currently in clinical development. [0004] Linkers covalently link the payload portion, for example, small molecule therapeutic agent of an ADC to its antibody. A significant challenge in linker design is in finding moieities that keep the payload stably attached to the antibody during storage, formulation, administration, and plasma circulation in the patient, yet allow efficient release upon the antibody binding its target, that allow facile conjugation to the payload under synthesis conditions, and that allow release of the intended payload without alteration in structure. [0005] Chemical site-selective protein modification has become increasingly popular for antibody-based bio-conjugates. Amongst all biorthogonal reactions developed to date, the [4+2] cycloaddition of 1,2,4,5-tetrazines (tetrazines, Tz) with various dienophiles, referred as inverse electron demand Diels-Alder (IEDDA) reactions, is one that satisfies most of the biorthogonal criteria (e.g., fast, selective, biocompatible, and catalyst-free) necessary for the conjugations (Chem. Rev.2021, 121, 12, 6850–6914). [0006] Described herein, tetrazine-linkers were designed to have two functions: (1) a tetrazine-linker as a handle that includes an additional chemical moiety (e.g., an amine) to be attached to an antibody while the tetrazine-moiety can react with a linker-payload to generate an ADC; and (2) a tetrazine-linker as a linker of a linker-payload that can be attached with an antibody-handle (FIG.13). There is a continuing need for linkers that possess these and other attributes. SUMMARY [0007] Provided herein are novel linkers, protein conjugates thereof, protein-drug conjugates thereof, and methods for treating a variety of diseases, disorders, and conditions including administering compounds or payloads via linker-payloads, and protein conjugates thereof. Also provided herein are methods of making the new linkers, protein conjugates thereof, and protein- drug conjugates thereof. Also included herein are linkers that bond with a hydroxyl, amino, or mercapto moiety of a payload or prodrug thereof that allow release of the payload under appropriate conditions with the hydroxyl, amino, or mercaptyl moiety intact. [0008] Provided herein are compounds, compositions, and methods useful for treating, for example, cancer, or managing symptoms of any diseases, disorders, or conditions associated with cancer, in a subject. [0009] In one embodiment, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein W is C1-C10 alkylene or C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene, -C(O)N(R3)-C1-C10 alkylene, -C(O)N(R3)-C1-C10 alkylene-C(O)-(AA)p-N(R3)-, -C(O)-(AA)p-N(R3)-, -C(O)N(R3)-, -N(R5)2, -C(O)N(R5)2, -CH(R5)(R6), -C(O)-(AA)p-N(R3)-C1-C10 alkylene-PEGn-C1-C10 alkylene- N(R5)2, -C(O)-(AA)p-N(R5)2, -C(O)-(AA)p-N(R3)-CH(R5)(R6), or -N(R5)(R6); Y when present is C1-C10 alkylene; Z is selected from the group consisting of , , , , , , and ; R1 and R2 are independently hydrogen, C1-C10 alkyl, or -C(O)-C1-C10 alkyl-COOH; R3 and R4 are independently hydrogen or C1-C10 alkyl; R5 is -Y-C(O)N(R3)-Y-Z, -Y-(R3)NC(O)-Y-Z, -(R3)NC(O)-Y-C(O)N(R3)-Y-Z, or -C(O)N(R3)-Y-Z; R6 is -Y-C(O)N(R3)-PEGn1-C1-C10 alkylene-N3, -C(O)N(R3)-PEGn1-C1-C10 alkylene-N3, or -C1-C10 alkylene-N3; AA is an amino acid residue and p is an integer from one to twenty; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is an integer from two to one hundred; wherein when W is C1- C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is an integer from one to three, five to seven, or nine to one hundred; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is an integer from one to one hundred; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; and Z is , then n is an integer from one to one hundred; and n1 is an integer from zero to one hundred. [0010] In one embodiment, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein W is C1-C10 alkylene or C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene, -C(O)N(R3)-, -N(R5)2, or -N(R5)(R6); Y when present is C1-C10 alkylene; Z is selected from the group consisting of , , , , , and ; R1 and R2 are independently hydrogen, C1-C10 alkyl, or -C(O)-C1-C10 alkyl-COOH; R3 and R4 are independently hydrogen or C1-C10 alkyl; R5 is -Y-C(O)N(R3)-Y-Z; R6 is -Y-C(O)N(R3)-PEGn1-C1-C10 alkylene-N3; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is then n is an integer from two to one hundred; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is an integer from one to three, five to seven, or nine to one hundred; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is an integer from one to one hundred; and n1 is an integer from zero to one hundred. [0011] In one embodiment, provided is a compound having the following chemical structure L10aE. [0012] In one embodiment, provided is a compound selected from the group consisting of LP1; LP7; LP15; LP16; and LP17. In one embodiment, the compound is LP1. In one embodiment, the compound is LP7. In one embodiment, the compound is LP15. In one embodiment, the compound is LP16. In one embodiment, the compound is LP17. [0013] In another embodiment, provided is a compound having the following structure (I) or (II) or a pharmaceutically acceptable salt thereof, wherein L is hydrogen or a linker comprising a moiety reactive with a binding agent; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent; SP is a spacer group; R1a and R1b are, independently, hydrogen or alkyl; R2 is hydrogen or an amino acid side chain; R3, R4, and R6 is hydrogen or alkyl; R5 is oxygen, NR6, or sulfur; R7 is an O-amino acid residue; R8 is C1-C10 alkylene or C1-C10 heteroalkylene; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein when m is four, then the moiety reactive with the binding agent is ; and wherein when n is four, then the moiety reactive with the binding agent is , with the proviso that the compound of Formula I is not GlyGlyDxd. [0014] In another embodiment, provided is a compound having the following structure (I) or (II) or a pharmaceutically acceptable salt thereof, wherein L is a linker comprising a moiety reactive with a binding agent; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent; SP is a spacer group; R1a and R1b are, independently, hydrogen or alkyl; R2 is hydrogen or an amino acid side chain; R3, R4, and R6 is hydrogen or alkyl; R5 is oxygen, NR6, or sulfur; R7 is an O-amino acid residue; R8 is C1-C10 alkylene or C1-C10 heteroalkylene; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein when m is four, then the moiety reactive with the binding agent Is ; and wherein when n is four, then the moiety reactive with the binding agent is . In one embodiment, D* is a residue of a therapeutic moiety, wherein the therapeutic moiety is a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, or an analogue or derivative thereof, or an imaging agent moiety. In one embodiment, D* is a residue of a maytansinoid. In one embodiment, D* is a residue of a tubulysin. In one embodiment, D* is a residue of an auristatin. In one embodiment, D* is a residue of a dolastatin. In one embodiment, D* is a residue of a camptothesin. In one embodiment, D* is a residue of a pyrrolobenzodiazepine. In one embodiment, D* is a residue of an antibiotic. In one embodiment, D* is a residue of an antiviral agent. In one embodiment, D* is a residue of an anti-inflammatory agent. In one embodiment, D* is a residue of an immunomodulator. In one embodiment, D* is a residue of an antifungal agent. In one embodiment, D* is a residue of a steroid. In one embodiment, D* is a residue of an imaging agent. In certain embodiments in this paragraph, D* is a residue of a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, or an imaging agent moiety, or an analogue or derivative thereof. In one embodiment, provided is a conjugate selected from the group consisting of and
. BRIEF DESCRIPTION OF THE DRAWING [0015] FIGS.1-10 show synthetic chemistry schemes for linkers including AL4, AL10, and L5; branched AL11, AL12, AL13, AL14, AL15, AL16, AL17, L10a, and L10aE; linear vcPABC linker-payloads LP4 and LP12-LP15; GGFG linker-paylaods LP2 and LP8; branched GGFG linker-payloads LP5 and LP10; and branched EvcPABC linker-payloads LP6 and LP11. [0016] FIG.11 shows 4DAR ADC conjugations. [0017] FIG.12 shows exemplary 4DAR, 8DAR, and 16DAR ADC conjugations. [0018] FIG.13 shows ADC generation via metal-free 9enzotriazol inverse electron demand Diels-Alder reactions [0019] FIG. 14 shows Her2-MMAE ADCs conjugated with amino-dienophile linkers via AL2 or AL3 versus Mc-vcPAB-MMAE that potently kill Her2 positive cells [0020] FIG. 15 shows the potency of 4DAR ADCs 14, 20-23, 32, and 33 conjugated via AL6 and AL7 Amino-tetrazine linkers or azido-PEGn-N3. [0021] FIG.16 shows the potency of 8DAR ADCs conjugated with branched linker-payload LP6 via AL6 and AL7 amino-tetrazine linkers or azido-PEGn-N3. [0022] FIG.17 shows the potency of 8DAR ADCs conjugated with branched linker-payload LP5 via AL6 and AL7 amino-tetrazine linker or azido-PEGn-N3. [0023] FIG. 18 shows 8DAR ADC potency via amino-tetrazine linker AL11 compared to AL6, AL7, and azido-PEGn-N3. [0024] FIG. 19 shows the potency of 15DAR ADC via amino-tetrazine linker AL11 compared to AL6, AL7, and azido-PEGn-N3. [0025] FIG.20 shows ADC synthetic Approaches I and II. [0026] FIG.21 shows stability data for Tz handles. [0027] FIG.22 shows aHer2-(AL11)4. [0028] FIG.23 shows ES-MS ADC characterization data for aHer2-(AL11)4, aHer2-(AL11- LP4)n, and FelD1-(AL11-LP4)n. [0029] FIG. 24 shows ES-MS ADC characterization data for aHer2-(AL11-LP6)4 and FelD1-(AL11-LP6)4. [0030] FIG.25 shows that both aHer2-(AL7-LP4)n and aHer2-(AL7-LP6)n are stable in all plasma for fourteen days. DESCRIPTION OF EXEMPLARY EMBODIMENTS Definitions [0031] When referring to the compounds provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term provided herein, these Definitions prevail unless stated otherwise. [0032] As used herein, “alkyl” refers to a monovalent and saturated hydrocarbon radical moiety. Alkyl is optionally substituted and can be linear, branched, or cyclic, (i.e., cycloalkyl). Alkyl includes, but is not limited to, those radicals having one to twenty carbon atoms, for example, C1-20 alkyl; one to twelve carbon atoms, for example, C1-12 alkyl; one to eight carbon atoms, for example, C1-8 alkyl; one to six carbon atoms, for example, C1-6 alkyl; and one to three carbon atoms for example, C1-3 alkyl. Examples of alkyl moieties include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, i-butyl, a pentyl moiety, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A pentyl moiety includes, but is not limited to, n-pentyl and i-pentyl. A hexyl moiety includes, but is not limited to, n-hexyl. [0033] As used herein, “alkylene” refers to a divalent alkyl group. Unless specified otherwise, alkylene includes, but is not limited to, one to twenty carbon atoms. The alkylene group is optionally substituted as described herein for alkyl. In some embodiments, alkylene is unsubstituted. Examples of alkylene moieties include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like. [0034] As used herein, “heteroalkylene” refers to a divalent alkyl group wherein one or more carbon atoms is replaced with a heteroatom. Unless specified otherwise, heteroalkylene includes, but is not limited to, one to twenty total atoms (i.e., carbons and heteroatoms). The heteroalkylene group is optionally substituted as described herein for alkyl. In some embodiments, heteroalkylene is unsubstituted. In some embodiments, heteroatoms contemplated within heteroalkylene moieties include oxygen, nitrogen, sulfur (i.e., including sulfoxide, sulphite, sulfate, and sulfone), silicon, and phosphorous (i.e., including phosphite and phosphate), and/or combinations thereof. Nonlimiting exemplary embodiments of heteroalkylene moieties include -CH2O-, -CH2OCH2-, -CH2OCH2CH2-, -CH2CH2CH2OCH2-, and the like; -CH2NR-, -CH2NRCH2-, -CH2NRCH2CH2-, -CH2CH2CH2NRCH2-, and the like; and -CH2S-, -CH2SCH2-, -CH2SCH2CH2-, -CH2CH2CH2SCH2-, and the like wherein R includes, but is not limited to, hydrogen or alkyl. [0035] Designation of an amino acid or an amino acid residue without specifying stereochemistry is intended to encompass the L- form of the amino acid or amino acid residue, the D- form of the amino acid or amino acid residue, or a racemic mixture thereof. [0036] As used herein, “haloalkyl” refers to alkyl, as defined above, wherein the alkyl includes at least one substituent selected from a halogen, for example, fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Examples of haloalkyl include, but are not limited to, –CF3, –CH2CF3, –CCl2F, –CHF2, and –CCl3. [0037] As used herein, “alkenyl” refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more non-aromatic carbon-carbon double bonds or olefins. Alkenyl is optionally substituted and can be linear, branched, or cyclic. Alkenyl includes, but is not limited to, those radicals having two to twenty carbon atoms, for example, C2-20 alkenyl; two to twelve carbon atoms, for example, C2-12 alkenyl; two to eight carbon atoms, for example, C2-8 alkenyl; two to six carbon atoms, for example, C2-6 alkenyl; and two to four carbon atoms for example, C2-4 alkenyl. Examples of alkenyl moieties include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl. [0038] As used herein, “alkynyl” refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl is optionally substituted and can be linear, branched, or cyclic. Alkynyl includes, but is not limited to, those radicals having two to twenty carbon atoms, for example, C2-20 alkynyl; two to twelve carbon atoms, for example, C2-12 alkynyl; two to eight carbon atoms, for example, C2-8 alkynyl; two to six carbon atoms, for example, C2-6 alkynyl; and two to four carbon atoms, for example, C2-4 alkynyl. Examples of alkynyl moieties include, but are not limited to ethynyl, propynyl, propargyl, and butynyl. [0039] As used herein, “alkoxy” refers to a monovalent and saturated hydrocarbon radical moiety wherein the hydrocarbon includes a single bond to an oxygen atom and wherein the radical is localized on the oxygen atom, for example, CH3CH2-O· for ethoxy. Alkoxy substituents bond to the compound which they substitute through this oxygen atom of the alkoxy substituent. Alkoxy is optionally substituted and can be linear, branched, or cyclic, for example, cycloalkoxy. Alkoxy includes, but is not limited to, those radicals having one to twenty carbon atoms, for example, C1-20 alkoxy; one to twelve carbon atoms, for example, C1-12 alkoxy; one to eight carbon atoms, for example, C1-8 alkoxy; one to six carbon atoms, for example, C1-6 alkoxy; and one to three carbon atoms, for example, C1-3 alkoxy. Examples of alkoxy moieties include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, i- butoxy, a pentoxy moiety, a hexoxy moiety, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. [0040] As used herein, “haloalkoxy” refers to alkoxy, as defined above, wherein the alkoxy includes at least one substituent selected from a halogen, for example, F, Cl, Br, or I. [0041] As used herein, “aryl” refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms are carbon atoms. Aryl is optionally substituted and can be monocyclic or polycyclic, for example, bicyclic or tricyclic. Examples of aryl moieties include, but are not limited to, those having six to twenty ring carbon atoms, for example, C6-20 aryl; six to fifteen ring carbon atoms, for example, C6-15 aryl, and six to ten ring carbon atoms, for example, C6-10 aryl. Examples of aryl moieties include, but are limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, and pyrenyl. [0042] As used herein, “arylalkyl” refers to a monovalent moiety that is a radical of an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent, for example, the aromatic compound includes a single bond to an alkyl group and wherein the radical is localized on the alkyl group. An arylalkyl group bonds to the illustrated chemical structure via the alkyl group. An arylalkyl can be represented by a structure, for example, , , , , or , wherein B is an aromatic moiety, for example, aryl or phenyl. Arylalkyl is optionally substituted, for example, the aryl group and/or the alkyl group can be substituted as disclosed herein. Examples of arylalkyl include, but are not limited to, benzyl. [0043] As used herein, “alkylaryl” refers to a monovalent moiety that is a radical of an aryl compound, wherein the aryl compound is substituted with an alkyl substituent, for example, the aryl compound includes a single bond to an alkyl group and wherein the radical is localized on the aryl group. An alkylaryl group bonds to the illustrated chemical structure via the aryl group. An alkylaryl can be represented by a structure, for example, , , , , or , wherein B is an aromatic moiety, for example, phenyl. Alkylaryl is optionally substituted, for example, the aryl group and/or the alkyl group can be substituted as disclosed herein. Examples of alkylaryl include, but are not limited to, toluyl. [0044] As used herein, “aryloxy” refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms are carbon atoms and wherein the ring is substituted with an oxygen radical, for example, the aromatic compound includes a single bond to an oxygen atom and wherein the radical is localized on the oxygen atom, for example, for phenoxy. Aryloxy substituents bond to the compound that they substitute through this oxygen atom. Aryloxy is optionally substituted. Aryloxy includes, but is not limited to, those radicals having six to twenty ring carbon atoms, for example, C6-20 aryloxy; six to fifteen ring carbon atoms, for example, C6-15 aryloxy, and six to ten ring carbon atoms, for example, C6-10 aryloxy. Examples of aryloxy moieties include, but are not limited to phenoxy, naphthoxy, and anthroxy. [0045] As used herein, “arylene” refers to a divalent moiety of an aromatic compound wherein the ring atoms are only carbon atoms. Arylene is optionally substituted and can be monocyclic or polycyclic, for example, bicyclic or tricyclic. Examples of arylene moieties include, but are not limited to those having six to twenty ring carbon atoms, for example, C6-20 arylene; six to fifteen ring carbon atoms, for example, C6-15 arylene, and six to ten ring carbon atoms, for example, C6-10 arylene. [0046] As used herein, “heteroalkyl” refers to an alkyl in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkenyl” refers to an alkenyl in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkynyl” refers to an alkynyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl. [0047] As used herein, “heteroaryl” refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms contain carbon atoms and at least one oxygen, sulfur, nitrogen, or phosphorus atom. Examples of heteroaryl moieties include, but are not limited to those having five to twenty ring atoms; five to fifteen ring atoms; and five to ten ring atoms. Heteroaryl is optionally substituted. [0048] As used herein, “heteroarylene” refers to a divalent heteroaryl in which one or more ring atoms of the aromatic ring are replaced with an oxygen, sulfur, nitrogen, or phosphorus atom. Heteroarylene is optionally substituted. [0049] As used herein, “heterocycloalkyl” or “heterocyclyl” refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms (i.e., including sulfoxide and sulfone). Heterocycloalkyl or heterocyclyl is optionally substituted. Examples of heterocycloalkyl and heterocyclyl moieties include, but are not limited to, morpholinyl, piperidinyl, tetrahydropyranyl, pyrrolidinyl, aziridnyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, dioxolanyl, dithiolanyl, oxanyl, and thianyl. [0050] As used herein, “Lewis acid” refers to a molecule or ion that accepts an electron lone pair. The Lewis acids used in the methods described herein are those other than protons. Lewis acids include, but are not limited to, non-metal acids, metal acids, hard Lewis acids, and soft Lewis acids. Lewis acids include, but are not limited to, Lewis acids of aluminum, boron, iron, tin, titanium, magnesium, copper, antimony, phosphorus, silver, ytterbium, scandium, nickel, and zinc. Illustrative Lewis acids include, but are not limited to, AlBr3, AlCl3, BCl3, boron trichloride methyl sulfide, BF3, boron trifluoride methyl etherate, boron trifluoride methyl sulfide, boron trifluoride tetrahydrofuran, dicyclohexylboron trifluoromethanesulfonate, iron (III) bromide, iron (III) chloride, tin (IV) chloride, titanium (IV) chloride, titanium (IV) isopropoxide, Cu(Otf)2, CuCl2, CuBr2, zinc chloride, alkylaluminum halides (RnAlX3-n, wherein R is hydrocarbyl), Zn(Otf)2, ZnCl2, Yb(Otf)3, Sc(Otf)3, MgBr2, NiCl2, Sn(Otf)2, Ni(Otf)2, and Mg(Otf)2. [0051] As used herein, “N-containing heterocycloalkyl” refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms and wherein at least one replacing heteroatom is a nitrogen atom. Suitable heteroatoms in addition to nitrogen include, but are not limited to, oxygen and sulfur atoms. N-containing heterocycloalkyl is optionally substituted. Examples of N-containing heterocycloalkyl moieties include, but are not limited to, morpholinyl, piperidinyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, or thiazolidinyl. [0052] As used herein, “optionally substituted” when used to describe a radical moiety, for example, optionally substituted alkyl, means that such moiety is optionally bonded to one or more substituents. Examples of such substituents include, but are not limited to, halo, cyano, nitro, amino, hydroxyl, optionally substituted haloalkyl, aminoalkyl, hydroxyalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, , , , , , , , , , , , , , or , wherein RA, RB, and RC are, independently at each occurrence, hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heteroaryl, or heterocycloalkyl, or RA and RB together with the atoms to which they are bonded, form a saturated or unsaturated carbocyclic ring, wherein the ring is optionally substituted, and wherein one or more ring atoms is optionally replaced with a heteroatom. In certain embodiments, when a radical moiety is optionally substituted with an optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring, the substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring, if they are substituted, are not substituted with substituents which are further optionally substituted with additional substituents. In some embodiments, when a group described herein is optionally substituted, the substituent bonded to the group is unsubstituted unless otherwise specified. [0053] As used herein, “binding agent” refers to any molecule, for example, protein, antibody, or antigen binding fragment thereof, capable of binding with specificity to a given binding partner, for example, an antigen. [0054] As used herein, “linker” refers to a divalent, trivalent, or multivalent moiety that covalently links, or is capable of covalently linking (e.g., via a reactive group), the binding agent to one or more compounds described herein, for instance, payload compounds and enhancement agents. [0055] As used herein, “amide synthesis conditions” refers to reaction conditions suitable to effect the formation of an amide, for example, by the reaction of a carboxylic acid, activated carboxylic acid, or acyl halide with an amine. In some examples, amide synthesis conditions refer to reaction conditions suitable to effect the formation of an amide bond between a carboxylic acid and an amine. In some of these examples, the carboxylic acid is first converted to an activated carboxylic acid before the activated carboxylic acid reacts with an amine to form an amide. Suitable conditions to effect the formation of an amide include, but are not limited to, those utilizing reagents to effect the reaction between a carboxylic acid and an amine including, but not limited to, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), (16enzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (16enzotriazole-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7- azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O-(16enzotriazole-1-yl)- N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU), O-(16enzotriazole-1-yl)- N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), and carbonyldiimidazole (CDI). In some examples, a carboxylic acid is first converted to an activated carboxylic ester before treating the activated carboxylic ester with an amine to form an amide bond. In certain embodiments, the carboxylic acid is treated with a reagent. The reagent activates the carboxylic acid by deprotonating the carboxylic acid and then forming a product complex with the deprotonated carboxylic acid as a result of nucleophilic attack by the deprotonated carboxylic acid onto the protonated reagent. The activated carboxylic esters for certain carboxylic acids are subsequently more susceptible to nucleophilic attack by an amine than the carboxylic acid is before activation. This results in amide bond formation. As such, the carboxylic acid is described as activated. Exemplary reagents include DCC and DIC. [0056] As used herein, “regioisomer,” “regioisomers,” or “mixture of regioisomers” refers to the product(s) of 1,3-cycloadditions or strain-promoted alkyne-azide cycloadditions (SPAACs)—otherwise known as click reactions—that derive from suitable azides (e.g.,-N3, or –PEG-N3 derivitized antibodies) treated with suitable alkynes; or refers to the product(s) of inverse electron demand Diels-Alder reactions that derive from suitable dienophiles (e.g., alkenes or alkynes) treated with suitable dienes (e.g., tetrazines), or suitable dienes (e.g., tetrazines) treated with suitable dienophiles. In certain embodiments, for example, regioisomers and mixtures of regioisomers are characterized by the click reaction products shown below: wherein represents attachment to a binding agent as described elsewhere herein. In certain embodiments, more than one suitable azide and more than one suitable alkyne can be utilized within a synthetic scheme en route to a product, where each pair of azide-alkyne can participate in one or more independent click reactions to generate a mixture of regioisomeric click reaction products. For example, a person of skill will recognize that a first suitable azide may independently react with a first suitable alkyne, and a second suitable azide may independently react with a second suitable alkyne, en route to a product, resulting in the generation of four possible click reaction regioisomers or a mixture of the four possible click reaction regioisomers. In certain embodiments, for example, regioisomers and mixtures of regioisomers are characterized by the inverse electron demand Diels-Alder reaction products below: or wherein represents attachment to a binding agent as described elsewhere herein. In certain embodiments, more than one suitable dienophile and more than one suitable diene can be utilized within a synthetic scheme en route to a product, where each pair of dienophile-diene can participate in one or more independent inverse electron demand Diels-Alder reactions to generate a mixture of regioisomeric Diels-Alder reaction products. For example, a person of skill will recognize that a first suitable dienophile may independently react with a first suitable diene, and a second suitable dienophile may independently react with a second suitable diene, en route to a product, resulting in the generation of four possible Diels-Alder reaction regioisomers or a mixture of the four possible Diels-Alder reaction regioisomers. [0057] As used herein, the term “residue” refers to the chemical moiety within a compound that remains after a chemical reaction. For example, the term “amino acid residue,” “peptide residue,” “O-amino acid residue,” “HO-amino acid residue,” or “N-alkyl amino acid residue” refers to the product of an amide coupling or peptide coupling of an amino acid, O-amino acid, OH-amino acid, or a N-alkyl amino acid to a suitable coupling partner; wherein, for example, a water molecule is expelled after the amide or peptide coupling of the amino acid, peptide, O- amino acid, OH-amino acid, or the N-alkylamino acid, resulting in the product having the amino acid residue, O-amino acid residue, OH-amino acid residue, or N-alkyl amino acid residue incorporated therein. [0058] As used herein, “therapeutically effective amount” refers to an amount (e.g., of a compound) that is sufficient to provide a therapeutic benefit to a patient in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. [0059] As used herein, “constitutional isomers” refer to compounds that have the same molecular formula, but different chemical structures resulting from the way the atoms are arranged. Exemplary constitutional isomers include n-propyl and isopropyl; n-butyl, sec-butyl, and tert-butyl; and n-pentyl, isopentyl, and neopentyl, and the like. [0060] Certain groups, moieties, substituents, and atoms are depicted with a wiggly line that intersects a bond or bonds to indicate the atom through which the groups, moieties, substituents, atoms are bonded. For example, a phenyl group that is substituted with a propyl group depicted as: or has the following structure: . As used herein, illustrations showing substituents bonded to a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) through a bond between ring atoms are meant to indicate, unless specified otherwise, that the cyclic group may be substituted with that substituent at any ring position in the cyclic group or on any ring in the fused ring group, according to techniques set forth herein or which are known in the field to which the instant disclosure pertains. For example, the group, or , wherein subscript q is an integer from zero to four and in which the positions of substituent R1 are described generically, for example, not directly attached to any vertex of the bond line structure, for example, a specific ring carbon atom, includes the following, non-limiting examples of groups in which the substituent R1 is bonded to a specific ring carbon atom: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and . [0061] As used herein, the phrase “reactive linker,” or the abbreviation “RL” refers to a monovalent group that includes a reactive group (“RG”) and spacer group (“SP”), depicted for example as , wherein RG is the reactive group and SP is the spacer group. As described herein, a reactive linker may include more than one reactive group and more than one spacer group. The spacer group is any divalent moiety that bridges the reactive group to another group, such as a payload (e.g., a biologically active compound). The reactive linkers (RLs), together with the payloads to which they are bonded, provide intermediates (“linker-payloads” or LPs) useful as synthetic precursors for the preparation of the conjugates described herein (e.g., antibody-drug conjugates (ADCs)). The reactive linker includes a reactive group, which is a functional group or moiety that is capable of reacting with a reactive portion of another group, for instance, a binding agent, an antibody, modified antibody, or antigen binding fragment thereof, or an enhancement group. The moiety resulting from the reaction of the reactive group with the binding agent, antibody, modified antibody, or antigen binding fragment thereof, together with the linking group, include the “binding agent linker” (“BL”) portion of the conjugate, described herein. In certain embodiments, the “reactive group” is a functional group or moiety (e.g., maleimide or N-hydroxysuccinimide (NHS) ester) that reacts with a cysteine or lysine residue of an antibody or antigen-binding fragment thereof. In certain embodiments, the “reactive group” is a functional group or moiety that is capable of undergoing a click chemistry reaction (see, e.g., click chemistry, Huisgen Proc. Chem. Soc.1961, Wang et al. J. Am. Chem. Soc. 2003, and Agard et al. J. Am. Chem. Soc. 2004). In some embodiments of said click chemistry reaction, the reactive group is an alkyne that is capable of undergoing a 1,3- cycloaddition reaction with an azide. Such suitable reactive groups include, but are not limited to, strained alkynes, for example, those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, for example, cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in the absence of copper catalysts. Suitable alkynes also include, but are not limited to, (TMTH); (COMBO); (PYRROC); cyclooctyne (OCT); (SNO-OCTs); azacyclooctyne (DIMAC); dibenzoazacyclooctyne or (DIBAC); dibenzocyclooctyne or (DIBO); biarylazacyclooctynone or (BARAC); monofluorinated cyclooctyne (MOFO); difluorinated cyclooctyne or , or , or (DIFO), substituted, for example, fluorinated alkynes, aza-cycloalkynes; bicycle[6.1.0]nonyne or (BCN, where R is alkyl, alkoxy, or acyl); and derivatives thereof. Particularly useful alkynes include and . Linker-payloads including such reactive groups are useful for conjugating binding agents or antibodies that have been functionalized with azido groups. Such functionalized binding agents or antibodies include antibodies functionalized with azido-polyethylene glycol groups. In certain embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an an amino group and an azide group, in the presence of the enzyme transglutaminase. In certain embodiments, the “reactive group” is a functional group or moiety that is capable of undergoing an inverse electron demand Diels-Alder reaction (see, e.g., Chem. Rev. 2021, 121, 12, 6850–6914). In some embodiments of said inverse electron demand Diels-Alder reaction, the reactive group is a dienophile (e.g., alkenes or alkynes) that is capable of undergoing an inverse electron demand Diels-Alder reaction with a diene (e.g., a tetrazine). Such suitable reactive groups include, but are not limited to, dienophiles or strained dienophiles suitable for the inverse electron demand Diels-Alder reaction, for example, or . Linker-payloads including such reactive groups are useful for conjugating antibodies that have been functionalized with diene groups. Such functionalized antibodies include antibodies functionalized with compounds described herein. In certain embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an an amino agroup and a diene group (e.g., a tetrazine), in the presence of the enzyme transglutaminase. In certain embodiments, the “reactive group” is a functional group or moiety that is capable of undergoing an inverse electron demand Diels-Alder reaction (see, e.g., Chem. Rev. 2021, 121, 12, 6850–6914). In some embodiments of said inverse electron demand Diels-Alder reaction, the reactive group is a diene (e.g., a tetrazine) that is capable of undergoing an inverse electron demand Diels-Alder reaction with a dieneophile (e.g., alkenes or alkynes). Such suitable reactive groups include, but are not limited to, dienes suitable for the inverse electron demand Diels-Alder reaction, for example, , , , and . Linker-payloads including such reactive groups are useful for conjugating antibodies that have been functionalized with dieneophile groups. Such functionalized antibodies include antibodies functionalized with compounds described herein. In certain embodiments, such a functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a compound bearing an an amino agroup and a dieneophile group, in the presence of the enzyme transglutaminase. [0062] In some examples, the reactive group is an alkyne, for example, , which can react via click chemistry with an azide, for example, , to form a click chemistry product, for example, or . In some examples, the group reacts with an azide on a modified antibody or antigen binding fragment thereof. In some examples, the reactive group is an alkyne, for example, , which can react via click chemistry with an azide, for example, to form a click chemistry product, for example, . In some examples, the reactive group is a dienophile, for example, or , which can react via an inverse electron demand Diels-Adler reaction with a diene, for example, , to form a Diels-Alder product, for example, or . In some examples, the group reacts with a diene (e.g., a tetrazine) on a modified antibody or antigen binding fragment thereof. In some examples, the reactive group is a diene, for example, , which can react via an inverse electron demand Diels-Alder reaction with a dienophile, for example, to form a Diels-Alder product, for example, or . In some examples, the group reacts with a dienophile on a modified antibody or antigen binding fragment thereof. In some examples, the reactive group is an alkyne, for example, , which can react via click chemistry with an azide, for example, , to form a click chemistry product, for example, or . In some examples, the reactive group is a functional group, for example, ,which reacts with a cysteine residue on an antibody or antigen-binding fragment thereof (i.e., Michael addition), to form a carbon-sulfur bond thereto, for example, , wherein Ab refers to an antibody or antigen-binding fragment thereof and sulphur S refers to the S atom on a cysteine residue through which the functional group bonds to the Ab. In some examples, the reactive group is a functional group, for example, ,which reacts with a lysine residue on an antibody or antigen-binding fragment thereof, to form an amide bond thereto, for example, , wherein Ab refers to an antibody or antigen-binding fragment thereof and NH refers to the NH moiety on a lysine side chain residue through which the functional group bonds to the Ab. [0063] As used herein, the phrase “biodegradable moiety” refers to a moiety that degrades in vivo to non-toxic, biocompatible components which can be cleared from the body by ordinary biological processes. In some embodiments, a biodegradable moiety completely or substantially degrades in vivo over the course of about ninety days or less, about sixty days or less, or about thirty days or less, where the extent of degradation is based on percent mass loss of the biodegradable moiety, and wherein complete degradation corresponds to 100% mass loss. Exemplary biodegradable moieties include, without limitation, aliphatic polyesters such as poly( ^-caprolactone) (PCL), poly(3-hydroxybutyrate) (PHB), poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and its copolymers with glycolic acid (i.e., poly(D,L-lactide- coglycolide) (PLGA) (Vert M, Schwach G, Engel R and Coudane J (1998) J Control Release 53(1-3):85-92; Jain R A (2000) Biomaterials 21(23):2475-2490; Uhrich K E, Cannizzaro S M, Langer R S and Shakesheff K M (1999) Chemical Reviews 99(11): 3181-3198; and Park T G (1995) Biomaterials 16(15):1123-1130, each of which are incorporated herein by reference in their entirety). [0064] As used herein, the phrase “binding agent linker,” or “BL” refers to any divalent, trivalent, or multi-valent group or moiety that links, connects, or bonds a binding agent (e.g., an antibody or an antigen-binding fragment thereof) with a payload compound set forth herein and, optionally, with one or more side chain compounds. Generally, suitable binding agent linkers for the antibody-drug conjugates described herein are those that are sufficiently stable to exploit the circulating half-life of the antibody-drug conjugates and, at the same time, capable of releasing a payload after antigen-mediated internalization of the conjugate. Linkers can be cleavable or non-cleavable. Cleavable linkers are linkers that are cleaved by intracellular metabolism following internalization, for example, cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers are linkers that release an attached payload via lysosomal degradation of the antibody following internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolytically-labile linkers, enzymatically cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, those that are or comprise peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units (e.g., PEGn, (CH2CH2O)n, and (CH2OCH2)n, and used interchangeably herein), hydrazones, mal-caproyl units, dipeptide units, valine-citruline units, para-aminobenzyloxycarbonyl (PABC), and para-aminobenzyl (PAB) units. In some embodiments, the binding agent linker (BL) includes a moiety that is formed by the reaction of the reactive group (RG) of a reactive linker (RL) and reactive portion of the binding agent, for example, antibody, modified antibody, or antigen binding fragment thereof. [0065] In some examples, the BL includes the following moiety: or , wherein is the bond to the binding agent. In some examples, the BL includes the following moiety: , wherein is the bond to the binding agent. In some examples, the BL includes the following moiety: or , wherein is the bond to the binding agent. In some examples, the BL includes the following moiety: , , , or , wherein is the bond to the binding agent. In some examples, the BL includes the following moiety: , wherein is the bond to the cysteine of the antibody or antigen-binding fragment thereof. In some examples, the BL includes the following moiety: , wherein is the bond to the lysine of the antibody or antigen-binding fragment thereof. [0066] As used herein, “amino acid side chain” refers to the additional chemical moiety on the same carbon that bears a primary or secondary amine and a carboxylic acid of an amino acid. As would be appreciated by a person of skill in the art, there are twenty-one “standard” amino acids. Exemplary “standard” amino acids include, without limitation, alanine, serine, proline, arginine, and aspartic acid. Other amino acids include, cysteine, selenocysteine, and glycine (e.g., wherein the additional chemical moiety on the same carbon that bears the primary amine and carboxylic acid of glycine is hydrogen). Exemplary amino acid side chains include, without limitation, methyl (i.e., alanine), sec-buytl (i.e., isoleucine), iso-butyl (i.e., leucine), –CH2CH2SCH3 (i.e., methionine), –CH2Ph (i.e., phenylalanine), (i.e., tryptophan), (i.e., tyrosine), iso-propyl (i.e., valine), hydroxymethyl (i.e., serine), –CH(OH)CH3 (i.e., threonine), –CH2C(O)NH2 (i.e., asparagine), –CH2CH2C(O)NH2 (i.e., glutamine), –CH2SH (i.e., cysteine), –CH2SeH (i.e., selenocysteine), –CH2NH2 (i.e., glycine), propylene or -CH2CH2CH2- (i.e., proline), –CH2CH2CH2NHC(=NH)NH2 (i.e., arginine), (i.e., histidine), –CH2CH2CH2CH2NH2 (i.e., lysine), –CH2COOH (i.e., aspartic acid), and –CH2CH2COOH (i.e., glutamic acid). [0067] As used herein, “biologically active compound” refers to a compound, prodrug, or payload that elicits a biological response when administered to a biological entity. Exemplary biological responses include, without limitation, increase or decrease in DNA or protein synthesis, up-regulation or down-regulation of signalling pathways, and increase or decrease in cell proliferation, and the like. [0068] As used herein, the term “O-amino acid” or “HO-amino acid” designates an amino acid wherein the native amino group at the N-terminus of an amino acid or an amino acid sequence has been replaced with an oxygen or hydroxyl group, respectively. For example, “O- AAAA” or “HO-AAAA” is intended to designate an amino acid sequence (AAAA) wherein the native amino group at the N-terminus has been replaced with an oxygen or hydroxyl group, respectively (e.g., , where each R is an amino acid side chain). Similarly, the terms “O-amino acid residue” or “HO-amino acid residue” refers to the chemical moiety within a compound that remains after a chemical reaction. For example, “O- amino acid residue” or “HO-amino acid residue” refers to the product of an amide coupling or peptide coupling of an O-amino acid or a HO-amino acid to a suitable coupling partner; wherein, for example, a water molecule is expelled after the amide or peptide coupling of the O-amino acid or a HO-amino acid, resulting in the product having the O-amino acid residue or a HO- amino acid residue incorporated therein. Compounds, Prodrugs, or Payloads [0069] In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is six In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ten. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eleven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twelve. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fourteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventeen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is nineteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty- five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is twenty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W- X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is thirty- nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N- W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty- seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is forty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W- X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is fifty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W- X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is sixty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy- one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy- seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is seventy-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty- three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W- X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eigty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is eighty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety- one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety- four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety- seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is ninety-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is one hundred. In one embodiment, R1 and R2 are hydrogen. In one embodiment, when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is two, three, eight, or twelve. In one embodiment, the compound is selected from the group consisting of AL4c; AL4d; and AL4e. In one embodiment, the compound is . In one embodiment, the compound is AL4c. In one embodiment, the compound is AL4d. In one embodiment, the compound is AL4e. [0070] In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ten. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eleven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twelve. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fourteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventeen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is nineteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is twenty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is thirty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is forty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is fifty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is sixty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is - C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is seventy-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is eighty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is ninety-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is one hundred. In one embodiment, R1 and R2 are hydrogen. In one embodiment, when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is an integer from one to three, five to seven, or nine to one hundred. In one embodiment, the compound is selected from the group consisting of ; ; ; ; ; ; ; ; ; and AL10c. In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is . In one embodiment, the compound is AL10c. [0071] In one embodiment, W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R5)2 or -N(R5)(R6); R5 is -Y-C(O)N(R3)-Y-Z; and R6 is -Y-C(O)N(R3)-PEGn1-C1-C10 alkylene-N3. In one embodiment, the compound is AL11. In one embodiment, the compound is AL12. In certain embodiments, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is an integer from one to ten. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is one. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is two. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is three. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is four. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is five. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is six. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is seven. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is eight. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is nine. In one embodiment, R1 and R2 are hydrogen; W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R5)2; R5 is -Y-(R3)NC(O)-Y-Z; Z is ; and n is ten. In one embodiment, the compound is AL13. In certain embodiments, R1 and R2 are hydrogen; W is C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene-C(O)-(AA)p-N(R3)- or -C(O)-(AA)P-N(R3)-; and Z is . In one embodiment, the compound is selected from the group consisting of AL14a; AL14b; and AL14c. In one embodiment, the compound is AL14a. In one embodiment, the compound is AL14b. In one embodiment, the compound is AL14c. In certain embodiments, R1 and R2 are hydrogen; W is C1-C10 alkylene; X is -CH(R5)(R6); R5 is -(R3)NC(O)-Y-C(O)N(R3)-Y-Z; R6 is -C(O)N(R3)-PEGn1-C1-C10 alkylene-N3; and Z is . In one embodiment, the compound is 513. In certain embodiments, R1 and R2 are hydrogen; W is C1-C10 alkylene; X is -C(O)-(AA)p-N(R3)-C1-C10 alkylene-PEGn-C1-C10 alkylene-N(R5)2; R5 is -Y-C(O)N(R3)-Y-Z; and Z is . In one embodiment, the compound is AL15. In certain embodiments, R1 and R2 are hydrogen; W is C1-C10 alkylene; X is -C(O)-(AA)p-N(R3)- ; and Z is . In one embodiment, the compound is 473. In certain embodiments, R1 and R2 are hydrogen; W is C1-C10 alkylene; X is -C(O)-(AA)p-N(R5)2; R5 is -Y-C(O)N(R3)-Y-Z; and Z is . In one embodiment, the compound is AL16. In certain embodiments, R1 and R2 are hydrogen; W is C1-C10 alkylene; X is -C(O)-(AA)p-N(R3)-CH(R5)(R6); and Z is . In one embodiment, the compound is AL17. [0072] In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ten. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eleven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twelve. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fourteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventeen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is nineteen. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is twenty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is thirty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is forty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N- W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X- Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is fifty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W- X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is sixty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is seventy-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is eighty-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-one. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-two. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-three. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-four. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-five. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-six. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-seven. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-eight. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is ninety-nine. In certain embodiments, provided are compounds having the following formula (R1)(R2)N-W-X-Y-Z wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is one hundred. In one embodiment, R1 is hydrogen and R2 is -C(O)-C1-C10 alkyl-COOH. In one embodiment, R1 is hydrogen and R2 is -C(O)CH2CH2COOH. In one embodiment, when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-C1-C10 alkylene; and Z is , then n is an integer from one to one hundred. In one embodiment, n is four, eight, or twelve. In one embodiment, the compound is selected from the group consisting of L5a; L5b; and L5c. In one embodiment, the compound is L5a. In one embodiment, the compound is L5b. In one embodiment, then compound is L5c. [0073] Provided herein are prodrugs or payloads. The payload can be any payload deemed suitable by the person of skill in the art. Without being bound by any particular theory of operation, the compounds or payloads include prodrugs thereof. The terms or phrases “compounds,” “biologically active compounds,” “prodrugs,” and “payloads” are used interchangeably throughout this disclosure. In certain embodiments, the biologically active compound (D*) or residue thereof includes hydroxyl functionality (e.g., D*–OH or D*–O–R). In certain embodiments herein, for example and convenience, R5 represents the hydroxyl, amino, and thiol functional groups within the biologically active compounds described herein, as would be appreciated by a person of skill, or a portion thereof such as -O-, -NI-, or -S-. Alternatively stated, a person of skill would recognize that R5 may be part of the biologically active compounds described herein (e.g., D*), and may be used as a functional group for conjugation purposes. In one embodiment, the hydroxyl functionality is a primary hydroxyl moiety (e.g., D*–CH2OH or D*–CH2O–R; or D*–C(O)CH2OH or D*–C(O)CH2O–R). In another embodiment, the hydroxyl functionality is a secondary hydroxyl moiety (e.g., D*–CH(OH)R or D*–CH(O–R)R; or D*–C(O)CHI(OH) or D*–C(O)CHI(O–R)). In another embodiment, the hydroxyl functionality is a tertiary hydroxyl moiety (e.g., D*–C(R1)(R2)(OH) or D*– C(R1)(R2)(O–R); or D*–C(O)C(R1)(R2)(OH) or D*–C(O)C(R1)(R2)(O–R)). Those of skill will recognize that each functional group in the previous sentences can be part of the biologically active compound D* and simultaneously be depicted in the formula for clarity, convenience, and/or emphasis. In another embodiment, the D* including the hydroxyl functionality is an aryl hydroxyl or phenolic hydroxyl (e.g., D*–Ar–OH, D*–Ar–O–R. In certain embodiments, the biologically active compound (D*) or residue thereof includes amino functionality (e.g., D*–NR2 or D*–N(R)–R). In one embodiment, the amino functionality is a primary amino moiety (e.g., D*–CH2NR2 or D*–CH2N(R)–R; or D*–C(O)CH2NR2 or D*–C(O)CH2N(R)–R). In another embodiment, the amino functionality is a secondary amino moiety (e.g., D*–CH(NR2)R or D*–CH(NR–R)R; or D*–C(O)CH(R)(NR2) or D*–C(O)CH(R)(NR–R)). In another embodiment, the amino functionality is a tertiary amino moiety (e.g., D*–C(R1)(R2)(NR2) or D*–C(R1)(R2)(N(R)–R); or D*–C(O)C(R1)(R2)(NR2) or D*–C(O)C(R1)(R2)(N(R)–R)). In another embodiment, the D* including the amino functionality is an aryl amine (e.g., D*–Ar–NR2, D*–Ar–N(R)–R. In certain embodiments, the payload is a cytotoxin. In certain embodiments, the payload is a calicheamicin. In certain embodiments, the payload is an auristatin, for instance, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In certain embodiments, the payload is a maytansinoid, for instance, DM1 or DM4. In certain embodiments, payload is a duocarmycin. In certain embodiments, payload is an amanitin. In certain embodiments, payload is a pyrrolobenzodiazepine. In certain embodiments, payload is an exatecan. In one embodiment, the biologically active compound (D*) including amino functionality is exatecan, and the residue including the amino functionality is , wherein indicates attachment to a prodrug moiety (as shown in Formulae I-IV), a linker, and/or binding agent, as described herein. In one embodiment, the biologically active compound (D*) including amino functionality is MMAE, and the residue including the amino functionality is , wherein indicates attachment to a prodrug moiety (as shown in Formulae I-IV), a linker, and/or binding agent, as described herein. In certain embodiments, the biologically active compound (D*) or residue thereof includes thiol functionality (e.g., D*–SH or D*–S–R). In one embodiment, the thiol functionality is a primary thiol moiety or sulfide (e.g., D*–CH2SH or D*–CH2S–R; or D*–C(O)CH2SH or D*–C(O)CH2S–R). In another embodiment, the thiol functionality is a secondary thiol moiety or sulfide (e.g., D*–CH(SH)R or D*–CH(S–R)R; or D*–C(O)CH(R)(SH) or D*–C(O)CH(R)(S–R)). In another embodiment, the thiol functionality is a tertiary thiol moiety or sulfide (e.g., D*–C(R1)(R2)(SH) or D*–C(R1)(R2)(S–R); or D*–C(O)C(R1)(R2)(SH) or D*– C(O)C(R1)(R2)(S–R)). In another embodiment, the D* including the thiol functionality is an aryl thiol or thiophenol or sulfide (e.g., D*–Ar–SH, D*–Ar–S–R. In certain embodiments, the compounds can be delivered to cells as part of a conjugate. In certain embodiments, the compounds are capable of carrying out any activity of exatecan or MMAE, or derivatives thereof at or in a target, for instance, a target cell. Certain compounds can have one or more additional activities. In certain embodiments within this paragraph, all diastereomers are contemplated. For example, in one embodiment, the stereochemistry at the exatecan primary amine or the exatecan hydroxyl is undefined or racemic. By way of further example, in one embodiment, the stereochemistry at the exatexan primary amine is (R)-. By way of further example, in one embodiment, the stereochemistry at the exatecan primary amine is (S)-. By way of further example, in one embodiment, the stereochemistry at the exatexan primary amine is (R)- in excess of (S)-. By way of further example, in one embodiment, the stereochemistry at the exatecan primary amine is (S)- in excess of (R)-. [0074] In certain embodiments, provided is a compound having the following structure (I) or a pharmaceutically acceptable salt thereof, wherein L is hydrogen; R1a and R1b are hydrogen; R2 is hydrogen or an amino acid side chain; R3, R4, and R6 is hydrogen or alkyl; R5 is NR6; R7 is an O-amino acid residue; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; and m is zero, one, two, three, four, five, or six. In certain embodiments, m is zero. In certain embodiments, m is one. In certain embodiments, m is two. In certain embodiments, m is three. In certain embodiments, m is four. In certain embodiments, m is five. In certain embodiments, m is six. In one embodiment, the compound is selected from the group consisting of SerDXd; GlnDXd; GluDXd;
LysDXd; PheDXd; DPheDXd; and GlyNMeCH2DXd. In one embodiment, the compound is SerDXd. In one embodiment, the compound is GlnDXd. In one embodiment, the compound is GluDXd. In one embodiment, the compound is LysDXd. In one embodiment, the compound is PheDXd. In one embodiment, the compound is DPheDXd. In one embodiment, the compound is GlyNMeCH2DXd. Binding agents [0075] Suitable binding agents for any of the conjugates provided in the instant disclosure include, but are not limited to, antibodies, lymphokines (e.g., IL-2 or IL-3), hormones (e.g., insulin and glucocorticoids), growth factors (e.g., EGF, transferrin, and fibronectin type III), viral receptors, interleukins, or any other cell binding or peptide binding molecules or substances. Binding agents also include, but are not limited to, ankyrin repeat proteins and interferons. [0076] In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof. The antibody can be in any form known to those of skill in the art. The term “antibody,” as used herein, refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments disclosed herein, the FRs of the antibodies (or antigen-binding portion thereof) suitable for the compounds herein may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from full antibody molecules using any suitable, standard technique(s) such as proteolytic digestion or recombinant genetic engineering technique(s) involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, for example, commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. Non- limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR- grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen- binding fragment,” as used herein. An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain. In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of this disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH- CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL- CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. As with full antibody molecules, antigen- binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art. In certain embodiments described herein, antibodies described herein are human antibodies. The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The term “human antibody” does not include naturally occurring molecules that normally exist without modification or human intervention/manipulation, in a naturally occurring, unmodified living organism. The antibodies of this disclosure may, in some embodiments, be recombinant human antibodies. The term “recombinant human antibody,” as used herein, is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification. The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant disclosure encompasses antibodies having one or more mutations in the hinge, CH2, or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form. The antibodies described herein may be isolated antibodies. An “isolated antibody,” as used herein, refers to an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody” for purposes of the instant disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals. The antibodies used herein can comprise one or more amino acid substitutions, insertions, and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. This disclosure includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first eight amino acids of FR1 or within the last eight amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, for example, wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure. Antibodies useful for the compounds herein also include antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. The term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen. [0077] In certain embodiments, the antibody comprises a light chain. In certain embodiments, the light chain is a kappa light chain. In certain embodiments, the light chain is a lambda light chain. In certain embodiments, the antibody comprises a heavy chain. In some embodiments, the heavy chain is an IgA. In some embodiments, the heavy chain is an IgD. In some embodiments, the heavy chain is an IgE. In some embodiments, the heavy chain is an IgG. In some embodiments, the heavy chain is an IgM. In some embodiments, the heavy chain is an IgG1. In some embodiments, the heavy chain is an IgG2. In some embodiments, the heavy chain is an IgG3. In some embodiments, the heavy chain is an IgG4. In some embodiments, the heavy chain is an IgA1. In some embodiments, the heavy chain is an IgA2. [0078] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is an Fv fragment. In some embodiments, the antibody fragment is a Fab fragment. In some embodiments, the antibody fragment is a F(ab′)2 fragment. In some embodiments, the antibody fragment is a Fab′ fragment. In some embodiments, the antibody fragment is an scFv (sFv) fragment. In some embodiments, the antibody fragment is an scFv-Fc fragment. [0079] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a bispecific antibody including a first antigen-binding domain (also referred to herein as “D1”), and a second antigen-binding domain (also referred to herein as “D2”). [0080] As used herein, the expression “antigen-binding domain” means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding a particular antigen of interest (e.g., PRLR, STEAP2, HER2, FelD1, and/or FGFR2). The term “specifically binds” or the like, as used herein, means that the antigen-binding domain forms a complex with a particular antigen characterized by a dissociation constant (KD) of 1 ^M or less, and does not bind other unrelated antigens under ordinary test conditions. “Unrelated antigens” are proteins, peptides, or polypeptides that have less than 95% amino acid identity to one another. [0081] Exemplary categories of antigen-binding domains that can be used in the context of the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and aptamers or portions thereof. [0082] Methods for determining whether two molecules specifically bind one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antigen-binding domain, as used in the context of the present disclosure, includes polypeptides that bind a particular antigen (e.g., a target molecule [T] or an internalizing effector protein [E]) or a portion thereof with a KD of less than about 1 ^M, less than about 500 nM, less than about 250 nM, less than about 125 nM, less than about 60 nM, less than about 30 nM, less than about 10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM, as measured in a surface plasmon resonance assay. [0083] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. [0084] In some embodiments, the antibody is an anti-PSMA, anti-PRLR, anti-MUC16, anti- HER2, Her2, or anti-Her2, anti-EGFRvIII, anti-FelD1, anti-FGFR2, FGFR2, or anti-STEAP2 antibody. In some embodiments, the antibody is an anti-PRLR or anti HER2 antibody. In some embodiments, the antibody, or antigen-binding fragment thereof, is anti-STEAP2. In some embodiments, the antibody, or antigen-binding fragment thereof, is anti-PRLR. In some embodiments, the antibody, or antigen-binding fragment thereof, is aHer2. In some embodiments, the antibody, or antigen-binding fragment thereof, is aFelD1. In some embodiments, the antibody, or antigen-binding fragment thereof, is aFGFR2. [0085] The antibody can have binding specificity for any antigen deemed suitable to those of skill in the art. In certain embodiments, the antigen is a transmembrane molecule (e.g., receptor). In one embodiment, the antigen is expressed on a tumor. In some embodiments, the binding agents interact with or bind to tumor antigens, including antigens specific for a type of tumor or antigens that are shared, overexpressed, or modified on a particular type of tumor. In one embodiment, the antigen is expressed on solid tumors.Exemplary antigens include, but are not limited to, lipoproteins; alpha1-antitrypsin; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; fibroblast growth factor receptor 2 (FGFR2), EpCAM, GD3, FelD1, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, STEAP2, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD134, CD137, CD138, CD152, or an antibody which binds to one or more tumor-associated antigens or cell-surface receptors disclosed in U.S. Publication No.2008/0171040 or U.S. Publication No.2008/0305044 each incorporated in their entirety by reference; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); T-cell receptors; surface membrane proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; a tumor associated antigen such as AFP, ALK, B7H4, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD123, CDK4, CLEC12A, c-kit, cMET, cyclin-B1, CYP1B1, EGFRvIII, endoglin, EphA2, ErbB2/Her2, ErbB3/Her3, ErbB4/Her4, ETV6-AML, Fra-1, FOLR1, GAGE proteins, GD2, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/EBNA1, HLA/k-ras, HLA/MAGE- A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins, MART-1, ML-IAP, CA-125, MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap- 1, Steap-2, STn, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, uroplakin-3, and fragments of any of the above-listed polypeptides; cell-surface expressed antigens; c-MET; molecules such as class A scavenger receptors including scavenger receptor A (SR-A), and other membrane proteins such as B7 family-related members including V-set and Ig domain-containing 4 (VSIG4), Colony stimulating factor 1 receptor (CSF1R), asialoglycoprotein receptor (ASGPR), and Amyloid beta precursor-like protein 2 (APLP-2); macrophage receptor with collagenous structure (MARCO), scavenger receptor with C-type lectin (SRCL), and scavenger receptor A-5 (SCARA5), COLEC12, class B macrophage scavenger receptors including CD36, LIMPII, SRBI, SRBII, class D scavenger receptor CD68, lysosomal membrane glycoprotein (LAMP), class E scavenger receptor including lectin-like oxidized low density lipoprotein receptor 1 LOX-1 and Dectin-1, class F scavenger receptors including scavenger receptor expressed by endothelial cells-I (SREC-I) and SREC-II as well as multiple epidermal growth factor (EGF)-like domains (MEGF)10, class G scavenger receptor CXC chemokine ligand 16 (CXCL16), class H scavenger receptors including Fasciclin, EGF- like, lamin type EGF-like and link domain-containing scavenger receptor-1 (FEEL-1) and -2 (FEEL-2), class I scavenger receptor CD163, and class J scavenger receptor receptor for advanced glycation end products (RAGE), other C-type lectin superfamily members including DEC205, CD206, Dectin-2, Mincle, DC-SIGN, and DNGR-1, and other membrane proteins such as B7 family-related member including V-set and Ig domain-containing 4 (VSIG4); AXL, BAFFR, BCR-list components, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD168, CD177, CD209, CD209L, CD226, CD248, CD27, CD274, CD276, CD300A, CD45, CD46, CD47, CD48, CD62E, CD69, CD74, CD79a, CD79b, CD86, CD90.2, CD96, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CXCR1/2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, LAG3, LIFR, MAG/Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, TACI, TCR-list components/assoc, PTCRA, TCRb, CD3z, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TNF-α, TROY, TSLPR, TYRO, VLDLR, and VTCN1. In some embodiments, the binding agent is adalimumab or infliximab. In some embodiments, the binding agent is alemtuzumab, muromonab, rituximab, tosituzumab, or agonistic antibodies (where immune stimulation might be part of the intended mechanism of action). In some embodiments, the antigen is PRLR or HER2. In some embodiments, the antigen is STEAP2. In some embodiments the antigen is human STEAP2. In some embodiments, the antigen is Her2. In some embodiments, the antigen is FelD1. In some embodiments, the antigen is FGFR2. In some examples, the MAGE proteins are selected from MAGE-1, -2, -3, -4, -6, and -12. In some examples, the GAGE proteins are selected from GAGE-1 and GAGE-2. [0086] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, GPNMB, MSR1, and UPK3A. Exemplary antigens also include, but are not limited to, MUC16, STEAP2, and HER2. [0087] In some embodiments, the antigens include MUC16. In some embodiments, the antigens include STEAP2. In some embodiments, the antigens include PSMA. In some embodiments, the antigens include MSR1. In some embodiments, the antigens include HER2. In some embodiments, the antigen is prolactin receptor (PRLR) or prostate-specific membrane antigen (PSMA). In some embodiments, the antigen is MUC16. In some embodiments, the antigen is HER2. In some embodiments, the antigen is STEAP2. In some embodiments, the antigen is MSR1. [0088] In certain embodiments, the antibody comprises a glutamine residue at one or more heavy chain positions numbered 295 in the EU numbering system. In the present disclosure, this position is referred to as glutamine 295, or as Gln295, or as Q295. Those of skill will recognize that this is a conserved glutamine residue in the wild type sequence of many antibodies. In other useful embodiments, the antibody can be engineered to comprise a glutamine residue. In certain embodiments, the antibody is glycosylated, for instance at N297. In certain embodiments, the antibody is deglycosylated. In certain embodiments, the antibody is aglycosylated. In certain embodiments, the antibody comprises one or more N297 mutations. In certain embodiments, the antibody comprises one or more N297Q mutations. Techniques for modifying an antibody sequence to include a glutamine residue are within the skill of those in the art (see, e.g., Ausubel et al. Current Protoc. Mol. Biol.). [0089] In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets STEAP2. Sutiable anti-STEAP antibodies or antigen-binding fragments thereof include those, for example, in International Publication No. WO 2018/058001 A1, including those comprising amino acid sequences disclosed in Table 1, on page 75 therein. In some embodiments, an anti- STEAP2 antibody is H1H7814N of WO 2018/058001 A1, comprising the CDRs of H1M7814N in the same publication. In some embodiments, an anti-STEAP2 antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 2; an HCDR2 comprising SEQ ID NO: 3; an HCDR3 comprising SEQ ID NO: 4; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 6; an LCDR2 comprising SEQ ID NO: 7; and an LCDR3 comprising SEQ ID NO: 8. In some embodiments, an anti-STEAP2 antibody comprises a heavy chain variable region (HCVR) comprising SEQ ID NO: 1 and a light chain variable region (LCVR) comprising SEQ ID NO: 5. In any of the foregoing embodiments, the anti-STEAP2 antibody can be prepared by site-directed mutagenesis to insert a glutamine residue at a site without resulting in disabled antibody function or binding. For example, in any of the foregoing embodiments, the anti-STEAP2 antibody can comprise an Asn297Gln (N297Q) mutation. Such antibodies having an N297Q mutation can also contain one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to, for example, transglutaminase and therefore capable of conjugation to a linker compound, payload, or a linker-payload (Table A). In certain embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO:1; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) amino acid sequence of SEQ ID NO:5. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR amino acid sequence of SEQ ID NO:1; and an LCVR amino acid sequence of SEQ ID NO:5. International Publication No. WO 2018/058001 A1 is hereby incorporated herein by reference in its entirety. [0090] In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets human prolactin receptor (PRLR). Suitable anti-PRLR antibodies or antigen-binding fragments thereof include those, for example, in International Publication No. WO 2015/026907 A1, including those comprising amino acid sequences disclosed in Table 1, on page 36 therein. In some embodiments, an anti-PRLR antibody is H1H6958N2 of WO 2015/026907 A1, comprising the CDRs of H2M6958N2 in the same publication The expression “PRLR” includes both monomeric and multimeric PRLR molecules, such as those described in WO 2015/026907. In some embodiments, an anti-PRLR antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 10; an HCDR2 comprising SEQ ID NO: 11; an HCDR3 comprising SEQ ID NO: 12; a light chain complementarity determining region (LCDR)-1 comprising SEQ ID NO: 14; an LCDR2 comprising SEQ ID NO: 15; and an LCDR3 comprising SEQ ID NO: 16. In some embodiments, an anti-PRLR antibody comprises a heavy chain variable region (HCVR) comprising SEQ ID NO: 9 and a light chain variable region (LCVR) comprising SEQ ID NO: 13. In any of the foregoing embodiments, the anti- PRLR antibody can be prepared by site-directed mutagenesis to insert a glutamine residue at a site without resulting in disabled antibody function or binding. For example, in any of the foregoing embodiments, the anti-PRLR antibody can comprise an Asn297Gln (N297Q) mutation. Such antibodies having an N297Q mutation can also contain one or more additional naturally occurring glutamine residues in their variable regions, which can be accessible to, for example, transglutaminase and therefore capable of conjugation to a linker compound, payload, or a linker-payload (Table A). In certain embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) amino acid sequence of SEQ ID NO:9; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) amino acid sequence of SEQ ID NO:13. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR amino acid sequence of SEQ ID NO:9; and an LCVR amino acid sequence of SEQ ID NO:13. International Publication No. WO 2015/026907 A1 is hereby incorporated herein by reference in its entirety. Table A. Sequences of Exemplary Antibodies H1H7814N (anti-STEAP2) and H1H6958N (anti-PRLR)
[0091] In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets Her2. In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets FelD1. In some embodiments, the antibody, or antigen-binding fragment thereof, conjugated to the linker compound, linker-payload, or payload can be an antibody that targets FGFR2. [0092] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table A, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. [0093] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table A, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. [0094] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table A paired with any of the LCVR amino acid sequences listed in Table A. According to certain embodiments, this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-STEAP2 antibodies listed in Table A. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of: 250/258; as described in International Publication No. WO 2018/058001 A1, the contents of which are incorporated herein by reference in its entirety. [0095] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a heavy chain CDR1 (HCDR1 ) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity [0096] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [0097] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [0098] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a light chain CDR1 (LCDR1 ) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [0099] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00100] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00101] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table A paired with any of the LCDR3 amino acid sequences listed in Table A. According to certain embodiments, this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-STEAP2 antibodies listed in Table A. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of: 256/254; as described in International Publication No. WO 2018/058001 A1, the contents of which are incorporated herein by reference in its entirety. [00102] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-STEAP2 antibodies listed in Table A. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of: 252-254-256-260-262-264; as described in International Publication No. WO 2018/058001 A1, the contents of which are incorporated herein by reference in its entirety. [00103] In a related embodiment, this disclosure provides antibodies, or antigen-binding fragments thereof that specifically bind STEAP2, comprising a set of six CDRs (i.e., HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-STEAP2 antibodies listed in Table A. For example, this disclosure includes antibodies or antigen-binding fragments thereof that specifically bind STEAP2, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of: 250/258; as described in International Publication No. WO 2018/058001 A1, the contents of which are incorporated herein by reference in its entirety. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol.273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody. [00104] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table A, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. [00105] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table A, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. [00106] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table A paired with any of the LCVR amino acid sequences listed in Table A. According to certain embodiments, this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-PRLR antibodies listed in Table A. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of: 18/26; 66/74; 274/282; 290/298; and 370/378; as described in International Publication No. WO 2015/026907 A1, the contents of which are incorporated herein by reference in its entirety. [00107] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a heavy chain CDR1 (HCDR1 ) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00108] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00109] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00110] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a light chain CDR1 (LCDR1 ) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00111] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00112] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table A or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00113] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table A paired with any of the LCDR3 amino acid sequences listed in Table A. According to certain embodiments, this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-PRLR antibodies listed in Table A. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of: 24/32; 72/80; 280/288; 296/304; and 376/384; as described in International Publication No. WO 2015/026907 A1, the contents of which are incorporated herein by reference in its entirety. [00114] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3) contained within any of the exemplary anti-PRLR antibodies listed in Table A. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of: 20-22-24-28-30-32; 68-70-72-76- 78-80; 276-278-280-284-286-288; 292-294-296-300-302-304; and 372-374-376-380-382-384; as described in International Publication No. WO 2015/026907 A1, the contents of which are incorporated herein by reference in its entirety. [00115] In a related embodiment, this disclosure provides antibodies, or antigen-binding fragments thereof that specifically bind PRLR, comprising a set of six CDRs (i.e., HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-PRLR antibodies listed in Table A. For example, this disclosure includes antibodies or antigen-binding fragments thereof that specifically bind PRLR, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of: 18/26; 66/74; 274/282; 290/298; and 370/378; as described in International Publication No. WO 2015/026907 A1, the contents of which are incorporated herein by reference in its entirety. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol.273:927- 948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody. [00116] In any of the compound or conjugate embodiments provided, BA is an antibody, or antigen binding fragment thereof, that binds PRLR. In any of the compound or conjugate embodiments provided, BA is an antibody, or antigen binding fragment thereof, that binds Her. In any of the compound or conjugate embodiments provided, BA is an antibody, or antigen binding fragment thereof, that binds FelD1. In any of the compound or conjugate embodiments provided, BA is an antibody, or antigen binding fragment thereof, that binds FGFR2. In any of the compound or conjugate embodiments provided, BA is an antibody or antigen-binding fragment thereof, and conjugation is through at least one Q295 residue. In any of the compound or conjugate embodiments provided, BA is an antibody or antigen-binding fragment thereof, and conjugation is through two Q295 residues. In any of the compound or conjugate embodiments provided, BA is a N297Q antibody or antigen-binding fragment thereof. In any of the compound or conjugate embodiments provided, BA is a N297Q antibody or antigen-binding fragment thereof, and conjugation is through at least one Q295 and at least one Q297 residue. In any of the compound or conjugate embodiments provided, BA is a N297Q antibody or antigen-binding fragment thereof, and conjugation is through two Q295 residues and two Q297 residues. In particular embodiments, numbering is according to the EU numbering system. [00117] In any of the embodiments above, BA is an anti-MSR1 antibody. In certain embodiments, BA is the anti-MSR1 antibody H1H21234N. In certain embodiments, BA is the anti-MSR1 antibody H1H21234N N297Q. In certain embodiments, BA is an anti-MSR1 antibody comprising an HCVR according to SEQ ID NO:19 and an LCVR according to SEQ ID NO: 27. In certain embodiments, BA is an anti-MSR1 antibody comprising one, two, three, four, five, or six of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 according to SEQ ID NOS: 21, 23, 25, 29, 31, and 33, respectively. In certain embodiments, the HCVR is encoded by SEQ ID NO:18. In certain embodiments, the LCVR is encoded by SEQ ID NO: 26. In certain embodiments, one, two, three, four, five, or six of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are encoded by the polynucleotide sequences SEQ ID NOS: 20, 22, 24, 28, 30, and 32, respectively. N297Q indicates that one or more residues 297 are mutated from asparagine (N) to glutamine (Q). In certain embodiments, each residue 297 is mutated to Q. In certain embodiments, numbering is according to the EU numbering system. In certain embodiments of this paragraph, the drug:antibody ratio (DAR) is from one to twenty-four. In certain embodiments, DAR is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, or twenty-four. In certain embodiments, DAR is two. In certain embodiments, DAR is four. In certain embodiments, DAR is eight. In certain embodiments, DAR is twelve. In certain embodiments, DAR is sixteen. In certain embodiments, DAR is twenty-four. [00118] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table B, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. [00119] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table B, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. [00120] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table B paired with any of the LCVR amino acid sequences listed in Table B. According to certain embodiments, this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-MSR1 antibodies listed in Table B. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of: 2/10, 23/42, 50/58, 90/98, and 282/290; as described in International Publication No. WO 2019/217597 A1, the contents of which are incorporated herein by reference in its entirety. [00121] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a heavy chain CDR1 (HCDR1 ) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00122] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00123] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00124] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a light chain CDR1 (LCDR1 ) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00125] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00126] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table B or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [00127] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table B paired with any of the LCDR3 amino acid sequences listed in Table B. According to certain embodiments, this disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-MSR1 antibodies listed in Table B. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of: 8/16, 40/48, 56/64, 96/104, and 288/296; as described in International Publication No. WO 2019/217591 A1, the contents of which are incorporated herein by reference in its entirety. [00128] This disclosure also provides antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3) contained within any of the exemplary anti-MSR1 antibodies listed in Table B. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of: 4-6-8-12-14-16; 36-38-40-44-46-48; 52- 54-56-60-62-64; 92-94-96-100-102-104, and 284-286-288-292-294-296; as described in International Publication No. WO 2019/217591 A1, the contents of which are incorporated herein by reference in its entirety. [00129] In a related embodiment, this disclosure provides antibodies, or antigen-binding fragments thereof that specifically bind MSR1, comprising a set of six CDRs (i.e., HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-MSR1 antibodies listed in Table B. For example, this disclosure includes antibodies or antigen-binding fragments thereof that specifically bind MSR1, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of: 2/10, 23/42, 50/58, 90/98, and 282/290; as described in International Publication No. WO 2019/217591 A1, the contents of which are incorporated herein by reference in its entirety. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol.273:927- 948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody. Table B. Sequences of Exemplary anti-MSR1 Antibodies SEQ Molecule / Region Sequence ID NO: Antibody S S D [00130] The binding agent linkers can be bonded to the binding agent, for example, antibody or antigen-binding molecule, through an attachment at a particular amino acid within the antibody or antigen-binding molecule. Exemplary amino acid attachments that can be used in the context of this embodiment of the disclosure include, for example, lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808; US 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), non- natural amino acids (see, e.g., WO 2013/068874, and WO 2012/166559), and acidic amino acids (see, e.g., WO 2012/05982). Linkers, including the linkers described herein, can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127- 130). [00131] In some examples, the binding agent is an antibody or antigen binding molecule, and the antibody is bonded to the linker through a lysine residue. In some embodiments, the antibody or antigen binding molecule is bonded to the linker through a cysteine residue. [00132] Linkers, including the linkers described herein, can also be conjugated to one or more glutamine residues via transglutaminase-based chemo-enzymatic conjugation (see, e.g., Dennler et al., Bioconjugate Chem. 2014, 25, 569-578). For example, in the presence of transglutaminase, one or more glutamine residues of an antibody can be coupled to a primary amine compound or a linker compound described herein. Primary amine compounds include, for example, payloads, linkers, linkers described herein, or linker-payloads, which directly provide antibody drug conjugates via transglutaminase-mediated coupling. Primary amine compounds also include linkers, including linkers described herein, and spacers that are functionalized with reactive groups that can be subsequently treated with further compounds towards the synthesis of antibody-drug conjugates (i.e., click chemistry and/or inverse electron deman Diels-Alder chemistry as described elsewhere herein). Antibodies comprising glutamine residues can be isolated from natural sources or engineered to comprise one or more glutamine residues. Techniques for engineering glutamine residues into an antibody polypeptide chain (glutaminyl- modified antibodies or antigen binding molecules) are within the skill of the practitioners in the art. In certain embodiments, the antibody is aglycosylated. [00133] In certain embodiments, the antibody or a glutaminyl-modified antibody or a transglutaminse-modified antibody or antigen binding molecule comprises at least one glutamine residue in at least one polypeptide chain sequence. In certain embodiments, the antibody or a glutaminyl-modified antibody or a transglutaminase-modified antibody or antigen binding molecule comprises two heavy chain polypeptides, each with one Gln295 or Q295 residue. In further embodiments, the antibody or a glutaminyl-modified antibody or transglutaminase- modified antibody or antigen binding molecule comprises one or more glutamine residues at a site other than a heavy chain 295. Included herein are antibodies of this section bearing N297Q mutation(s) described herein. Primary Amine Compounds [00134] In certain embodiments, primary amine compounds useful for the transglutaminase- mediated coupling of an antibody (or antigen binding compound) comprising a glutamine can be any primary amine compound deemed useful by the practitioner of ordinary skill. Generally, the primary amine compound has the formula H2N-R, where R can be any group compatible with the antibody and reaction conditions. In certain embodiments, R is alkyl, substituted alkyl, heteroalkyl, or substituted heteroalkyl. [00135] In some embodiments, the primary amine compound comprises a reactive group or protected reactive group. Useful reactive groups include azides, alkynes, dienophiles, dienes, cycloalkynes, thiols, alcohols, ketones, aldehydes, carboxylic acids, esters, amides, hydrazides, anilines, and amines. In certain embodiments, the reactive group is selected from the group consisting of azide, alkyne, sulfhydryl, cycloalkyne, aldehyde, dienophile, diene, and carboxyl. [00136] In certain embodiments, the primary amine compound is according to the formula H2N-LL-X, where LL is a divalent spacer and X is a reactive group or protected reactive group. In particular embodiments, LL is a divalent polyethylene glycol (PEG) group. In certain embodiments, X is selected from the group consisting of –SH, –N3, alkyne, aldehyde, and tetrazole. In particular embodiments, X is -N3. In certain embodiments, the primary amine compound is according to the formula (R1)(R2)N-W-X-Y-Z as described elsewhere herein. [00137] In certain embodiments, the primary amine compound is according to one of the following formulae: H2N-(CH2)n-X; H2N-(CH2CH2O)n-(CH2)p-X; H2N-(CH2)n-N(H)C(O)-(CH2)m-X; H2N-(CH2CH2O)n-N(H)C(O)-(CH2CH2O)m-(CH2)p-X; H2N-(CH2)n-C(O)N(H)-(CH2)m-X; H2N-(CH2CH2O)n-C(O)N(H)-(CH2CH2O)m-(CH2)p-X; H2N-(CH2)n-N(H)C(O)-(CH2CH2O)m-(CH2)p-X; H2N-(CH2CH2O)n-N(H)C(O)-(CH2)m-X; H2N-(CH2)n-C(O)N(H)-(CH2CH2O)m-(CH2)p-X; and H2N-(CH2CH2O)n-C(O)N(H)-(CH2)m-X; where n is an integer selected from one to twelve; m is an integer selected from zero to twelve; p is an integer selected from zero to two; and X is selected from the group consisting of –SH, -N3, –C≡CH, –C(O)H, tetrazole, and any of and wherein R4 is described elsewhere herein within the context of X-Y-Z as described elsewhere herein. [00138] In the above, any of the alkyl or alkylene (i.e., –CH2–) groups can optionally be substituted, for example with C1-8 alkyl, methylformyl, or –SO3H. In certain embodiments, the alkyl groups are unsubstituted. [00139] In certain embodiments, the primary amine compound is selected from the group consisting of:
[00140 [ ] p y above reactions are provided in the Examples below. Linkers [00142] In certain embodiments, the linker L portion of the conjugates described herein is a moiety, for instance a divalent moiety, that covalently links a binding agent to a payload compound described herein. In other instances, the linker L is a trivalent or multivalent moiety that covalently links a binding agent to a payload compound described herein. Suitable linkers may be found, for example, in Antibody-Drug Conjugates and Immunotoxins; Phillips, G. L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates; Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates; Wang, J., Shen, W.-C., and Zaro, J. L., Eds.; Springer International Publishing, 2015, the contents of each incorporated herein in their entirety by reference. In certain embodiments, the linker L portion of the linker-payloads described herein is a moiety covalently linked to a payload compound described herein, capable of divalently and covalently linking a binding agent to a payload compound described herein. In other instances, the linker L portion of the linker-payloads described herein is a moiety covalently linked to a payload compound described herein, capable of covalently linking, as a trivalent or multivalent moiety, a binding agent to a payload compound described herein. Payload compounds include MMAE, exatecan, DXd, and the like, and their residues following bonding or incorporation with linker L are linker-payload compounds. The linker-payloads can be further bonded to binding agents such as antibodies or antigen binding fragments thereof to form antibody-drug conjugates. Those of skill in the art will recognize that certain functional groups of payload moieties are convenient for linking to linkers and/or binding agents. For example, in certain embodiments, the linker is absent and payloads are directly bonded to binding agents. In certain embodiments, prodrugs or payloads include hydroxyl, amine, or thiol functionality capable of bonding with linkers and/or peptide residues within binding agents. [00143] In certain embodiments, the linkers are stable in physiological conditions. In certain embodiments, the linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value. In some embodiments, a linker comprises an enzyme-cleavable moiety. Illustrative enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages. In some embodiments, the linker comprises a cathepsin-cleavable linker. In some embodiments, the linker comprises a moiety that is stable at certain pHs and cleavable to release the payload portion at other pHs. For instance, in certain embodiments, the linker is stable at physiological pH and capable of releasing the payload portion at a local pH in the vicinity of a target. [00144] In some embodiments, the linker comprises a non-cleavable moiety. In some embodiments, the non-cleavable linker is derived from maleimide. In some embodiments, the non-cleavable linkers are derived from an ester. In some embodiments, the non-cleavable linker is derived from an N-hydroxysuccinimide ester. In some embodiments, the non-cleavable linkers are derived from a dienophile (e.g., alkenes or alkynes). In some embodiments, the non-cleavable linkers are derived from a diene (e.g., a tetrazine). In some embodiments, the non-cleavable linker is derived from or a residue thereof. In some embodiments, the non-cleavable linker-payload residue is , or a regioisomer thereof. In some embodiments, the non-cleavable linker is derived from or a residue thereof. In some embodiments, the non-cleavable linker-payload residue is , or a regioisomer thereof. In one embodiment, the linker is maleimide cyclohexane carboxylate or 4-(N- maleimidomethyl)cyclohexanecarboxylic acid (MCC), where the payload can be added to either end of the MCC linker. In another embodiment, the linker is , where the payload can be added to either end of this linker. In certain embodiments, the linker is a self-stabilizing maleimide. In one exemplary embodiment, the self-stabilizing maleimide linker is , where the bond from the amide nitrogen to the payload can be a direct bond to the payload; or the bond from the amide nitrogen to the payload, as shown, contemplates the remainder of the linker. In another exemplary embodiment, the self-stabilizing linker manifests as , where the bond from the amide nitrogen to the payload can be a direct bond to the payload; or the bond from the amide nitrogen to the payload, as shown, contemplates the remainder of the linker. Without being bound by any particular theory, the self-stabilizing linker includes moieties that stabilize the bond from the self-stabilizing linker to a binding agent. For example, in some embodiments, when the bond from a binding agent to a self-stabilizing linker is a carbon-sulfur bond (e.g., following a Michael addition of a binding agent cysteine to the self-stabilizing maleimide linker), the self-stabilizing linker mitigates retro-Michael additions. More specifically, in the self-stabilizing maleimide (or succinimide) linkers shown, the aminomethyl functionality facilitates rapid hydrolysis of the succinimide Michael addition product to provide , where the bond from the amide nitrogen to the payload can be a direct bond to the payload; or the bond from the amide nitrogen to the payload, as shown, contemplates the remainder of the linker; thus, decreasing susceptibility to retro-Michael additions. Moieties other than aminomethyl within self-stabilizing maleimide linkers that stablilize conjugates will be appreciated by those of skill in the art. In the structures, indicates a bond to a binding agent. In the structures, in some examples, indicates a click chemistry residue which results from the reaction of, for example, a binding agent having an azide or alkyne functionality and a linker-payload having a complementary alkyne or azide functionality. In the structures, in other examples, indicates a divalent sulfide which results from the reaction of, for example, one or more binding agent cysteines with one or more linkers or linker-payloads having maleimide functionality via Michael addition reactions. In the structures, in other examples, indicates an amide bond which results from the reaction of, for example, one or more binding agent lysines with one or more linkers or linker-payloads having activated or unactivated carboxyl functionality, as would be appreciated by a person of skill in the art. In one embodiment, indicates an amide bond which results from the reaction of, for example, one or more binding agent lysines with one or more linkers or linker-payloads having activated carboxyl functionality, as would be appreciated by a person of skill in the art. In the structures, in some examples, indicates an inverse electron demand Diels-Alder residue which results from the reaction of, for example, a binding agent having dienophile or diene functionality and a linker-payload having a complementary dienophile or diene functionality. [00145] In some embodiments, suitable linkers include, but are not limited to, those that are chemically bonded to two cysteine residues of a single binding agent, for example, an antibody or antigen binding fragment thereof. Such linkers can serve to mimic the antibody’s disulfide bonds that are disrupted as a result of the conjugation process. [00146] In some embodiments, the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D- α-amino acids. In some embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or any combination thereof (e.g., dipeptides, tripeptides, oligopeptides, polypeptides, and the like). In certain embodiments, one or more side chains of the amino acids are linked to a side chain group, described below. In some embodiments, the linker is a peptide comprising or consisting of the amino acids valine and citrulline (e.g., divalent –Val-Cit– or divalent –VCit–). In some embodiments, the linker is a peptide comprising or consisting of the amino acids alanine and alanine, or divalent –AA–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid and alanine, or –EA–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid and glycine, or –EG–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glycine and glycine, or –GG–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamine, valine, and citrulline, or –Q-V- Cit– or –QVCit–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids glutamic acid, valine, and citrulline, or –E-V-Cit– or –EVCit–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –GGGGS–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –GGGGG–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –GGGGK–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –GFGG–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids lysine, valine, and citrulline, or –KVCit–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –KVA–. In some embodiments, the linker is a peptide comprising or consisting of the amino acids –VA–. In any of the embodiments in this paragraph, and throughout this disclosure, the standard three-letter or one-letter amino acid designations are used, as would be appreciated by a person of skill in the art. Exemplary single- letter amino acid designations include, G for glycine, K for lysine, S for serine, V for valine, A for alanine, and F for phenylalanine. [00147] In some embodiments, the linker comprises a self-immolative group. The self-immolative group can be any such group known to those of skill. In particular embodiments, the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those of skill will recognize that a self-immolative group is capable of carrying out a chemical reaction which releases the remaining atoms of a linker from a payload. [00148] In some embodiments, the linker is wherein SP1 is a spacer; SP2 is a spacer; is one or more bonds to the binding agent; is one or more bonds to the payload; each AA is an amino acid residue; and n is an integer from zero to ten. In some embodiments, the linker is , , or wherein SP is a spacer SP1 is a spacer; SP2 is a spacer; SP3 is a spacer; is one or more bonds to the binding agent; is one or more bonds to the payload; each AA is an amino acid residue; R8 is alkylene or heteroalkylene; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent;and p is an integer from zero to ten. [00149] In one embodiment, SP is . The SP1 spacer is a moiety that connects the (AA)n moiety or residue to the binding agent (BA) or to a reactive group residue which is bonded to BA. Suitable SP1 spacers include, but are not limited to, those comprising alkylene or polyether, or both. The ends of the spacers, for example, the portion of the spacer bonded to the BA or an AA, can be moieties derived from reactive moieties that are used for purposes of coupling the antibody or an AA to the spacer during chemical synthesis of the conjugate. In certain embodiments, n is one, two, three, or four. In particular embodiments, n is two. In particular embodiments, n is three. In particular embodiments, n is four. In certain embodiments, when n is zero, then (AA)n is a bond. In certain embodiments, p is one, two, three, or four. In particular embodiments, p is two. In particular embodiments, p is three. In particular embodiments, p is four. In certain embodiments, when p is zero, then (AA)n is a bond. [00150] In some embodiments, the SP1 spacer comprises an alkylene. In some embodiments, the SP1 spacer comprises a C5-7 alkylene. In some embodiments, the SP1 spacer comprises a polyether. In some embodiments, the SP1 spacer comprises a polymer of ethylene oxide such as polyethylene glycol. [00151] In some embodiments, the SP1 spacer is , , , or wherein RG' is a reactive group residue following reaction of a reactive group RG with a binding agent; is a bond to the binding agent; is a bond to (AA)n wherein n is an integer from zero to ten; and b is an integer from two to eight. [00152] The reactive group RG can be any reactive group known to those of skill in the art to be capable of forming one or more bonds to the binding agent. The reactive group RG is a moiety comprising a portion in its structure that is capable of reacting with the binding agent (e.g., reacting with an antibody at its cysteine or lysine residues; or at an azide moiety, for example, a PEG-N3 functionalized antibody at one or more glutamine residues; or at a dienophile moiety, for example, a dienophile functionalized antibody at one or more glutamine residues; or a diene moiety, for example, a diene functionalized antibody at one or more glutamine residues) to form a conjugate. Following conjugation to the binding agent, the reactive group becomes the reactive group residue (RG′). Illustrative reactive groups include, but are not limited to, those that comprise haloacetyl, isothiocyanate, succinimide, N-hydroxysuccinimide, dienophile, diene, or maleimide portions that are capable of reacting with the binding agent or functionalized binding agent. [00153] In certain embodiments, reactive groups include, but are not limited to, alkynes, dienophiles, and dienes. In certain embodiments, the alkynes are alkynes capable of undergoing 1,3-cycloaddition reactions with azides in the absence of copper catalysts, such as strained alkynes. Strained alkynes are suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), and include cycloalkynes, for example, cyclooctynes and benzannulated alkynes. Suitable alkynes include, but are not limited to, dibenzoazacyclooctyne or (DIBAC); dibenzocyclooctyne or (DIBO); biarylazacyclooctynone or (BARAC); difluorinated cyclooctyne or , or , or (DIFO); substituted, for example, fluorinated alkynes, aza-cycloalkynes, bicycle[6.1.0]nonyne or (BCN); and derivatives thereof. Particularly useful alkynes include , , and . In certain embodiments, the dienophiles are dienophiles capable of undergoing inverse electron demand Diels-Alder reactions with dienes. In certain embodiments, the dienes are dienes (e.g., tetrazines) capable of undergoing inverse electron demand Diels- Alder reactions with dienophiles. Suitable dienophiles include, but are not limited to, , , and . Suitable dienes include, but are not limited to, and . [00154] In certain embodiments, the binding agent is bonded directly to RG′. In certain embodiments, the binding agent is bonded to RG′ via a spacer, for instance SP4, located between and RG′. In particular embodiments, the binding agent is bonded indirectly to RG′ via SP4, for example, a PEG spacer. As discussed in detail below, in certain embodiments, the binding agent is prepared by functionalizing with one or more azido groups. Each azido group is capable of reacting with RG to form RG′. In particular embodiments, the binding agent is derivatized with –PEG-N3 linked to a glutamine residue. Exemplary -N3 derivatized binding agents, methods for their preparation, and methods for their use in reacting with RG are provided herein. In certain embodiments, RG is an alkyne suitable for participation in 1,3-cycloadditions, and RG′ is a regioisomeric 1,2,3-triazolyl moiety formed from the reaction of RG with an azido- functionalized binding agent. By way of further example, in certain embodiments, RG′ is linked to the binding agent as shown in or , or a mixture of each regioisomer. Each R and R′ is as described or exemplified herein. [00155] The SP2 spacer, when present, is a moiety that connects the (AA)n or (AA)p moiety to the binding agent. The SP2 spacer, when present, can be any spacer suitable for connection of the (AA)n or (AA)p moiety to the binding agent, for example, those comprising alkylene or polyether, or both. In one embodiment, the SP2 spacer, when present, is where b is an integer from one to ten. [00156] The SP3 spacer, when present, is a moiety that connects the (AA)n or (AA)p moiety to the payload. Suitable spacers include, but are not limited to, those described above as SP1 spacers. Further suitable SP3 spacers include, but are not limited to, those comprising alkylene or polyether, or both. The ends of the SP3 spacers, for example, the portion of the spacer directly bonded to the payload or an AA, can be moieties derived from reactive moieties that are used for purposes of coupling the payload or AA to the SP3 spacer during the chemical synthesis of the conjugate. In some examples, the ends of the SP3 spacers, for example, the portion of the SP3 spacer directly bonded to the payload or an AA, can be residues of reactive moieties that are used for purposes of coupling the payload or an AA to the spacer during the chemical synthesis of the conjugate. [00157] In some embodiments, the SP3 spacer, when present, is selected from the group consisting of –NH-(p-C6H4)-CH2–, –NH-(p-C6H4)-CH2OC(O)–, an amino acid, a dipeptide, a tripeptide, an oligopeptide, –O–, –N(H)–, , , , , , , , , , , , , , , and any combinations thereof. In certain embodiments, each is a bond to the payload, and each is a bond to (AA)n or (AA)p [00158] In the above formulae, each (AA)n or (AA)p is an amino acid or, optionally, a p- aminobenzyloxycarbonyl residue (PABC). If PABC is present, in certain embodiments, then only one PABC is present. In certain embodiments, the PABC residue, if present, is bonded to a terminal AA in the (AA)n group, proximal to the payload. Suitable amino acids for each AA include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D- α-amino acids. In some embodiments, the AA comprises alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or any combinations thereof (e.g., dipeptides, tripeptides, and oligopeptides, and the like). In certain embodiments, one or more side chains of the amino acids is linked to a side chain group, described below. In some embodiments, n or p is two. In some embodiments, the (AA)n or (AA)p is valine-citrulline. In some embodiments, (AA)n or (AA)p is citrulline- valine. In some embodiments, (AA)n or (AA)p is valine-alanine. In some embodiments, (AA)n or (AA)p is alanine-valine. In some embodiments, (AA)n or (AA)p is valine-glycine. In some embodiments, (AA)n or (AA)p is glycine-valine. In some embodiments, n or p is three. In some embodiments, the (AA)n or (AA)p is valine-citrulline-PABC. In some embodiments, (AA)n or (AA)p is citrulline-valine-PABC. In some embodiments, (AA)n or (AA)p is glutamate-valine- citrulline. In some embodiments, (AA)n or (AA)p is glutamine-valine-citrulline. In some embodiments, (AA)n or (AA)p is lysine-valine-alanine. In some embodiments, (AA)n or (AA)p is lysine-valine-citrulline. In some embodiments, n or p is four. In some embodiments, (AA)n or (AA)p is glutamate-valine-citrulline-PABC. In some embodiments, (AA)n or (AA)p is glutamine- valine-citrulline-PABC. Those of skill will recognize PABC as a residue of p- aminobenzyloxycarbonyl with the following structure: . The PABC residue has been shown to facilitate cleavage of certain linkers in vitro and in vivo. Those of skill will recognize PAB as a divalent residue of p-aminobenzyl or –NH-(p-C6H4)-CH2–. Linker-Payloads [00159] In certain embodiments, linker-payloads include any specific compound, prodrug, or payload described herein, bonded to a linker, wherein the linker(s) described herein include a moiety that is reactive with an antibody or antigen binding fragment thereof described herein. In one embodiment, the compound or linker-payload is a structure of Formula III or IV (III) or (IV) or a pharmaceutically acceptable salt thereof, wherein X is or ; SP1, SP2, and SP3, when present, are in each instance independent spacer groups wherein SP1 further comprises the moiety reactive with the binding agent; each AA is an amino acid; and p is an integer from zero to ten. In any embodiment in this paragraph, the linker further comprises . In one embodiment, p is zero. In one embodiment, p is one. In one embodiment, p is two. In one embodiment, p is three. In one embodiment, p is four. In one embodiment, p is five. In one embodiment, p is six. In one embodiment, p is seven. In one embodiment, p is eight. In one embodiment, p is nine. In one embodiment, p is ten. In one embodiment, SP1 comprises a reactive group that comprises an alkene, alkyne, , and/or . In one embodiment, the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction. In one embodiment, the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction to form regioisomeric Diels-Alder adduct moieties, wherein the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent. In one embodiment, SP3 comprises . In one embodiment, SP3 comprises , and R2 is hydrogen or benzyl. In one embodiment, the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels- Alder reaction to form regioisomeric Diels-Alder adduct moieties, the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent, and SP3 comprises . In one embodiment, the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction to form regioisomeric Diels- Alder adduct moieties, the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent, and SP3 comprises , and R2 is hydrogen or benzyl. In certain embodiments in this paragraph, the binding agent is an antibody or antigen binding fragment thereof. [00160] In one embodiment, the compound or linker-payload is a structure of Formula III (III) wherein R1a, R1b, R2, and R3 are hydrogen; and m is one. In certain embodiments, the compound or linker-payload is a structure of Formula III (III) wherein R1a, R1b, and R3 are hydrogen; R2 is an amino acid side chain; and m is one. In certain embodiments, the compound or linker- payload is a structure of Formula III (III) wherein R1a, R1b, R2, and R3 are hydrogen; R4 is alkyl; and m is one. In one embodiment, R1a, R1b, R2, and R3 are hydrogen; and m is two or four. In one embodiment, m is two. In one embodiment, m is four. In one embodiment, the compound or linker-payload is a structure of Formula IV (IV) wherein R1a, R1b, and R3 are hydrogen; R2 is hydrogen or benzyl; and n is one or four. In one embodiment, D* is a residue of a biologically active compound or a therapeutic moiety, wherein the therapeutic moiety is a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, an imaging agent moiety, or an analogue or derivative thereof, comprising amino. In one embodiment, D* is a residue of a maytansinoid. In one embodiment, D* is a residue of a tubulysin. In one embodiment, D* is a residue of an auristatin. In one embodiment, D* is a residue of a dolastatin. In one embodiment, D* is a residue of a camptothesin. In one embodiment, D* is a residue of a pyrrolobenzodiazepine. In one embodiment, D* is a residue of an antibiotic. In one embodiment, D* is a residue of an antiviral agent. In one embodiment, D* is a residue of an anti-inflammatory agent. In one embodiment, D* is a residue of an immunomodulator. In one embodiment, D* is a residue of an antifungal agent. In one embodiment, D* is a residue of a steroid. In one embodiment, D* is a residue of an imaging agent. In certain embodiments in this paragraph, D* is a residue of a maytansinoid, a tubulysin, an auristatin, a dolastatin, a camptothesin, a pyrrolobenzodiazepine, an antibiotic, an antiviral agent, an anti-inflammatory agent, an immunomodulator, an antifungal agent, a steroid, or an imaging agent moiety, or an analogue or derivative thereof. In one embodiment, the compound of linker-payload is selected from the group consisting of LP3; LP9; LP12; LP13; LP14; LP18; LP19; and LP20. In one embodiment, the compound or linker-payload is LP3. In one embodiment, the compound or linker-payload is LP9. In one embodiment, the compound or linker-payload is LP12. In one embodiment, the compound or linker-payload is LP13. In one embodiment, the compound or linker-payload is LP14. In one embodiment, the compound or linker-payload is LP18. In one embodiment, the compound or linker-payload is LP19. In one embodiment, the compound or linker-payload is LP20. In one embodiment, the compound or linker-payload is LP21. In one embodiment, the compound or linker payload is LP8. In one embodiment, the compound or linker-payload is LP22. In one embodiment, the compound or linker-payload is selected from the group consisting of LP10; and
LP11. In one embodiment, the compond or linker-payload is LP10. In one embodiment, the compound or linker-payload is LP11. In certain embodiments in this paragraph, the binding agent is an antibody or antigen binding fragment thereof. Conjugates/Antibody-Drug Conjugates (ADCs) [00161] Provided herein are binding agents and antibodies or antigen binding fragments thereof, wherein said binding agent or antibody is conjugated to one or more compounds described herein. Provided herein is a compound wherein the compound is a binding agent or transglutaminase-modified binding agent comprising a compound of the formula (R1)(R2)N-W-X-Y-Z wherein W is C1-C10 alkylene or C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene, -C(O)N(R3)-C1-C10 alkylene-C(O)-(AA)p-N(R3)-, -C(O)- (AA)p-N(R3)-, -C(O)N(R3)-, -N(R5)2, -C(O)N(R5)2, -CH(R5)(R6), -C(O)-(AA)p-N(R3)-C1-C10 alkylene-PEGn-C1-C10 alkylene-N(R5)2, -C(O)-(AA)p-N(R5)2, -C(O)-(AA)p-N(R3)-CH(R5)(R6), or -N(R5)(R6); Y when present is C1-C10 alkylene; Z is selected from the group consisting of , , , , , , and ; R1, R2, R3, and R4 are independently hydrogen or C1-C10 alkyl; R5 is -Y-C(O)N(R3)-Y-Z, -Y-(R3)NC(O)-Y-Z, -(R3)NC(O)-Y-C(O)N(R3)-Y-Z, or -C(O)N(R3)-Y-Z; R6 is -Y-C(O)N(R3)-PEGn1-C1-C10 alkylene-N3, -C(O)N(R3)-PEGn1-C1-C10 alkylene-N3, or -C1-C10 alkylene-N3; AA is an amino acid residue and p is an integer from one to twenty; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -N(R3)C(O)-C1-C10 alkylene; and Z is , then n is an integer from two to one hundred; wherein when W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-; Y is C1-C10 alkylene; and Z is , then n is an integer from one to three, five to seven, or nine to one hundred; wherein when W is C1-C10 alkylene-PEGn- C1-C10 alkylene; X is -C(O)N(R5)2; and Z is , then n is an integer from one to one hundred; and n1 is an integer from zero to one hundred. In one embodiment, the binding agent further comprises a compound of (I) or (II) or a pharmaceutically acceptable salt thereof, wherein L is a linker comprising a moiety reactive with a binding agent; X is or , wherein T is part of the linker and comprises a moiety reactive with a binding agent; SP is a spacer group; R1a and R1b are, independently, hydrogen or alkyl; R2 is hydrogen or an amino acid side chain; R3, R4, and R6 is hydrogen or alkyl; R5 is oxygen, NR6, or sulfur; R7 is an O-amino acid residue; R8 is C1-C10 alkylene or C1-C10 heteroalkylene; D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein when m is four, then the moiety reactive with the binding agent is ; and wherein when n is four, then the moiety reactive with the binding agent is ; or LP1; LP7; LP15; LP16; or LP17. In one embodiment, the binding agent or transglutaminase-modified binding agent compound is , wherein BA is the binding agent. In any embodiment in this paragraph, the binding agent is an antibody or transglutaminase-modified antibody, or antigen binding fragments thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof are selected from an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti-EGFRvIII antibody, an anti-MUC16 antibody, an anti-prolactin receptor (PRLR) antibody, an anti-prostate- specific membrane antigen (PSMA) antibody, an anti-FGFR2 antibody, an anti-FOLR1 antibody, an anti-HER2/HER2 bispecific antibody, an anti-MET/MET bispecific antibody, or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase- modified antibody, or antigen binding fragments thereof is an anti-HER2 antibody or an antigen- binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-STEAP2 antibody or an antigen- binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MET antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-EGFRvIII antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MUC16 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-prolactin receptor (PRLR) antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-prostate-specific membrane antigen (PSMA) antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase- modified antibody, or antigen binding fragments thereof is an anti-FGFR2 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase- modified antibody, or antigen binding fragments thereof is an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase- modified antibody, or antigen binding fragments thereof is an anti-HER2/HER2 bispecific antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MET/MET bispecific antibody or an antigen-binding fragment thereof. [00162] In one embodiment, provided is a conjugate comprising (R1)(R2)N-W-X-Y-Z wherein R1, R2, W, X, Y, and Z are as described elsewhere herein; LP1; LP7; (I); or (II) wherein L, X, T, SP, R1a, R1b, R2, R3, R4, R5, R6, R7, R8, D*, m, and each n are as described elsewhere herein. In another embodiment, the conjugate is selected from the group consisting of ADC10; ADC11; ADC12; ADC13; ADC17; ADC22; ADC23;
ADC24; ADC25;
; and wherein BA is a binding agent. In one embodiment, the binding agent is an antibody or transglutaminase-modified antibody, or antigen binding fragments thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof are selected from an anti-HER2 antibody, an anti-STEAP2 antibody, an anti-MET antibody, an anti- EGFRvIII antibody, an anti-MUC16 antibody, an anti-prolactin receptor (PRLR) antibody, an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-FGFR2 antibody, an anti- FOLR1 antibody, an anti-HER2/HER2 bispecific antibody, an anti-MET/MET bispecific antibody, or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-HER2 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-STEAP2 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MET antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-EGFRvIII antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MUC16 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-prolactin receptor (PRLR) antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti- prostate-specific membrane antigen (PSMA) antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-FGFR2 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-FOLR1 antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-HER2/HER2 bispecific antibody or an antigen-binding fragment thereof. In one embodiment, the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof is an anti-MET/MET bispecific antibody or an antigen-binding fragment thereof. Methods of Preparing Compounds, Prodrugs, or Payloads, and Linker-Payloads [00163] The compounds provided herein can be prepared, isolated, or obtained by any method apparent to those of skill in the art. Exemplary methods of preparation are described in detail in the Examples below. In one embodiment, provided is a method of making a conjugate comprising the steps of (a) treating a binding agent with H2N-PEGn-N3 in the presence of microbial transglutaminase to provide a transglutaminase-modified binding agent; (b) treating the transglutaminase-modified binding agent with a compound or linker-payload described herein; and (c) purifying the conjugate, wherein n is an integer from one to one hundred. In one embodiment, the compound or linker-payload used in step (b) is LP1. [00164] In one embodiment, provided is a method of making a conjugate comprising the steps of (a) treating a binding agent with a compound of (R1)(R2)N-W-X-Y-Z wherein R1, R2, W, X, Y, and Z are as described elsewhere herein, in the presence of microbial transglutaminase to provide a transglutaminase-modified binding agent; (b) treating the transglutaminase-modified binding agent with a complementary compound LP1; LP3; LP7; LP8; LP9; LP10; LP11; LP12; LP13; LP14; LP15; LP16; LP17; LP18; LP19; LP20; LP21; LP22; (I); or (II) wherein L, X, T, SP, R1a, R1b, R2, R3, R4, R5, R6, R7, R8, D*, m, and each n are as described elsewhere herein; and (c) purifying the conjugate. In one embodiment, the compound or linker-payload used in step (b) is selected from the group consisting of LP7; LP3; LP9; LP8;
LP10; and LP11. In one embodiment, the compound or linker-payload used in step (b) is LP7. In one embodiment, the compound or linker-payload used in step (b) is LP3. In one embodiment, the compound or linker-payload used in step (b) is LP9. In one embodiment, the compound or linker-payload used in step (b) is LP8. In one embodiment, the compound or linker-payload used in step (b) is LP10. In one embodiment, the compound or linker-payload used in step (b) is LP11. [00165] The conjugates described herein can be synthesized by coupling the linkers or linker- payloads described herein with a binding agent, for example, an antibody under standard conjugation conditions (see, e.g., Doronina et al. Nature Biotechnology 2003, 21, 778, which is incorporated herein by reference in its entirety). When the binding agent is an antibody, the antibody may be coupled to a linker or linker-payload via one or more cysteine or lysine residues of the antibody. Linker-payloads can be coupled to cysteine residues, for example, by subjecting the antibody to a reducing agent, for example, dithiotheritol, to cleave the disulfide bonds of the antibody, purifying the reduced antibody, for example, by gel filtration, and subsequently treating the antibody with a linker-payload containing a suitable reactive moiety, for example, a maleimido group (i.e., via Michael addition). Suitable solvents include, but are not limited to water, DMA, DMF, and DMSO. Linkers or Linker-payloads containing a reactive group, for example, an activated ester or acid halide group, can be coupled to lysine residues of the antibody. Suitable solvents include, but are not limited to, water, DMA, DMF, and DMSO. Conjugates can be purified using known protein techniques, including, for example, size exclusion chromatography, dialysis, and ultrafiltration/diafiltration. [00166] Binding agents, for example antibodies, can also be conjugated via click chemistry reactions. In some embodiments of said click chemistry reactions, the linker-payload includes a reactive group, for example an alkyne, that is capable of undergoing a regioisomeric 1,3-cycloaddition reaction with an azide. Such suitable reactive groups are described above. The antibody includes one or more azide groups. Such antibodies include antibodies functionalized with, for example, azido-polyethylene glycol groups. In certain embodiments, such functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a primary amine compound in the presence of the enzyme transglutaminase. In certain embodiments, such functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln297, with a primary amine compound in the presence of the enzyme transglutaminase. Such antibodies include Asn297Gln (N297Q) mutants. In certain embodiments, such functionalized antibody is derived by treating an antibody having at least two glutamine residues, for example, heavy chain Gln295 and heavy chain Gln297, with a primary amine compound in the presence of the enzyme transglutaminase. Such antibodies include Asn297Gln (N297Q) mutants. In certain embodiments, the antibody has two heavy chains as described in this paragraph for a total of two or a total of four glutamine residues. Binding agents, for example antibodies, can also be conjugated via inverse electron demand Diels-Alder reactions. In some embodiments of said Diels-Alder reactions, the linker-payload includes a reactive group, for example an alkyne, that is capable of undergoing a regioisomeric Diels-Alder reaction with a tetrazine. Such suitable reactive groups are described above. The antibody includes one or more dienophile or diene compounds. Such antibodies include antibodies functionalized with, for example, (R1)(R2)N-W- X-Y-Z as described elsewhere herein. In certain embodiments, such functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln295, with a primary amine compound (e.g., (R1)(R2)N-W-X-Y-Z as described elsewhere herein) in the presence of the enzyme transglutaminase. In certain embodiments, such functionalized antibody is derived by treating an antibody having at least one glutamine residue, for example, heavy chain Gln297, with a primary amine compound (e.g., (R1)(R2)N-W-X-Y-Z as described elsewhere herein) in the presence of the enzyme transglutaminase. Such antibodies include Asn297Gln (N297Q) mutants. In certain embodiments, such functionalized antibody is derived by treating an antibody having at least two glutamine residues, for example, heavy chain Gln295 and heavy chain Gln297, with a primary amine compound (e.g., (R1)(R2)N-W-X-Y-Z as described elsewhere herein) in the presence of the enzyme transglutaminase. Such antibodies include Asn297Gln (N297Q) mutants. In certain embodiments, the antibody has two heavy chains as described in this paragraph for a total of two or a total of four glutamine residues. [00167] In certain embodiments, the antibody comprises two glutamine residues, one in each heavy chain. In particular embodiments, the antibody comprises a Q295 residue in each heavy chain. In further embodiments, the antibody comprises one, two, three, four, five, six, seven, eight, or more glutamine residues. These glutamine residues can be in heavy chains, light chains, or in both heavy chains and light chains. These glutamine residues can be wild-type residues, or engineered residues, or the antibody can comprise both wild-type and engineered glutamine residues. In certain embodiments, the antibody is glycosylated. In certain embodiments, the antibody is aglycosylated. In certain embodiments, the antibody is deglycosylated. The antibodies can be prepared according to standard techniques. [00168] Those of skill will recognize that antibodies are often glycosylated at residue N297, near residue Q295 in a heavy chain sequence. Glycosylation at residue N297 can interfere with a transglutaminase at residue Q295 (Dennler et al., supra). Accordingly, in advantageous embodiments, the antibody is not glycosylated. In certain embodiments, the antibody is deglycoslated or aglycosylated. In particular embodiments, an antibody heavy chain has an N297 mutation. Alternatively stated, the antibody is mutated to no longer have an asparagine residue at position 297. In particular embodiments, an antibody heavy chain has an N297Q mutation. Such an antibody can be prepared by site-directed mutagenesis to remove or disable a glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at a site apart from any interfering glycosylation site or any other interfering structure. Such an antibody also can be isolated from natural or artificial sources. [00169] The antibody without interfering glycosylation is then treated with a primary amine compound (e.g., (R1)(R2)N-W-X-Y-Z as described elsewhere herein). In certain embodiments, an aglycosylated antibody is reacted or treated with a primary amine compound to produce a glutaminyl-modified antibody. In certain embodiments, a deglycosylated antibody is reacted or treated with a primary amine compound to produce a glutaminyl-modified antibody. [00170] The primary amine can be any primary amine that is capable of forming a covalent bond with a glutamine residue in the presence of a transglutaminase. Useful primary amines are described herein (e.g., (R1)(R2)N-W-X-Y-Z). The transglutaminase can be any transglutaminase deemed suitable by those of skill in the art. In certain embodiments, the transglutaminase is an enzyme that catalyzes the formation of an isopeptide bond between a free amine group on the primary amine compound and the acyl group on the side chain of a glutamine residue. Transglutaminase is also known as protein-glutamine-γ-glutamyltransferase. In particular embodiments, the transglutaminase is classified as EC 2.3.2.13. The transglutaminase can be from any source deemed suitable. In certain embodiments, the transglutaminase is microbial. Useful transglutaminases have been isolated from Streptomyces mobaraense, Streptomyces cinnamoneum, Streptomyces griseo-carneum, Streptomyces lavendulae, and Bacillus subtilis. Non-microbial transglutaminases, including mammalian transglutaminases, can also be used. In certain embodiments, the transglutaminase can be produced by any technique or obtained from any source deemed suitable by the practitioner of skill. In particular embodiments, the transglutaminase is obtained from a commercial source. [00171] In particular embodiments, the primary amine compound (e.g., (R1)(R2)N-W-X-Y- Z as described elsewhere herein) comprises a reactive group capable of further reaction after transglutamination. In these embodiments, the glutaminyl-modified antibody can be treated with a reactive payload compound or a reactive linker-payload compound to form an antibody- payload conjugate. In certain embodiments, the primary amine compound comprises an azide. In certain embodiment, the primary amine compound is (R1)(R2)N-W-X-Y-Z as described elsewhere herein. [00172] In certain embodiments, the glutaminyl-modified antibody is treated with a reactive linker-payload to form an antibody-payload conjugate. The reaction can proceed under conditions deemed suitable by those of skill in the art. In certain embodiments, the glutaminyl- modified antibody is contacted with the reactive linker-payload compound under conditions suitable for forming a bond between the glutaminyl-modified antibody and the linker-payload compound. Suitable reaction conditions are well known to those in the art. Exemplary reactions are provided in the Examples below. Accordingly, provided herein is a method of preparing an antibody-drug conjugate comprising contacting a binding agent, as described herein, with a linker or linker-payload, also as described herein. Pharmaceutical Compositions and Methods of Treatment [00173] Provided herein are methods of treating and preventing diseases, conditions, or disorders comprising administering a therapeutically or prophylactically effective amount or one or more of the compounds disclosed herein, for example, one or more of the compounds of a formula provided herein (e.g., (R1)(R2)N-W-X-Y-Z or Formula I-IV each as described elsewhere herein). Diseases, disorders, and/or conditions include, but are not limited to, those associated with the antigens described herein. [00174] The compounds described herein can be administered alone or together with one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered just prior to, concurrent with, or shortly after the administration of the compounds described herein. The present disclosure also includes pharmaceutical compositions comprising any of the compounds described herein in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combinations to subjects in need thereof. [00175] Suitable additional therapeutic agents include, but are not limited to, a second glucocorticoid, steroid, LXR modulator, an inflammatory therapeutic agent, an autoimmune therapeutic agent, a hormone, a biologic, or a monoclonal antibody. Suitable therapeutic agents also include, but are not limited to any pharmaceutically acceptable salts, acids, or derivatives of a compound set forth herein. The compounds described herein can also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma, and/or NSAIDs. [00176] In some embodiments of the methods described herein, multiple doses of a compound described herein (or a pharmaceutical composition comprising a combination of a compound described herein and any of the additional therapeutic agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this embodiment of the disclosure comprise sequentially administering to a subject multiple doses of a compound described herein. As used herein, “sequentially administering” means that each dose of the compound is administered to the subject at a different point in time, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of a compound described herein, followed by one or more secondary doses of the compound, and optionally followed by one or more tertiary doses of the compound. [00177] The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the compounds described herein. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses can all include the same amount of a compound described herein, but generally can differ from one another in terms of frequency of administration. In certain embodiments, the amount of the compound included in the initial, secondary, and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”). [00178] In certain exemplary embodiments of the present disclosure, each secondary and/or tertiary dose is administered one to twenty-six (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein means, in a sequence of multiple administrations, the dose the compound which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses. [00179] The methods according to this embodiment of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of the compound. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient. The administration regimen may be carried out indefinitely over the lifetime of a particular subject, or until such treatment is no longer therapeutically needed or advantageous. [00180] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient one to two weeks or one to two months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient two to twelve weeks after the immediately preceding dose. In certain embodiments of the disclosure, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination. [00181] The present disclosure includes administration regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by administration of two or more maintenance doses to the patient on a less frequent basis. For example, according to this embodiment of the disclosure, if the loading doses are administered at a frequency of once a month, then the maintenance doses may be administered to the patient once every six weeks, once every two months, once every three months, etc. [00182] The present disclosure includes pharmaceutical compositions of the compounds and/or conjugates described herein, for example, the (R1)(R2)N-W-X-Y-Z compounds and/or compounds of Formulae I-IV, for example, compositions comprising a compound described herein, a salt, stereoisomer, regioisomer, polymorph thereof, and a pharmaceutically acceptable carrier, diluent, and/or excipient. Examples of suitable carriers, diluents and excipients include, but are not limited to, buffers for maintenance of proper composition pH (e.g., citrate buffers, succinate buffers, acetate buffers, phosphate buffers, lactate buffers, oxalate buffers, and the like), carrier proteins (e.g., human serum albumin), saline, polyols (e.g., trehalose, sucrose, xylitol, sorbitol, and the like), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxolate, and the like), antimicrobials, and antioxidants. In one embodiment, provided is a pharmaceutical composition including the compounds of (R1)(R2)N-W-X-Y-Z and Formulae I-IV, and a pharmaceutically acceptable excipient, carrier, or diluent. [00183] In some examples, set forth herein is a method of treating a disease, disorder or condition including administering to a patient having said disorder a therapeutically effective amount of a compound set forth herein, or a pharmaceutical composition thereof. [00184] In some examples, set forth herein is a method of treating a disease, disorder or condition including administering to a patient having said disorder a therapeutically effective amount of (R1)(R2)N-W-X-Y-Z and/or Formulae I-IV, or a pharmaceutical composition thereof. [00185] The present disclosure includes methods of preventing certain disorders or conditions comprising administering a therapeutically effective amount of one or more of the compounds disclosed herein (i.e., prophylactic uses). Examples include, but are not limited to preventing cytokine release syndrome for CD3 bispecifics, and adoptive cellular therapies such as CAR T cells, systemic IL-2 administration, graft-versus-host disease, and post-operative nausea and vomiting. Examples also include, but are not limited to, therapeutic antibodies such as alemtuzumab, muromonab, rituximab, tosituzumab, and agonistic antiboides where immune stimulation might be part of the intended mechanism of action.. [00186] In some embodiments, the disease, disorder, or condition is an allergic state, including, but not limited to, asthma, atopic dermatitis, contact dermatitis, drug hypersensitivity reactions, anaphylactic rhinitis, perennial or seasonal allergic rhinitis, and serum sickness; dermatologic diseases and conditions including, but not limited to, skin itching, seborrheic dermatitis neurodermatitis bullous dermatitis herpetiformis exfoliative erythroderma mycosis fungoides, pemphigus, and severe erythema multiforme (Stevens-Johnson syndrome); endocrine disorders including, but not limited to, primary or secondary adrenocortical insufficiency, congenital adrenal hyperplasia, hypercalcemia associated with cancer, and nonsuppurative thyroiditis; gastrointestinal diseases; hematologic disorders including, but not limited to, acquired (autoimmune) hemolytic anemia, congenital (erythroid) hypoplastic anemia (Diamond- Blackfan anemia), idiopathic thrombocytopenic purpura in adults, pure red cell aplasia, and secondary thrombocytopenia; trichinosis; tuberculous meningitis with subarachnoid block or impending block; neoplastic diseases including, but not limited to, leukemias and lymphomas; nervous system disorders including, but not limited to, acute exacerbations of multiple sclerosis, cerebral edema associated with primary or metastatic brain tumor, craniotomy, or head injury; ophthalmic diseases including, but not limited to, sympathetic ophthalmia, temporal arteritis, uveitis, and ocular inflammatory conditions unresponsive to topical corticosteroids; renal diseases including, but not limited to, for inducing a diuresis or remission of proteinuria in idiopathic nephrotic syndrome or that due to lupus erythematosus; respiratory diseases including, but not limited to, berylliosis, fulminating or disseminated pulmonary tuberculosis when used concurrently with appropriate antituberculous chemotherapy, idiopathic eosinophilic pneumonias, symptomatic sarcoidosis; and Rheumatic disorders including, but not limited to, use as adjunctive therapy for short-term administration (to tide the patient over an acute episode or exacerbation) in acute gouty arthritis, acute rheumatic carditis, ankylosing spondylitis, psoriaticarthritis, rheumatoid arthritis, including juvenile rheumatoid arthritis, and for use in dermatomyositis, polymyositis, and systemic lupus erythematosus. [00187] In some examples, set forth herein is a method for treating a disease, disorder, or condition selected from an autoimmune disease, an allergy, arthritis, asthma, a breathing disorder, a blood disorder, a cancer, a collagen disease, a connective tissue disorder, a dermatological disease, an eye disease, an endocrine problem, an immunological disease, an inflammatory disease, an intestinal disorder, a gastrointestinal disease, a neurological disorder, an organ transplant condition, a rheumatoid disorder, a skin disorder, a swelling condition, a wound healing condition, and combinations thereof, comprising administering a payload or conjugate thereof described herein. [00188] In some examples, the autoimmune disorder is selected from multiple sclerosis, autoimmune hepatitis, shingles, systemic lupus erythematosus (i.e., lupus), myasthenia gravis, Duchenne muscular dystrophy, and sarcoidosis. In some examples, the breathing disorder is selected from asthma, chronic respiratory disease, chronic obstructive pulmonary disease, bronchial inflammation, and acute bronchitis. In some examples, the cancer is selected from leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma (NHL), and multiple myeloma. In some examples, the collagen disease is systemic lupus erythematosus. In some examples, the eye disease is keratitis. In some examples, the endocrine problem is selected from Addison's Disease, adrenal insufficiency, adrenal cortical dysfunction, adrenocortical, and congenital adrenal hyperplasia. In some examples, the inflammatory disease is selected from inflammation after cataract surgery, joint inflammation, immune inflammation, tendon inflammation, bursitis, epicondylitis, Crohn's disease, inflammatory bowels disease, lipid pneumonitis thyroiditis, urticaria (hives), pericarditis, nephrotic syndrome, and uveitis. In some examples, the intestinal disorder is selected from ulcerative colitis, Crohn’s disease, and inflammatory bowel disease. In some examples, the rheumatoid disorder is selected from rheumatoid arthritis, polymyalgia rheumatic, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus. In some examples, the skin disorder is selected from psoriasis, eczema, and poison ivy. In some examples, the neurological disorder is chronic inflammatory demyelinating polyradiculoneuropathy. [00189] In some embodiments, the compounds described herein are administered to a patient to treat an acute inflammatory event including, but not limited to, shock, brain edema, and graft- vs-host disease. In some embodiments, the compounds described herein are administered to treat lympholytic effects including, but not limited to, those associated with hematological malignancies, for example, leukemias, lymphomas, and myelomas. [00190] In some examples, set forth herein is a method for reducing inflammation in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein. In some examples, set forth herein is a method for modulating the immune system in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a conpound or conjugate thereof described herein. In some examples, set forth herein is a method for modulating cortisol levels in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein. In some examples, set forth herein is a method of reducing lymphocyte migration in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or conjugate thereof described herein. In some examples, set forth herein is a method of treating hypercalcemia due to cancer, Meniere's disease, a migraine headache, a cluster headache, a severe aphthous ulcer, laryngitis, severe tuberculosis, a Herxheimer reaction to syphilis, a decompensated heart failure, allergic rhinitis or nasal polyps, comprising administering to a subject in need thereof a payload or conjugate thereof described herein. In some examples, the compounds disclosed herein can be used for treating inflammatory bowel disease, Crohn's disease, or ulcerative colitis. In some examples, the disease, disorder, or condition is a chronic inflammatory condition including, but not limited to, asthma, skin infections, and ocular infections. In some examples, compounds described herein are used for immunosuppression in patients undergoing organ transplantation. [00191] In some embodiments, the payloads and conjugates thereof described herein are administered to a patient to treat a nervous disorders associated with GR signalling including, but not limited to, psychiatric disorders such as schizophrenia, drug addiction, post-traumatic stress disorder (PTSD), and mood disorders, substance abuse, stress, and anxiety. In some embodiments, the payloads and conjugates thereof described herein are administered to a patient to treat a visual system disorder including, but not limited to, ocular inflammation (e.g., conjunctivitis, keratitis, uveitis), macular edema, and macular degeneration. In some embodiments, the payloads and conjugates thereof described herein are administered to a patient to treat a cardiovascular disorder. In some embodiments, the payloads and conjugates thereof described herein are administered to a patient to treat a glucose and/or liver metabolism disorder. In some embodiments, the payloads and conjugates thereof described herein are administered to a patient to treat a musculoskeletal system disorder. In some embodiments, the payloads and conjugates thereof described herein are administered to a patient to treat a cutaneous inflammatory condition, such as eczema and psoriasis. [00192] The protein conjugates described herein provide a means for targeted delivery of its payload to particular cells or organ systems, thereby reducing or preventing side effects that result from administration of the free unconjugated payload. Examples of such potential side effects to be reduced or prevented include those listed in the approved drug label for Decadron® (dexamethasome), which is incorporated herein by reference in its entirety. In some embodiments, the side effect to be reduced or prevented is selected from elevation of blood pressure; sodium retention; water/fluid retention (edema, angioedema, pulmonary edema); increased excretion of potassium; reversible hypothalamic-pituitary adrenal (HPA) axis suppression; potential corticosteroid insufficiency after withdrawal of treatment; susceptibility to infecctions; exacerbation of systemic fungal infections; worsening of severity of chickenpox in pediatric and adult patients; worsening of severity of measles in pediatric and adult patients; posterior subcapsular cataracts; glaucoma with possible damage to the optic nerves; enhancement of the establishment of secondary ocular infections due to bacteria, fungi, or viruses; increase in new episodes of optic neuritis; Kaposi’s sarcoma; drug-induced secondary adrenocortical insufficiency; increased risk of a perforation when active or latent peptic ulcers, diverticulitis, fresh intestinal anastomoses, and nonspecific ulcerative colitis, are present; peritoneal irritation following gastrointestinal perforation; decreased bone formation; increased bone resorption; inhibition of osteoblast function; inhibition of bone growth in pediatric patients; development of osteoporosis at any age; acute myopathy (possibly involving ocular and respiratory muscles, and potentially resulting in quadriparesis); elevation of creatinine kinase; psychic derangements, ranging from euphoria, insomnia, mood swings, personality changes, and severe depression, to frank psychotic manifestations; aggravation of existing emotional instability or psychotic tendencies; elevated intraocular pressure; bradycardia; cardiac arrest; cardiac arrhythmias; cardiac enlargement; circulatory collapse; congestive heart failure; fat embolism; hypertension; hypertrophic cardiomyopathy in premature infants; myocardial rupture following recent myocardial infarction; syncope; tachycardia; thromboembolism; thrombophlebitis; vasculitis; acne; allergic dermatitis; dry scaly skin; ecchymoses and petechiae; erythema; impaired wound healing; increased sweating; rash; striae; suppression of reactions to skin tests; thin fragile skin; thinning scalp hair; urticarial; decreased carbohydrate and glucose tolerance; development of cushingoid state; hyperglycemia; glycosuria; hirsutism; hypertrichosis; increased requirements for insulin or oral hypoglycemic agents in diabetes (insulin resistance); manifestations of latent diabetes mellitus; menstrual irregularities; secondary adrenocortical and pituitary unresponsiveness (particularly in times of stress; as in trauma; surgery; or illness); suppression of growth in pediatric patients; congestive heart failure in susceptible patients; fluid retention; hypokalemic alkalosis; potassium loss; sodium retention; abdominal distention; elevation in serum liver enzyme levels (usually reversible upon discontinuation); hepatomegaly; increased appetite; nausea; pancreatitis; peptic ulcer with possible perforation and hemorrhage; perforation of the small and large intestine (particularly in patients with inflammatory bowel disease); ulcerative esophagitis; negative nitrogen balance due to protein catabolism; aseptic necrosis of femoral and humeral heads; loss of muscle mass; muscle weakness; osteoporosis; pathologic fracture of long bones; steroid myopathy; tendon rupture; vertebral compression fractures; convulsions; depression; emotional instability; euphoria; headache; increased intracranial pressure with papilledema (pseudotumor cerebri) usually following discontinuation of treatment; insomnia; mood swings; neuritis; neuropathy; paresthesia; personality changes; psychic disorders; vertigo; exophthalmos; glaucoma; increased intraocular pressure; posterior subcapsular cataracts; abnormal fat deposits; decreased resistance to infection; hiccups; increased or decreased motility and number of spermatozoa; malaise; moon face; and weight gain; and those side effects associated with drug-drug interactions. In some embodiments, the side effect to be reduced or prevented are those associated with drug-drug interactions. In some embodiments, the side effect to be reduced or prevented is associated with drug-drug interactions from the use of a corticosteroid with aminoglutethimide including diminishment of adrenal suppression by corticosteroids; amphotericin B injection and potassium-depleting agents, including development of hypokalemia, cardiac enlargement, and congestive heart failure; antibiotics including a significant decrease in corticosteroid clearance; anticholinesterases including producing severe weakness in patients with myasthenia gravis; oral anticoagulants including inhibition of response to warfarin; antidiabetics including increased blood glucose concentrations; antitubercular drugs including decreased serum concentrations of isoniazid; cholestyramine including increased clearance of corticosteroids; cyclosporine including increased activity of both cyclosporine and corticosteroids, and incidence of convulsions; dexamethasone suppression test (DST) interference including false-negative results in patients being treated with indomethacin; digitalis glycosides including increased risk of arrhythmias due to hypokalemia; ephedrine including enhancement of the metabolic clearance of corticosteroids, resulting in decreased blood levels and lessened physiologic activity; estrogens, including oral contraceptives, including decreased hepatic metabolism of certain corticosteroids and associated increase in their effect; hepatic enzyme inducers, inhibitors and substrates (drugs which induce cytochrome P4503A4 (CYP 3A4) enzyme activity, for example, barbiturates, phenytoin, carbamazepine, rifampin), including enhancing of metabolism of corticosteroids; drugs which inhibit CYP 3A4 (e.g., ketoconazole, macrolide antibiotics such as erythromycin) including the potential for increased plasma concentrations of corticosteroids; drugs that are metabolized by CYP 3A4 (e.g., indinavir, erythromycin), including increase in their clearance, resulting in decreased plasma concentration; ketoconazole including decreased metabolism of certain corticosteroids by up to 60%, leading to increased risk of corticosteroid side effects, and inhibition of adrenal corticosteroid synthesis potentially causing adrenal insufficiency during corticosteroid withdrawal; nonsteroidal anti-inflammatory agents (NSAIDS), including increased risk of gastrointestinal side effects and increased clearance of salicylates; phenytoin, including increases or decreases in phenytoin level, altered seizure control; skin tests, including suppression of reactions to skin tests; thalidomide including toxic epidermal necrolysis; and vaccines including a diminished response to toxoids and live or inactivated vaccines due to inhibition of antibody response or potentiation of the replication of some organisms contained in live attenuated vaccines). Thus, provided herein are methods for treating a disease, disorder, or condition associated with the glucocorticoid receptor comprising administering a conjugate of (R1)(R2)N-W-X-Y-Z and/or Formulae I-IV, to a patient having said disease, disorder, or condition, wherein the side effects associated with administration of the free steroid payload of said conjugate is reduced. Furthermore, provided herein are methods of delivering a compound of (R1)(R2)N-W-X-Y-Z and/or Formulae I-IV, to a cell comprising contacting said cell with a protein conjugate the compound of (R1)(R2)N-W-X-Y-Z and/or Formulae I-IV, wherein the protein conjugate comprises an antibody or antigen binding fragment thereof that binds a surface antigen of said cell. [00193] In some examples, set forth herein is a method of treating a disease, disorder or condition selected from the group consisting of an immunological disease, autoimmune disease, inflammation, asthma, or an inflammatory bowel disorder, Crohn's disease, ulcerative colitis. [00194] In some examples, set forth herein is a method of treating a disease, disorder or condition by targeting an antigen, for example, cell-surface expressing antigen, to which compound delivery can achieve a therapeutic benefit comprising administering the conjugates described herein. In some embodiments, the antigen is AXL, BAFFR, BCMA, BCR–list components, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD168, CD177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD47, CD46, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1/2, CXCR4, CXCR5, DDR1, DDR2, DEC–205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG/Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR-list components/assoc, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4, or VTCN1. In some embodiments, the antigen is IL2R–γ. [00195] In some examples, set forth herein is a method for treating a disease, disorder, or condition selected from an immunological disease, an autoimmune disease, an inflammatory disease, a dermatological disease, or a gastrointestinal disease. [00196] In some examples, the disease is Crohn’s disease, ulcerative colitis, Cushing's syndrome, adrenal insufficiency, or congenital adrenal hyperplasia. [00197] In some examples, the disease is inflammation, asthma, or an inflammatory bowel disorder. [00198] In some examples, the disease is an autoimmune diseases selected from multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, psoriasis, or eczema. [00199] In some examples, the disease is a cancer. [00200] In some examples, set forth herein is a method for reducing or ameliorating the side effects of chemotherapy, wherein the method includes administering to a patient having said disorder a therapeutically effective amount of a compound or a composition described herein. [00201] In some examples, set forth herein is a method for reducing or ameliorating the side effects of immunosuppressive therapy, wherein the method includes administering to a patient having said disorder a therapeutically effective amount of a compound or a composition described herein. [00202] In some examples, set forth herein is a method for treating cancer, wherein the method includes administering to a patient having said disorder a therapeutically effective amount of a compound or a composition described herein. In some examples, the cancer is selected from acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin’s lymphoma, Non–Hodgkin’s lymphoma (NHL), or multiple myeloma, as well as others. [00203] In some examples, set forth herein are methods for treating or preventing any disease, disorder, or condition responsive to modulation of LXR signalling. In some examples, the disease or disorder is associated with LXR function, LXR polymorphisms, LXR agonist activity, or LXR antagonist activity. In some examples, set forth herein is a method of treating or preventing a disease, disorder, or condition selected from the group consisting of a proliferative disorder, a neurodegenerative disorder, an immunological disorder, an autoimmune disease, an inflammatory disorder, a dermatological disease, a metabolic disease, cardiovascular disease, and a gastrointestinal disease. [00204] The proliferative disorder can be any proliferative disorder known to those of skill. In certain embodiments, proliferative disorders include, without limitation, oncology disorders, where the oncology disorder can be any cancer disorder known to those of skill. In certain embodiments, provided herein are methods of treating or preventing a melanoma. In certain embodiments, provided herein are methods of treating or preventing metastatic melanoma. In certain embodiments, provided herein are methods of treating or preventing lung cancer. In certain embodiments, provided herein are methods of treating or preventing EGFR-tyrosine kinase inhibitor resistant lung cancer. In certain embodiments, provided herein are methods of treating or preventing oral cancer. In certain embodiments, provided herein are methods of treating or preventing oral squamous cell carcinoma. In certain embodiments, provided herein are methods of treating or preventing prostate cancer. In certain embodiments, provided herein are methods of treating or preventing Hodgkin’s lymphoma. In certain embodiments, provided herein are methods of treating or preventing breast cancer. In certain embodiments, provided herein are methods of treating or preventing gastric cancer. [00205] The neurodegenerative disorder can be any neurodegenerative disorder known to those of skill. In certain embodiments, provided herein are methods of treating or preventing Alzheimer’s disease. In certain embodiments, provided herein are methods of treating or preventing Parkinson’s disease. In certain embodiments, provided herein are methods of treating or preventing Huntington’s disease. In certain embodiments, provided herein are methods of treating or preventing amyotrophic lateral sclerosis. In certain embodiments, provided herein are methods of treating or preventing myelin gene expression. In certain embodiments, provided herein are methods of treating or preventing myelination and remyelination conditions, diseases, or disorders. [00206] The immunological disorder can be any immunological disorder known to those of skill. In certain embodiments, provided herein are methods of treating or preventing imflammatory bowel disease. In certain embodiments, provided herein are methods of treating or preventing ulcerative colitis. In certain embodiments, provided herein are methods of treating or preventing Crohn’s disease. [00207] The inflammatory disorder can be any inflammatory disorder known to those of skill. In certain embodiments, provided herein are methods of treating or preventing arthritis. In certain embodiments, provided herein are methods of treating or preventing rheumatoid arthritis. [00208] The metabolic disease can be any metabolic disease known to those of skill. In certain embodiments, the metabolic disease is dyslipidemia. Dyslipidemia can be any dyslipidemia known to those of skill. In certain embodiments, dyslipidemia is selected from the group consisting of hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, HDL deficiency, ApoA-I deficiency, and cardiovascular disease such as coronary artery disease (including, for example, treatment and prevention of angina, myocardial infarction, and sudden cardiac death); atherosclerosis (including, for example, treatment and prevention of atherosclerosis); and restenosis (including, for example, preventing or treating atherosclerotic plaques which develop as a consequence of medical procedures such as balloon angioplasty). In certain embodiments, provided herein are methods of treating or preventing diabetes. [00209] The cardiovascular disease can be any cardiovascular disease known to those of skill. In certain embodiments, provided herein are methods of treating or preventing atherosclerosis. In certain embodiments, provided herein are methods of treating or preventing atherosclerosis derived from abnormal macrophage processing. In certain embodiments, provided herein are methods of treating or preventing atherosclerosis derived from the formation of oxidized low- density lipoproteins (oxLDLs), where marcrophages fail to process oxLDLs. In certain embodiments provided herein are methods of treating or preventing ischemic heart disease In certain embodiments, provided herein are methods of treating or preventing stroke. In certain embodiments, provided herein are methods of treating or preventing hypertensive heart disease. In certain embodiments, provided herein are methods of treating or preventing aortic aneurysm. In certain embodiments, provided herein are methods of treating or preventing endocarditis. In certain embodiments, provided herein are methods of treating or preventing peripheral artery disease. In certain embodiments, provided herein are methods of treating or preventing combinations of any of the diseases provided in this paragraph. [00210] In some examples, set forth herein is a method for modulating the function of a nuclear receptor. By way of non-limiting example, the function may be selected from expression/secretion of inflammatory mediators (e.g. cytokines, chemokines), cholesterol regulation, cholesterol intake, cholesterol efflux, cholesterol oxidation, migration, chemotaxis, apoptosis and necrosis, an inflammatory activity, lipid regulation, apoptosis, migration, chemotaxis, gene transcription, and protein expression. EXAMPLES [00211] Provided herein are novel linkers, and protein conjugates thereof, and methods for treating a variety of diseases, disorders, and conditions including administering compounds or payloads via linker-payloads, and protein conjugates thereof. Abbreviations ADC Antibody-drug conjugate s
O'
H2N' vcPABC-MMAE !*< NH 644981-35-1 Commercial L >°H
N-r NH2
[00212] Synthesis of Linker AL4 (FIG.1). [00213] General Procedure for Synthesis of Linker AL4 [00214] To a solution of compound L1E (1.0 equiv) in DMF (0.2 M) was added H2N-PEGn- NH2 (2.0 equiv) and DIPEA (2.0 equiv) and the reaction was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-40% acetonitrile with aq. TFA (0.1%)) twice to give amino-linker AL4 as an oil. [00215] N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)-2-(cyclooct-2-yn-1- yloxy)acetamide (AL4c) [00216] Following the general procedure for AL4, linker AL4c (60 mg, 27% yield as a TFA salt) was obtained as yellow oil. ESI m/z 357 (M+H)+.1H NMR (400 MHz, CDCl3) δ 8.24 (s, 3H), 6.99 (t, J = 6.0 Hz, 1H), 4.29-4.23 (m, 1H), 4.08-4.02 (m, 1H), 3.91-3.85 (m, 1H), 3.82-3.77 (m, 2H), 3.73-3.68 (m, 2H), 3.66-3.60 (m, 6H), 3.60-3.55 (m, 2H), 3.53-3.46 (m, 2H), 3.18 (br s, 2H), 2.27-2.11 (m, 3H), 2.03-1.90 (m, 2H), 1.89-1.76 (m, 2H), 1.73-1.58 (m, 2H), 1.50-1.40 (m, 1H) ppm.19F NMR (377 MHz, CDCl3) δ -75.59 ppm. [00217] N-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosan-1-yl)-2-(cyclooct-2-yn-1- yloxy)acetamide (AL4d) [00218] Following the general procedure for AL4, linker AL4d (50 mg, 14% yield as TFA salt) was obtained as yellow oil. ESI m/z 577 (M+H)+. [00219] N-(38-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxaoctatriacontan-1-yl)-2- (cyclooct-2-yn-1-yloxy)acetamide (AL4e) [00220] Following the general procedure for AL4, linker AL4e (30 mg, 14% yield as TFA salt) was obtained as yellow oil. ESI m/z 753 (M+H)+. [00221] Synthesis of Linker AL10 and L5 (FIG.2). [00222] General Procedure for Synthesis of Linker AL10 [00223] To a solution of linker N-Boc-amino-PEGn-acid (1.0 equiv) in dry DMF (20-25 mM) was added DIPEA (5.0 equiv) and HATU (1.5 equiv), and the reaction mixture was stirred at room temperature for fifteen minutes before the addition of AL7 (1.0 equiv, HCl salt). The reaction mixture was then stirred at room temperature for sixteen hours, which was monitored by LCMS. The resulting mixture was separated by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.01%)) to give Boc-AL10 as a red oil, which was dissolved in DCM (0.1 M). To the solution was added TFA (1/3 volume of DCM) and the reaction mixture was stirred at room temperature for an hour until Boc was completely removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.01%)) to give AL10 as a light-red oil. [00224] 1-amino-N-{[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}-3,6,9,12- tetraoxapentadecan-15-amide (AL10a) Commercially available. ESI m/z 449 (M+H)+. [00225] 1-amino-N-{[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}- 3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide (AL10b) [00226] Following the general procedure for AL10, linker AL10b (55 mg, 74% yield as TFA salt) was obtained as a light-red oil. ESI m/z 625 (M+H)+. [00227] 1-amino-N-{[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}- 3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-amide (AL10c) [00228] Following the general procedure for AL10, linker AL10c (19 mg, 21% yield as TFA salt) was obtained as a light-red oil. ESI m/z 801 (M+H)+. [00229] General Procedure for Synthesis of Linker L5 [00230] To a solution of linker AL10 (1.0 equiv, obtained above) in DCM (20 mM) was added succinic anhydride (1.1 equiv) and the mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was concentrated in vacuo, and the residue was separated by prep-HPLC (5-95% acetonitrile with aq. TFA (0.5%)) to give linker L5 as a red solid. [00231] 3-{[14-({[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}carbamoyl)-3,6,9,12- tetraoxatetradecan-1-yl]carbamoyl}propanoic acid (L5a) [00232] Following the general procedure for L5, linker L5a (47 mg, 85% yield) was obtained as a red solid. ESI m/z: 549 (M+H)+.1H NMR (500 MHz, DMSO-d6) δ 12.03 (s, 1H), 8.51 (t, J = 7.0 Hz, 1H), 8.42 (d, J = 10.5 Hz, 2H), 7.90 (t, J = 7.5 Hz, 1H), 7.52 (d, J = 10.0 Hz, 2H), 4.40 (d, J = 9.0 Hz, 2H), 3.68 (t, J = 8.0 Hz, 2H), 3.52-3.48 (m, 12H), 3.37 (t, J = 8.0 Hz, 2H), 3.20-3.15 (m, 2H), 3.0 (s, 3H), 2.45-2.35 (m, 4H), 2.30 (t, J = 9.0 Hz, 2H) ppm. [00233] 3-{[26-({[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}carbamoyl)- 3,6,9,12,15,18,21,24-octaoxahexacosan-1-yl]carbamoyl}propanoic acid (L5b) [00234] Following the general procedure for L5, linker L5b (19 mg, 52% yield) was obtained as a red solid. ESI m/z: 725 (M+H)+. [00235] 3-{[38-({[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}carbamoyl)- 3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxaoctatriacontan-1-yl]carbamoyl}propanoic acid (L5c) [00236] Following the general procedure for L5, linker L5b (12 mg, 27% yield) was obtained as a light-red solid. ESI m/z: 901 (M+H)+. [00237] Synthesis of Branched Linker AL11 (FIG.3) 1-amino-N-{[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}-12-[({[4-(6-methyl-1,2,4,5- tetrazin-3-yl)phenyl]methyl}carbamoyl)methyl]-3,6,9-trioxa-12-azatetradecan-14-amide (AL11) [00238] To a solution of L9 (0.15 g, 0.37 mmol) in DMF (4.0 mL) was added HATU (0.31 g, 0.81 mmol), DIPEA (0.51 mL, 2.9 mmol), and AL7 (0.29 g, 0.92 mmol, TFA salt) and the mixture was stirred at 20 °C for sixteen hours, which was monitored by LCMS. The resulting mixture was then poured into water (30 mL) and the aqueous mixture was extracted with ethyl acetate (30 mL x 3). The combined organic solution was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give Boc-AL11 (0.14 g, 90% purity, ESI m/z 775 (M+H)+) as a pink oil, which was dissolved in DCM (3.0 mL). To the solution was added TFA (0.6 mL, 8.1 mmol) and the reaction mixture was stirred at 20 °C for two hours until Boc was totally removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by prep-HPLC (18-38% acetonitrile with aq. TFA (0.01%)) to give linker AL11 (74 mg, 25% yield) as a pink solid. ESI m/z 675 (M+H)+. [00239] Boc-AL11: 1H NMR (500 MHz, DMSO-d6) δ 8.68 (t, J = 6.1 Hz, 2H), 8.35 (d, J = 8.3 Hz, 4H), 7.50 (d, J = 8.4 Hz, 4H), 6.73 (br s, 1H), 4.45 (d, J = 6.1 Hz, 4H), 3.56 (t, J = 5.5 Hz, 2H), 3.49-3.42 (m, 8H), 3.34 (br s, 4H), 3.05-3.00 (m, 2H), 2.98 (s, 6H), 2.93 (br s, 2H), 2.77 (t, J = 5.6 Hz, 2H), 1.35 (s, 9H) ppm. [00240] AL11: 1H NMR (500 MHz, DMSO-d6) δ 8.89 (br s, 2H), 8.38 (d, J = 8.1 Hz, 4H), 7.76 (br s, 3H), 7.53 (d, J = 8.3 Hz, 4H), 4.47 (br d, J = 5.9 Hz, 4H), 3.65 (br s, 6H), 3.59-3.48 (m, 12H), 3.03-2.91 (m, 8H) ppm. [00241] Synthesis of Branched Linker AL12 (FIG.4). [00242] Benzyl 1-{[(tert-butoxy)carbonyl]amino}-3,6,9-trioxa-12-azatetradecan-14-oate (4-2) [00243] To a stirred solution of N-Boc-amino-PEG3-amine (3.8 g, 13 mmol) in 1,4-dioxane (30 mL) was added a solution of compound 4-1 (1.0 g, 4.4 mmol) in 1,4-dioxane (15 mL) dropwise over a period of 1.5 hours at 0 °C. The reaction mixture was then stirred at room temperature for 10.5 hours, which was monitored by TLC. The resulting mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give compound 4-2 (1.8 g, 84% yield) as a colorless oil.1H NMR (400 MHz, CDCl3) δ 7.40-7.29 (m, 5H), 5.34-5.27 (m, 1H), 5.16 (s, 2H), 3.67-3.57 (m, 10H), 3.55-3.50 (m, 2H), 3.49 (s, 2H), 3.36-3.19 (m,2H), 2.82 (t, J = 5.2 Hz, 2H), 2.08-2.03 (m, 1H), 1.50-1.34 (m, 9H) ppm. [00244] Ethyl 12-[2-(benzyloxy)-2-oxoethyl]-1-{[(tert-butoxy)carbonyl]amino}-3,6,9- trioxa-12-azatetradecan-14-oate (4-3)
[00245] To a solution of 4-2 (1.8 g, 4.1 mmol) in acetonitrile (5.0 mL) was added sodium carbonate (0.52 g, 4.9 mmol) and ethyl 2-bromoacetate (0.54 mL, 4.9 mmol). The reaction mixture was stirred at 50 °C for twelve hours, which was monitored by TLC. The resulting mixture was then filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel flash chromatography (0-10% methanol in DCM) to give compound 4-3 (1.7 g, 71% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.38-7.28 (m, 5H), 5.17-5.11 (m, 2H), 5.06 (br s, 1H), 4.24- 4.14 (m, 2H), 3.71-3.66 (m, 2H), 3.64-3.53 (m, 12H), 3.50 (t, J = 5.1 Hz, 2H), 3.34-3.23 (m, 2H), 2.96 (t, J = 5.6 Hz, 2H), 1.46-1.38 (m, 9H), 1.26 (t, J = 7.1 Hz, 3H) ppm. [00246] 1-{[(tert-butoxy)carbonyl]amino}-12-(2-ethoxy-2-oxoethyl)-3,6,9-trioxa-12- azatetradecan-14-oic acid (4-4) [00247] To a solution of compound 4-3 (1.7 g, 3.2 mmol) in methanol (20 mL) was added Palladium on carbon (10% Pd, 0.30 g, 3.2 mmol) and the suspension was stirred under a hydrogen balloon at room temperature for three hours, which was monitored by TLC. The resulting suspension was then filtered and the filtrate was concentrated in vacuo to give compound 4-4 (1.4 g, crude) as a colorless oil, which was used in the next step without purification. [00248] Ethyl 12-[({2-[2-(2-azidoethoxy)ethoxy]ethyl}carbamoyl)methyl]-1-{[(tert- butoxy)carbonyl]amino}-3,6,9-trioxa-12-azatetradecan-14-oate (4-5) [00249] To a solution of compound 4-4 (1.4 g, obtained above) in DMF (10 mL) was added HATU (2.4 g, 6.4 mmol), DIPEA (1.7 g, 13 mmol) and amino-PEG3-azide (0.84 g, 4.8 mmol) and the reaction mixture was stirred at 20 °C for three hours, which was monitored by TLC. The resulting mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by prep-HPLC (28-48% acetonitrile with aq. formic acid (0.1%)) to give compound 4-5 (1.1 g, 52% yield in two steps from 4-3) as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ 7.93 (br s, 1H), 6.74 (br t, J = 5.3 Hz, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.65-3.57 (m, 4H), 3.57-3.54 (m, 2H), 3.54-3.47 (m, 12H), 3.46-3.41 (m, 4H), 3.40 (br s, 4H), 3.29-3.24 (m, 3H), 3.17 (s, 1H), 3.10-2.97 (m,2H), 1.37 (s, 9H), 1.20 (t, J = 7.1 Hz, 3H) ppm. [00250] 12-[({2-[2-(2-azidoethoxy)ethoxy]ethyl}carbamoyl)methyl]-1-{[(tert- butoxy)carbonyl]amino}-3,6,9-trioxa-12-azatetradecan-14-oic acid (4-6) [00251] To a solution of compound 4-5 (1.1 g, 1.9 mmol) in THF (2.0 mL) was added aq. sodium hydroxide (1 M, 2.0 mL) and the mixture was stirred at 20 °C for four hours, which was monitored by LCMS. The resulting mixture was concentrated in vacuo to remove THF. The residual aqueous solution was neutralized with aq. hydrochloride (1 M) to pH 7. The mixture was directly separated by prep-HPLC (20-40% acetonitrile with aq. Formic acid (0.1%)) to give compound 4-6 (0.59 g, 51% yield) as a yellow oil. ESI m/z 565 (M+H)+. 1H NMR (400 MHz, DMSO-d6) δ 12.90-11.56 (m, 1H), 7.97-7.81 (m, 1H), 6.74 (br s, 1H), 3.64-3.58 (m, 2H), 3.55 (br d, J = 2.4 Hz, 2H), 3.53-3.45 (m, 12H), 3.43 (br d, J = 3.4 Hz, 3H), 3.41-3.34 (m, 6H), 3.29-3.21 (m, 5H), 3.05 (q, J = 5.8 Hz,2H), 2.77 (br t, J = 5.2 Hz, 2H), 1.37 (s, 9H) ppm. [00252] tert-butyl N-[1-({2-[2-(2-azidoethoxy)ethoxy]ethyl}carbamoyl)-2-[({[4-(6-methyl- 1,2,4,5-tetrazin-3-yl)phenyl]methyl}carbamoyl)methyl]-5,8,11-trioxa-2-azatridecan-13- yl]carbamate (4-7)
[00253] To a solution of compound 4-6 (0.10 g, 0.18 mmol) in DMF (1.0 mL) was added AL7 (43 mg, 0.21 mmol, TFA salt), HATU, (74 mg, 0.19 mmol), and DIPEA (92 mg, 0.71 mmol) and the reaction mixture was stirred at 20 °C for twelve hours, which was monitored by LCMS. The resulting mixture was diluted with water (30 mL) and the aqueous mixture was extracted with 2- methyl tetrahydrofuran (30 mL x 3). The combined organic solution was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-10% methanol in DCM) to give compound 4-7 (0.11 g, 82% yield) as a pink oil. ESI m/z 748 (M+H)+.1H NMR (400MHz, DMSO-d6) δ 8.64 (t, J = 6.1 Hz, 1H), 8.42 (d, J = 8.4 Hz, 2H), 8.04 (t, J = 5.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 6.74 (br t, J = 5.5 Hz, 1H), 4.45 (d, J = 6.1 Hz, 2H), 3.60-3.55 (m, 2H), 3.54-3.49 (m, 5H), 3.47 (br d, J = 5.5 Hz, 7H), 3.42 (t, J = 5.9 Hz, 2H), 3.36 (dd, J = 5.3, 9.8 Hz, 4H), 3.30-3.23(m, 4H), 3.21 (s, 2H), 3.03 (q, J = 6.0 Hz, 2H), 2.72 (t, J = 5.6 Hz, 2H), 2.52-2.50 (m, 4H), 1.36 (s, 9H) ppm. [00254] 1-amino-N-{2-[2-(2-azidoethoxy)ethoxy]ethyl}-12-[({[4-(6-methyl-1,2,4,5- tetrazin-3-yl)phenyl]methyl}carbamoyl)methyl]-3,6,9-trioxa-12-azatetradecan-14-amide (AL12) [00255] To a solution of compound 4-7 (0.10 g, 0.13 mmol) in DCM (2.0 mL) was added TFA (0.5 mL) and the reaction mixture was stirred at 20 °C for an hour until Boc was totally removed, as monitored by LCMS. The volatiles were removed in vacuo and the residue was purified by prep-HPLC (18-38% acetonitrile with aq. TFA (0.01%)) to give linker AL12 (55 mg, 53% yield, TFA salt) as a red oil. ESI m/z 648 (M+H)+. 1H NMR (400 MHz, DMSO-d6) δ 8.89 (br s, 1H), 8.43 (d, J = 8.3 Hz, 2H), 8.33 (br s, 1H), 7.79 (br s, 3H), 7.56 (d, J = 8.3 Hz, 2H), 4.47 (br d, J = 5.9 Hz, 2H), 3.63 (br s, 3H), 3.61-3.56 (m, 6H), 3.55-3.49 (m, 14H), 3.31-3.19 (m, 6H), 2.99 (s, 3H), 2.98-2.91 (m, 2H), 1.37-1.18 (m, 2H) ppm. [00256] Synthesis of Branch Linker AL13 (FIG.5A) [00257] Tert-butyl N-{2-[N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)-1-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)-3,6,9,12-tetraoxapentadecan-15-amido]ethyl}carbamate (11- 2) [00258] To a solution of N-Fmoc-PEG4-acid (1.7 g, 3.5 mmol) in DMF (20 mL) was added compound 11-1 (1.2 g, 3.8 mmol), DIPEA (0.9 g, 7.0 mmol), HOBt (0.57 g, 4.2 mmol), and EDCI (1.0 g, 5.2 mmol) and the reaction mixture was stirred at room temperature for twenty-four hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-70% acetonitrile in aq. TFA (0.1%)) to give compound 11-2 (2.2 g, 81% yield) as an oil. ESI m/z: 773.3 (M + H)+. [00259] (9H-fluoren-9-yl)methyl N-{14-[bis({2-[2-(cyclooct-2-yn-1- yloxy)acetamido]ethyl})carbamoyl]-3,6,9,12-tetraoxatetradecan-1-yl}carbamate (11-4) [00260] To a stirred solution of compound 11-2 (2.2 g, 2.9 mmol) in ethyl acetate (10 mL) was added hydrochloride in ethyl acetate (4 N, 2.8 mL, 11 mmol) and the reaction mixture was stirred at room temperature for four hours. The reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo to give an oil (1.6 g, ESI m/z: 573.3 (M + H)+), which was dissolved in DMF (20 mL). To the solution was added compound 11-3 (1.6 g, 5.7 mmol) and DIPEA (0.90 g, 7.0 mmol), and the reaction mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-70% acetonitrile in aq. TFA (0.1%)) to give compound 11-4 (2.3 g, 91% yield) as an oil. ESI m/z: 901.3 (M + H)+. [00261] 1-amino-N,N-bis({2-[2-(cyclooct-2-yn-1-yloxy)acetamido]ethyl})-3,6,9,12- tetraoxapentadecan-15-amide (AL13) [00262] To a stirred solution of compound 11-4 (2.3 g, 2.6 mmol) in DMF (20 mL) was added diethylamine (0.37 g, 5.1 mmol), and the reaction mixture was stirred at room temperature. The reaction was monitored by LCMS. The resulting mixture was diluted with ethyl acetate (100 mL) and washed with water. The organic layer was collected and the aqueous solution was extracted with ethyl acetate (100 mL x 4). The combined organic solutions were dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (0- 50% methanol in DCM) to give AL13 (0.78 g, 45% yield) as a light yellow oil. ESI m/z: 679.3 (M + H)+. 1H NMR (400 MHz, CDCl3) δ 7.05 (t, J = 4.8 Hz, 1H), 6.97-6.89 (m, 1H), 4.24 (s, 2H), 4.09-4.01 (m, 2H), 3.90-3.81 (m, 2H), 3.79 (t, J = 6.2 Hz, 2H), 3.65-3.62 (m, 10H), 3.58-3.46 (m, 8H), 2.91 (t, J = 5.2 Hz, 2H), 2.67 (t, J = 6.2 Hz, 2H), 2.28-1.78 (m, 20H), 1.71-1.61 (m, 4H), 1.52-1.42 (m, 2H) ppm. [00263] Synthesis of Branch Linker AL14 (FIG.5B) [00264] (2S)-2-(2-{[(tert-butoxy)carbonyl]amino}acetamido)-5- carbamimidamidopentanoic acid (12-2b) [00265] To a solution of N-Boc-Gly-OH (0.50 g, 2.9 mmol) and HOSu (0.39 g, 3.4 mmol) in DCM (5 mL) was added EDCI (0.82 g, 4.3 mmol) at room temperature, and the reaction mixture was stirred at room temperature for sixteen hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-35% acetonitrile in aq. formic acid (0.1%)) to give N-Boc-Gly-OSu (0.62 g, ESI m/z: 295.0 (M + Na)+) as a white solid. [00266] To a solution of L-arginine-OH (0.16 g, 0.92 mmol) in mixed dioxane (1 mL) and water (1 mL) was added sodium bicarbonate (0.19 g, 2.3 mmol) and N-Boc-Gly-OSu (0.25 g, 0.92 mmol) at room temperature. The mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-10% acetonitrile in aq. ammonium bicarbonate (0.01%)) to give compound 12-2b (0.19 g, 49% yield) as a white solid. ESI m/z: 332.2 (M + H)+.1H NMR (400 MHz, DMSO- d6) δ 9.47 (s, 1H), 7.89 (br s, 2H), 7.39 (d, J = 6.8 Hz, 2H), 7.17 (s, 1H), 3.87 (q, J = 5.6 Hz, 1H), 3.48 (d, J = 4.8 Hz, 2H), 3.01 (s, 2H), 1.54-1.65 (m, 2H), 1.36-1.47 (m, 11H) ppm. [00267] Tert-butyl N-({[(1S)-4-carbamimidamido-1-({[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}carbamoyl)butyl]carbamoyl}methyl)carbamate (12-3b) [00268] To a solution of compound 12-2b (0.19 g, 0.56 mmol) in THF (2 mL) was added successively AL7 (0.32 g, 0.67 mmol), DIPEA (0.18 g, 1.4 mmol), and T3P (50% in ethyl acetate, 0.27 mg, 0.84 mmol) at room temperature, and the reaction mixture was stirred at room temperature for four hours. The reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo at low temperature and the residue was separated by reversed phase flash chromatography (35% acetonitrile in aq. TFA (0.01%)) to give 12-3b (90 mg, 31% yield) as a red solid. ESI m/z: 515.3 (M + H)+.1H NMR (400 MHz, methanol-d4) δ 8.65 (t, J = 5.2 Hz, 1H), 8.50 (d, J = 8.4 Hz, 2H), 8.20 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 4.52 (d, J = 5.2 Hz, 2H), 4.43-4.49 (m, 1H), 3.68-3.78 (m, 2H), 3.16-3.24 (m, 2H), 3.03 (s, 3H), 1.91-2.01 (m, 1H), 1.58- 1.78 (m, 3H), 1.42 (s, 9H) ppm. [00269] (2S)-2-(2-aminoacetamido)-5-carbamimidamido-N-{[4-(6-methyl-1,2,4,5- tetrazin-3-yl)phenyl]methyl}pentanamide (AL14b) [00270] To a solution of compound 12-3b (90 mg, 0.16 mmol) in DCM (5 mL) was added TFA (1 mL), and the reaction mixture was stirred at room temperature for an hour. The reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo and lyophilized then to give AL14b (50 mg, 67% yield) as a red solid. ESI m/z: 415.1 (M + H)+. 1H NMR (400 MHz, methanol-d4) δ 8.49 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 4.45-4.57 (m, 3H), 3.79 (br s, 2H), 3.21 (s, 2H), 3.03 (s, 3H), 1.90-1.91 (m, 1H), 1.64-1.76 (m, 3H) ppm. [00271] 6-amino-N-({[(1S)-4-carbamimidamido-1-({[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}carbamoyl)butyl]carbamoyl}methyl)hexanamide (AL14a) [00272] Following a similar procedure as AL14b except starting from 12-1a instead of 12-1b, compound AL14a (17 mg, 1.3% total yield) was obtained as a red solid. ESI m/z: 528.3 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (t, J = 5.9 Hz, 1H), 8.51 (d, J = 8.3 Hz, 2H), 8.28-8.18 (m, 2H), 7.76 (s, 2H), 7.70-7.65 (m, 1H), 7.61 (d, J = 8.2 Hz, 2H), 4.54-4.47 (m, 2H), 4.42-4.37 (m, 1H), 3.87-3.81 (m, 2H), 3.23-3.17 (m, 2H), 3.09 (s, 3H), 2.90-2.81 (m, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.90-1.81 (m, 1H), 1.72-1.50 (m, 8H), 1.42-1.28 (m, 4H) ppm. [00273] (2S)-2-[2-(2-aminoacetamido)acetamido]-5-carbamimidamido-N-{[4-(6-methyl- 1,2,4,5-tetrazin-3-yl)phenyl]methyl}pentanamide (AL14c) [00274] Following a similar procedure as AL14b except starting from 12-1c instead of 12-1b, compound AL14c (60 mg, 15% total yield) was obtained as a red solid. ESI m/z: 472.1 (M + H)+. 1H NMR (400 MHz, methanol-d4) δ 8.50 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 4.41-4.56 (m, 3H), 4.01 (s, 2H), 3.76 (s, 2H), 3.21 (t, J = 6.4 Hz, 2H), 3.03 (s, 3H), 1.91-1.96 (m, 1H), 1.64- 1.80 (m, 3H) ppm. [00275] Synthesis of Branch Linker AL15 (FIG.5C) [00276] 1-[(2S)-2-[2-(6-aminohexanamido)acetamido]-5- carbamimidamidopentanamido]-N-{[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}-12- [({[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl}carbamoyl)methyl]-3,6,9-trioxa-12- azatetradecan-14-amide (AL15) [00277] To a solution of compound 12-2a (43 mg, 97 μmol) in DMF (3.0 mL) was added AL11 (72 mg, 0.11 mmol), PyAOP (61 mg, 0.12 mmol), and DIPEA (19 mg, 0.15 mmol) and the reaction mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-65% acetonitrile in aq. TFA (0.01%)) to give Boc-AL15 (38 mg, ESI m/z: 551.4 (M/2 + H)+) as a red solid. Boc-AL15 was dissolved in DCM (2 mL). To the solution was added TFA (0.4 mL) and the reaction mixture was stirred at room temperature for an hour. The reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by prep- HPLC (5-95% acetonitrile in aq. TFA (0.1%)) to give AL15 (10 mg, 11% yield) as a red solid. ESI m/z: 1002.5 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.96 (d, J = 1.8 Hz, 2H), 8.37 (d, J = 8.2 Hz, 4H), 8.10 (t, J = 5.6 Hz, 1H), 8.04 (t, J = 5.4 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 5.6 Hz, 4H), 7.53 (d, J = 8.2 Hz, 4H), 4.47 (d, J = 5.7 Hz, 4H), 4.23 (br s, 2H), 3.85-3.62 (m, 9H), 3.48 (d, J = 7.9 Hz, 8H), 3.37 (t, J = 5.9 Hz, 2H), 3.22-3.16 (m, 2H), 3.10-3.02 (m, 2H), 2.98 (s, 6H), 2.85-2.65 (m, 2H), 2.12 (t, J = 7.3 Hz, 2H), 1.76-1.59 (m, 2H), 1.56-1.35 (m, 8H), 1.32- 1.21 (m, 3H) ppm.19F NMR (377 MHz, DMSO-d6) δ -73.91 (s) ppm. [00278] Synthesis of Branch Linker AL16 (FIG.5D) [00279] Ethyl 2-[2-(2-{[(tert-butoxy)carbonyl]amino}acetamido)-N-(2-ethoxy-2- oxoethyl)acetamido]acetate (14-3) [00280] To a solution of compound Boc-Gly-Gly-OH (14-2) (5.0 g, 22 mmol) in THF (50 mL) was added HATU (9.8 g, 26 mmol) and DIPEA (5.6 mL, 32 mmol) at room temperature, and the mixture was stirred for fifteen minutes before compound 14-1 (4.1 g, 22 mol) was added into the reaction mixture. The mixture was then stirred at room temperature for two hours, and was monitored by LCMS. The resulting mixture was diluted with ethyl acetate (150 mL), washed with sat. aq. sodium bicarbonate (50 mL x 3), aq. HCl (0.1 N, 50 mL x 3), brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered,, and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 40% ethyl acetate in petroleum ether) to give compound 14-3 (6.0 g, 69% yield) as a colorless oil. ESI m/z: 426.3 (M + Na)+.1H NMR (400 MHz, CDCl3) δ 6.97 (br s, 1H), 5.12 (br s, 1H), 4.17-4.25 (m, 6H), 4.11-4.12 (m, 4H), 3.87 (d, J = 5.2 Hz, 2H), 1.46 (s, 9H), 1.29 (t, 7.2 Hz, 6H) ppm. [00281] 2-[2-(2-{[(tert-butoxy)carbonyl]amino}acetamido)-N- (carboxymethyl)acetamido]acetic acid (14-4) [00282] To a solution of compound 14-3 (6 g, 15 mmol) in dioxane (30 mL) was added aq. lithium hydroxide (1 M, 37 mL, 27 mmol), and the reaction mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The resulting mixture was acidified via aq. HCl (1 N) to pH 6~7 at 0 °C, and was then lyophilized. The obtained solids were extracted with DCM (50 mL). The organic solution was concentrated in vacuo to give 14-4 (4.5 g, 87% yield) as a white solid. ESI m/z: 370.1 (M + Na)+. [00283] Tert-butyl N-{[({bis[({[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}carbamoyl)methyl]carbamoyl}methyl)carbamoyl]methyl}carbamate (14- 5) [00284] To a solution of compound 14-4 (0.20 g, 0.58 mmol) in acetonitrile (4 mL) was added AL7 (0.34 g, 1.4 mmol), water (4 mL), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMTMM) (0.42 g, 1.4 mmol) at room temperature and the reaction mixture was stirred at room temperature for sixteen hours. The reaction was monitored by LCMS. The resulting mixture was filtered. The solid was collected, washed with water (1 mL x 2), and lyophilized overnight to give compound 14-5 (0.25 g, 61% yield) as a purple solid. ESI m/z: 614.2 (M – Boc + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.31 (t, J = 6.0 Hz, 1H), 8.89 (t, J = 6.0 Hz, 1H), 8.38 (dd, J = 8.4 Hz, 3.2 Hz, 4H), 7.83 (t, J = 4.8 Hz, 1H), 7.53 (d, J = 12.4 Hz, 8.4 Hz, 4H), 7.08 (t, J = 6.0 Hz, 1H), 4.48 (d, J = 5.6 Hz, 2H), 4.44 (d, J = 5.6 Hz, 2H), 4.27 (s, 2H), 4.09 (m, 2H), 4.00 (d, J = 4.4 Hz, 2H), 3.59 (d, J = 6.0 Hz, 2H), 2.98 (s, 6H), 1.37 (s, 9H) ppm. [00285] 2-amino-N-({bis[({[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}carbamoyl)methyl]carbamoyl}methyl)acetamide (AL16) [00286] To a solution of compound 14-5 (0.10 g, 0.14 mmol) in DCM (1 mL) was added TFA (1 mL) at room temperature, and the reaction mixture was stirred at room temperature for an hour. The reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was lyophilized overnight to give AL16 (98 mg, 96% yield as TFA salt) as a purple solid. ESI m/z: 614.3 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.35 (t, J = 6.0 Hz, 1H), 8.89 (t, J = 6.0 Hz, 1H), 8.57 (t, J = 5.0 Hz, 1H), 8.38 (dd, J = 8.2 Hz, 5.6 Hz, 4H), 8.00 (s, 3H), 7.55 (d, 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 4.46 (m, 4H), 4.29 (s, 2H), 4.12 (d, J = 4.0 Hz, 4H), 3.61 (m, 2H), 2.99 (s, 6H) ppm.19F NMR (376 MHz, DMSO-d6) δ -73 ppm. [00287] Synthesis of Branch Linker AL17 (FIG.5E) [00288] (9H-fluoren-9-yl)methyl N-[(1S)-5-azido-1-({[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}carbamoyl)pentyl]carbamate (15-2)
[00289] To a solution of compound 15-1 (0.30 g, 0.76 mmol) in DMF (10 mL) was added compound AL7 (0.18 g, 0.76 mmol), HATU (0.43 g, 1.1 mmol), and DIPEA (0.20 g, 1.5 mmol) and the reaction mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-65% acetonitrile in aq. TFA (0.01%)) to give compound 15-2 (0.35 g, 80% yield) as a red solid. ESI m/z: 578.3 (M + H)+. [00290] (2S)-2-amino-6-azido-N-{[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}hexanamide (15-3) [00291] To a solution of compound 15-2 (0.35 g, 0.61 mmol) in DMF (5 mL) was added diethylamine (89 mg, 1.2 mmol), and the reaction mixture was stirred at room temperature for one hour. The reaction was monitored by LCMS. The resulting mixture was separated by reversed phase flash chromatography (0-55% acetonitrile in aq. TFA (0.01%)) to give 15-3 (0.21 g, 91% yield as TFA salt) as a red solid. ESI m/z: 356.2 (M + H)+. [00292] (2S)-2-[2-(6-{[(tert-butoxy)carbonyl]amino}hexanamido)acetamido]-5- carbamimidamidopentanoic acid (15-5) [00293] To a solution of compound 15-4 (synthesized according to Biochemistry (2009), 48(33), 8077-8082, CAS 748151-75-9) (0.90 g, 3.1 mmol) in DMF (10 mL) was added L-arginine (0.60 g, 3.4 mmol), PyAOP (2.0 g, 3.8 mmol), and DIPEA (0.61 g, 4.7 mmol) and the reaction mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-50% acetonitrile in aq. TFA (0.01%)) to give compound 15-5 (0.26 mg, 18% yield) as a white solid. ESI m/z: 445.4 (M + H)+. [00294] Tert-butyl N-{5-[({[(1S)-1-{[(1S)-5-azido-1-({[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}carbamoyl)pentyl]carbamoyl}-4- carbamimidamidobutyl]carbamoyl}methyl)carbamoyl]pentyl}carbamate (15-6) [00295] To a solution of compound 15-5 (75 mg, 0.17 mmol) in DMF (3 mL) was added compound 15-3 (89 mg, 0.19 mmol), PyAOP (0.11 g, 0.20 mmol), and DIPEA (33 mg, 0.25 mmol) and the reaction mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-50% acetonitrile in aq. TFA (0.01%)) to give compound 15-6 (99 mg, 75% yield) as a red solid. ESI m/z: 782.4 (M + H)+. [00296] (2S)-2-[(2S)-2-[2-(6-aminohexanamido)acetamido]-5- carbamimidamidopentanamido]-6-azido-N-{[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}hexanamide (AL17)
[00297] To a solution of compound 15-6 (99 mg, 0.13 mmol) in DCM (3 mL) was added TFA (0.6 mL), and the reaction mixture was stirred at room temperature for an hour. The reaction was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)) to give compound AL17 (47 mg, 45% yield as TFA salt) as a red solid. ESI m/z:682.3 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.54 (t, J = 6.0 Hz, 1H), 8.42 (d, J = 8.3 Hz, 2H), 8.13 (m, 2H), 8.06 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 0.7 Hz, 2H), 7.60 (t, J = 5.5 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 4.47-4.23 (m, 5H), 3.72 (m, 2H), 3.31 (t, J = 6.8 Hz, 2H), 3.08 (m, 2H), 3.00 (s, 3H), 2.80-2.71 (m, 2H), 2.12 (t, J = 7.4 Hz, 2H), 1.80- 1.58 (m, 4H), 1.58-1.42 (m, 10H), 1.41-1.22 (m, 5H) ppm.19F NMR (377 MHz, DMSO-d6) δ -73 ppm. [00298] Synthesis of Branched Linkers L10a and L10aE (FIG.5). [00299] 3-[3-(2-carboxyethoxy)-2-{2-[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]acetamido}propoxy]propanoic acid (L10a) [00300] To a stirred mixture of compound L3E (0.28 g, 0.87 mmol) in DMF (10 mL) was added commercially available compound L10 (0.21 g, 0.87 mmol) and DIPEA (0.34 g, 2.6 mmol) and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.1%)) to give compound L10a (65 mg, 17% yield) as a red solid. ESI m/z 448 (M+H)+. [00301] 2,3,4,5,6-pentafluorophenyl 3-(2-{2-[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]acetamido}-3-[3-oxo-3-(2,3,4,5,6- pentafluorophenoxy)propoxy]propoxy)propanoate (L10aE) [00302] To a solution of compound L10a (65 mg, 0.15 mmol) in DCM (10 mL) was added pentafluorophenol (PFP) (54 mg, 0.29 mmol) and N,N’-diisopropylcarbodiimide (DIC) (37 mg, 0.29 mmol) and the mixture was stirred at room temperature for two hours, which was monitored by LCMS. The resulting solution was concentrated in vacuo to give crude compound L10aE (65 mg, 57% yield) as a red solid, which was used in the next step without further purification. ESI m/z 780 (M+H)+. [00303] Synthesis of Linear vcPABC Linker-payloads LP1, LP2, LP3, LP7, and LP8 (FIG. 6). [00304] {4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5- (carbamoylamino)pentanamido]phenyl}methyl N-[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamate (6-1b, vcPABC-Exatecan) [00305] To a stirred suspension of Exatecan mesylate (50 mg, 94 μmol) and HOBt (13 mg, 94 μmol) in DMF (3.6 mL) was added commercially available linker Fmoc-vcPAB-PNP (L7) (79 mg, 0.10 mmol) and DIPEA (36 mg, 0.28 mmol) successively. The mixture turned clear and was stirred at room temperature for sixteeen hours, which was monitored by LCMS. The resulting solution was directly purified by reversed phase flash chromatography (0-70% methanol with aq. ammonium bicarbonate (10 mM)) to give Fmoc-vcPABC-Exatecan (64 mg mixed with an E-ring- open product (7%), ESI m/z 532.2 (M/2+H)+) as a pale yellow solid. The pale yellow solid was dissolved in DMF (2 mL). To the solution was added piperidine (0.4 mL), and the reaction mixture was stirred at room temperature for half an hour until Fmoc was totally removed according to LCMS. The resulting solution was directly purified by reversed phase flash chromatography (0- 100% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give a yellow solid containing vcPABC-Exatecan and its E-ring-open product. The mixture was suspended in water (5 mL) and acidified with aq. TFA (1 M) to pH 4.0. The aqueous solution was then lyophilized to give pure vcPABC-Exatecan 6-1b (39 mg, 43% yield) as a pale yellow solid. ESI m/z 841 (M+H)+.1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.69 (d, J = 7.2 Hz, 1H), 8.08-8.06 (m, 4H), 7.79 (d, J = 11.2 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.32 (s, 1H), 6.54 (br s, 1H), 6.07- 6.04 (m, 1H), 5.49 (br s, 1H), 5.45 (s, 2H), 5.29 (d, J = 6.0 Hz, 2H), 5.09 (s, 2H), 4.55-4.49 (m, 1H), 3.87-3.63 (m, 2H), 3.27-3.22 (m, 1H), 3.17-2.94 (m, 3H), 2.38 (s, 3H), 2.25-2.05 (m, 3H), 1.94-1.82 (m, 2H), 1.74-1.60 (m, 2H), 1.51-1.39 (m, 2H), 0.97-0.93 (m, 6H), 0.88 (t, J = 7.6 Hz, 3H) ppm. [00306] {4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5- (carbamoylamino)pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (6-1c, vcPABC-ProDXd) [00307] To a solution of commercially available Fmoc-vcPAB-PNP (L7) (0.36 g, 0.47 mmol, 1.0 equiv,) in DMF (2 mL) was added ProDXd (0.27 g, 0.47 mmol, 1.0 equiv), HOAt (95 mg, 0.70 mmol, 1.5 equiv), and DIPEA (0.12 mg, 0.94 mmol, 2.0 equiv) and the reaction mixture was stirred at room temperature for four hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography to give Fmoc-vcPABC-ProDXd (0.22 mg, ESI m/z: 1207 (M+H)+) as a yellow solid, which was dissolved in DMF (2 mL). To the solution was added diethylamine (0.2 mL), and the reaction mixture was stirred at room temperature for two hours, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography to give vcPABC-ProDXd 6-1c (0.16 g, 28% yield based on ProDXd) as a white solid. ESI m/z: 1085 (M+H)+. [00308] LP1 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12- tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N- [(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamate (LP1) [00309] To a stirred solution of compound 6-1b (30 mg, 36 μmol) in DMF (2 mL) was added linker L2E (19 mg, 36 μmol) and DIPEA (14 mg, 0.11 mmol) successively, and the clear yellow solution was stirred at room temperature for 16 hours which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-70% methanol with aq. TFA (0.01%)) to give crude LP1 (54 mg, 80% purity). Crude LP1 was purified by prep- HPLC twice to give LP1 (36 mg, 80% yield) as a white solid. ESI m/z 627 (M/2+H)+.1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.13 (d, J = 6.8 Hz, 1H), 8.07 (d, J = 9.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.63-7.59 (m, 3H), 7.36 (d, J = 8.4 Hz, 2H), 7.32 (s, 1H), 6.54 (s, 1H), 6.00 (s, 1H), 5.45 (s, 3H), 5.29 (s, 3H), 5.08 (s, 2H), 4.41-4.35 (m, 1H), 4.29- 4.21 (m, 2H), 3.87 (d, J = 14.4 Hz, 1H), 3.75 (d, J = 14.4 Hz, 1H), 3.62-3.57 (m, 2H), 3.50-3.48 (m, 14 H), 3.27-3.23 (m, 3H), 3.15-2.90 (m, 4H), 2.38-2.33 (m, 4H), 2.24-2.07 (m, 5H), 1.96-1.67 (m, 9H), 1.59-1.53 (m, 3H), 1.46-1.33 (m, 3H), 0.89-0.82 (m, 9H) ppm. [00310] LP3 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[1-({[(4E)-cyclooct-4-en-1-yloxy]carbonyl}amino)- 3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP3) [00311] To a yellow solution of linker L6 (8.5 mg, 20 µmol) in dry DMF (1.5 mL) was added DIPEA (7.8 mg, 61 µmol) and HATU (9.2 mg, 24 µmol) and the reaction mixture was stirred at room temperature for half an hour before the addition of vcPABC-ProDXd (6-1c) (20 mg, 18 μmol). The reaction mixture was stirred at room temperature for two hours and was monitored by LCMS. The resulting mixture was separated by prep-HPLC (5-95% acetonitrile with aq. formic acid (0.1%)) to give LP3 (9.7 mg, 35% yield) as a white solid. ESI m/z 693 (M/2+H)+.1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.80 (s, 1H), 8.56-8.45 (m, 2H), 8.14 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 10.7 Hz, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.43 (s, 1H), 7.35- 7.21 (m, 3H), 6.94 (s, 1H), 6.53 (s, 1H), 6.01 (s, 1H), 5.60 (s, 2H), 5.48-5.39 (m, 5H), 5.20 (s, 2H), 4.92 (s, 2H), 4.62 (d, J = 6.6 Hz, 2H), 4.37 (s, 1H), 4.27-4.17 (m, 2H), 4.01 (s, 2H), 3.61 (d, J = 6.1 Hz, 4H), 3.60-3.40 (m, 13H), 3.36 (s, 2H), 3.15-3.00 (m, 4H), 2.45-2.34 (m, 5H), 2.25 (s, 3H), 2.18 (s, 2H), 2.03-1.80 (m, 6H), 1.64 (br s, 2H), 1.60-1.50 (m, 3H), 1.48-1.41 (m, 3H), 0.89-0.79 (m, 9H) ppm. [00312] LP4 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12- tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N- ({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP4) [00313] To a solution of linker L2b (63 mg, 0.15 mmol) in DMF (2 mL) was added HATU (83 mg, 0.22 mmol) and DIPEA (58 mg, 0.45 mmol) and the reaction mixture was stirred at room temperature for an hour before the addition of vcPABC-ProDXd (6-1c) (0.16 g, 0.15 mmol). The reaction mixture was stirred at room temperature for four hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC to give LP4 (20 mg, 10% yield) as a white solid. ESI m/z 1396 (M+H)+. An alternative synthesis is further described elsewhere herein. [00314] LP7 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-3-methyl-2-(3-{[14-({[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]methyl}carbamoyl)-3,6,9,12-tetraoxatetradecan-1- yl]carbamoyl}propanamido)butanamido]pentanamido]phenyl}methyl N-[(1S)-1-{[(1S)-1- {[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-2-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]carbamoyl}-1- methoxy-2-methylethyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4- yl](methyl)carbamoyl}-2-methylpropyl]carbamoyl}-2-methylpropyl]-N-methylcarbamate (LP7) [00315] To a stirred mixture of linker L5a (7.2 mg, 13 µmol) and HATU (5.0 mg, 13 µmol) in DMF (1 mL) was added a solution of vc-PAB-MMAE (6-1a) (10 mg, 8.9 µmol) in DMF (1 mL) and DIPEA (2.2 mg, 18 µmol) successively. The reaction mixture was stirred at room temperature for two hours and was monitored by LCMS. The volatiles were removed in vacuo and the residue was directly separated by prep-HPLC (5-95% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give LP7 (10 mg, 68% yield) as a white solid. ESI m/z: 827.5 (M/2+H)+.1H NMR (400 MHz, methanol-d4) δ 8.53-8.51 (m, 2H), 7.69-7.67 (m, 2H), 7.58-7.56 (m, 2H), 7.39-7.17 (m, 7H), 5.37-5.04 (m, 3H), 4.67-4.30 (m, 11H), 4.29-4.05 (m, 4H), 3.89-3.74 (m, 3H), 3.68-3.36 (m, 17H), 3.29-3.12 (m, 7H), 3.05-3.04 (m, 3H), 2.96-2.93 (m, 3H), 2.67-2.43 (m, 8H), 2.30-2.15 (m, 3H), 2.06-1.42 (m, 11H), 1.19-1.13 (m, 6H), 1.02-0.76 (m, 25H) ppm. [00316] LP9 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-3-methyl-2-{1-[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenoxy]-3,6,9,12-tetraoxapentadecan-15- amido}butanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP9) [00317] To a yellow solution of linker L4 (11 mg, 25 µmol) in dry DMF (2 mL) was added DIPEA (10 mg, 76 µmol) and HATU (12 mg, 30 µmol) and the mixture was stirred at room temperature for half an hour until the mixture turned clear. To the solution was added vcPABC- ProDXd (6-1c) (25 mg, 23 µmol) and the reaction mixture was stirred at room temperature for two hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (5-95% acetonitrile with aq. TFA (0.1%)) to give linker-payload LP9 (20 mg, 55% yield) as a red solid. ESI m/z 702 (M/2+H)+.1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.80 (d, J = 6.6 Hz, 1H), 8.50 (d, J = 9.0 Hz, 1H), 8.40 (d, J = 8.9 Hz, 2H), 8.11 (d, J = 7.5 Hz, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 11.1 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.42 (t, J = 5.7 Hz, 1H), 7.33-7.24 (m, 3H), 7.21 (t, J = 7.1 Hz, 2H), 5.99 (br s, 1H), 5.60 (br s, 1H), 5.46-5.29 (m, 2H), 5.19 (s, 2H), 4.92 (s, 2H), 4.62 (d, J = 6.4 Hz, 2H), 4.38 (d, J = 5.1 Hz, 1H), 4.28-4.17 (m, 4H), 4.01 (br s, 2H), 3.83-3.76 (m, 3H), 3.63-3.59 (m, 4H), 3.56-3.40 (m, 9H), 3.23-3.13 (m, 2H), 3.05-2.93 (m, 5H), 2.41-2.33 (m, 5H), 2.18 (s, 2H), 1.91-1.71 (m, 4H), 1.64-1.50 (m, 3H), 1.50-1.20 (m, 4H), 0.88- 0.80 (m, 9H) ppm. [00318] LP16 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-3-methyl-2-(1-{2-[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]acetamido}-3,6,9,12-tetraoxapentadecan-15- amido)butanamido]pentanamido]phenyl}methyl N-[(5S)-5-amino-5-{[({[(10S,23S)-10-ethyl- 18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (LP16) [00319] Following a similar procedure as LP9, linker-payload LP16 (15 mg, 17% yield) was obtained as a red solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 758 (M/2 + H)+. [00320] Alternative Synthesis of LP4 (FIG.7). [00321] LP4 {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12- tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N- ({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP4) [00322] To a yellow solution of Fmoc-ProDXd (0.13 g, 0.16 mmol), linker L8 (0.16 g, 0.17 mmol), and 4-methyl-2-hydroxyquinoline (MeHYQ) (13 mg, 82 μmol) in DMF (1.3 mL) was added DBU (2.4 mg, 16 μmol) and triethylamine (32 mg, 0.32 mmol) at 25 °C. The clear solution was stirred at 50 °C for six hours, which was monitored by LCMS. After cooling to room temperature, the resulting mixture was poured into stirred MTBE (6 mL) at 0-10 °C and a brown oil appeared, which was collected after separation to remove the MTBE layer. The oil was then purified by reversed phase flash chromatography (5-95% acetonitrile in aq. TFA (0.01%)) to give LP4 (0.13 g, 58% yield) as a white solid. ESI m/z: 1396 (M+H)+.1H NMR (400 MHz, DMSO-d6) δ 10.00 (t, J = 6.4 Hz, 1H), 8.80 (t, J = 6.4 Hz, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.65-7.50 (m, 3H), 7.43 (t, J = 6.0 Hz, 1H), 7.31 (s, 1H), 7.27 (d, J = 8.8 Hz, 2H), 6.54 (s, 1H), 5.98 (t, J = 5.2 Hz, 1H), 5.63-5.57 (m, 1H), 5.41 (s, 4H), 5.21 (s, 2H), 4.92 (s, 2H), 4.62 (d, J = 6.4 Hz, 2H), 4.43-4.33 (m, 1H), 4.31-4.17 (m, 2H), 4.01 (s, 2H), 3.86 (d, J = 14.4 Hz, 1H), 3.75 (d, J = 14.8 Hz, 1H), 3.67-3.54 (m, 4H), 3.53-3.46 (m, 12H), 3.44-3.39 (m, 2H), 3.27-3.10 (m, 4H), 3.06-2.90 (m, 2H), 2.47-2.32 (m, 5H), 2.26-1.64 (m, 14H), 1.63-1.52 (m, 3H), 1.47-1.32 (m, 3H), 0.90-0.80 (m, 9H) ppm.19F NMR (376 MHz, DMSO-d6) δ -111 ppm. [00323] Synthesis of Other ProDXds and Corresponding Linker-payloads (FIG.7A) [00324] General Procedures for the Synthesis of Compounds 16-2 [00325] To a stirred solution of Fmoc protected amino-acid 16-1 (1 equiv) in DCM (0.2 M) was added HOSu (2.2 equiv) and EDCI (2.2 equiv), and the reaction mixture was stirred at room temperature for one hour. The reaction was monitored by LCMS. The mixture was diluted with DCM, washed with water (3x) and brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was dissolved in DMF (0.2 M). To the solution was added a corresponding amino-acid (R4NHCR2R3COOH) (1.0 equiv) and DIPEA (3.0 equiv.), and the reaction mixture was stirred at room temperature for an hour. The reaction was monitored by LCMS. The volatiles were removed in vacuo and the residue solution was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.03%)) to give compound 16-2 (37-70% yield) as a white solid. [00326] 2-[(2S)-3-[(tert-butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)propanamido]acetic acid (16-2b) [00327] Following the General Procedures for the Synthesis of Compounds 16-2, compound 16-2b (0.50 g, 54% yield) was obtained as a white solid. ESI m/z: 499 (M + H)+. [00328] 2-[(2S)-5-(benzyloxy)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5- oxopentanamido]acetic acid (16-2c) [00329] Following the General Procedures for the Synthesis of Compounds 16-2, compound 16-2c (0.65 g, 58% yield) was obtained as a white solid. ESI m/z: 517 (M + H)+. [00330] 2-[(2S)-6-azido-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)hexanamido]acetic acid (16-2d) [00331] Following the General Procedures for the Synthesis of Compounds 16-2, compound 16-2d (0.66 g, 70% yield) was obtained as a white solid. ESI m/z: 452 (M + H)+. [00332] 2-[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3- phenylpropanamido]acetic acid (16-2e) [00333] Following the General Procedures for the Synthesis of Compounds 16-2, compound 16-2e (0.43 g, 37% yield) was obtained as a white solid. ESI m/z: 445 (M + H)+. [00334] 2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]acetic acid (16-2f) [00335] Following the General Procedures for the Synthesis of Compounds 16-2, compound 16-2f (2.6 g, 72% yield) was obtained as a white solid. ESI m/z: 369 (M + H)+. [00336] 2-[(2R)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3- phenylpropanamido]acetic acid (16-2h) [00337] Following the General Procedures for the Synthesis of Compounds 16-2, compound 16-2h (0.43 g, 37% yield) was obtained as a white solid. ESI m/z: 445 (M + H)+. [00338] General Procedure for the Synthesis of Compounds 16-3 [00339] To a 10 L reaction flask was added THF (0.25-0.30 M) and compound 16-2 (1.0 equiv) at 25-30 °C, and to the resulting suspension was added pyridine (2.0 equiv) at 25-30 °C. After the mixture was stirred and became clear, cupric acetate (zero or 0.3 equiv) was added into the solution. The reaction mixture was then cooled to 0-5 °C and lead (IV) acetate (1.5 equiv) was added at 0-5 °C. The mixture was then stirred at 0-5 °C for an hour and was then allowed to warm to 25-30 °C. The reaction mixture was stirred at 25-30 °C for sixteen hours until most of compound 16-2 was consumed, as monitored by LCMS. The resulting mixture was filtered through a short silica gel plug and the silica gel was washed with ethyl acetate (2x). The combined filtrate was diluted with ethyl acetate and water. After careful neutralization to pH 7 with sodium bicarbonate powder, the mixture was separated and the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give a brown crude product. The crude was dissolved into DCM (3 L). The mixture was filtered through short silica gel plug eluting with DCM (3x) until all of compound 16-3 was collected. The collected solution was concentrated. To the residue was added MTBE and a white solid was precipitated at 25-30 °C, which was collected by filtration. The solid was dried with nitrogen blowing at 25-30 °C for more than sixteen hours to give pure compound 16-3 as a white solid. Or, the brown crude product was purified by reversed phase flash chromatography or prep-HPLC to give pure compound 16-3 as a white solid. [00340] [(2S)-3-[(tert-butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)propanamido]methyl acetate (16-3b) [00341] Following the General Procedure for the Synthesis of Compounds 16-3 except without cupric acetate, compound 16-3b (0.19 g, 45% yield) was obtained as a white solid after purification by prep-HPLC (0-100% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 535 (M + Na)+. [00342] Benzyl (4S)-4-{[(acetyloxy)methyl]carbamoyl}-4-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)butanoate (16-3c) [00343] Following the General Procedure for the Synthesis of Compounds 16-3 except without cupric acetate, compound 16-3c (0.20 g, 30% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-60% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 553 (M + Na)+. [00344] [(2S)-6-azido-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)hexanamido]methyl acetate (16-3d) [00345] Following the General Procedure for the Synthesis of Compounds 16-3 except without cupric acetate, compound 16-3d (0.57 g, 84% yield) was obtained as a white solid after purification by prep-HPLC (0-100% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 488 (M + Na)+. [00346] [(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3- phenylpropanamido]methyl acetate (16-3e) [00347] Following the General Procedure for the Synthesis of Compounds 16-3 except without cupric acetate, compound 16-3c (0.36 g, 81% yield) was obtained as a white solid after purification by prep-HPLC (0-100% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 481 (M + Na)+.1H NMR (400 MHz, DMSO-d6) δ 9.13 (t, J = 6.9 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 8.7 Hz, 1H), 7.67-7.58 (m, 2H), 7.46-7.36 (m, 2H), 7.35-7.23 (m, 6H), 7.19 (t, J = 7.1 Hz, 1H), 5.18-5.04 (m, 2H), 4.32-4.21 (m, 1H), 4.21-4.07 (m, 3H), 3.05-2.73 (m, 2H), 2.00 (s, 3H) ppm. [00348] [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-N-methylacetamido]methyl acetate (16-3f) [00349] Following the General Procedure for the Synthesis of Compounds 16-3 except without cupric acetate, compound 16-3f (1.65 g, 60% yield) was obtained as a white solid. ESI m/z: 405 (M + Na)+. [00350] [(2R)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3- phenylpropanamido]methyl acetate (16-3h) [00351] Following the General Procedure for the Synthesis of Compounds 16-3 (catalyzed with cupric acetate (0.3 equiv)), compound 16-3h (0.34 g, 80% yield) was obtained as a white solid after purification by prep-HPLC (0-100% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 481 (M + Na)+. [00352] General Procedure for the Synthesis of Compounds 16-4 [00353] To a reaction flask was added 1,2-dichloroethane (0.10-0.15 M), compound 16-3 (1.0 equiv), glycolic acid (0.6 equiv), and pyridium p-toluenesulfonate (PPTS) (0.2 equiv) at room temperature. The reaction mixture was heated to 45-50 °C and stirred for an hour. To the hot solution was added glycolic acid (0.6 equiv, 2x) once per hour. The mixture was then stirred at 45- 50 °C for sixteen hours and monitored by LCMS. After the reaction was cooled to 25-30 °C, the precipitates were filtered and collected. The solid was dissolved into aqueous sodium bicarbonate (3%) having a pH 7-8 at 5-10 °C, which was washed with ethyl acetate:THF (v:v = 1, 3x). To the aqueous layer was added MTBE at 5-10 °C and the aqueous layer was acidified with sat. aq. citric acid to pH 3-4 to precipitate a large amount of solids. The mixture was filtered and the cake was washed with water (1x) and MTBE (2x) and dried under nitrogen blow at 25-30 °C for over forty- eight hours to give wet compound 16-4 (75% yield) as a white solid (containing 3% water according to HNMR). The product was dried again under vacuum for over forty-eight hours to give dry compound 16-4 (73% yield) as a white solid. Or, the reaction mixture was directly purified by reversed phase flash chromatography or prep-HPLC to give pure compound 16-4 as a white solid. [00354] 2-{[(2S)-3-[(tert-butyldimethylsilyl)oxy]-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)propanamido]methoxy}acetic acid (16-4b) [00355] Following the General Procedure for the Synthesis of Compounds 16-4, compound 16- 4b (57 mg, 31% yield) was obtained as a yelllow solid after purification by prep-HPLC (0-100% acetonitrile in aq. TFA (0.05%)). ESI m/z: 551 (M + Na)+. [00356] 2-{[(2S)-5-(benzyloxy)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-5- oxopentanamido]methoxy}acetic acid (16-4c) [00357] Following the General Procedure for the Synthesis of Compounds 16-4, compound 16- 4c (0.13 g, 65% yield) was obtained as a white solid after purification by prep-HPLC (0-90% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 569 (M + Na)+. [00358] 2-{[(2S)-6-azido-2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)hexanamido]methoxy}acetic acid (16-4d) [00359] Following the General Procedure for the Synthesis of Compounds 16-4, compound 16- 4d (0.30 g, 51% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. ammonium bicarbonate (10 mM)). ESI m/z: 504 (M + Na)+. [00360] 2-{[(2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3- phenylpropanamido]methoxy}acetic acid (16-4e) [00361] Following the General Procedure for the Synthesis of Compounds 16-4, compound 16- 4e (0.14 g, 38% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-25% acetonitrile in water). ESI m/z: 474 (M + Na)+. [00362] 2-{[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-N- methylacetamido]methoxy}acetic acid (16-4f) [00363] Following the General Procedure for the Synthesis of Compounds 16-4, compound 16- 4f (1.0 g, 50% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)). ESI m/z: 421 (M + Na)+. [00364] 2-{[(2R)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3- phenylpropanamido]methoxy}acetic acid (16-4h) [00365] Following the General Procedure for the Synthesis of Compounds 16-4, compound 16- 4h (0.14 g, 38% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)). ESI m/z: 474 (M + Na)+. [00366] General Procedure for the synthesis of Compounds 16-5 [00367] To a solution of compound 16-4 (1.1 equiv) in DMF (5-8 mL per gram of 16-4) was added HATU (1.1 equiv) and DIPEA (1.0 equiv), and the reaction mixture was stirred at room temperature for fifteen minutes. To the stirred solution was then added a mixture of Exatecan (1.0 equiv) and DIPEA (2.0 equiv.) in DMF (10 mL per gram of Exatecan). The reaction mixture was stirred at room temperature for four hours and monitored by LCMS. The resulting mixture was diluted with ethyl acetate and washed with brine (2x). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was co-evaporated with ethyl acetate (4x) in vacuo to provide crude product 16-5. Ethyl acetate was added to crude 16-5. The suspension was refluxed for around twenty minutes until the suspension turned clear and was then cooled to 25 °C naturally. The reaction then stood for half an hour. The white precipitates were collected by filtration, washed with ethyl acetate (2x), and dried under vacuum to give 16-5 as a white solid. Or, the crude product 16-5 was purified by reversed phase flash chromatography to give pure compound 16-5 as a solid. [00368] (9H-fluoren-9-yl)methyl N-[(1S)-2-[(tert-butyldimethylsilyl)oxy]-1-{[({[(10S,23S)- 10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}ethyl]carbamate (16-5b) [00369] Following the General Procedure for the synthesis of Compounds 16-5, compound 16- 5b (0.12 g, 53% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)). ESI m/z: 946 (M + H)+. [00370] Benzyl (4S)-4-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo- 8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)- heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-4-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)butanoate (16-5c) [00371] Following the General Procedure for the synthesis of Compounds 16-5, compound 16- 5c (0.21 g, 74% yield) was obtained as a yellow solid after purification by reversed phase flash chromatography (0-70% acetonitrile in aq. formic acid (0.1%)). ESI m/z: 964 (M + H)+. [00372] (4S)-4-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa- 4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen- 23-yl]carbamoyl}methoxy)methyl]carbamoyl}-4-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)butanoic acid (16-5ca)
[00373] To a stirred solution of compound 16-5a (0.21 g, 0.22 mmol) in methanol (20 mL) was added palladium on carbon (36 mg, containing 10% palladium) under the protection of nitrogen. The reaction mixture was then stirred at room temperature under a hydrogen atmosphere for four hours and monitored by LCMS. The mixture was filtered through Celite and the filtrate was concentrated in vacuo to give crude compound 16-5ca (0.10 g, 53% yield) as a yellow solid. The crude was used in the next step without further purification. ESI m/z: 874 (M + H)+. [00374] (9H-fluoren-9-yl)methyl N-[(1S)-3-carbamoyl-1-{[({[(10S,23S)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}propyl]carbamate (16-5cb) [00375] To a solution of compound 16-5ca (50 mg, 57 µmol) in DMF (1 mL) was added ammonium chloride (3.0 mg, 57 µmol), HATU (32 mg, 85 µmol), and DIPEA (22 mg, 0.17 mmol) and the reaction mixture was stirred at room temperature for three hours. The reaction was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile in aq. formic acid (0.1%)) to give compound 16-5cb (40 mg, 81% yield) as a white solid. ESI m/z: 873 (M + H)+. [00376] (9H-fluoren-9-yl)methyl N-[(1S)-5-azido-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (16-5d) [00377] Following the General Procedure for the synthesis of Compounds 16-5, compound 16- 5d (85 mg, 91% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 900 (M + H)+. [00378] (9H-fluoren-9-yl)methyl N-[(1S)-5-amino-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (16-5da) [00379] To a stirred solution of compound 16-5d (45 mg, 50 µmol) in methanol (20 mL) was added palladium on carbon (10 mg, containing 10% palladium) under the protection of nitrogen. The reaction mixture was then stirred at room temperature under a hydrogen atmosphere for two hours and monitored by LCMS. The mixture was filtered through Celite and the filtrate was concentrated in vacuo. The residue was purified by prep-HPLC (5-95% acetonitrile in aq. TFA (0.03%)) to give compound 16-5da (38 mg, 86% yield) as a yellow solid. ESI m/z: 873 (M + H)+. [00380] (9H-fluoren-9-yl)methyl N-[(1S)-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2- phenylethyl]carbamate (16-5e) [00381] Following the General Procedure for the synthesis of Compounds 16-5, compound 16- 5e (24 mg, 64% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 892 (M + H)+. [00382] (9H-fluoren-9-yl)methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19- methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl](methyl)carbamoyl}methyl)carbamate (16-5f)
[00383] Following the General Procedure for the synthesis of Compounds 16-5, compound 16- 5f (50 mg, 61% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.01%)). ESI m/z: 816 (M + H)+. [00384] (9H-fluoren-9-yl)methyl N-[(1R)-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}-2- phenylethyl]carbamate (16-5h) [00385] Following the General Procedure for the synthesis of Compounds 16-5, compound 16- 5h (92 mg, 73% yield) was obtained as a yellow solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 892 (M + H)+. [00386] General Procedure for Fmoc Deprotection to Obtain ProDXds [00387] To a solution of compound 16-5 (1.0 equiv) in THF (20 mL per gram of 16-5) was added diethylamine (2 mL per gram of 16-5), and the reaction mixture was stirred at room temperature for 2-48 hours until Fmoc was totally removed according to LCMS. The volatiles were removed thoroughly in vacuo and the residue was diluted with water (5 mL). The aqueous mixture was adjusted to pH 2 with the addition of aq. TFA (10%) and was washed with MTBE (20 mL x 2). The aqueous layer was then stirred at room temperature for sixteen hours until the ring-open form turned to the lactone form, as monitored by LCMS. The resulting aqueous mixture was lyophilized to give crude ProDXds and purified by reversed phase flash chromatography (0- 100% acetonitrile in aq. TFA (0.03%)) to give pure ProDXds (TFA salt) as a solid; or prep-HPLC (5-95% acetonitrile in aq. formic acid (0.1%)) to give pure payloads (free base) as a solid. [00388] SerDXd [00389] (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-3- hydroxypropanamide (SerDXd) [00390] To a solution of compound 16-5b (0.12 g, 0.12 mmol) in DMF (1 mL) was added diethylamine (0.1 mL) and the mixture was stirred at room temperature for two hours until Fmoc was totally removedas monitored by LCMS. The resulting mixture was directly purified by reserved phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to give a de-Fmoc product (68 mg, ESI m/z: 724 (M + H)+) as a yellow solid). The de-Fmoc product was dissolved in DMF (1 mL). To the solution was added cesium fluoride (31 mg, 0.20 mmol) at 0 °C. The mixture was then stirred at room temperature for an hour and monitored by LCMS. The mixture was separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.05%)) to give SerDXd (17 mg, 22% yield) as a white solid. ESI m/z: 610 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.26 (t, J = 6.4 Hz, 1H), 8.58 (d, J = 8.8 Hz, 1H), 8.14 (s, 2H), 7.79 (d, J = 10.8 Hz, 1H), 7.33 (s, 1H), 6.56 (s, 1H), 5.60-5.53 (m, 2H), 5.42 (s, 2H), 5.20-5.18 (m, 2H), 4.80-4.76 (m, 1H), 4.67-4.63 (m, 1H), 4.05 (s, 2H), 3.90-3.89 (m, 1H), 3.77-3.76 (m, 2H), 3.27-3.14 (m, 2H), 2.39 (s, 3H), 2.17-2.16 (m, 2H), 1.90-1.83 (m, 2H), 0.87 (t, J = 6.8 Hz, 3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -74 (TFA), -111 (Ar-F) ppm. [00391] GlnDXd [00392] (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]pentanediamide (GlnDXd) [00393] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, GlnDXd (20 mg, 49% yield) was obtained as a light-yellow solid. ESI m/z: 651 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.95-8.80 (m, 1H),8.80 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 7.81 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 7.29-7.20 (m, 1H), 6.73 (s, 1H), 6.52 (s, 1H), 5.65-5.56 (m, 1H), 5.42 (s, 2H), 5.21 (s, 2H), 4.63 (br s, 2H), 4.01 (s, 1H), 3.25-3.13 (m, 2H), 3.06-2.90 (m, 2H), 2.40 (s, 3H), 2.25- 2.05 (m, 4H), 1.93-1.74 (m, 3H), 1.64-1.50 (m, 1H), 0.87 (t, J = 6.8 Hz, 1H) ppm.19F NMR (376 MHz, DMSO-d6) δ -111 (Ar-F) ppm. [00394] GluDXd [00395] (4S)-4-amino-4-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}butanoic acid (GluDXd) [00396] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, GluDXd (20 mg, 49% yield) was obtained as a light-yellow solid. ESI m/z: 652 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.95-8.82 (m, 1H),8.55 (d, J = 9.2 Hz, 1H), 8.30 (s, 1H), 7.79 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 5.65-5.56 (m, 1H), 5.42 (s, 2H), 5.21 (s, 2H), 4.62 (br s, 2H), 4.00 (s, 2H), 3.25- 3.10 (m, 4H), 3.06-2.90 (m, 2H), 2.32 (s, 3H), 2.27-2.12 (m, 4H), 1.92-1.71 (m, 3H), 1.60-1.50 (m, 1H), 0.87 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -111 (Ar-F) ppm. [00397] LysDXd [00398] (2S)-2,6-diamino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]hexanamide (LysDXd) [00399] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, LysDXd (13 mg, 43% yield) was obtained as a white solid. ESI m/z: 651 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.33 (t, J = 6.5 Hz, 1H), 8.61 (d, J = 8.8 Hz, 1H), 8.18 (s, 3H), 7.81 (d, J = 10.9 Hz, 1H), 7.72 (s, 2H), 7.34 (s, 1H), 6.57 (s, 1H), 5.63-5.57 (m, 1H), 5.43 (s, 2H), 5.26-5.15 (m, 2H), 4.79-4.67 (m, 2H), 4.11-4.01 (m, 2H), 3.86-3.80 (m, 1H), 3.25-3.10 (m, 2H), 2.81-2.72 (m, 2H), 2.40 (s, 3H), 2.22-2.13 (m, 2H), 1.92-1.83 (m, 2H), 1.80-1.71 (m, 2H), 1.60-1.50 (m, 2H), 1.40- 1.31 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -74 (TFA), -111 (Ar-F) ppm. [00400] Lys(N3)DXd [00401] (2S)-2-amino-6-azido-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl- 5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]hexanamide (Lys(N3)DXd) [00402] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, Lys(N3)DXd (27 mg, 89% yield) was obtained as a white solid. ESI m/z: 677 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.92-8.78 (m, 1H), 8.57 (d, J = 8.8 Hz, 1H), 8.23 (s, 1H), 7.77 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.66-5.54 (m, 1H), 5.41 (s, 2H), 5.19 (s, 2H), 4.60 (d, J = 1.8 Hz, 2H), 4.00 (s, 2H), 3.26 (t, J = 6.8 Hz, 3H), 3.21-3.11 (m, 3H), 2.38 (s, 3H), 2.24-2.12 (m, 2H), 1.92-1.80 (m, 2H), 1.54-1.41 (m, 3H), 1.36-1.24 (m, 3H), 0.87 (t, J = 7.3 Hz, 3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -111 (Ar-F) ppm. [00403] PheDXd [00404] (2S)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-3- phenylpropanamide (PheDXd) [00405] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, PheDXd (15 mg, 62% yield) was obtained as a white solid. ESI m/z: 670 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.86-8.77 (m, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.29 (s, 1H), 7.78 (d, J = 11.0 Hz, 1H), 7.29 (s, 1H), 7.21 (t, J = 7.2 Hz, 2H), 7.12 (t, J = 8.5 Hz, 3H), 6.52 (s, 1H), 5.64-5.56 (m, 1H), 5.45-5.34 (m, 2H), 5.25-5.12 (m, 2H), 4.58 (s, 2H), 3.96 (s, 2H), 3.25-3.08 (m, 4H), 2.85-2.70 (m, 1H), 2.39 (s, 3H), 2.28-2.08 (m, 3H), 1.87-1.76 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -111 (Ar-F) ppm. [00406] DPheDXd [00407] (2R)-2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]-3- phenylpropanamide (DPheDXd) [00408] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, DPheDXd (36 mg, 52% yield, TFA salt) was obtained as a white solid. ESI m/z: 670 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.29-9.26 (m, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.19 (s, 3H), 7.80 (d, J = 12 Hz, 1H), 7.33-7.23 (m, 6H), 6.55 (s, 1H), 5.62-5.57 (m, 1H), 5.45-5.35 (m, 2H), 5.20 (s, 2H), 4.69 (d, J = 8.0 Hz, 1H), 4.07-3.96 (m, 3H), 3.11-2.96 (m, 3H), 2.40 (s, 3H), 2.21-2.16 (m, 2H), 1.92-1.82 (m, 2H), 1.27-1.23 (m, 1H), 0.88 (t, J = 8.0 Hz,3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -73 (TFA), -111 (Ar-F) ppm. [00409] GlyGlyDXd [00410] 2-amino-N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8- oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)- heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)acetamide (GlyGlyDXd) [00411] To a solution of N-Fmoc-glycine (16.2 mg, 0.054 mmol) in DMF (1 mL) was added HATU (30.9 mg, 0.081 mmol) and DIPEA (21 mg, 0.108 mmol), and the reaction mixture was stirred at room temperature for fifteen minutes. To the stirred mixture was then added compound GlyDXd (30 mg, 0.054 mmol, TFA salt) and the reaction mixture was stirred at room temperature for an hour. The reaction was monitored by LCMS. To the resulting mixture was then added diethylamine (1 mL) and the mixture was stirred at room temperature for an hour until Fmoc was totally removed according to LCMS. The volatiles were removed in vacuo and the residual mixture was directly separated by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)) to give GlyGlyDXd (11 mg, 31% yield, TFA salt) as a light-yellow solid. ESI m/z: 637 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.88 (t, J = 6.4 Hz, 1H), 8.65 (t, J = 5.6 Hz, 1H), 8.54 (d, J = 8.8 Hz, 1H), 8.01 (br s, 3H), 7.80 (d, J = 10.4 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.62-5.56 (m, 1H), 5.42 (s, 2H), 5.19 (s, 2H), 4.65 (d, J = 6.4 Hz, 2H), 4.01 (s, 2H), 3.86 (d, J = 5.6 Hz, 2H), 3.67 (s, 2H), 3.24-3.09 (m, 2H), 2.39 (s, 3H), 2.24-2.11 (m, 2H), 1.94-1.79 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -73 (TFA), -111 (Ar-F) ppm. [00412] GlyNMeCH2DXd [00413] 2-amino-N-[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8- oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)- heptaen-23-yl]carbamoyl}methoxy)methyl]-N-methylacetamide (GlyNMeCH2DXd) [00414] Following the General Procedure for Fmoc Deprotection to Obtain ProDXds, GlyNMeCH2DXd (22 mg, 60% yield) was obtained as a white solid. ESI m/z: 594 (M + H)+.1H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J = 8.9 Hz, 0.5H), 8.61 (d, J = 8.9 Hz, 0.5H), 8.07 (s, 2H), 7.82-7.78 (m, 1H), 7.33 (d, J = 1.8 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 5.67-5.53 (m, 1H), 5.43 (s, 2H), 5.29-5.10 (m, 2H), 4.96-4.87 (m, 2H), 4.20-3.90 (m, 4H), 3.19 (d, J = 6.6 Hz, 2H), 3.02 (s, 1.5H), 2.99 (s, 1.5H), 2.40 (s, 3H), 2.19-2.17 (m, 2H), 1.93-1.81 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H) ppm.19F NMR (376 MHz, DMSO-d6) δ -73 (TFA), -111 (Ar-F) ppm. [00415] General Procedure for Linker-ProDXds [00416] To a solution of intermediate L8 (1.0-1.2 equiv.) in DMF (0.15 mM) was added HOBt (0.5 equiv) or HOAt (0.5 equiv), DIPEA (3.0 equiv), and ProDXds described above (1.0 equiv). The reaction mixture was then stirred at room temperature for two hours and monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography to give linker- ProDXds as white solids. [00417] LP12 [00418] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(1S)-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}-2-hydroxyethyl]carbamate (LP12) [00419] Following the General Procedure for Linker-ProDXds starting from payload SerDXd (18 mg, 29 µmol) catalyzed by HOAt, linker-payload LP12 (19 mg, 45% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 714 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.82 (t, J = 6.7 Hz, 1H), 8.50 (d, J = 8.6 Hz, 1H), 8.14 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.66- 7.54 (m, 3H), 7.35-7.24 (m, 3H), 7.18 (d, J = 8.2 Hz, 1H), 6.59-6.46 (m, 1H), 5.99 (s, 1H), 5.65- 5.55 (m, 1H), 5.42 (s, 3H), 5.21 (s, 2H), 4.97-4.83 (m, 2H), 4.67-4.57 (m, 2H), 4.41-4.34 (m, 1H), 4.30-4.21 (m, 2H), 4.00 (s, 2H), 3.99-3.65 (m, 3H), 3.62-3.57 (m, 2H), 3.53-3.46 (m, 12H), 3.28- 3.16 (m, 4H), 3.06-2.81 (m, 3H), 2.39 (s, 3H), 2.26-2.02 (m, 6H), 2.02-1.80 (m, 6H), 1.78-1.66 (m, 3H), 1.62-1.52 (m, 3H), 1.48-1.22 (m, 8H), 0.92-0.77 (m, 9H) ppm. [00420] LP13 [00421] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(1S)-3-carbamoyl-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo- 8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)- heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}propyl]carbamate (LP13)
[00422] Following the General Procedure for Linker-ProDXds starting from payload GlnDXd (14 mg, 22 µmol) catalyzed by HOAt, linker-payload LP13 (16 mg, 49% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 734 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.83 (t, J = 6.5 Hz, 1H), 8.54 (d, J = 8.6 Hz, 1H), 8.14 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.66- 7.51 (m, 3H), 7.44 (d, J = 7.4 Hz, 1H), 7.31-7.23 (m, 3H), 6.78 (s, 1H), 6.59-6.48 (m, 1H), 6.03- 5.94 (m, 1H), 5.64-5.56 (m, 1H), 5.42 (s, 3H), 5.22 (s, 2H), 4.95-4.81 (m, 2H), 4.68-4.56 (m, 2H), 4.42-4.34 (m, 1H), 4.31-4.19 (m, 2H), 4.01 (s, 2H), 3.87 (d, J = 14.8 Hz, 2H), 3.75 (d, J = 14.8 Hz, 1H), 3.64-3.55 (m, 2H), 3.53-3.44 (m, 12H), 3.28-3.20 (m, 4H), 3.08-2.90 (m, 3H), 2.40 (s, 3H), 2.24-2.02 (m, 8H), 2.01-1.50 (m, 16H), 1.46-1.29 (m, 4H), 0.90-0.76 (m, 9H) ppm. [00423] LP14 [00424] (4S)-4-{[({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1- yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3- methylbutanamido]pentanamido]phenyl}methoxy)carbonyl]amino}-4-{[({[(10S,23S)-10- ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}butanoic acid (LP14) [00425] Following the General Procedure for Linker-ProDXds starting from payload GluDXd (16 mg, 25 µmol) catalyzed by HOAt, linker-payload LP14 (12 mg, 35% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 735 (M/2 + H)+. 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.89-8.82 (m, 1H), 8.53 (d, J = 8.5 Hz, 1H), 8.12 (d, J = 7.5 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.78 (d, J = 11.1 Hz, 1H), 7.65-7.51 (m, 3H), 7.44 (d, J = 7.4 Hz, 1H), 7.33-7.22 (m, 2H), 6.53 (s, 1H), 6.04-5.95 (m, 1H), 5.64-5.54 (m, 1H), 5.42 (s, 3H), 5.21 (s, 2H), 4.97-4.81 (m, 2H), 4.67-4.56 (m, 2H), 4.41-4.33 (m, 1H), 4.31- 4.18 (m, 2H), 4.00 (s, 2H), 3.99-3.65 (m, 3H), 3.63-3.55 (m, 2H), 3.54-3.42 (m, 12H), 3.27-3.17 (m, 4H), 3.09-2.87 (m, 3H), 2.39 (s, 3H), 2.27-1.13 (m, 28H), 0.98-0.64 (m, 9H) ppm. [00426] LP15 [00427] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(5S)-5-amino-5-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8- oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)- heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (LP15) [00428] Following the General Procedure for Linker-ProDXds starting from compound 5da (32 mg, 37 µmol) with intermediate L8 catalyzed by HOBt, Fmoc-LP15 (35 mg, 56% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). [00429] To a solution of Fmoc-LP15 (35 mg, 21 µmol) in DMF (2 mL) was added diethylamine (7.6 mg, 0.10 mmol), and the reaction mixture was stirred at room temperature for two hours until Fmoc was totally removed according to LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (5-95% acetonitrile in aq. TFA (0.01%)) to give LP15 (9.6 mg, 31% yield) as a white solid. ESI m/z: 734 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 10.00 (d, J = 13.5 Hz, 1H), 9.19-9.04 (m, 1H), 9.01-8.87 (m, 1H), 8.65-8.55 (m, 1H), 8.20-8.08 (m, 1H), 7.92-7.86 (m, 1H), 7.85-7.75 (m, 1H), 7.71 (s, 1H), 7.63-7.54 (m, 2H), 7.33-7.15 (m, 3H), 6.54 (s, 1H), 6.06-5.97 (m, 1H), 5.63-5.56 (m, 1H), 5.45-5.39 (m, 2H), 5.20 (s, 1H), 5.14-5.04 (m, 1H), 4.90 (d, J = 6.1 Hz, 2H), 4.69-4.57 (m, 2H), 4.40-4.33 (m, 1H), 4.31-4.18 (m, 2H), 4.10-3.96 (m, 2H), 3.99-3.65 (m, 3H), 3.65-3.55 (m, 4H), 3.53-3.44 (m, 12H), 3.29-3.19 (m, 4H), 3.03-2.35 (m, 7H), 2.27-1.17 (m, 32H), 0.91-0.63 (m, 9H) ppm. [00430] LP19 [00431] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(1S)-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (LP19) [00432] Following the General Procedure for Linker-ProDXds starting from payload PheDXd (17 mg, 25 µmol) catalyzed by HOAt, linker-payload LP19 (17 mg, 46% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 744 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.97 (t, J = 6.8 Hz, 1H), 8.54 (d, J = 8.7 Hz, 1H), 8.13 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 11.0 Hz, 1H), 7.62 (t, J = 5.5 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.2 Hz, 1H), 7.30 (s, 1H), 7.26-7.20 (m, 4H), 7.18-7.12 (m, 2H), 6.03-5.95 (m, 1H), 5.64-5.56 (m, 1H), 5.47-5.34 (m, 3H), 5.26-5.14 (m, 2H), 4.86-4.75 (m, 2H), 4.70-4.57 (m, 2H), 4.41-4.33 (m, 1H), 4.31-4.12 (m, 4H), 3.99 (s, 2H), 3.99- 3.65 (m, 3H), 3.63-3.57 (m, 3H), 3.50-3.47 (m, 12H), 3.28-3.10 (m, 4H), 2.98-2.90 (m, 1H), 2.39 (s, 3H), 2.28-1.16 (m, 25H), 0.87-0.80 (m, 9H) ppm. [00433] LP20 [00434] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-[(1R)-1-{[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}-2-phenylethyl]carbamate (LP20) [00435] Following the General Procedure for Linker-ProDXds starting from payload DPheDXd (19 mg, 28 µmol) catalyzed by HOBt, linker-payload LP20 (16 mg, 39% yield) was obtained as a white solid after purification by reversed phase flash chromatography (0-70% acetonitrile in aq. TFA (0.1%)). ESI m/z: 743.7 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.96 (t, J = 6.4 Hz, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 11.2 Hz, 1H), 7.60 (t, J = 5.6 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.29-7.22 (m, 4H), 7.15-7.13 (m, 2H), 6.54 (s, 1H), 5.99 (s, 1H), 5.63-5.58 (m, 1H), 5.44-5.31 (m, 3H), 5.27-5.17 (m, 2H), 4.88-4.78 (m, 2H), 4.68-4.60 (m, 2H), 4.40-4.35 (m, 1H), 4.29-4.15 (m, 3H), 4.00 (s, 2H), 3.89-3.73 (m, 2H), 3.63-3.57 (m, 3H), 3.51-3.47 (m, 12H), 3.27- 3.22 (m, 4H), 3.05-3.00 (m, 1H), 2.99-2.89 (m, 2H), 2.74-2.68 (m, 1H), 2.46 (d, J = 6.8 Hz, 1H), 2.38 (s, 3H), 2.28-2.13 (m, 4H), 2.12-1.65 (m, 11H), 1.64-1.31 (m, 7H), 1.28-1.23 (m, 1H), 0.87- 0.82 (m, 9H) ppm.19F NMR (377 MHz, DMSO-d6) δ -73.72 (s), -111.28 (s) ppm. [00436] LP21 [00437] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-{[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]methyl}carbamate (LP21) [00438] Following the General Procedure for Linker-ProDXds starting from payload GlyGlyDXd (20 mg, 32 µmol) catalyzed by HOBt, linker-payload LP21 (14 mg, 29% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 727 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.72-8.68 (m, 1H), 8.51 (d, J = 8.5 Hz, 1H), 8.19-8.12 (m, 2H), 7.88 (d, J = 9.1 Hz, 1H), 7.80 (d, J = 11.2 Hz, 1H), 7.63- 7.55 (m, 3H), 7.47-7.43 (m, 1H), 7.31 (s, 1H), 7.28 (d, J = 8.3 Hz, 2H), 6.53 (s, 1H), 6.00-5.95 (m, 1H), 5.62-5.58 (m, 1H), 5.42 (d, J = 4.9 Hz, 3H), 5.21 (s, 2H), 4.94 (s, 2H), 4.63 (d, J = 6.4 Hz, 2H), 4.39-4.35 (m, 1H), 4.28-4.22 (m, 2H), 4.01 (s, 2H), 3.85 (s, 1H), 3.77 (s, 1H), 3.74-3.70 (m, 2H), 3.66-3.57 (m, 4H), 3.52-3.39 (m, 14H), 3.28-3.21 (m, 4H), 3.04-2.97 (m, 2H), 2.40 (s, 3H), 2.25-2.13 (m, 6H), 2.01-1.82 (m, 9H), 1.61-1.55 (m, 3H), 1.49-1.36 (m, 5H), 0.91-0.78 (m, 9H) ppm. [00439] LP22 [00440] {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]- 3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl](methyl)carbamoyl}methyl)carbamate (LP22) [00441] Following the General Procedure for Linker-ProDXds starting from payload GlyNMeCH2DXd (6.0 mg, 10 µmol) catalyzed by HOBt, linker-payload LP22 (5.0 mg, 36% yield) was obtained as a white solid after purification by prep-HPLC (5-95% acetonitrile in aq. TFA (0.1%)). ESI m/z: 706 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.63-8.50 (m, 1H), 8.15-8.08 (m, 1H), 7.88-7.78 (m, 2H), 7.59-7.57 (m, 3H), 7.30-7.22 (m, 4H), 6.52 (s, 1H), 5.98 (s, 1H), 5.61 (s, 1H), 5.42 (br s, 4H), 5.22 (s, 2H), 4.88-4.82 (m, 4H), 4.42-4.20 (m, 4H), 4.09- 3.74 (m, 8H), 3.59-3.49 (m, 13H), 3.25-3.23 (m, 4H), 3.00-2.88 (m, 6H), 2.39-2.38 (m, 4H), 2.20- 2.16 (m, 3H), 1.97-1.73 (m, 9H), 1.56-1.35 (m, 7H), 0.86-0.83 (m, 9H) ppm. [00442] Synthesis of EvcPAB-Linker-payloads (FIG.7B) [00443] LP17 [00444] (4S)-4-(1-amino-3,6,9,12-tetraoxapentadecan-15-amido)-4-{[(1S)-1-{[(1S)-4- (carbamoylamino)-1-{[4-({[(4- nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2- methylpropyl]carbamoyl}butanoic acid (17-4) [00445] To a solution of compound 17-1 (0.30 g, 0.38 mmol) in dry DMF (4 mL) was added diethylamine (56 mg, 0.76 mmol) and the mixture was stirred at room temperature for two hours until Fmoc was totally removed as monitored by LCMS. The reaction mixture was purified by prep-HPLC to give a white solid (0.13 g, ESI m/z: 587.3 (M + Na)+), which was dissolved in dry DMF (3 mL). To the solution was added N-Boc-PEG4-acid (70 mg, 0.19 mmol), HATU (87 mg, 0.23 mmol), and DIPEA (99 mg, 0.23 mmol). The reaction mixture was stirred at room temperature for two hours and monitored by LCMS. The mixture was directly purified by prep-HPLC to give 17-2 (0.15 g, 43% yield) as a white solid. ESI m/z: 913.3 (M + H)+. [00446] To a solution of 17-2 (0.14 g, 0.15 mmol) in DMF (1.5 mL) was added DIPEA (50 mg, 0.38 mmol) and bis(4-nitrophenyl) carbonate (70 mg, 0.23 mmol), and the reaction mixture was stirred at room temperature for two hours. The reaction was monitored by LCMS. The mixture was separated by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound 17-3 (0.19 g, 93% yield) as a white solid. ESI m/z: 1100 (M + Na)+. [00447] To a solution of compound 17-3 (0.18 g, 0.17 mmol) in acetonitrile (2.0 mL) was added HCl in ethyl acetate (4 M, 2 mL). The reaction mixture was stirred at room temperature for 3 hours. The mixture was purified by reversed phase flash chromatography (0-100% acetonitrile in aq. TFA (0.01%)) to give compound 17-4 (0.10 g, 58% yield) as a white solid. ESI m/z: 943.5 (M + Na)+. [00448] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4- nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2- methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2-yn-1-yloxy)acetamido]-3,6,9,12- tetraoxapentadecan-15-amido}butanoic acid (17-5) [00449] To a mixture of intermediate A (39 mg, 0.10 mmol) in DMF (1 mL) was added DIPEA (28 mg, 0.22 mmol) and a solution of compound 17-4 (90 mg, 87 µmol) in DMF (1.5 mL). The reaction mixture was stirred at room temperature for an hour and monitored by LCMS. After the reaction completed, the mixture was immediately separated by prep-HPLC to give compound 17- 5 (30 mg, 29% yield) as a white solid. ESI m/z: 1085 (M + H)+, 1107 (M + Na)+. [00450] (4S)-4-{[(1S)-1-{[(1S)-1-({4-[({[(5S)-5-amino-5-{[({[(10S,23S)-10-ethyl-18-fluoro- 10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamoyl}oxy)methyl]phenyl}carbamoy l)-4-(carbamoylamino)butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2- yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoic acid (LP17) [00451] Following a similar procedure as LP15 except using compound 17-5 instead of intermediate L8, linker-payload LP17 (7 mg, 14% yield) was obtained as a light yellow solid. ESI m/z: 799 (M/2 + H).1H NMR (400 MHz, DMSO-d6) δ 12.09 (br s, 1H), 10.02 (s, 1H), 9.29 (br s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.25-7.99 (m, 5H), 7.80 (d, J = 10.9 Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.62-7.58 (m, 3H), 7.34 (s, 1H), 7.31-7.10 (m, 3H), 6.55 (s, 1H), 5.98 (m, 1H), 5.61 (br s, 1H), 5.42 (br s, 4H), 5.21 (s, 2H), 4.91 (s, 2H), 4.81-4.64 (m, 2H), 4.38-4.14 (m, 4H), 4.06 (s, 2H), 3.82-3.77 (m, 2H), 3.62-3.58 (m, 2H), 3.53-3.44 (m, 12H), 3.41 (m, 2H), 3.28-3.14 (m, 4H), 3.05- 2.96 (m, 4H), 2.44-2.29 (m, 6H), 2.28-2.13 (m, 6H), 2.07 (br s, 1H), 1.98-1.83 (m, 5H), 1.80-1.65 (m, 6H), 1.61-1.53 (m, 3H), 1.44-1.37 (m, 5H), 1.32-1.22 (m, 3H), 0.91-0.77 (m, 9H) ppm. 19F NMR (376 MHz, DMSO-d6) δ -73, -111 ppm. [00452] LP18 [00453] (9H-fluoren-9-yl)methyl N-[(5S)-5-azido-5-{[({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (16-5e)
[00454] Following a similar procedure for 17-5d except starting from N3-Lys(Fmoc) instead of N-Fmoc-Lys(N3)-OH, compound 16-5e (0.15 g, 85% yield from exatecan) was obtained as a white solid. ESI m/z: 899.4 (M + H)+. [00455] (9H-fluoren-9-yl)methyl N-[(5S)-5-amino-5-{[({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamate (16-5ea) [00456] Following a similar procedure as 16-5da except starting from 16-5e instead of starting from 16-5d, compound 16-5ea (0.12 g.67% yield) was obtained as a yellow solid. ESI m/z: 873.2 (M + H)+. [00457] (4S)-4-{[(1S)-1-{[(1S)-1-({4-[({[(1S)-5-amino-1-{[({[(10S,23S)-10-ethyl-18-fluoro- 10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}pentyl]carbamoyl}oxy)methyl]phenyl}carbamoy l)-4-(carbamoylamino)butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-{1-[2-(cyclooct-2- yn-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}butanoic acid (LP18) [00458] Following a similar procedure for LP17 except using 16-5ea instead of 16-5da, LP18 (35 mg, 41% yield) was obtained as a white solid. ESI m/z: 798.8 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.85 (t, J = 6.8 Hz, 1H), 8.54 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 6.4 Hz, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 10.8 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.70-7.55 (m, 6H), 7.41 (d, J = 7.2 Hz, 1H), 7.31 (s, 1H), 7.26 (d, J = 8.8 Hz, 2H), 6.54 (br s, 1H), 6.10-5.90 (m, 1H), 5.67-5.56 (m, 1H), 5.51-5.34 (m, 3H), 5.20 (s, 2H), 4.99-4.81 (m, 4H), 4.72-4.55 (m, 2H), 4.44-4.31 (m, 2H), 4.29-4.25 (m, 1H), 4.23-4.15 (m, 1H), 4.02 (s, 2H), 3.91-3.83 (m, 2H), 3.80- 3.73 (m, 1H), 3.62-3.56 (m, 3H), 3.52-3.46 (m, 16H), 3.43-3.40 (m, 3H), 3.27-3.20 (m, 3H), 3.19- 3.11 (m, 1H), 3.07-2.90 (m, 2H), 2.80-2.70 (m, 2H), 2.39 (s, 3H), 2.37-2.32 (m, 1H), 2.26-2.15 (m, 5H), 2.10-2.02 (m, 1H), 2.00-1.83 (m, 5H), 1.76-1.66 (m, 3H), 1.63-1.55 (m, 3H), 1.54-1.46 (m, 3H), 0.90-0.79 (m, 9H) ppm. [00459] Synthesis of Linear GGFG Linker-payloads LP2 and LP8 (FIG.8). [00460] LP2 (2S)-2-(2-{2-[2-(Cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)-3- phenylpropanoic acid (8-2a) [00461] To a solution of compound L1E (29 mg, 0.10 mmol) and peptide H-Gly-Gly-Phe-OH (29 mg, 0.10 mmol) in DMF (3.0 mL) was added DIPEA (54 µL, 0.31 mmol) and the reaction mixture was stirred at room temperature for half an hour, which was monitored by LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.01%)) to give compound 8-2a (45 mg, 98% yield) as a white solid. ESI m/z: 444 (M+H)+. [00462] (2S)-2-(2-{2-[2-(Cyclooct-2-yn-1-yloxy)acetamido]acetamido}acetamido)-N- ({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)-3-phenylpropanamide (LP2) [00463] To a yellow solution of compound 8-2a (23 mg, 52 μmol) in dry DMF (4 mL) was added DIPEA (20 mg, 0.16 mmol) and HATU (22 mg, 58 μmol) and the mixture was stirred at room temperature for thirty minutes before the addition of ProDXd (30 mg, 52 μmol). The reaction mixture was stirred at room temperature for two hours until most of the starting materials were consumed according to LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.01%)) to give linker-payload LP2 (3.0 mg, 5% yield, TFA salt) as a white solid. ESI m/z: 1005 (M+H)+. 1H NMR (400 MHz, DMSO-d6) δ 8.70-8.62 (m, 1H), 8.58-8.51 (m, 1H), 8.39-8.31 (m, 1H), 8.21-8.06 (m, 2H), 7.90-7.84 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.28-7.13 (m, 3H), 6.73-6.66 (m, 1H), 6.60-6.52 (m, 1H), 5.64- 5.57 (m, 1H), 5.42 (s, 1H), 5.35-5.30 (m, 1H), 5.21 (s, 1H), 4.63 (d, J = 5.3 Hz, 1H), 4.51-4.42 (m, 1H), 4.34-4.25 (m, 1H), 4.02 (s, 1H), 3.94-3.86 (m, 1H), 3.83-3.64 (m, 4H), 2.42-2.36 (m, 3H), 2.23-2.12 (m, 3H), 2.05-1.94 (m, 3H), 1.92-1.63 (m, 6H), 1.61-1.52 (m, 2H), 1.49-1.33 (m, 3H), 1.23 (s, 8H), 0.92-0.80 (m, 3H) ppm. [00464] LP8 (2S)-2-[2-(2-{2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetamido}acetamido)acetamido]- 3-phenylpropanoic acid (8-2b) [00465] Following the procedure for 8-2a except starting from L3E instead of L1E, compound 8-2b (40 mg, 81% yield) was obtained as a red solid. ESI m/z 492 (M+H)+. [00466] (2S)-N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa- 4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen- 23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)-2-[2-(2-{2-[4-(6-methyl-1,2,4,5- tetrazin-3-yl)phenyl]acetamido}acetamido)acetamido]-3-phenylpropanamide (LP8) [00467] Following the procedure for LP2 except starting from 8-2b instead of 8-2a, linker- payload LP8 (10 mg, 12% yield) was obtained as a red solid. ESI m/z 1053 (M+H)+. [00468] Synthesis of Branch GGFG Linker-payloads LP5 and LP10 (FIG.9). [00469] LP5 (2S)-2-[2-(2-Aminoacetamido)acetamido]-N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)-3- phenylpropanamide (9-1) [00470] To a yellow solution of Fmoc-Gly-Gly-Phe-OH (CAS: 160036-44-2, 50 mg, 0.10 mmol) in dry DMF (1 mL) was added HATU (46 mg, 0.12 mmol), and the mixture was stirred at room temperature for five minutes until the mixture turned clear. To the mixture was then added a solution of ProDXd (TFA salt, 69 mg, 0.10 mmol) and DIPEA (39 mg, 0.30 mmol) in dry DMF (1 mL). The reaction mixture was stirred at room temperature for two hours, which was monitored by LCMS. To the resulting solution was added diethylamine (0.2 mL), and the reaction mixture was stirred at room temperature for half an hour, which was monitored by LCMS. The resulting mixture was separated by reversed phase flash chromatography (0-50% acetonitrile with aq. TFA (0.01%)) to give compound 9-1 (30 mg, 36% yield) as a white solid. ESI m/z: 841 (M+H)+. 1H NMR (400 MHz, DMSO-d6) δ 8.70 (t, J = 5.2 Hz, 1H), 8.54 (dd, J = 6.8, 2.4 Hz, 1H), 8.49 (t, J = 4.4 Hz, 1H), 8.39 (t, J = 4.4 Hz, 1H), 8.35-8.31 (m, 1H), 8.28 (br s, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.32 (s, 1H), 7.27-7.20 (m, 4H), 7.19-7.16 (m, 1H), 6.54 (s, 1H), 5.62-5.58 (m, 1H), 5.42 (s, 2H), 5.21 (s, 2H), 4.65 (d, J = 5.2 Hz, 2H), 4.57-4.50 (m, 1H), 4.03 (s, 2H), 3.89-3.84 (m, 1H), 3.79- 3.68 (m, 3H), 3.58 (s, 2H), 3.25-3.11 (m, 2H), 3.06-3.01 (m, 1H), 2.78-2.72 (m, 1H), 2.39 (s, 3H), 2.23-2.13 (m, 2H), 1.92-1.81 (m, 2H), 1.28-1.24 (m, 1H), 0.87 (t, J = 5.6 Hz,, 3H ) ppm. [00471] (2S)-2-[2-(2-{3-[2-(2-Aminoethoxy)ethoxy]propanamido}acetamido)acetamido]- N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)-3-phenylpropanamide (9-2) [00472] To a solution of compound 9-1 (58 mg, 69 μmol) in DMF (5 mL) was successively added Fmoc-PEG2-acid (CAS: 872679-70-4, 28 mg, 69 µmol), DIPEA (18 mg, 0.14 mmol), and HATU (40 mg, 0.10 mmol) and the reaction mixture was stirred at room temperature for four hours, which was monitored by LCMS. The resulting mixture was separated by prep-HPLC (0- 100% acetonitrile with aq. TFA (0.05%)) to give a white solid (54 mg, ESI m/z: 729.3 (M – MDxd + H)+), which was dissolved in DMF (5 mL). To the solution was added diethylamine (16 mg, 0.22 mmol), and the mixture was stirred at room temperature for two hours until Fmoc was totally removed according to LCMS. The resulting mixture was directly purified by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.01%)) to give compound 9-2 (40 mg, 57% yield) as a white solid. ESI m/z: 1001 (M+H)+, 501 (M/2+H)+. [00473] 1-[2-(Cyclooct-2-yn-1-yloxy)acetamido]-N-(2-{2-[2-({[({[(1S)-1-[({[({[(10S,23S)- 10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]-2- phenylethyl]carbamoyl}methyl)carbamoyl]methyl}carbamoyl)ethoxy]ethoxy}ethyl)-12- {[(2-{2-[2-({[({[(1S)-1-[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo- 8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)- heptaen-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]-2- phenylethyl]carbamoyl}methyl)carbamoyl]methyl}carbamoyl)ethoxy]ethoxy}ethyl)carbam oyl]methyl}-3,6,9-trioxa-12-azatetradecan-14-amide (LP5) [00474] To a mixture of compound 9-2 (40 mg, 40 μmol) in DMF (5 mL) was added compound L9E (16 mg, 20 μmol) and DIPEA (8.0 mg, 62 μmol) and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (0-100% acetonitrile with aq. TFA (0.01%)) to give linker-payload LP5 (12 mg, 24% yield) as a white solid. ESI m/z: 813 (M/3+H)+.1H NMR (400 MHz, DMSO-d6) δ 8.64 (t, J = 6.6 Hz, 2H), 8.52 (d, J = 8.7 Hz, 2H), 8.31 (t, J = 5.9 Hz, 2H), 8.19-8.09 (m, 4H), 8.05-7.98 (m, 3H), 7.77 (d, J = 10.9 Hz, 2H), 7.65-7.56 (m, 2H), 7.31 (s, 2H), 7.29-7.12 (m, 8H), 6.53 (s, 2H), 5.65-5.56 (m, 3H), 5.42 (s, 3H), 5.20-5.15 (m, 3H), 4.70-4.60 (m, 3H), 4.50-4.44 (m, 2H), 4.30-4.22 (m, 2H), 4.02 (s, 3H), 3.86 (d, J = 14.8 Hz, 2H), 3.78-3.66 (m, 9H), 3.63-3.55 (m, 6H), 3.53-3.40 (m, 27H), 3.27-3.19 (m, 6H), 3.15 (s, 4H), 3.09-3.00 (m, 2H), 2.81-2.72 (m, 2H), 2.70-2.63 (m, 2H), 2.42-2.32 (m, 8H), 2.25-2.11 (m, 6H), 2.09-1.97 (m, 3H), 1.94-1.68 (m, 9H), 1.63-1.51 (m, 3H), 1.43-1.34 (m, 2H), 1.29-1.20 (m, 3H), 0.87 (t, J = 7.3 Hz, 6H) ppm.19F NMR (376 MHz, DMSO-d6) δ -111.24 ppm. [00475] LP10 (2S)-N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)-2-(2-{2-[3-(2-{2-[2-(N-{[(2-{2-[2- ({[({[(1S)-1-[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]-2- phenylethyl]carbamoyl}methyl)carbamoyl]methyl}carbamoyl)ethoxy]ethoxy}ethyl)carbam oyl]methyl}-2-[4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl]acetamido)acetamido]ethoxy}ethoxy)propanamido]acetamido}acetamido)-3- phenylpropanamide (LP10) [00476] Following the procedures for LP5 except starting from L10aE (16 mg, 21 μmol) instead of L9E, linker-payload LP10 (8 mg, 17% yield) was obtained as a red solid. ESI m/z 770 (M/3+H)+. [00477] Synthesis of Branch EvcPABC-linker-payloads LP6 and LP11 (FIG.10). [00478] LP6 The synthesis of linker-payload LP6 can be found in WO 2022/015656. Another route is described in FIG.10. [00479] tert-butyl (4S)-4-amino-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2- methylpropyl]carbamoyl}butanoate (10-1) [00480] To a solution of Fmoc-Glu(OtBu)-OH (CAS: 71989-18-9, 1.6 g, 3.8 mmol) in DMF (10 mL) was added HATU (2.9 g, 7.7 mmol) and DIPEA (0.99 g, 7.7 mmol), and the mixture was stirred at room temperature for fifteen minutes before the addition of vcPAB (CAS: 159857-79-1, 1.6 g, 4.2 mmol). The reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was separated by reversed phase flash chromatography (0-100% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give Fmoc-10-1 (1.8 g, ESI m/z 787 (M+H)+) as a white solid, which was dissolved in DCM (10 mL). To the solution was added diethylamine (0.65 g, 8.9 mmol), and the reaction mixture was stirred at room temperature for eighteen hours until Fmoc was totally removed according to LCMS. The volatiles were removed in vacuo and the residue was purified by reversed phase flash chromatography (10-40% acetonitrile with aq. TFA (0.01%)) to give compound 10-1 (1.0 g, 47% yield) as a white solid. ESI m/z 565 (M+H)+. [00481] tert-butyl (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4- (hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2- [2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoate (10-2) [00482] To a solution of Fmoc-PEG2-acid (0.42 g, 1.0 mmol) in DMF (5 mL) was added HATU (0.48 g, 1.3 mmol) and DIPEA (0.27 g, 2.1 mmol), and the reaction mixture was stirred at room temperature for fifteen minutes before the addition of compound 10-1 (0.59 g, 1.0 mmol). The reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture as directly separated by reversed phase flash chromatography (0-100% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give compound 10-2 (0.73 g, 73% yield) as a white solid. ESI m/z 946 (M+H)+. [00483] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[(4- nitrophenoxy)carbonyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2- methylpropyl]carbamoyl}-4-(3-{2-[2-({[(9H-fluoren-9- yl)methoxy]carbonyl}amino)ethoxy]ethoxy}propanamido)butanoic acid (10-3) [00484] To a solution of compound 10-2 (0.50 g, 0.53 mmol) in DMF (5 mL) was added DMAP (0.13 g, 1.1 mmol), DIPEA (0.68 g, 5.3 mmol), and bis(4-nitrophenyl) carbonate (1.6 g, 5.3 mmol) and the reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (40- 80% acetonitrile in water) to give a white solid (0.44 g, ESI m/z 1112 (M+H)+), which was dissolved in acetonitrile (4 mL). To this solution was added a solution of hydrochloride in ethyl acetate (4 N, 4 mL) at 0 °C. The reaction mixture was stirred at 0 °Cfor two hours, which was monitored by LCMS. The volatiles were removed in vacuo at room temperature and the residue was purified by reversed phase flash chromatography (0-100% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give compound 10-3 (0.20 g, 36% yield) as a light yellow solid. ESI m/z 1055 (M+H)+. [00485] (4S)-4-{3-[2-(2-aminoethoxy)ethoxy]propanamido}-4-{[(1S)-1-{[(1S)-4- (carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9- dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamo yl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}butanoic acid (10-4) [00486] To a solution of compound 10-3 (0.11 g, 0.10 mmol) in DMF (2 mL) was added ProDXd (70 mg, 0.12 mmol), HOBt (0.1 to 0.5 equiv), and DIPEA (26 mg, 0.20 mmol). The reaction mixture was stirred at room temperature for an hour, which was monitored by LCMS. The resulting mixture was directly separated by reversed phase flash chromatography (30-75% acetonitrile in water) to give Fmoc-10-4 (0.15 g, ESI m/z 749 (M/2 + H)+) as a yellow solid, which was dissolved in DMF (2 mL). To this solution was added diethylamine (26 mg, 0.35 mmol), and the reaction mixture was stirred at room temperature for two hours, which was monitored by LCMS. The volatiles were removed in vacuo and the residual solution was separated by reversed phase flash chromatography (0-100% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give compound 10-4 (85 mg, 67% yield from 10-2, 56% yield from ProDXd) as a white solid. ESI m/z 637 (M/2 + H)+. [00487] (4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18- fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamo yl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-(3-{3-[2-({2-[2-(2-{[(1S)-1- {[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamo yl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-3- carboxypropyl]carbamoyl}ethoxy)ethoxy]ethyl}carbamoyl)ethoxy]-2-[3-(2-{2-[2-(cyclooct- 2-yn-1- yloxy)acetamido]ethoxy}ethoxy)propanamido]propoxy}propanamido)ethoxy]ethoxy}propa namido)butanoic acid (LP6) [00488] To a solution of compound 10-4 (90 mg, 71 μmol) in DMF (2 mL) was added compound L10bE (24 mg, 27 μmol) and DIPEA (21 mg, 0.16 mmol) and the reaction mixture was stirred at room temperature for two hours, which was monitored by LCMS. The resulting mixture was separated by reversed phase flash chromatography (0-100% acetonitrile with aq. ammonium bicarbonate (10 mM)) to give LP6 (45 mg, 21% yield) as a white solid. ESI m/z 1024 (M/3 + H)+. 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 2H), 8.80 (t, J = 6.5 Hz, 2H), 8.51 (d, J = 8.8 Hz, 2H), 8.19 (d, J = 7.2 Hz, 2H), 8.10 (d, J = 7.8 Hz, 2H), 7.93 (t, J = 5.4 Hz, 2H), 7.76 (s, 1H), 7.78-7.73 (m, 4H), 7.65-7.55 (m, 5H), 7.42 (d, J = 5.6 Hz, 2H), 7.31 (s, 2H), 7.27 (d, J = 8.4 Hz, 4H), 6.54 (s, 2H), 6.01 (br s, 2H), 5.62-5.57 (m, 2H), 5.45-5.36 (m, 8H), 5.26-5.10 (m, 4H), 4.92 (s, 4H), 4.63 (d, J = 6.5 Hz, 4H), 4.41-4.30 (m, 4H), 4.29-4.25 (m, 1H), 4.22-4.15 (m, 2H), 4.02 (s, 4H), 3.89-3.84 (m, 1H), 3.78-3.73 (m, 1H), 3.66-3.54 (m, 17H), 3.50-3.14 (m, 22H), 3.08-2.99 (m, 2H), 2.96-2.92 (m, 2H), 2.38 (s, 8H), 2.36-2.29 (m, 10H), 2.25-2.13 (m, 12H), 1.99-1.94 (m, 2H), 1.90- 1.81 (m, 8H), 1.76-1.67 (m, 6H), 1.61-1.55 (m, 4H), 1.46-1.35 (m, 6H), 0.89-0.81 (m, 22H) ppm. [00489] LP11
(4S)-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10- hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15- diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamo yl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-4-(3-{2-[2-(3-{3-[2-({2-[2-(2-{[(1S)-1- {[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy- 19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0²,¹⁴.0⁴,¹³.0⁶,¹¹.0²⁰,²⁴]tetracosa- 1,6(11),12,14,16,18,20(24)-heptaen-23- yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamoyl]oxy}methyl)phenyl]carbamo yl}butyl]carbamoyl}-2-methylpropyl]carbamoyl}-3- carboxypropyl]carbamoyl}ethoxy)ethoxy]ethyl}carbamoyl)ethoxy]-2-{2-[4-(6-methyl- 1,2,4,5-tetrazin-3- yl)phenyl]acetamido}propoxy}propanamido)ethoxy]ethoxy}propanamido)butanoic acid (LP11) [00490] Following the procedure for LP6 except starting from L10aE (65 mg, 83 μmol) instead of L10bE, linker-payload LP11 (99 mg, 40% yield) was obtained as a red solid. ESI m/z 1479 (M/2 + H)+.1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 2H), 8.80 (t, J = 6.3 Hz, 2H), 8.50 (d, J = 8.6 Hz, 2H), 8.39 (d, J = 8.2 Hz, 2H), 8.20-8.12 (m, 3H), 8.08 (d, J = 7.8 Hz, 2H), 7.94 (t, J = 5.5 Hz, 2H), 7.77 (t, J = 10.1 Hz, 4H), 7.61-7.49 (m, 6H), 7.43 (t, J = 5.9 Hz, 2H), 7.32-7.21 (m, 6H), 6.53 (s, 2H), 5.98 (t, J = 5.5 Hz, 2H), 5.63-5.56 (m, 2H), 5.48-5.35 (m, 8H), 5.19 (s, 4H), 4.92 (s, 4H), 4.62 (d, J = 6.5 Hz, 4H), 4.41-4.30 (m, 4H), 4.21-4.16 (m, 2H), 4.01 (s, 4H), 3.98-3.92 (m, 1H), 3.66-3.52 (m, 16H), 3.47 (s, 12H), 3.21-3.17 (m, 6H), 2.98 (s, 3H), 2.26-2.14 (m, 9H), 2.08 (s, 9H), 2.03-1.79 (m, 12H), 1.77-1.52 (m, 9H), 1.50-1.34 (m, 6H), 1.24 (s, 6H), 0.88-0.80 (m, 18H) ppm. [00491] Conjugations [00492] Generic Procedures for Making Site-specific Conjugates [00493] Aglycosylated human antibody IgG (IgG1, IgG4, etc.) containing an N297Q or N297D mutation were used in ADC conjugations. Two Approaches (I & II) were conducted via a two- step process (FIG. 11), and the conjugation results with the structures and MS-DAR values are summarized in Table 7. [00494] Step 1: Site-specific Conjugation of Handle-functionalized Amine With an Antibody Generated Drug Conjugate Containing Two, Four, or Eight Handles per Antibody [00495] Aglycosylated human antibody IgG containing an N297Q mutation or an N297D mutation in BupH buffer (pH 7.4) was mixed with ≥ 100 molar equivalents of non-branched Handle-amine or branched Handle-amine (AL). The resulting solution was mixed with transglutaminase (350 U/mL; 1 U mTG per mg of antibody, SLCK1576, Sigma; or 25 U/mL; 1 U mTG per mg of antibody, Zedira, Darmstadt, Germany; or 10 U/mL; 0.06 mg mTG per mg of antibody, Modernist Pantry-ACTIVA TI contains Maltodextrin from Ajinomoto, Japan) resulting in a final concentration of the antibody at 0.5-20 mg/mL. The reaction mixture was incubated at 25-37 °C for twenty-four hours while gently shaking and monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC) or protein A column chromatography. The conjugate was characterized by UV-Vis, SEC, and ESI- MS. [00496] Step 2: Click Reactions Between Handle-functionalized Antibodies and a Linker- Payload in Table 2 to Generate the Site-specific ADCs [00497] The Handle-functionalized antibody (Ab-(AL)n, 1-20 mg/mL) in PBS (pH 7.4) was incubated with ≥2-10 molar equivalents of a linker-payload (LP) dissolved in an organic solvent such as DMSO or DMA (10 mg/mL) to have the overall reaction mixture containing 5-15% organic solvent (v/v), at 25-37 °C for 1-48 hours while gently shaking. The reaction was monitored by ESI-MS. Upon reaction completion, the excess amount of LP and organic solvent were removed via a desalting column with BupH (pH 7.4), and protein aggregates (if any) were removed by size exclusion chromatography (SEC). The purified conjugate, Ab-(AL-LP)n ADC or Ab-(AL- (LP)2)4 ADC, was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. Conjugate monomer purity was >95% by SEC. [00498] All ADCs were purified by SEC using an ÄKTA instrument from Cytiva, using a 16/600 Superdex® 200 column, eluting with DPBS, at a flow rate of 1.0 mL/min at pH 7.4. The DAR values of the ADCs were measured by ESI-MS. A mass increase of 4 x AL-LP (i.e., AL6- LP1) from Ab to Ab-[AL-LP]4 was observed, correlating to 4DAR ADC. Shown in FIG. 12, a mass increase of 4 x AL-(LP)2 from Ab to Ab-[AL-(LP)2]4 was observed: where AL is a branched Handle and clicked with 2x LP (e.g., LP1), indicating an 8DAR ADC was generated (i.e., Ab- [AL11-(LP1)2]4); where AL is a branched Handle and LP is a branched linker-payload, indicating an ~16DAR ADC was generated after Ab-(AL)n was clicked with an LP (i.e., Ab-[AL11- (LP6)2]4). See examples of 4DAR, 8DAR, and 16DAR ADC conjugations in FIG.12. [00499] Detailed Conjugation Procedure [00500] Conjugation of 4DAR Ab-(AL7-LP4)n (here n = 4) [00501] Representative 4DAR ADC from Approach I. An aglycosylated anti-Her2 human IgG antibody containing an N297Q mutation was mixed with 100 molar equivalents of a HCl salt of (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine (AL7, MW 237.7 g/moL). The resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 11.5 mg/mL. The reaction mixture was incubated at 32 °C for one hour while gently shaking and was monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS. The tetrazine-modified antibody resulted in a 745Da mass increase compared to mAb, indicating four AL7 were conjugated to the antibody (Ab- (AL7)4) with four tetrazine handles. The site-specific antibody tetrazine conjugate (11.5 mg/mL) in PBS (pH 7.4) was mixed with 5.1 molar equivalents of linker-payload (LP4) in 0.057 mM of DMA to result in a reaction mixture containing 11% organic solvent (v/v), and the solution was set at 25 °C for two hours while gently shaking. The reaction was monitored by ESI-MS. Upon reaction completion, the excess amount of linker-payload and protein aggregates were removed by size exclusion chromatography (SEC). The purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. Conjugate monomer purity was 99.8% by SEC. The ADC resulted in a 5475 Da mass increase for the 4DAR conjugate. Conjugate monomer purity was >99% by SEC. [00502] Conjugation of 8DAR Ab-(AL7-LP6)n (here n = 4) [00503] Representative 8DAR ADC from Approach I. An aglycosylated anti-Her2 human IgG antibody containing an N297Q mutation was mixed with 100 molar equivalents of a HCl salt of (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine (AL7, MW 237.7 g/moL). The resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 11.5 mg/mL. The reaction mixture was incubated at 32 °C for one hour while gently shaking and monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS. The tetrazine-modified antibody resulted in a 745Da mass increase compared to mAb, indicating four AL7 were conjugated to the antibody (Ab-(AL7)4) with four tetrazine handles. The site-specific antibody tetrazine conjugate (11.5 mg/mL) in PBS (pH 7.4) was mixed with 7.7 molar equivalents of linker-payload (LP6) in 0.057 mM of DMA resulting in the reaction mixture containing 11 % organic solvent (v/v), and the solution was set at 25 °C for two hours while gently shaking. The reaction was monitored by ESI- MS. Upon reaction completion, the excess amount of linker-payload and protein aggregates were removed by size exclusion chromatography (SEC). The purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. Conjugate monomer purity was 99.8% by SEC. The ADC resulted in a 12164 Da mass increase for the DAR7.5 conjugate. Conjugate monomer purity was >99% by SEC. [00504] A representative 8DAR ADC from Approach I. An aglycosylated anti-Her2 human IgG antibody containing an N297Q mutation was mixed with 100 molar equivalents of a TFA salt of 14-amino-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3-(2-((4-(6-methyl-1,2,4,5-tetrazin-3- yl)benzyl)amino)-2-oxoethyl)-6,9,12-trioxa-3-azatetradecanamide (AL11, MW 788.8 g/moL). The resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 9.2 mg/mL. The reaction mixture was incubated at 37 °C for seven hours while gently shaking and monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS. The tetrazine-modified antibody resulted in a 2626Da mass increase compared to mAb, indicating four AL11 were conjugated to the antibody (Ab-(AL11)4) with eight tetrazine handles. The site-specific antibody tetrazine conjugate (8.56 mg/mL) in PBS (pH 7.4) was mixed with twelve molar equivalents of linker-payload (LP6) in 7.2 mM of DMA resulting in the reaction mixture containing 8.2 % organic solvent (v/v), and the solution was set at 37 °C for twenty-one hours while gently shaking. The reaction was monitored by ESI-MS. Upon reaction completion, the excess amount of linker-payload and protein aggregates were removed by size exclusion chromatography (SEC). The purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. Conjugate monomer purity was 99.8% by SEC. The ADC resulted in average 10959 Da mass increase for the 8DAR conjugate. [00505] Conjugation of 8DAR Ab-(AL11-(LP4)2)4 [00506] Representative 8DAR ADC from Approach II. An aglycosylated anti-Her2 human IgG antibody containing an N297Q mutation was mixed with 100 molar equivalents of a TFA salt of 14-amino-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3-(2-((4-(6-methyl-1,2,4,5-tetrazin-3- yl)benzyl)amino)-2-oxoethyl)-6,9,12-trioxa-3-azatetradecanamide (AL11, MW 674.77 g/moL for its free base). The resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 9.2 mg/mL. The reaction mixture was incubated at 37 °C for seven hours while gently shaking and monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS. The tetrazine-modified antibody resulted in a 2626Da mass increase compared to mAb, indicating four AL11 were conjugated to the antibody (Ab-(AL11)4) with eight tetrazine handles. The site-specific antibody tetrazine conjugate (8.56 mg/mL) in PBS (pH 7.4) was mixed with twelve molar equivalents of linker- payload (LP4) in 7.2 mM of DMA resulting in the reaction mixture containing 8.2% organic solvent (v/v), and the solution was set at 37 °C for twenty-one hours while gently shaking. The reaction was monitored by ESI-MS. Upon reaction completion, the excess amount of linker- payload and protein aggregates were removed by size exclusion chromatography (SEC). The purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI- MS. Conjugate monomer purity was 99.8% by SEC. The ADC resulted in a 10959 Da mass increase for the 7.75DAR conjugate. [00507] Conjugations of 16 DAR Ab-(AL11-(LP6)2)4 [00508] Representative 8DAR ADC from Approach I. An aglycosylated anti-Her2 human IgG antibody containing an N297Q mutation was mixed with 100 molar equivalents of 14-amino-N- (4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3-(2-((4-(6-methyl-1,2,4,5-tetrazin-3- yl)benzyl)amino)-2-oxoethyl)-6,9,12-trioxa-3-azatetradecanamide (AL11, MW 674.77 g/moL). The resulting solution was mixed with microbial transglutaminase (350 U/mL; Sigma) resulting in a final concentration of the antibody at 10 mg/mL. The reaction mixture was incubated at 37 °C for seven hours while gently shaking and monitored by ESI-MS. Upon reaction completion, the excess amine and mTG were removed by size exclusion chromatography (SEC). The conjugate was characterized by UV-Vis, SEC, and ESI-MS. The tetrazine-modified antibody resulted in a 2626Da mass increase compared to mAb, indicating four AL11 were conjugated to the antibody (Ab-(AL11)4) with eight tetrazine handles. The site-specific antibody tetrazine conjugate (8.56 mg/mL) in PBS (pH 7.4) was mixed with twelve molar equivalents of linker-payload (LP6) in 0.0065 mM of DMA resulting in the reaction mixture containing 8.6% organic solvent (v/v), and the solution was set at 37 °C for four hours while gently shaking. The reaction was monitored by ESI-MS. Upon completion, the excess amount of linker-payload and protein aggregates were removed by size exclusion chromatography (SEC). The purified conjugate was concentrated, sterile filtered, and characterized by UV-Vis, SEC, and ESI-MS. Conjugate monomer purity was 99.8% by SEC. The ADC resulted in an average 24338 Da mass increase for the 15DAR conjugate. Conjugate monomer purity was > 99% by SEC. [00509] SDS-PAGE for Analysis of ADC Integrity and Purity [00510] In one method, SDS-PAGE conditions included non-reduced and reduced samples (1- 2 µg) along with Precision Plus Protein Dual Color Standards (Bio-Rad, 500 µL, Cat# 1610374) loaded per lane in (1.0 mm × 10 well) Novex 4-20% No Tris-Glycine Gel and and run at 180V, 300 mA, for eighty minutes. A non-reduced sample was prepared using NuPAGE® LDS Sample Buffer (4X) (Thermo Fisher Scientific, Cat#1887691) and the reduced sample was prepared with SDS sample buffer (4X) containing 10% sample reducing agent (10X) (Thermo Fisher Scientific, Cat#1769410). [00511] Molecular weights of the antibodies and ADCs on SDS-PAGE were determined under non-reducing and reducing conditions. The mass shifts may not be obvious under non-reducing conditions due to relatively small percentages of mass changes. However, the masses of the heavy chains are increased from the naked antibodies to the azido-functionalized antibodies, and further to the ADC conjugates. [00512] Size Exclusion Chromatography (SEC) for ADC Analysis and Purification [00513] To determine the purity of antibody-drug conjugates, size exclusion chromatography was performed. Analytical SEC experiments were run using a Thermo UltiMate™ 3000 instrument, on a XBridge Protein BEH SEC Column (Waters, 200 Å, 3.5 µm, 7.8 mm X 300 mm), and each sample (30-40 µg, 20 µL) was run at a flow rate of 0.5 mL/min using PBS pH 7.4 with 15% 2-propanol, and monitored at λ 280 nm using Thermo DAD-3000 RS Rapid Separation Diode Array Detector. [00514] ADCs were purified by SEC and concentrated by using ultra centrifugation. To separate the antibody-drug conjugates from the reaction mixture, preparative SEC purifications were performed using the ÄKTA instrument from GE Healthcare, on a Superdex® 200 increase 10/300 GL (1.0 × 30cm) column, at a flow rate of 0.6 mL/min eluting with BupH at pH 7.4, and monitored at λ 280 nm. To concentrate the product Amicon ® Ultra-4 Centrifugal Filters (Ultracel-10K) were used in an Allegra x-12r centrifuge, and the solution was stirred after each concentration to avoid high aggregation. [00515] Plasma Stability [00516] Both aHer2-(AL7-LP4)n and aHer2-(AL7-LP6)n are stable in all plasma for fourteen days.
aHer2-(AL7-LP6)n Time Human Monkey Mouse Rat (Days) DAR DAR DAR DAR 0 7.410 7.512 7.385 7.409 1 7.410 7.445 7.398 7.447 2 7.396 7.423 7.421 7.449 3 7.413 7.438 7.469 7.425 7 7.458 7.437 7.335 7.421 14 7.357 7.460 7.118 7.379 aHer2-(AL7-LP4)n Time Human Monkey Mouse Rat (Days) DAR DAR DAR DAR 0 3.821 3.820 3.801 3.810 1 3.808 3.810 3.809 3.814 2 3.805 3.806 3.803 3.807 3 3.817 3.804 3.809 3.810 7 3.795 3.797 3.773 3.801 14 3.752 3.683 3.730 3.782 [00517] Cell Viability Assay [00518] Cell Types and Growth Media: • SK-BR-3, human breast cancer cell line, ACL1657. Her2 is highly expressed in this cell line. o Growth media: McCoy’s, 10% FBS, 100 units/mL Penicillin, 100 µg/mL Streptomycin, 50 µg/mL glutamine. • NCI-N87, human gastric carcinoma cell line, ACL6581. Her2 is highly expressed in this cell line. o Growth media: RPMI, 10% FBS, 100 units/mL Penicillin, 100 µg/mL Streptomycin, 50 µg/mL glutamine. • Calu-3, human lung adenocarcinoma cell line, ACL15056. Her2 is highly expressed in this cell line. o Growth media: MEM, 10% FBS, 100 units/mL Penicillin, 100 µg/mL Streptomycin, 50 µg/mL glutamine, 1 mM sodium pyruvate, 100 µM non-essential amino acids. • JIMT-1, human breast ductal carcinoma, ACL14141. Her2 is moderately expressed in this cell line. o Growth media: DME, 100 units/mL Penicillin, 100 µg/mL Streptomycin, 50 µg/mL glutamine. • NCI-H1975, human lung adenocarcinoma, non-small cell lung cancer, ACL6573. This line expresses Her2 at very low level and serves as a negative cell line. o Growth media: RPMI, 10% FBS, 100 units/mL Penicillin, 100 µg/mL Streptomycin, 50 µg/mL glutamine. [00519] An in vitro cytotoxicity assay was performed to test the potency of Her2-DXd ADCs, described herein and conjugated by an IEDDA method, in killing human cell lines. In vitro cytotoxicity of the ADCs described herein as well as reference ADCs and compounds were evaluated using the CellTiter-Glo Assay Kit (Promega, Cat# G9243), in which the quantity of ATP present was used to determine the number of viable cells in culture. For the assay, the SK-BR-3 (1000/well), NCI-N87 (1000/well), Calu-3 (1000/well), JIMT-1 (1000/well), and NCI-H1975 (800/well) were seeded into white 96-well plates (ThermoFisher, #136101) in complete growth medium and grown overnight at 37 °C in 5% in CO2 to allow the cells attach to the bottom of the plates. To obtain the dose-response curves, ADCs described herein and control ADCs were serially diluted at 1:4 starting from 400 nM in assay media (Opt-MEM+0.1% BSA) for ten points, leaving the last point as blank. 20 µL diluted ADCs were added to the cells, yielding serial final concentrations of 400 nM, 100 nM, 25 nM, …6.1 pM and zero. Free payloads were the first ten points serially diluted starting from 50 µM in 100% DMSO at 1:4, leaving the last well as blank (i.e., containing only DMSO). 10 µL of DMSO-diluted compounds were transferred to 990 µL assay media (Opti-MEM, 0.1% BSA) and mixed well. 20 µL media-diluted payloads were transferred to cells, yielding serial final concentrations of 100 nM, 25 nM, 6.25 nM…1.5 pM and zero. [00520] After the 6-day incubation at 37 °C in 5% CO2, cells were incubated at room temperature with 100 µL of CellTiter-Glo reagents for ten minutes. Relative luminescence units (RLU) were determined on an Envision plate reader (PerkinElmer). The IC50 values were determined from a four-parameter logistic equation over a 10-point response curve (GraphPad Prism). All IC50 values are expressed in molar (M) concentration. The % viable cells remaining after ADC/payload treatment is expressed as a percentage of the untreated well (% viable cells). Averages ^ standard deviation (SD) are included where replicate experiments were performed. [00521] As shown in Table 9 and FIG. 14, ADCs described herein with amino-dienophile linkers demonstrated potent killing of Her2 positive SK-BR-3, NCI-N87, and Calu-3 cells with IC50 values between 0.0015 nM and 0.013 nM. The potency differences between ADCs and their corresponding isotype controls were >1000 fold, so the killing is highly target specific. Notably, ADC (26 or 27) derived from the same payload MMAE and same linker and conjugated via cysteine is 7-25 fold less potent than the ADCs described herein with amino-dienophile linkers and conjugated to glutamine. All the ADCs described herein and the reference(s) weakly killed the moderate Her2 expression in JIMT-1 cells. Table 9. Potency of ADCs With Amino-dienophile Linkers Antigen SK-BR-3 NCI-N87 Calu-3 JIMT-1 NCI-H1975 (Antibody ill 8 1 7 6 5 7 [00522] ADCs described herein with amino tetrazine linkers in comparison with the azido PEG3-amine linker are summarized in Table 10. Both AL7 and AL6 amino-tetrazine linkers support single chain linkers and branched linkers. When AL7 and AL6 were conjugated with a single chain linker payload, the DAR values were comparable to those ADCs conjugated via the azido-PEG3-amine linker (e.g., see AL7-LP4, AL6-LP4). When conjugated with a branched linker-payload, the DAR values can get close to the maximum capacity of eight (e.g., see AL7- LP6, AL6-LP6, AL6-LP5). The payloads were efficiently released from ADCs conjugated via amino-tetrazine linkers and their potencies were similar to those ADCs conjugated via the azido- PEG3-amine linkers via click chemistry (FIG.15, FIG.16, FIG.17).
Table 10. Potency of ADCs With Amino-Tetrazine Linkers SKBR3 NCIN87 Calu3 JIMT1 NCIH1975 [00523] Among the amino-tetrazine linkers, the efficiency of payload release from conjugates via AL11 was similar in SK-BR-3 and Calu-3 cells, and therefore, the cell killing potency was similar. But in NCI-N87 cells, ADCs with the AL11 linker worked better than AL6 and AL7, and the AL11 ADCs were apparently more potent than others in killing N87 cells (FIG. 18). In addition, many reported studies state that high DAR ADCs usually result in aggregates that cause solubility issues. A 15DAR ADC was made using the AL11 linker and aggregation was not observed. In the cell killing assay, the 15DAR ADC worked slightly better than DAR8 ADCs made from all amino-tetrazine linkers herein (Table 2 and FIG.19). [00524] The embodiments and examples described above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of this patent application and are encompassed by the scope of the appended claims.

Claims

WHAT IS CLAIMED IS
1. A compound of the following formula
(R1)(R2)N-W-X-Y-Z wherein
W is C1-C10 alkylene or C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -N(R3)C(0)- C1-C10 alkylene, -C(O)N(R3)- C1-C10 alkylene, -C(0)N(R3)-C1-C10 alkylene-C(O)-(AA)P-N(R3)-, -C(O)-(AA)P-N(R3)-, -C(O)N(R3)-, -N(R5)2, -C(O)N(R5)2, -CH(R5)(R6), -C(0)-(AA)P-N(R3)- C1-C10 alkylene-PEGn-C1-C10 alkylene-N(R5)2, -C(O)-(AA)P-N(R5)2, -C(O)-(AA)P-N(R3)-CH(R5)(R6), or -N(R5)(R6);
Y when present is C1-C10 alkylene;
Z is selected from the group consisting of
R1 and R2 are independently hydrogen, C1-C10 alkyl, or -C(O)-C1-C10 alkyl-COOH;
R3 and R4 are independently hydrogen or C1-C10 alkyl;
R5 is -Y-C(O)N(R3)-Y-Z, -Y-(R3)NC(O)-Y-Z, -(R3)NC(O)-Y-C(O)N(R3)-Y-Z, or -C(O)N(R3)-Y-Z;
R6 is -Y-C(0)N(R3)-PEGn1-C1-C10 alkylene-N3, -C(0)N(R3)-PEGn1-C1-C10 alkylene-N3, or -C1-C10 alkylene-N3;
AA is an amino acid residue and p is an integer from one to twenty; wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -N(R3)C(0)-C1-C10 alkylene; and
Z is , then n is an integer from two to one hundred; wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene; X is -C(O)N(R3)-;
Y is C1-C10 alkylene; and
Z is then n is an integer from one to three, five to seven, or nine to one hundred; wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -C(0)N(R3)-C1-C10 alkylene; and
Z is then n is an integer from one to one hundred; wherein when
W is C1-C10 alkyl ene-PEGn-Ci-C 10 alkylene;
X is -C(O)N(R5)2; and
Z is then n is an integer from one to one hundred; and nl is an integer from zero to one hundred.
2. The compound of claim 1, having the following formula
(R1)(R2)N-W-X-Y-Z wherein
W is C1-C10 alkylene or C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -N(R3)C(0)-C1-C10 alkylene, -C(0)N(R3)-C1-C10 alkylene, -C(O)N(R3)-, -N(R5)2, or -N(R5)(R6);
Y when present is C1-C10 alkylene; Z is selected from the group consisting of
R1 and R2 are independently hydrogen, C1-C10 alkyl, or -C(0)-C1-C10 alkyl-COOH;
R3 and R4 are independently hydrogen or C1-C10 alkyl;
R5 is -Y-C(O)N(R3)-Y-Z;
R6 is -Y-C(O)N(R3)-PEGn1-C1-C10 alkylene-N3; wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -N(R3)C(0)-C1-C10 alkylene; and
Z is then n is an integer from two to one hundred; wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -C(O)N(R3)-;
Y is C1-C10 alkylene; and
Z is then n is an integer from one to three, five to seven, or nine to one hundred; wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -C(0)N(R3)-C1-C10 alkylene; and Z is then n is an integer from one to one hundred; and nl is an integer from zero to one hundred.
3. The compound of claim 2, wherein R1 and R2 are hydrogen.
4. The compound of claim 3, wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -N(R3)C(0)-C1-C10 alkylene; and
Z is then n is two, three, eight, or twelve.
5. The compound of claim 4, wherein the compound is selected from the group consisting of
6. The compound of claim 3, wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -C(O)N(R3)-;
Y is C1-C10 alkylene; and Z is then n is an integer from one to three, five to seven, or nine to one hundred.
7. The compound of claim 6, wherein the compound is selected from the group consisting of
8. The compound of claim 3, wherein
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -N(R5)2 or -N(R5)(R6);
R5 is -Y-C(O)N(R3)-Y-Z; and
R6 is -Y-C(O)N(R3)-PEGn1-C1-C10 alkylene-N3.
9. The compound of claim 8, wherein the compound is
ALII
10. The compound of claim 8, wherein the compound is 3
AL12
11. The compound of claim 1, wherein
R1 and R2 are hydrogen;
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -C(O)N(R5)2;
R5 is -Y-(R3)NC(0)-Y-Z;
Z is and n is an integer from one to ten.
12. The compound of claim 11, wherein the compound is AL13
13. The compound of claim 1, wherein R1 and R2 are hydrogen;
W is C1-C10 alkylene;
X is -C(0)N(R3)-C1-C10 alkylene-C(O)-(AA)P-N(R3)- or -C(O)-(AA)p-N(R3)-; and
Z is
14. The compound of claim 13, wherein the compound is selected from the group consisting
15. The compound of claim 1, wherein
R1 and R2 are hydrogen;
W is C1-C10 alkylene;
X is -CH(R5)(R6);
R5 is -(R3)NC(O)-Y-C(O)N(R3)-Y-Z;
R6 is -C(0)N(R3)-PEGn1-C1-C10 alkylene-Ns; and Z is
16. The compound of claim 15, wherein the compound is 513
17. The compound of claim 1, wherein
R1 and R2 are hydrogen;
W is C1-C10 alkylene;
X is -C(0)-(AA)P-N(R3)-C1-C10 alkylene-PEGn-C1-C10 alkylene-N(R5)2;
R5 is -Y-C(O)N(R3)-Y-Z; and
18. The compound of claim 17, wherein the compound is
AL15
19. The compound of claim 1, wherein
R1 and R2 are hydrogen;
W is C1-C10 alkylene;
X is -C(0)-(AA)P-N(R3)-; and Z is
20. The compound of claim 19, wherein the compound is
473.
21. The compound of claim 1, wherein
R1 and R2 are hydrogen;
W is C1-C10 alkylene;
X is -C(O)-(AA)P-N(R5)2;
R5 is -Y-C(O)N(R3)-Y-Z; and
Z is
22. The compound of claim 21, wherein the compound is
H2
AL16
23. The compound of claim 1, wherein
R1 and R2 are hydrogen;
W is C1-C10 alkylene;
X is -C(O)-(AA)P-N(R3)-CH(R5)(R6); and Z is
24. The compound of claim 23, wherein the compound is
AL17
25. The compound of claim 2, wherein R1 is hydrogen and R2 is -C(0)-C1-C10 alkyl-COOH.
26. The compound of claim 25, wherein R1 is hydrogen and R2 is -C(O)CH2CH2COOH.
27. The compound of claim 25 or 26, wherein when
W is C1-C10 alkylene-PEGn-C1-C10 alkylene;
X is -C(0)N(R3)-C1-C10 alkylene; and
Z is then n is an integer from one to one hundred.
28. The compound of claim 27, wherein n is four, eight, or twelve.
29. The compound of claim 28, selected from the group consisting of
L5a; L5b; and
L5c
30. A compound having the following chemical structure
L10aE
31. A compound selected from the group consisting of LP7;
LP15; F
LP16; and
LP17
32. A compound having the following structure or a pharmaceutically acceptable salt thereof, wherein L is hydrogen or a linker comprising a moiety reactive with a binding agent;
X is wherein T is part of the linker and comprises a moiety reactive with a binding agent;
SP is a spacer group; R1a and R1b are, independently, hydrogen or alkyl;
R2 is hydrogen or an amino acid side chain;
R3, R4, and R6 is hydrogen or alkyl;
R5 is oxygen, NR6, or sulfur;
R7 is an 0-amino acid residue;
R8 is C1-C10 alkylene or C1-C10 heteroalkylene;
D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein when m is four, then the moiety reactive with the binding agent is and wherein when n is four, then the moiety reactive with the binding agent is with the proviso that the compound of Formula I is not
GlyGlyDxd.
33. The compound of claim 32, having the following structure
(II) or a pharmaceutically acceptable salt thereof, wherein L is a linker comprising a moiety reactive with a binding agent;
X is wherein T is part of the linker and comprises a moiety reactive with a binding agent;
SP is a spacer group;
R1a and R1b are, independently, hydrogen or alkyl;
R2 is hydrogen or an amino acid side chain;
R3, R4, and R6 is hydrogen or alkyl;
R5 is oxygen, NR6, or sulfur;
R7 is an 0-amino acid residue;
R8 is C1-C10 alkylene or C1-C10 heteroalkylene;
D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; m is zero, one, two, three, four, five, or six; and each n is zero, one, two, three, four, five, or six; wherein when m is four, then the moiety reactive with the binding agent is and wherein when n is four, then the moiety reactive with the binding agent is
34. The compound of claim 32, wherein
L is hydrogen;
R1a and R1b are hydrogen;
R2 is hydrogen or an amino acid side chain;
R3, R4, and R6 is hydrogen or alkyl;
R5 is NR6;
R7 is an 0-amino acid residue;
D* is a residue of a biologically active compound comprising hydroxyl, amino, or thiol; and m is zero, one, two, three, four, five, or six.
35. The compound of claim 34, wherein the compound is selected from the group consisting of SerDXd;
GlnDXd;
GlyNMeCH2DXd
36. The compound of claim 33, wherein the compound is or a pharmaceutically acceptable salt thereof, wherein
X is
SP1, SP2, and SP3, when present, are in each instance independent spacer groups wherein SP1 further comprises the moiety reactive with the binding agent; each AA is an amino acid; and p is an integer from zero to ten.
37. The compound of claim 36, wherein SP1 comprises a reactive group that comprises an alkene, alkyne,
38. The compound of claim 31 or 37, wherein the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction.
39. The compound of claim 38, wherein the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction to form regioisomeric Diels- Alder adduct moieties, wherein the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent.
40. The compound of claim 36, wherein SP3 comprise
41. The compound of claim 36, wherein SP3 comprise and R2 is hydrogen or benzyl.
42. The compound of claim 39, wherein the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction to form regioisomeric Diels- Alder adduct moieties, the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent, and SP3 comprise
43. The compound of claim 39, wherein the alkene, alkyne, and/or tetrazine are capable of participating in an inverse electron demand Diels-Alder reaction to form regioisomeric Diels- Alder adduct moieties, the alkene and/or tetrazine comprise an alkene or tetrazine functionalized binding agent, and SP3 comprises , and R2 is hydrogen or benzyl.
44. The compound of claim 36, having the following formula (III) wherein
R1a, R1b, R2, and R3 are hydrogen; and m is one.
45. The compound of claim 36, having the following formula *
(Ill) wherein
R1a, R1b, and R3 are hydrogen;
R2 is an amino acid side chain; and m is one.
46. The compound of claim 36, having the following formula (III) wherein
R1a, R1b, R2, and R3 are hydrogen;
R4 is alkyl; and m is one.
47. The compound of claim 44, wherein
R1a, R1b, R2, and R3 are hydrogen; and m is two or four.
48. The compound of claim 36, having the following formula (IV) wherein
R1a, R1b, and R3 are hydrogen; R2 is hydrogen or benzyl; and n is one or four.
49. The compound of any one of claims 44-48, wherein D* is a residue of a biologically active compound comprising amino.
50. The compound of claim 49, selected from the group consisting of
LP3; ;
LP19; and
LP20
51. The compound of claim 49, wherein the compound is
LP21; or
LP8
52. The compound of claim 49, wherein the compound is
LP22
53. The compound of claim 49, selected from the group consisting of LP10; and LPll
54. The compound of any one of claims 33-53, wherein the binding agent is an antibody or antigen binding fragment thereof.
55. A compound wherein the compound is a binding agent or transglutaminase-modified binding agent comprising a compound of claim 1.
56. The compound of claim 55, wherein the binding agent further comprises a compound of claim 31.
57. The compound of claim 55 or 56, wherein the compound is
and wherein BA is the binding agent.
58. The compound of any one of claims 55-57, wherein the binding agent is an antibody or transglutaminase-modified antibody, or antigen binding fragments thereof.
59. The compound of claim 58, wherein the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof are selected from an anti-HER2 antibody, an anti-STEAP2 antibody, an anti -MET antibody, an anti-EGFRvIII antibody, an anti-MUC16 antibody, an antiprolactin receptor (PRLR) antibody, an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-FGFR2 antibody, an anti-FOLRl antibody, an anti-HER2/HER2 bispecific antibody, an anti-MET/MET bispecific antibody, or an antigen-binding fragment thereof.
60. A conjugate comprising the compound of any preceding claim.
61. The conjugate of claim 60, selected from the group consisting of
ADC10;
ADC11;
ADC25;
wherein BA is a binding agent.
62. The conjugate of claim 60 or 61, wherein the binding agent is an antibody or transglutaminase-modified antibody, or antigen binding fragments thereof.
63. The conjugate of claim 62, wherein the antibody or transglutaminase-modified antibody, or antigen binding fragments thereof are selected from an anti-HER2 antibody, an anti-STEAP2 antibody, an anti -MET antibody, an anti-EGFRvIII antibody, an anti-MUC16 antibody, an anti- prolactin receptor (PRLR) antibody, an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-FGFR2 antibody, an anti-FOLRl antibody, an anti-HER2/HER2 bispecific antibody, an anti-MET/MET bispecific antibody, or an antigen-binding fragment thereof.
64. A method of making a conjugate comprising the steps of
(a) treating a binding agent with H2N-PEGn -N3 in the presence of microbial transglutaminase to provide a transglutaminase-modified binding agent; (b) treating the transglutaminase-modified binding agent with a compound of claim
31; and
(c) purifying the conjugate, wherein n is an integer from one to one hundred.
65. The method of claim 64, wherein the compound of claim 31 is
LP1
66. A method of making a conjugate comprising the steps of
(a) treating a binding agent with a compound of claim 1 in the presence of microbial transglutaminase to provide a transglutaminase-modified binding agent;
(b) treating the transglutaminase-modified binding agent with a complementary compound of any one of claims 31 or 50-53; and
(c) purifying the conjugate.
67. The method of claim 66, wherein the compound of any one of claims 31 or 50-53 is selected from the group consisting of LP3;
EP24714268.0A 2023-02-09 2024-02-08 Antibody-drug conjugates via inverse electron demand diels-alder reactions Pending EP4661914A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363484136P 2023-02-09 2023-02-09
PCT/US2024/015073 WO2024168199A1 (en) 2023-02-09 2024-02-08 Antibody-drug conjugates via inverse electron demand diels-alder reactions

Publications (1)

Publication Number Publication Date
EP4661914A1 true EP4661914A1 (en) 2025-12-17

Family

ID=90473326

Family Applications (1)

Application Number Title Priority Date Filing Date
EP24714268.0A Pending EP4661914A1 (en) 2023-02-09 2024-02-08 Antibody-drug conjugates via inverse electron demand diels-alder reactions

Country Status (3)

Country Link
EP (1) EP4661914A1 (en)
CN (1) CN121001751A (en)
WO (1) WO2024168199A1 (en)

Family Cites Families (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US5714586A (en) 1995-06-07 1998-02-03 American Cyanamid Company Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates
US20070258987A1 (en) 2000-11-28 2007-11-08 Seattle Genetics, Inc. Recombinant Anti-Cd30 Antibodies and Uses Thereof
AR048098A1 (en) 2004-03-15 2006-03-29 Wyeth Corp CALIQUEAMYCIN CONJUGATES
NZ579482A (en) 2004-06-01 2011-02-25 Genentech Inc Antibody drug conjugates and methods
JP2008521828A (en) 2004-11-29 2008-06-26 シアトル ジェネティックス， インコーポレイテッド Engineered antibodies and immunoconjugates
US7750116B1 (en) 2006-02-18 2010-07-06 Seattle Genetics, Inc. Antibody drug conjugate metabolites
WO2008122039A2 (en) 2007-04-02 2008-10-09 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Selenocysteine mediated hybrid antibody molecules
EP2167963B1 (en) 2007-05-23 2019-04-17 Ventana Medical Systems, Inc. Polymeric carriers for immunohistochemistry and in situ hybridization
SG189817A1 (en) 2008-04-30 2013-05-31 Immunogen Inc Potent conjugates and hydrophilic linkers
SI2528625T1 (en) 2010-04-15 2013-11-29 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
US20130244905A1 (en) 2010-07-06 2013-09-19 Ed Grabczyk Reporter for RNA Polymerase II Termination
CN110078789A (en) 2011-05-27 2019-08-02 Ambrx 公司 Composition of aplysiatoxin derivative, the method for being related to the aplysiatoxin derivative containing unnatural amino acid connection and application thereof
US8815226B2 (en) 2011-06-10 2014-08-26 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
CA2850373C (en) 2011-10-14 2019-07-16 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
CN103997893B (en) 2011-10-14 2019-04-12 西雅图基因公司 Pyrrolobenzodiazepines and targeted conjugates
EP2751111B1 (en) 2011-10-14 2017-04-26 MedImmune Limited Asymmetrical bis-(5H-Pyrrolo[2,1-c][1,4]benzodiazepin-5-one) derivatives for the treatment of proliferative or autoimmune diseases
BR112014009070B1 (en) 2011-10-14 2021-11-23 Medimmune Limited SYNTHESIS METHOD AND INTERMEDIATES USEFUL IN THE PREPARATION OF PYROLOBENZO-DIAZEPINES
WO2013068874A1 (en) 2011-11-11 2013-05-16 Pfizer Inc. Antibody-drug conjugates
CN105142672B (en) 2012-10-23 2019-04-05 西纳福克斯股份有限公司 Modified antibodies, antibody-conjugates and methods of making the same
TWI641620B (en) 2013-08-21 2018-11-21 再生元醫藥公司 Anti-prlr antibodies and uses thereof
MY199468A (en) 2016-09-23 2023-10-31 Regeneron Pharma Anti-steap2 antibodies, antibody-drug conjugates, and bispecific antigen-binding molecules that bind steap2 and cd3, and uses thereof
AR111963A1 (en) * 2017-05-26 2019-09-04 Univ California METHOD AND MOLECULES
WO2019217597A1 (en) 2018-05-08 2019-11-14 Nike Innovate C.V. Bonding to polyolefin plates and articles of footwear formed therefrom
CN118955710A (en) 2018-05-09 2024-11-15 里珍纳龙药品有限公司 Anti-MSR1 antibodies and methods of use thereof
WO2019240219A1 (en) * 2018-06-14 2019-12-19 持田製薬株式会社 Novel crosslinked alginic acid
BR112021013464A2 (en) * 2019-01-08 2021-09-21 Regeneron Pharmaceuticals, Inc. TRACELESS BINDERS AND PROTEIN CONJUGATES THEREOF
EP3936501B1 (en) * 2019-03-08 2025-12-31 ABTIS Co., Ltd. Site-specific antibody conjugation and antibody-drug conjugate as a specific example thereof
KR20230065935A (en) * 2020-06-03 2023-05-12 스타파마 피티와이 리미티드 therapeutic conjugate
CN116390771A (en) 2020-07-13 2023-07-04 瑞泽恩制药公司 Camptothecin analogs conjugated to glutamine residues in proteins and uses thereof
CN113248408B (en) * 2021-04-30 2022-03-04 南京大学 A kind of multimodal molecular imaging probe P-FFGd-TCO and its preparation method and application
CA3241734A1 (en) * 2022-01-12 2023-07-20 Amy Han Camptothecin analogs conjugated to a glutamine residue in a protein, and their use

Also Published As

Publication number Publication date
WO2024168199A1 (en) 2024-08-15
CN121001751A (en) 2025-11-21

Similar Documents

Publication Publication Date Title
KR102877468B1 (en) Traceless linker and its protein-conjugate
KR102786381B1 (en) Steroids and their antibody-conjugates
US11491237B2 (en) Cyclodextrin protein drug conjugates
EP3883926B1 (en) Bis-octahydrophenanthrene carboxamide derivatives and protein conjugates thereof for use as lxr agonists
KR102911341B1 (en) Tubulisin and protein-tubulin conjugates
CN115867563B (en) Tubulolysin and protein-tubulolysin conjugates
CA2970565A1 (en) Antibody-drug conjugates (adcs) of ksp inhibitors with aglycosylated anti-tweakr antibodies
CA2934617A1 (en) Antibody drug conjugates (adcs) with kinesin spindle protein (ksp)
US20230119539A1 (en) Traceless linkers and protein-conjugates thereof
WO2018213082A1 (en) Bis-octahydrophenanthrene carboxamides and protein conjugates thereof
US20230414775A1 (en) Tubulysins and protein-tubulysin conjugates
EP4661914A1 (en) Antibody-drug conjugates via inverse electron demand diels-alder reactions
US20240269308A1 (en) Prodrugs of topoisomerase i inhibitor for adc conjugations and methods of use thereof
HK40116735A (en) Bis-octahydrophenanthrene carboxamide derivatives and protein conjugates thereof
HK40060716A (en) Bis-octahydrophenanthrene carboxamide derivatives and protein conjugates thereof for use as lxr agonists
HK40060716B (en) Bis-octahydrophenanthrene carboxamide derivatives and protein conjugates thereof for use as lxr agonists
EA049882B1 (en) BIS-OCTAHYDROPHENANTHRENECARBOXAMIDE DERIVATIVES AND THEIR PROTEIN CONJUGATES
EA047625B1 (en) TUBULISINS AND PROTEIN-TUBULYSIN CONJUGATES
EA045111B1 (en) STEROIDS AND THEIR ANTIBODY CONJUGATES
EA046545B1 (en) CYCLODEXTRIN-PROTEIN-DRUG CONJUGATES

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250806

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40128141

Country of ref document: HK