WO2023118961A1 - Antibody drug conjugates comprising toxins with polar groups and uses thereof - Google Patents

Antibody drug conjugates comprising toxins with polar groups and uses thereof Download PDF

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Publication number
WO2023118961A1
WO2023118961A1 PCT/IB2022/000772 IB2022000772W WO2023118961A1 WO 2023118961 A1 WO2023118961 A1 WO 2023118961A1 IB 2022000772 W IB2022000772 W IB 2022000772W WO 2023118961 A1 WO2023118961 A1 WO 2023118961A1
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Prior art keywords
drug conjugate
alkyl
aryl
heteroaryl
compound
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Ceased
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PCT/IB2022/000772
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English (en)
French (fr)
Inventor
Taekyo PARK
Yosup Rew
Sunyoung Kim
Doohwan JUNG
Donghoon SEO
Sangkwang LEE
Jihyeon HA
Jihye CHOI
Cheolmin Jeon
Myeonghwa JEONG
Hyewon Kim
Eun Hye YANG
Ya Gob KIM
Chohee Lee
Hyang Sook LEE
Beomseok Seo
Jina SONG
Sena Kim
Jae Do YOO
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Intocell Inc
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Intocell Inc
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Priority to JP2024537359A priority Critical patent/JP2025500342A/ja
Priority to CN202280084624.9A priority patent/CN118414170A/zh
Priority to CN202411879063.9A priority patent/CN119838020A/zh
Priority to KR1020247024345A priority patent/KR20240127409A/ko
Priority to EP22910281.9A priority patent/EP4452328A1/en
Publication of WO2023118961A1 publication Critical patent/WO2023118961A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D495/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • the drug conjugate is a compound represented by Formula (V): (V); or a pharmaceutically acceptable salt thereof.
  • targeted drug conjugates of Formula (VI) comprising a targeting moiety conjugated to any one of the drug conjugates of the present disclosure: (VI); wherein TM is a targeting moiety.
  • targeted drug conjugates of Formula (VIb) comprising a targeting moiety conjugated to the drug conjugates of the present disclosure: R O R 1 R 2 O O R O O z1 Q (Y) R z O O O R TM W 2 Z (L) y W 1 W a1 X n T 1-n (VIb); wherein: TM is a targeting moiety; R is hydrogen or a hydroxy protection group; X is -C(O)-, -NH-, -O-, or -S-; Q is an active agent substituted with a saccharide, a sulfonate, or a sulfate; T is ; n is an integer selected from 0 or 1; Y is hydrogen, haloC 1 -C 8 alkyl, halogen, cyano or nitro; z is an integer selected from 1-3, and Y may be the same or different from each other, if z is an integer of not less than 2; z
  • the compound is a compound of Formula (VII): (VII); or a pharmaceutically acceptable salt thereof.
  • the compound is a compound of formula (VIII): (VIII); or a pharmaceutically acceptable salt thereof.
  • drug conjugates comprising any one of the disclosed compounds and a linker group.
  • the drug conjugate is a compound of formula (IX), (X), or (XI): (IX); (X); or
  • targeted drug conjugates comprising any one of the drug conjugates provided herein and a targeting moiety.
  • the drug conjugate is a compound of formula (XII), (XIII) or (XIV): (XII); (XIII); or (XIV); or a pharmaceutically acceptable salt thereof; wherein TM is a targeting moiety.
  • methods of treating a cancer comprising administering any one of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutically compositions provided herein to a subject in need thereof.
  • the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney cancer, renal pelvis cancer, oral cavity cancer, pharynx cancer, uterine corpus cancer, or melanoma.
  • methods of treating autoimmune diseases or inflammatory diseases comprising administering any one of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutically compositions provided herein to a subject in need thereof.
  • the compounds, the drug conjugates, and targeted drug conjugates may be derived from genrerally toxin payloads which have been modified with a saccharide, a sulfate, or sulfonate.
  • the compounds, the drug conjugates, and targeted drug conjugates may significantly reduce non-specific uptake of drugs.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • preventing is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.
  • a condition such as a local recurrence (e.g., pain)
  • a disease such as cancer
  • a syndrome complex such as heart failure or any other medical condition
  • the term “optionally substituted” refers to the replacement of one to six hydrogen atoms in a given structure with a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH 2 -O-alkyl, - OP(O)(O-alkyl) 2 or –CH 2 -OP(O)(O-alkyl) 2 .
  • alkyl group may be optionally substituted.
  • acyl is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.
  • acylamino is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.
  • acyloxy is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.
  • alkoxy refers to an alkyl group having an oxygen attached thereto.
  • alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.
  • alkoxyalkyl refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.
  • alkyl refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1- 30 for straight chains, C 3-30 for branched chains), and more preferably 20 or fewer.
  • the term “lower alkyl” refers to the alkyl group with 1-6 carbon atoms.
  • the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc.
  • aryl as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon.
  • the ring is a 5- to 7-membered ring, more preferably a 6-membered ring.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • heteroaryl and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
  • heterocyclyl refers to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
  • polycyclyl refers to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”.
  • Each of the rings of the polycycle can be substituted or unsubstituted.
  • each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.
  • sulfate is art-recognized and refers to the group –OSO3H, or a pharmaceutically acceptable salt thereof.
  • sulfonamide is art-recognized and refers to the group represented by the general formulae or , wherein R 9 and R 10 independently represents hydrogen or hydrocarbyl.
  • sulfoxide is art-recognized and refers to the group–S(O)-.
  • sulfonate is art-recognized and refers to the group SO 3 H, or a pharmaceutically acceptable salt thereof.
  • bisulfite is art-recognized and refers to the group -OS(O)OH, or a pharmaceutically acceptable salt thereof.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an iminec a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic mo
  • the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms.
  • the selection of the appropriate salt will be known to one skilled in the art.
  • Other non-pharmaceutically acceptable salts e.g., oxalates, may be used, for example, in the isolation of compounds of the disclosure for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
  • pharmaceutically acceptable basic addition salt as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds represented by Formula I or any of their intermediates.
  • Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide.
  • Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia.
  • the selection of the appropriate salt will be known to a person skilled in the art.
  • Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30.
  • the disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers).
  • compounds of the disclosure may be racemic. In certain embodiments, compounds of the disclosure may be enriched in one enantiomer. For example, a compound of the disclosure may have greater than about 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, 95% ee, 96% ee, 97% ee, 98% ee, 99% ee, or greater ee. As is generally understood in the art, single bonds drawn without stereochemistry do not indicate the stereochemistry of the compound. The compound of formula I provides an example of a compound for which no stereochemistry is indicated.
  • compositions or compounds contains 98 grams of a first enantiomer and 2 grams of a second enantiomer, it would be said to contain 98 mol percent of the first enantiomer and only 2 mol% of the second enantiomer.
  • certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (ent ought) isomers. In each instance, the disclosure includes both mixture and separate individual isomers. Some of the compounds may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.
  • Prodrug or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of formula I).
  • Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound.
  • Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound.
  • log of solubility is used in the art to quantify the aqueous solubility of a compound.
  • the aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption.
  • LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol/liter.
  • glycosyl refers to a monovalent substituent formed from any natural sugar, a metabolite/catabolite thereof, a prodrug thereof, or a combination thereof.
  • the toxin payload is selected from a chemotherapeutic agent substituted with a saccharide, a sulfate, or a sulfonate or a toxin substituted with a saccharide, a sulfate, or a sulfonate.
  • the active agent is a chemotherapeutic agent substituted with a saccharide, a sulfate, or a sulfonate.
  • the toxin payload is independently selected from an immunomodulatory compound substituted with a saccharide, a sulfate, or a sulfonate, an anticancer agent substituted with a saccharide, a sulfate, or a sulfonate, an antiviral agent substituted with a saccharide, a sulfate, or a sulfonate, an antibacterial agent substituted with a saccharide, a sulfate, or a sulfonate, an antifungal agent substituted with a saccharide, a sulfate, or a sulfonate, or an antiparasitic agent substituted with a saccharide, a sulfate, or a sulfonate.
  • the toxin payload may be functionalized at one or more functional groups selected from -C(O)-, -O-, -NH-, -S-, and –C(O)O-.
  • said functional groups are functionalized by a saccharide, a sulfate, or a sulfonate.
  • the toxin payload may comprise a modified moiety bound to a saccharide through a functional group selected from ester, amide, thio, carbamate, oxime, hydrazone, and the like.
  • the toxin payload may comprise a modified moiety bound to a polar group such as a sulfonate (see, e.g., WO 2006/111759 A1), a sulfate, a sulfite, and the like.
  • a polar group such as a sulfonate (see, e.g., WO 2006/111759 A1), a sulfate, a sulfite, and the like.
  • each L’ is a C 10 –C 100 linear or branched, saturated, or unsaturated alkylene moiety, optionally comprising one or more double bonds and/or triple bonds.
  • each p and each d is independently an integer from 0-1.
  • the compound is represented by Formula (VII): (VII); or a pharmaceutically acceptable salt thereof.
  • A is 5- to 6-membered heterocycle.
  • R c ’ is hydroxyl.
  • R b ’ together with the intervening atoms, complete an aryl or heteroaryl. In certain embodiments, two R b ’, together with the intervening atoms, complete an aryl.
  • R e ’ is hydrogen, C 1-6 alkyl, C 3-10 cycloalkyl, 3- to 10- membered heterocycloalkyl, C 6-10 aryl, or 5- to 10-membered heteroaryl. In yet further embodiments, R e ’ is hydrogen, C 1-6 alkyl, or C 3-10 cycloalkyl. In still further embodiments, R e ’ is hydrogen.
  • the compound is selected from: , , , , or ; or a pharmaceutically acceptable salt thereof.
  • the compound is represented by Formula (VIII): (VIII); or a pharmaceutically acceptable salt thereof.
  • Cy is phenyl.
  • Cy is pyrrolidine or pyrrole.
  • the compound is represented by Formula (VIIIa) or (VIIIb): (VIIIa); (VIIIb); or a pharmaceutically acceptable salt thereof.
  • the DBD-(L’’’)r-X’’’-Gly unit is selected from: , , , or ; or a pharmaceutically acceptable salt thereof; wherein: Y’’ is C or N; X′′ is selected from –NR-, -S-, or –O-; R is hydrogen or alkyl; r is an integer selected from 0-1; each R b ’’ is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L’’’)r-X’’-Gly; R k is alkyl, preferably C 1 -C 3 alkyl; q is an integer selected from 0-3; and is a single bond or a double bond.
  • R d ’ is hydrogen, C 1-6 alkyl, C 3-10 cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 aryl, or 5- to 10-membered heteroaryl.
  • R d ’ is L’’-Gly.
  • the compound is selected from: , , , or ; or a pharmaceutically acceptable salt thereof; wherein is a single bond or a double bond.
  • R a ’ is halogen, amino, hydroxyl, alkoxy, cyano, nitro, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 aryl, or 5- to 10-membered heteroaryl.
  • the compound is selected from: , , , , or ; or a pharmaceutically acceptable salt thereof.
  • the compound is represented by Formula (VIII): (VIII); or a pharmaceutically acceptable salt thereof.
  • Cy is phenyl.
  • Cy is pyrrolidine or pyrrole.
  • the compound is represented by Formula (VIIIa) or (VIIIb): (VIIIa); (VIIIb); or a pharmaceutically acceptable salt thereof.
  • At least one R a ’’ is alkoxy, e.g. methoxy, ethoxy, or propoxy.
  • X’ is Cl.
  • X’ is Br.
  • Y’’ is C.
  • Y’’ is N.
  • R e ’ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • R e ’’ is hydrogen, C 1-6 alkyl, C 3-10 cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 aryl, or 5- to 10-membered heteroaryl.
  • R e ’’ is hydrogen, C 1-6 alkyl, or C 3-10 cycloalkyl.
  • R e ’’ is hydrogen.
  • at least one of R a ’’ is at the 8- position.
  • Z’ is a coupling group
  • Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl
  • Y’ is -(CR b 2 ) y N(R a )-, -(CR b 2 ) y O-, or -(CR b 2 ) y S-, positioned such that the N, O, or S atom is attached to TG if y is 1
  • TG is a triggering group that, when activated, generates an N, O, or S atom capable of reacting with the SO 2 to displace (Q) q -(L’) w and form a 5- to 6-membered ring including X-SO 2 and the intervening atoms of Ar
  • X is -O-, -C(R b ) 2 -, or -N(R c )-
  • L’ is a spacer moiety
  • Ar is 5- to 10-membered heteroaryl.
  • Y’ is - (CR b 2 ) y N(R a )- or -(CR b 2 ) y O-. In still further embodiments, Y’ is -(CR b 2 ) y O-. In certain embodiments, y is 0. In further embodiments, y is 1. In yet further embodiments, X is -O-, C(R b )(R c )- or -N(R c )-. In still further embodiments, X is -O-.
  • L’ is a spacer moiety, and forms an -O-, an -OC(O)-, an -OC(O)O-, a -NHC(O)O-, or an - OC(O)NH- linkage including the heteroatom of the active agent.
  • L’ is .
  • drug conjugates comprising any one of the toxin payload compounds of the present disclosure (an active agent) and a linking group; wherein the active agent is substituted with a polar group.
  • the polar group is selected from a saccharide, sulfate, or sulfonate.
  • drug conjugates comprising an active agent and a linking group are the drug conjugates of Formula (I): (I), or a pharmaceutically acceptable salt thereof; wherein: Z’ is a coupling group; Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y’ is -(CR b 2 ) y N(R a )-, -(CR b 2 ) y O-, or -(CR b 2 ) y S-, positioned such that the N, O, or S atom is attached to TG if y is 1; TG is a triggering group that, when activated, generates an N, O, or S atom capable of reacting with the SO 2 to displace (Q)q-(L’)w and form a 5- to 6-membered ring including X-SO 2 and the intervening atoms of Ar; X is -O-, -C(R b ) 2 -, or -N
  • each Q is independently selected from a chemotherapeutic agent substituted with a saccharide, a sulfate, or a sulfonate or a toxin substituted with a saccharide, a sulfate, or a sulfonate.
  • the active agent is a chemotherapeutic agent substituted with a saccharide, a sulfate, or a sulfonate.
  • each Q is independently selected from an immunomodulatory compound substituted with a saccharide, a sulfate, or a sulfonate, an anticancer agent substituted with a saccharide, a sulfate, or a sulfonate, an antiviral agent substituted with a saccharide, a sulfate, or a sulfonate, an antibacterial agent substituted with a saccharide, a sulfate, or a sulfonate, an antifungal agent substituted with a saccharide, a sulfate, or a sulfonate, or an antiparasitic agent substituted with a saccharide, a sulfate, or a sulfonate.
  • Q may be comprising modified moiety bonded with a saccharide through a functional group selected from ester, amide, thio, carbamate, oxime, hydrazone, and the like.
  • Q may be comprising modified moiety bonded with polar groups such as a sulfonate, a sulfate, a sulfite, and the like.
  • each Q is independently represented by: or ; wherein: Z 2 is a linking group; Z 3 is a linking group; t is an integer from 1-5; e is an integer from 1-5; and each Q’ is independently a modified benzodiazepine.
  • R b ’ one of the instances of R b ’ (or two germinal R b ’ taken together as described above), will serve as the attachment point to the remainder of the conjugate (e.g., where Q is ).
  • the remainder of the conjugate may be understood as a substituent on that (or those) instance(s) of R b ’.
  • Such R b ’ instance(s) are thus selected from the substituents listed above that may be made bivalent, e.g.
  • R f amino, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or a heterocyclic ring, preferably a five- or six-membered ring optionally, fused to or substituted with one or more aryl or heteroaryl rings.
  • Formulae (IIIa) and (IIIb) are referred to as Formulae (IX) and (X), respectively, as will be apparent from their context.
  • A is 5- to 6-membered heterocycle.
  • R c ’ is hydroxyl.
  • R c ’ is sulfonate or sulfate.
  • R d ’ is –L ” -Gly.
  • R d ’ is hydrogen.
  • each Q’ is independently selected from: , , , , , , , or ; or a pharmaceutically acceptable salt thereof; wherein is a single bond or a double bond.
  • R a ’ is halogen, amino, hydroxyl, alkoxy, cyano, nitro, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 aryl, or 5- to 10-membered heteroaryl.
  • one R b ’ is alkyl or two geminal R b ’ are taken together to form an alkenyl group.
  • two R b ’, together with the intervening atoms, complete a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; preferably wherein the aryl or heteroaryl is a 6-membered aryl or heteroaryl, optionally substituted with one or more R f ’.
  • two R b ’, together with the intervening atoms, complete an aryl or heteroaryl; preferably wherein the aryl or heteroaryl is a 6-membered aryl or heteroaryl, optionally substituted with one or more R f ’.
  • two R b ’, together with the intervening atoms complete an aryl.
  • each Q’ is independently selected from: , , , , , , , , , or ; or a pharmaceutically acceptable salt thereof; wherein is a single bond or a double bond; and wherein g is an integer from 0-4.
  • Z 3 is selected from: , , , or ; wherein: X 5 is -O- or -NR x -; Y 1 is CR y , or N; R y is hydrogen or C 1-6 alkyl; each b is independently an integer from 1-3; and c is an integer from 1-5.
  • Z 3 is selected from: or .
  • Z 2 is methylene.
  • Z 2 is , wherein: Y 5 is CR Y1 or N, provided that only one Y 5 is N; R Y1 is H, hydroxyl, amino, amido, or (CH 2 )y(R Y1a ); R Y1a is amino (e.g., secondary or tertiary amino), aryl (e.g., phenyl), or heteroaryl; and y is an integer having a value of 1 to about 10.
  • Z 2 is , , , or .
  • Z 2 is: , wherein: Y 6 is CR Y2 or N; R Y2 is H or alkyl, preferably lower alkyl; R Z2 is (CH 2 )zR Z2a ; R Z2a is amino (preferably tertiary amino), aryl (e.g., phenyl), or heteroaryl; and z is an integer having a value of 0 to about 10.
  • Z 2 is: , , or .
  • each L’ is a C 10 –C 100 linear or branched, saturated, or unsaturated alkylene moiety, optionally comprising one or more double bonds and/or triple bonds.
  • each p and each d is independently an integer from 0-1.
  • Cy is phenyl.
  • Cy is pyrrolidine or pyrrole.
  • each Q is independently a group of Formula (IVa) or (IVb): (IVa); (IVb); or a pharmaceutically acceptable salt thereof.
  • the DBD–(L’’’)r–X’’’-Gly unit is selected from: , , , or ; or a pharmaceutically acceptable salt thereof; wherein: Y’’ is C or N; X’’ is selected from –NR-, -S-, or -O-; each R b ’’ is independently halogen, amino, hydroxyl, acetyl, hydroyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or –(L”’)r-X”-Gly; R k is alkyl or hydroxyalkyl, preferably C 1 -C 3 alkyl; q is an integer selected from 0-3; and is a single bond or a double bond.
  • the DBD-(L’’’) r -X’’’-Gly unit is selected from: , , , or ; or a pharmaceutically acceptable salt thereof; wherein: Y’’ is C or N; each R b ’’ is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R k is alkyl or hydroxyalkyl, preferably C 1 -C 3 alkyl; q is an integer selected from 0-3; and is a single bond or a double bond.
  • the drug conjugate is selected from a group of Formula (Vc), (Vd), (Ve), or (Vf): (Vc);
  • the drug conjugate is represented by formula (Vc) or (Vd):
  • Gly is a monosaccharide. In further embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In yet further embodiments, Gly is or optionally wherein 1 or more of the –OH groups is masked by a protecting group. In still further embodiments, Gly is or . In certain embodiments, Gly is a disaccharide. In further embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof.
  • Gly is or optionally wherein 1 or more of the –OH groups is masked by a protecting group. In still further embodiments, Gly is or . In certain embodiments, X’’ or L’’ is coupled to Gly at the anomeric position. In further embodiments, Ar is aryl. In yet further embodiments, Ar is C 6-10 aryl. In still further embodiments, Ar is phenyl. In certain embodiments, Ar is heteroaryl. In further embodiments, Ar is 5- to 10-membered heteroaryl. In yet further embodiments, Y’ is - (CR b 2 ) y N(R a )- or -(CR b 2 ) y O-.
  • the drug conjugate is not selected from: In certain embodiments, the drug conjugate is not a compound disclosed in US2022/0047717.
  • Targeted Drug Conjugates comprising the drug conjugate comprising compounds of the present disclosure, a linker group, and a targeting moiety. In certain embodiments, the targeted drug conjugate is a compound represented by Formula (XII), (XIII) or (XIV):
  • TM is a targeting moiety conjugated to the drug conjugates of the present disclosure: (VIb); wherein: TM is a targeting moiety; R is hydrogen or a hydroxy protection group; X is -C(O)-, -NH-, -O-, or -S-; Q is an active agent substituted with a saccharide, a sulfonate, or a sulfate; T is ; n is an integer selected from 0 or 1; Y is hydrogen, haloC 1 -C 8 alkyl, halogen, cyano or nitro; z is an integer selected from 1-3, and Y may be the same or different from each other, if z is an integer of not less than 2; z1 is an integer selected from 0 or 1; W1 is ; W 2 is ; W a
  • targeted drug conjugates of Formula (VIc) comprising a targeting moiety conjugated to the drug conjugates of the present disclosure: (VIc); wherein: TM is a targeting moiety; G is a glucuronic acid moiety or a derivative thereof; Q is an active agent substituted with a saccharide, a sulfonate or a sulfate; W is an electron withdrawing group; Z is hydrogen, C 1 -C 8 alkyl, halogen, cyano, or nitro; n is an integer selected from 1-3, and when n is an integer of 2 or more, each of the Z(s) are the same as or different from each other; L is a linker connecting TM and W; and R 1 and R 2 are each independently hydrogen, C 1 -C 8 alkyl, or C 3 -C 8 cylcoalkyl In certian aspects, provided herein are targeted drug conjugate of Formula (VId) comprising a targeting moiety conjugated to the drug
  • the compounds and conjugates disclosed herein are capable of dissociating one or more active agents through an intramolecular cyclization reaction following a chemical reaction that activates the triggering group.
  • the chemical reaction is a physicochemical reaction and/or a biochemical reaction.
  • the compounds and conjugates disclosed herein comprise a nucleophilic functional group (Y or Y’) introduced at an atom on Ar adjacent to X (e.g., O).
  • X e.g., O
  • the nucleophilic functional group is masked by a triggering group (TG), as further detailed below.
  • the triggering group Upon activation, the triggering group releases the nucleophilic functional group to react with the nearby SO 2 moiety in an intramolecular cyclization, ultimately releasing the one or more compounds of Formula (II), (IIa), or (IIb).
  • one or more active agents are released through an intramolecular cyclization reaction after a chemical reaction, a physicochemical reaction and/or a biochemical reaction (see, for example, Reaction Scheme 1), or the active agent is released through 1,6-elimination or 1,4-elimination after the intramolecular cyclization reaction (see, for example, Reaction Scheme 2).
  • Q 1 is conjugated to a compound as described herein by the -C(O)-, -OH, -NH-, -SH, -COH, and –COOH, for instance through a functional group selected from ester, amide, thioester, carbamate, urea, oxime, hydrazone, etc.
  • Q 2 is used in place of Q 1 , and Q 2 is an amine group-containing drug.
  • Q 2 is an active agent capable of binding with an ammonium unit.
  • Q 2 is capable of being dissociated in its original form having an amine group upon release of Q 2 release, wherein the active agent may be a drug, a toxin, an affinity ligand, a probe for detection, or a combination thereof.
  • the compounds and conjugates disclosed herein are chemically and physiologically stable. In some such embodiments, the compounds and conjugates disclosed herein reach a desired target cell in a state wherien there is little dissociation of the active agent in the blood, thereby selectively releasing the drug.
  • Triggering Groups TGs
  • the conjugates of the present disclosure include a triggering group (TG).
  • TGs are groups capable of being cleaved, preferably selectively cleaved, by a chemical reaction, such as a biological reaction.
  • triggering groups serve to mask the nucleophilic nature of the Y’ group, thereby providing stability (e.g., by preventing self- immolation or intramolecular cyclization prior to the conjugate reaching a target location or experiencing a predetermined trigger condition) to the compounds and conjugates disclosed herein.
  • the triggering group releases the nucleophilic Y group and allows for self-immolation or intramolecular cyclization to occur, as described above.
  • the TG comprises a sequence (such as a peptide sequence) or a moiety recognized by TEV, trypsin, thrombin, cathepsin B, cathespin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), and the like, which can be hydrolyzed by an enzyme (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, etc.) and/or may include a moiety selected from a sulfate, a phosphodiester, a phospholipid, an ester, a ⁇ - galactose, a ⁇ -glucose, a fucose, an oligosugar, and the like.
  • an enzyme e.g., an oxidoreductase, a transferase, a hydrolase, a lyase,
  • the TG comprises a reactive chemical moiety or functional group that can be cleaved under nucleophilic reagent conditions (e.g., a silyl ether, a 2-N-acyl nitrobenzenesulfonamide, an unsaturated vinyl sulfide, a sulfonamide after activation, a malondialdehyde-indole derivative, a levulinoyl ester, a hydrazone, or an acyl hydrazone).
  • nucleophilic reagent conditions e.g., a silyl ether, a 2-N-acyl nitrobenzenesulfonamide, an unsaturated vinyl sulfide, a sulfonamide after activation, a malondialdehyde-indole derivative, a levulinoyl ester, a hydrazone, or an acyl hydrazone.
  • the TG may comprise a reactive chemical moiety or functional group that can be cleaved under basic reagent conditions (e.g., a 2-cyanoethyl ester, an ethylene glycolyl disuccinate, a 2-sulfonylethyl ester, an alkyl thioester, or a thiophenyl ester).
  • basic reagent conditions e.g., a 2-cyanoethyl ester, an ethylene glycolyl disuccinate, a 2-sulfonylethyl ester, an alkyl thioester, or a thiophenyl ester.
  • the TG may comprise a reactive chemical moiety or functional group that can be cleaved by photo-irradiation (e.g., 2-nitrobenzyl derivative, phenacyl ester, 8-quinolinyl benzenesulfonate, coumarin, phosphotriester, bis-arylhydrazone, or bimane bi- thiopropionic acid derivative).
  • the TG may comprise a reactive chemical moiety or functional group that can be cleaved by reducing agent conditions (e.g., hydroxylamine, disulfide, levulinate, nitro, or 4-nitrobenzyl derivative).
  • the TG may comprise a reactive chemical moiety or functional group that can be cleaved under oxidative conditions (e.g., a boronate, a vicinal diol, paramethoxybenzyl derivative, or a selenium compound).
  • oxidative conditions e.g., a boronate, a vicinal diol, paramethoxybenzyl derivative, or a selenium compound.
  • the TG comprises a saccharide, which can be cleaved under acidic or enzymatic conditions.
  • the triggering group is -NO 2 , which can be cleaved under reducing conditions.
  • the triggering group is a boronate, which can be cleaved under oxidative conditions.
  • the hydroxy protecting group is capable of being used in organic synthesis, including but not limited to: methyl ether, methoxymethyl ether, methylthiomethyl ether, 2-methoxyethoxymethyl ether, bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether, tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4- methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl- 1-methoxyethyl ether, 2-(phenylselenyl)ethyl ether, t-butyl ether, allyl ether, benzyl ether, o- nitrobenzyl ether, triphenyl methyl ether, ⁇ -naphthyldiphenyl methyl ether, p- methoxyphenyldip
  • TG is a monosaccharide. In further embodiments, TG is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In yet further embodiments, TG is or optionally wherein 1 or more of the –OH groups is masked by a protecting group. In still further embodiments, TG is or . In certain embodiments, TG is a disaccharide. In further embodiments, TG is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In yet further embodiments, TG is or optionally wherein 1 or more of the –OH groups is masked by a protecting group.
  • TG is or .
  • Y’ or L’ is coupled to TG at the anomeric position.
  • Protecting Groups as Triggering Groups In some embodiments, TG is a group that is capable of being cleaved by a chemical reaction, a physicochemical reaction, and/or a biological reaction. In certain embodiments, TG is a protecting group. In some such embodiments, the protecting group is an amine group protecting group, an alcohol protecting group, or a thiol protecting group.
  • the amine protecting group is a general protecting group that is capable of being used in organic synthesis, including but not limited to: m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, phenyl(o- nitrophenyl)methyl carbamate, alkyl carbamate, 9-fluorenylmethyl carbamate, 2,2,2- trichloroethyl carbamate, 2-trimethylsilylethyl carbamate(Teoc), t-butyl carbamate(Boc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, diphenyl
  • the alcohol protecting group is a general protecting group that is capable of being used in organic synthesis, including but not limited to: methyl ether, methoxymethyl ether (MOM ether), benzyloxymethyl ether (BOM ether), 2- (trimethylsilyl)ethoxymethyl ether (SEM ether), phenylthiomethyl ether (PTM ether), 2,2- dichloro-1,1-difluoroethyl ether, p-bromophenacyl ether, chloropropylmethyl ether, isopropyl ether, cyclohexyl ether, 4-methoxybenzyl, 2,6-dichlorobenzyl ether, 4- (dimethylaminocarbonyl)benzyl ether, 9-anthrylmethyl ether, 4-picolyl ether, methylthiomethyl ether (MTM ether), 2-methoxyethoxymethyl ether (MEM ether), bis(2- chloroethoxymethyl ether
  • the compounds and conjugates disclosed herein comprise a linking group connecting each TM and Ar through covalent bonds.
  • Typical linking groups are stable, non-hydrolyzable moieties, such as, for example a C 10 –C 100 linear or branched, saturated or unsaturated alkylene.
  • the linking group connecting each TM and Ar comprises a functional group produced through a click chemical reaction.
  • the linking unit comprises a reactive functional group capable of participating in a click chemical reaction.
  • a click chemical reaction is a reaction that can be performed under mild conditions, and is extremely selective for functional groups that are not commonly found in biological molecules (e.g., an azide group, an acetylene group, etc.). Accordingly, this reaction can be carried out in the presence of complex triggering groups, targeting moieties, etc. Further, click chemistry has high reaction specificity. For example, the click chemical reaction between an azide group and an acetylene group proceeds selectively without interference from other functional groups present in the molecule.
  • the linking group connecting each TM and Ar comprises , or V may be a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, - NR 23 C(O)-, -NR 24 SO 2 -, or -SO 2 NR 25 -, R 21 to R 25 may be each independently hydrogen, (C 1 - C 6 )alkyl, (C 1 -C 6 )alkyl(C 6 -C 20 )aryl, or (C 1 -C 6 )alkyl(C 3 -C 20 )heteroaryl, r may be an integer having a value of 1 to about 10, p may be an integer having a value of 0 to about 10, q may be an integer having a value of 1 to about 10, and L” may be a single bond.
  • R za is H or methyl
  • n and m are each independently an integer selected from 1-10
  • x is an integer selected from 1-2
  • a'' represents the bond between Z’ and the drug conjugate
  • b'' represents the bond between Z’ and TM
  • Z is selected from and .
  • Targeting Moieties The compounds and conjugates of the present disclosure can further comprise one or more ligand or targeting moiety, TM.
  • the antibody is selected from Muromonab-CD3, Abciximab, Rituximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab (herceptin), Etanercept, Basiliximab, Gemtuzumab ozogamicin, Alemtuzumab, Ibritumomab tiuxetan, Adalimumab, Alefacept, Omalizumab, Efalizumab, Tositumomab-I 131 , Cetuximab, Bevacizumab, Natalizumab, Ranibizumab, Panitumumab, Eculizumab, Rilonacept, Certolizumab pegol, Romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA- 29Y, Belimumab, TACI-Ig, Second generation anti-CD3, Abcixim
  • TM comprises two or more independently selected natural amino acids or non-natural amino acids conjugated by covalent bonds (e.g., peptide bonds), and the peptide may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more natural amino acids or non-natural amino acids that are conjugated by peptide bonds.
  • the ligand comprises shorter amino acid sequences (e.g., fragments of natural proteins or synthetic polypeptide fragments) as well as full-length proteins (e.g., pre- engineered proteins).
  • TM is selected from peptides, tumor cell-specific peptides, tumor cell-specific aptamers, tumor cell-specific carbohydrates, tumor cell-specific monoclonal antibodies, polyclonal antibodies, and antibody fragments that are identified in a library screen.
  • Exemplary ligands or targeting moieties include, but are not limited to, carnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B12, other water-soluble vitamins (vitamin B), fat-soluble vitamins (vitamin A, D, E, K), RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), transferein, VIP (vasoactive intestinal peptide) receptor, APRPG (Ala-Pro-Arg-Pro-Gly) peptide, TRX-20 (thioredoxin- 20), integrin, nucleolin, aminopeptidase N (CD13), endoglin, vascular epithelial growth factor receptor, low density lipoprotein receptor, transferrin receptor, somatostatin receptor, bombesin, neuropeptide Y, luteinizing hormone releasing hormone receptor, folic
  • the target or targets of the molecular recognition element are specifically associated with one or more particular cell or tissue types.
  • targets are specifically associated with one or more particular disease states.
  • targets are specifically associated with one or more particular developmental stages.
  • a cell type specific marker is typically expressed at levels at least 2 fold greater in that cell type than in a reference population of cells.
  • the cell type specific marker is present at levels at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, or at least 1,000 fold greater than its average expression in a reference population.
  • a target can comprise a protein, a carbohydrate, a lipid, and/or a nucleic acid, as described herein.
  • a substance is considered to be “targeted” if it specifically binds to a targeting moiety, such as a nucleic acid targeting moiety.
  • a targeting moiety such as a nucleic acid targeting moiety, specifically binds to a target under stringent conditions.
  • the conjugates and compounds described herein comprise a targeting moiety that specifically binds to one or more targets (e.g., antigens) associated with an organ, tissue, cell, extracellular matrix component, and/or intracellular compartment.
  • targets e.g., antigens
  • the conjugates and compounds described herein comprise a targeting moiety that specifically binds to targets associated with a particular organ or organ system.
  • the conjugates and compounds described herein comprise a targeting moiety that specifically binds to one or more intracellular targets (e.g., organelle, intracellular protein).
  • the conjugates and compounds described herein comprise a targeting moiety which specifically binds to targets associated with diseased organs, tissues, cells, extracellular matrix components, and/or intracellular compartments.
  • the conjugates and compounds described herein comprise a targeting moiety that binds to a target that is specific for one or more particular disease states (e.g., tumor cells vs. healthy cells).
  • the conjugates and compounds described herein comprise a targeting moiety that binds to a target that is specific for one or more particular developmental stages (e.g., stem cells vs. differentiated cells).
  • a target may be a marker that is exclusively or primarily associated with one or a few cell types, with one or a few diseases, and/or with one or a few developmental stages.
  • a cell type specific marker is typically expressed at levels at least 2 fold greater in that cell type than in a reference population of cells which may consist, for example, of a mixture containing cells from a plurality (e.g., 5–10 or more) of different tissues or organs in approximately equal amounts.
  • the cell type specific marker is present at levels at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, or at least 1000 fold greater than its average expression in a reference population. Detection or measurement of a cell type specific marker may make it possible to distinguish the cell type or types of interest from cells of many, most, or all other types.
  • Exemplary receptors include, but are not limited to, the transferrin receptor; LDL receptor; growth factor receptors such as epidermal growth factor receptor family members (e.g., EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules.
  • the marker can be a molecule that is present exclusively or in higher amounts on a malignant cell, e.g., a tumor antigen.
  • ADCs Antibody-Drug Conjugates
  • TM is an antibody
  • Q is a drug.
  • the compounds and conjugates disclosed herein may be used to conjugate an antibody to a drug moiety to form targeted drug conjugates which are antibody-drug conjugates (ADC).
  • ADCs antibody-drug conjugates
  • ADCs may increase therapeutic efficacy in treating disease, e.g., cancer, due to the ability of the ADC to selectively deliver one or more drug moiety(s) to target tissues, such as a tumor-associated antigen.
  • the disclosure provides ADCs for therapeutic use, e.g., treatment of cancer.
  • ADCs of the disclosure comprise an antibody linked to one or more drug moieties.
  • the specificity of the ADC is defined by the specificity of the antibody.
  • the ADCs selectively target specific cells of interest, such as, for example, tumor cells.
  • the specific antigen, and hence antibody selected will depend upon the identity of the desired target cell of interest.
  • the antibody of the ADC is an antibody suitable for administration to humans.
  • Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • VH refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab.
  • References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
  • antibody herein is used in the broadest sense and refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to murine, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including e.g., Fab’, F(ab’) 2 , Fab, Fv, rIgG, and scFv fragments.
  • Fab fragment antigen binding fragments of antibodies
  • one antigen may have more than one corresponding antibody.
  • An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease.
  • the immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • the immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.
  • antibody fragment refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab′, F(ab ′) 2 and Fv fragments.
  • An “Fv” fragment is the minimum antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding.
  • Single domain antibodies are composed of a single VH or VL domains which exhibit sufficient affinity to the target.
  • the single domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).
  • the Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • F(ab′) fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab ′) 2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • CDRs complementarity determining regions
  • FR framework
  • the amino acid position/boundary delineating a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art.
  • Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria.
  • One or more of these positions can also be found in extended hypervariable regions.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987). As used herein, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. In certain embodiments, the antibodies of the ADCs of the present disclosure are monoclonal antibodies.
  • mAb refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • a monoclonal antibody of the disclosure exists in a homogeneous or substantially homogeneous population.
  • Monoclonal antibody includes both intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab’) 2 fragments), which are capable of specifically binding to a protein.
  • Fab and F(ab’) 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal, and may have less non-specific tissue binding than an intact antibody (Wahl et al., 1983, J. Nucl. Med 24:316).
  • Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • the antibodies of the disclosure include chimeric, primatized, humanized, or human antibodies. While in most instances antibodies are composed of only the genetically-encoded amino acids, in some embodiments non-encoded amino acids may be incorporated at specific.
  • the antibody of the ADCs described herein is a chimeric antibody.
  • chimeric antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as rat or mouse antibody, and human immunoglobulin constant regions, typically chosen from a human immunoglobulin template.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties.
  • the antibody of the ADCs described herein is a humanized antibody.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’) 2 or other target-binding subdomains of antibodies), which contain minimal sequences derived from non-human immunoglobulin.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
  • Fc immunoglobulin constant region
  • Methods of antibody humanization are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No.6,180,370 to Queen et al.; EP239400; PCT publication WO 91/09967; U.S. Pat. No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol.
  • the antibody of the ADCs described herein is a human antibody. Completely “human” antibodies can be desirable for therapeutic treatment of human patients.
  • human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 4,716,111, 6,114,598, 6,207,418, 6,235,883, 7,227,002, 8,809,151 and U.S. Published Application No. 2013/189218, the contents of which are incorporated herein by reference in their entireties.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 7,723,270; 8,809,051 and U.S. Published Application No. 2013/117871, which are incorporated by reference herein in their entireties.
  • primary antibody refers to an antibody comprising monkey variable regions and human constant regions.
  • Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Pat. Nos.5,658,570; 5,681,722; and 5,693,780, which are incorporated herein by reference in their entireties.
  • the antibody of the ADCs described herein is a bispecific antibody or a dual variable domain antibody (DVD).
  • Bispecific and DVD antibodies are monoclonal, often human or humanized, antibodies that have binding specificities for at least two different antigens. DVDs are described, for example, in U.S. Pat. No. 7,612,181, the disclosure of which is incorporated herein by reference.
  • the antibody of the ADCs described herein is a derivatized antibody.
  • derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • the derivative can contain one or more non-natural amino acids, e.g., using ambrx technology (see, e.g., Wolfson, 2006, Chem. Biol.13(10):1011- 2).
  • the antibody of the ADCs described herein has a sequence that has been modified to alter at least one constant region-mediated biological effector function relative to the corresponding wild type sequence.
  • the antibody can be modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody, e.g., reduced binding to the Fc receptor (FcR).
  • FcR Fc receptor
  • FcR binding can be reduced by mutating the immunoglobulin constant region segment of the antibody at particular regions necessary for FcR interactions (see, e.g., Canfield and Morrison, 1991, J. Exp. Med 173:1483-1491; and Lund et al., 1991, J. Immunol.147:2657-2662).
  • the antibody of the ADCs described herein is modified to acquire or improve at least one constant region-mediated biological effector function relative to an unmodified antibody, e.g., to enhance Fc ⁇ R interactions (see, e.g., US 2006/0134709).
  • an antibody with a constant region that binds Fc ⁇ RIIA, Fc ⁇ RIIB and/or Fc ⁇ RIIIA with greater affinity than the corresponding wild type constant region can be produced according to the methods described herein.
  • the antibody of the ADCs described herein is an antibody that binds tumor cells, such as an antibody against a cell surface receptor or a tumor- associated antigen (TAA).
  • TAA tumor-associated antigen
  • tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to the surface of the no-cancerous cells.
  • Such cell surface receptor and tumor- associated antigens are known in the art, and can prepared for use in generating antibodies using methods and information which are well known in the art.
  • Exemplary Cell Surface Receptors and TAAs Examples of cell surface receptor and TAAs to which the antibody of the ADCs described herein may be targeted include, but are not limited to, the various receptors and TAAs listed below in Table 1.
  • nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available in public databases such as GenBank. Table I.
  • Exemplary antibodies to be used with ADCs of the disclosure include but are not limited to 3F8 (GD2), Abagovomab (CA-125 (imitation)), Adecatumumab (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), ALD518 (IL-6), Alemtuzumnab (CD52), Altumomab pentetate (CEA), Amatuximab (Mesothelin), Anatumomnab mafenatox (TAG- 72), Apolizumab (HLA-DR), Arcitumomab (CEA), Bavituximab (Phosphatidylserine), Bectumomab (CD22), Belimumab (BAFF), Besilesomab (CEA-related antigen), Bevacizumab (VEGF-A), Bivatuzumab mertansine (GD2), Ab
  • the antibody of an ADC can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
  • a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered.
  • Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., Greene Publishing Associates, 1989) and in U.S. Pat. No.4,816,397.
  • the Fc variant antibodies are similar to their wild-type equivalents but for changes in their Fc domains.
  • a DNA fragment encoding the Fc domain or a portion of the Fc domain of the wild- type antibody can be synthesized and used as a template for mutagenesis to generate an antibody as described herein using routine mutagenesis techniques; alternatively, a DNA fragment encoding the antibody can be directly synthesized.
  • these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the constant region genes to full-length antibody chain genes.
  • a CH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker.
  • the term “operatively linked,” as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • a variant antibody light chain gne and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • the expression vector Prior to insertion of the variant Fc domain sequences, the expression vector can already carry antibody variable region sequences.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vectors carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • promoters e.g., promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all to Axel et al.).
  • the selectable marker gene confers resistance to drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR ⁇ host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • transfection are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, and the like. It is possible to express the antibodies in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of antibodies is performed in eukaryotic cells, e.g., mammalian host cells, for optimal secretion of a properly folded and immunologically active antibody.
  • eukaryotic cells e.g., mammalian host cells
  • Exemplary mammalian host cells for expressing the recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including DHFR ⁇ CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.159:601-621), NS0 myeloma cells, COS cells, 293 cells and SP2/0 cells.
  • Chinese Hamster Ovary CHO cells
  • DHFR ⁇ CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.159:601-621
  • NS0 myeloma cells COS cells
  • 293 cells 293 cells and SP2/0 cells.
  • the antibodies When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. In some embodiments, the antibody of an ADC can be a bifunctional antibody.
  • Such antibodies in which one heavy and one light chain are specific for one antigen and the other heavy and light chain are specific for a second antigen, can be produced by crosslinking an antibody to a second antibody by standard chemical crosslinking methods.
  • Bifunctional antibodies can also be made by expressing a nucleic acid engineered to encode a bifunctional antibody.
  • dual specific antibodies i.e. antibodies that bind one antigen and a second, unrelated antigen using the same binding site, can be produced by mutating amino acid residues in the light chain and/or heavy chain CDRs.
  • Exemplary second antigens include a proinflammatory cytokine (such as, for example, lymphotoxin, interferon- ⁇ , or interleukin-1).
  • Dual specific antibodies can be produced, e.g., by mutating amino acid residues in the periphery of the antigen binding site (see, e.g., Bostrom et al., 2009, Science 323:1610- 1614). Dual functional antibodies can be made by expressing a nucleic acid engineered to encode a dual specific antibody. Antibodies can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Antibodies can also be generated using a cell-free platform (see, e.g., Chu et al., Biochemia No.2, 2001 (Roche Molecular Biologicals)).
  • an antibody can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for antigen after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for antigen after Protein A or Protein G selection, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • an antibody can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology (Work and Burdon, eds., Elsevier, 1980)), or by gel filtration chromatography on a SuperdexTM 75 column (Pharmacia Biotech AB, Uppsala, Sweden).
  • high performance liquid chromatography see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology (Work and Burdon, eds., Elsevier, 1980)
  • Gel filtration chromatography on a SuperdexTM 75 column
  • General method for preparing antibodies Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a given target, such as, for example, B7-H3, a tumor associated antigen or other target, or against derivatives, fragments, analogs homologs or orthologs thereof.
  • Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D.
  • the antibodies which may be used in embodiments disclosed herein are monoclonal antibodies.
  • Monoclonal antibodies are generated, for example, by using the procedures set forth in the Examples provided herein.
  • Antibodies are also generated, e.g., by immunizing BALB/c mice with combinations of cell transfectants expressing high levels of a given target on their surface. Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity to the selected target.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).
  • This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • IRES can greatly differ in their length and ribosome recruiting efficiency. Furthermore, it is possible to further tune the activity by introducing multiple copies of an IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541). The modulation of the expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus modify their relative expressions.
  • the Examples provided herein demonstrate that controlling the relative expression of the different chains is critical for maximizing the assembly and overall yield of the bispecific antibody.
  • the co-expression of the heavy chain and two light chains generates a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody.
  • the latter has to be purified from the mixture to obtain the molecule of interest.
  • the method described herein greatly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland).
  • affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland).
  • This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies which may be used in embodiments disclosed herein. This is in sharp contrast to specific purification methods that have to be developed and optimized for each bispecific antibodies derived from quadromas or other cell lines expressing antibody mixtures.
  • antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH 2 , and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol.152:5368 (1994). Antibodies with more than two valencies are contemplated.
  • the antibodies can be prepared in vitro using synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.4,676,980.
  • conjugated antibodies also referred to herein as immunoconjugates, comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • the toxin is a microtubule inhibitor or a derivative thereof.
  • the toxin is a dolastatin or a derivative thereof.
  • the toxin is auristatin E, auristatin F, AFP, MMAF, MMAE, MMAD, DMAF, or DMAE. In some embodiments, the toxin is a maytansinoid or maytansinoid derivative. In some embodiments, the toxin is DM1 or DM4. In some embodiments, the toxin is a nucleic acid damaging toxin. In some embodiments, the toxin is a duocarmycin or derivative thereof. In some embodiments, the toxin is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine or a derivative thereof.
  • radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re. Conjugates of the antibody and cytotoxic agent can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate),
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.
  • MX-DTPA 1-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid
  • Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies which may be used in embodiments disclosed herein. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings.
  • Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • the antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody can be prepared by any suitable methods, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG- derivatized phosphatidylethanolamine
  • Efficaciousness of treatment can be determined in association with any suitable method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the conjugate confers a clinical benefit. Conjugates directed against a target such as B7-H3, a tumor associated antigen or other antigen may be used in methods relating to the localization and/or quantitation of these targets, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • conjugates specific for any of these targets, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen-binding domain can be utilized as pharmacologically active compounds (referred to hereinafter as “Therapeutics”).
  • a conjugate of the disclosure can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation.
  • Conjugates of the disclosure can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • Conjugates of the disclosure may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology associated with aberrant expression or activation of a given target in a subject.
  • a conjugate preparation preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Administration of the conjugate may abrogate or inhibit or interfere with the signaling function of the target. Administration of the conjugate may abrogate or inhibit or interfere with the binding of the target with an endogenous ligand to which it naturally binds.
  • a therapeutically effective amount of a conjugate of the disclosure relates generally to the amount needed to achieve a therapeutic objective.
  • this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target and/or the effect of an active agent conjugated to the antibody.
  • the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen and/or the potency of the active agent, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
  • Common ranges for therapeutically effective dosing of a conjugate of the disclosure may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight.
  • Common dosing frequencies may range, for example, from twice daily to once a week.
  • Conjugates of the disclosure can be administered for the treatment of a variety of diseases and disorders in the form of pharmaceutical compositions.
  • Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol.4), 1991, M. Dekker, New York.
  • the formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • cytotoxic agent such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • the formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations can be prepared.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • a conjugate according to the disclosure can be used as an agent for detecting the presence of a given target (or a protein fragment thereof) in a sample.
  • the conjugate contains a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2 ) can be used.
  • the term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system.
  • the pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the disclosure. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any suitable method in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • compositions include the step of bringing into association an active compound, such as a compound of the disclosure, with the carrier and, optionally, one or more accessory ingredients.
  • active compound such as a compound of the disclosure
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • intravenous, intraocular such as intravitreal
  • intramuscular intraarterial
  • intrathecal intracapsular
  • intraorbital intracardiac
  • intradermal intraperitoneal
  • transtracheal subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • microorganisms Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
  • isotonic agents such as sugars, sodium chloride, and the like into the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • the rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • biocompatible polymers including hydrogels
  • biodegradable and non-degradable polymers can be used to form an implant for the sustained release of a compound at a particular target site.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • terapéuticaally effective amount is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the disclosure. A larger total dose can be delivered by multiple administrations of the agent. Many methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).
  • a suitable daily dose of an active compound used in the compositions and methods of the disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the patient receiving this treatment may be any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.
  • compounds of the disclosure may be used alone or conjointly administered with another type of therapeutic agent.
  • antioxidants examples include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • compositions may be prepared in an injectable form, either as a liquid solution or as a suspension.
  • Solid forms suitable for injection may also be prepared, e.g., as emulsions, or with the antibody-drug conjugate encapsulated in liposomes.
  • Antibody-drug conjugates may be combined with a pharmaceutically acceptable carrier, which includes any carrier that does not induce the production of antibodies harmful to the subject receiving the carrier.
  • Suitable carriers typically comprise large macromolecules that are slowly metabolized, for example, proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.
  • compositions may also contain diluents, for example, water, saline, glycerol, and ethanol.
  • diluents for example, water, saline, glycerol, and ethanol.
  • auxiliary substances for example, wetting or emulsifying agents, pH buffering substances, and the like may also be present therein.
  • the compositions may be parenterally administered by injection, wherein such injection may be either subcutaneous or intramuscular injection.
  • a composition may be administered into a tumor.
  • the composition may be inserted (e.g., injected) into a tumor. Additional formulations are suitable for other forms of administration, such as suppository or oral administration.
  • Oral compositions may be administered as a solution, suspension, tablet, pill, capsule, or sustained release formulation.
  • compositions may be administered in a manner compatible with a dose and a formulation.
  • the composition preferably comprises a therapeutically effective amount of the antibody-drug conjugate.
  • a dose may vary, depending on the subject to be treated, the subject's health and physical conditions, a degree of protection to be desired, and other relevant factors.
  • the exact amount of an active ingredient e.g., the antibody-drug conjugate
  • a therapeutically effective amount of the antibody-drug conjugate or composition containing the same may be administered to a patient suffering from a cancer or tumor to treat the cancer or tumor.
  • the antibody-drug conjugate according to the present disclosure or the composition containing the same may be administered in the form of a pharmaceutically acceptable salt thereof.
  • the antibody-drug conjugate according to the present disclosure or the composition containing the same may be administered with a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and/or a pharmaceutically acceptable additive.
  • the effective amount and the type of the pharmaceutically acceptable salt, excipient and additive may be measured using standard methods (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th Edition, 1990).
  • the disclosure relates to a method of treating cancer in a subject, comprising administering a pharmaceutical composition comprising an antibody-drug conjugate as described herein to the subject.
  • the subject is a mammal.
  • the subject may be selected from rodents, lagomorphs, felines, canines, porcines, ovines, bovines, equines, and primates.
  • the subject is a human.
  • the conjugates of the disclosure also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the conjugate and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, and subcutaneous administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ⁇ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • compositions can be prepared according to suitable methods, for example, as described in U.S. Patent No.4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Methods of Treatment
  • the compounds and conjugates disclosed herein may be used in methods to induce apoptosis in cells.
  • Agents useful to treat or ameliorate specific diseases that may be administered adjunctive to, or with, the compounds and compositions disclosed herein will be apparent to those of skill in the art. Although absolute cure is always desirable in any therapeutic regimen, achieving a cure is not required to provide therapeutic benefit. Therapeutic benefit may include halting or slowing the progression of the disease, regressing the disease without curing, and/or ameliorating or slowing the progression of symptoms of the disease. Prolonged survival as compared to statistical averages and/or improved quality of life may also be considered therapeutic benefit.
  • One particular class of diseases that involve dysregulated apoptosis and that are significant health burden world-wide are cancers. In a specific embodiment, the compounds and compositions disclosed herein may be used to treat cancers.
  • the cancer may be, for example, solid tumors or hematological tumors.
  • Cancers that may be treated with the compounds and compositions disclosed herein include, but are not limited to bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, myeloma, prostate cancer, small cell lung cancer and spleen cancer.
  • the compounds and compositions disclosed herein may be especially beneficial in the treatment of cancers because the antibody can be used to target the tumor cell specifically, thereby potentially avoiding or ameliorating undesirable side-effects and/or toxicities that may be associated with systemic administration of unconjugated inhibitors.
  • One embodiment pertains to a method of treating a disease involving dysregulated intrinsic apoptosis, comprising administering to a subject having a disease involving dysregulated apotosis an amount of a compound and composition disclosed herein effective to provide therapeutic benefit, wherein the ligand of the compounds and compositions disclosed herein binds a cell surface receptor on a cell whose intrinsic apoptosis is dysregulated.
  • the compounds and conjugates disclosed herein may be administered as monotherapy to provide therapeutic benefit, or may be administered adjunctive to, or with, other chemotherapeutic agents and/or radiation therapy.
  • Chemotherapeutic agents to which the compounds and compositions disclosed herein may be utilized as adjunctive therapy may be targeted (for example, ADCs, protein kinase inhibitors, etc.) or non-targeted (for example, non-specific cytotoxic agents such as radionucleotides, alkylating agents and intercalating agents).
  • Non-targeted chemotherapeutic agents with which the compounds and compositions disclosed herein may be adjunctively administered include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, Cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asperaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and
  • the compounds and conjugates disclosed herein that may not be effective as monotherapy to treat cancer may be administered adjunctive to, or with, other chemotherapeutic agents or radiation therapy to provide therapeutic benefit.
  • One embodiment pertains to a method in which a compound or composition disclosed herein is administered in an amount effective to sensitize the tumor cells to standard chemotherapy and/or radiation therapy.
  • therapeutic benefit includes administering the compounds and compositions disclosed herein adjunctive to, or with, chemotherapeutic agents and/or radiation therapy, either in patients who have not yet begin such therapy or who have but have not yet exhibited signs of resistance, or in patients who have begun to exhibit signs of resistance, as a means of sensitizing the tumors to the chemo and/or radiation therapy.
  • the present disclosure provides pharmaceutical compositions comprising an antibody drug conjugate as described herein, optionally further comprising a therapeutically effective amount of a chemotherapeutic agent.
  • methods of treating a cancer comprising administering one or more of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions of the present disclosure to a subject in need thereof.
  • the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney cancer, renal pelvis cancer, oral cavity cancer, pharynx cancer, uterine corpus cancer, or melanoma.
  • methods of treating autoimmune diseases or inflammatory diseases comprising administering one or more of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions of the present disclosure to a subject in need thereof.
  • the autoimmune disease or the inflammatory disease is selected from B-cell mediated autoimmune diseases or inflammatory diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom’s hypergammaglobulinaemia, Sjogren’s syndrome, multiple sclerosis (MS), or lupus nephritis.
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • ITP idiopathic thrombocytopenic purpura
  • MS multiple sclerosis
  • lupus nephritis lupus nephritis.
  • reaction mixture was purified by prep HPLC to obtain compound T-Int-5-3 (22 mg, 67%); EI-MS m/z: 1255(M + /2), 2509 (M + +1).
  • Preparation of compound T-Int-5-4 To a solution of compound T-Int-5-3 (22 mg, 0.001 mmol) in dry DCM (2.0 ml) at 0°C under N 2 atmosphere was treated with Dess Martin periodinane (8.6 mg, 0.02 mmol) and stirred overnight at room temperature. The reaction mixture was extracted with EA (10 mL X 2), H 2 O (3 mL). The organic layer was dried over anhydrous N a2 SO 4 , filtered, concentrated under reduced pressure.
  • reaction mixture was purified by prep- HPLC (0.1% formic acid in water/0.1% formic acid in ACN) to obtain compound T-23 (1.8 mg, 61%); EI-MS m/z: 1267 (M + /2), 2534 (M + +1).
  • Preparation of Compound T-7 T-7 was synthesized via a similar synthetic route as described in document WO2020/089687. Yield 92%: ESI-MS m/z: 2580(M +1 ).
  • Preparation of Additional Compounds Table 2 Compounds synthesized via a similar synthetic route as described in Example 33.
  • Compound DB-2-2 and DB-2-3 were synthesized in a way similar to the preparation method of compound DB-1-2 in Example 41.
  • Compound DB-4-2 Yield 75% 1 H NMR (400 Hz, CDCl 3 ) ⁇ 7.80-7.76 (m, 1H), 7.29 (s, 1H), 6.88 (s, 1H), 6.86 (s, 1H), 4.34- 4.31 (m, 2H), 4.27-4.25 (m, 2H), 3.91 (s, 3H).
  • Compound DB-4-3 Yield 65% 1 H NMR (400 Hz, CDCl 3 ) ⁇ 7.22-7.21 (m, 1H), 6.93-6.87 (m, 2H), 4.41-4.39 (m, 2H), 4.32- 4.30 (m, 2H), 3.93 (s, 3H); EI-MS m/z: 467 (2M).
  • reaction mixture was stirred at same temperature for 1 hour.
  • reaction mixture was filtered with DCM and the filterate was diluted with DCM and H 2 O.
  • the aqueous layer was washed further DCM.
  • the organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • the resiude was purified by column chromatography to obtain compound Int-TG19-1 (650 mg, 68%).
  • the reaction mixture was stirred at room temperature for 18 hours.
  • the reaction mixture diluted with H 2 O (150 mL).
  • the resulting mixture was extracted with EA (2 ⁇ 200 mL).
  • the combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated.
  • the residue was purified by column chromatography to produce the compound DB-14-2 (4.54 g, 49 %).
  • Compound T-124-2 Yield 87% ESI-MS m/z: 1526 (M + ).
  • Compound T-124-3 Yield 84% ESI-MS m/z: 1246 (M + ).
  • Compound T-124 Yield 86% ESI-MS m/z: 1646 (M + ).
  • Preparation of Compound T-125 Compound T-125 was synthesized via a similar synthetic route as described in Example 74.
  • Compound T-125-1 Yield 82% ESI-MS m/z: 1529 (M + ).
  • Compound T-125-2 Yield 61% ESI-MS m/z: 1249 (M + ).
  • Compound T-125 Yield 95% ESI-MS m/z: 1649 (M + ).
  • Lithium diisopropylamide (LDA) solution was stirred at -78 °C for 40 min and then t-butyl acetate was added to LDA solution at -78 °C.
  • the LDA and t-butyl acetate mixture was stirred at-78 °C for 1 hour and then added compound Int-4-1 and CDI mixture at -78 °C.
  • the reaction mixture was stirred at -78 °C for 3 hours.
  • the reaction mixture was quenched H 2 O (20 mL) and extracted with EA (20 mL ⁇ 3). The organic layer was dried over anhydrous N a2 SO 4 , filtered and concentrated under reduced pressure.
  • reaction mixture was quenched H 2 O (10 ml) at 0 °C.
  • the crud mixture was diluted with NaCl solution (10 ml) and extracted with EA (20 ml ⁇ 3).
  • the organic layer was dried over N a2 SO 4 , filtered and concentrated under reduced pressure.
  • Cysteine engineered monoclonal antibodies were reduced with about a 20 folds excess of TCEP (tris(2-carboxyethyl) phosphine hydrochloride in 4 mM Tris-HCl pH 7.3 with 1 mM EDTA for 1 hours at 37 °C.
  • Cysteine engineered monoclonal antibodies (B7H3, DLL3, HER2) were reduced with about a 5000 folds excess of L-Cysteine in 20 mM sodium phosphate pH 6.5 for 1 hours at 37 °C.
  • the reduced thiomab was diluted and loaded onto a PD-10 column or vivaspin (MWCO, 30kDa) in PBS, pH 6.5 and eluted with 20 mM PBS.
  • the eluted reduced thiomab was stored at 4 °C overnight for effective refolding.
  • the thiol/Ab value was checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, CAS No D8130) and determination of the absorbance at 412 nm.
  • Conjugation Method 1 (Thiomab conjugation) Stock solution of linker-toxin was made up in dimethylsulfoxide (DMSO) at concentrations of 1-3 mM. To a solution of the reduced antibody in 20mM sodium phosphate (pH 6.5) was added about 1.5-2.5 molar excess relative to cysteines per antibody of a linker toxin, such as T-2-AB ⁇ T-6-AB, T-10-AB, T-107-AB ⁇ T-113-AB with a thiol reactive functional group such as maleimide. The conjugation reaction was allowed to proceed at 40 °C for 1 h.
  • DMSO dimethylsulfoxide
  • Conjugation Method 3 (2-step conjugation method) A solution of cysteine-engineered antibody (1-3mmol in buffer system, pH6.5) was diluted with PBS buffer, pH7.4. DMSO was added followed by a solution of linker-toxin (T-7 of Example 36) in DMSO. The final concentration of DMA was 4-10%. The resulting mixture was agitated gently for 3 hours at room temperature.
  • the reduced thiomab was diluted and loaded onto a PD-10 column or vivaspin (MWCO, 30kDa) in PBS, pH 7.4 and eluted with PBS, pH 7.4.
  • MWCO molecular weight
  • the thiol/Ab value was checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, CAS No D8130) and determination of the absorbance at 412 nm.

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CN202280084624.9A CN118414170A (zh) 2021-12-21 2022-12-20 包含具有极性基团的毒素的抗体药物缀合物及其用途
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US12274898B2 (en) 2018-10-31 2025-04-15 Intocell, Inc. Substituted benzodiazepines as antibody-drug conjugates
WO2025155901A1 (en) 2024-01-19 2025-07-24 24M Technologies, Inc. Systems and methods for preparing semi-solid electrodes
WO2025214466A1 (en) * 2024-04-12 2025-10-16 BeiGene Guangzhou Biologics Manufacturing Co., Ltd. Targeted pyrrolobenzodiazapine conjugates
WO2026059501A1 (en) * 2024-09-13 2026-03-19 Hummingbird Bioscience Pte. Ltd. Linker moieties

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WO1997044000A2 (en) * 1996-05-23 1997-11-27 Panorama Research, Inc. Dna-binding indole derivatives, their prodrugs and immunoconjugates as anticancer agents
WO2014080251A1 (en) * 2012-11-24 2014-05-30 Hangzhou Dac Biotech Co., Ltd. Hydrophilic linkers and their uses for conjugation of drugs to cell binding molecules
KR20190064619A (ko) * 2016-10-14 2019-06-10 메디뮨 리미티드 피롤로벤조디아제핀 컨쥬게이트
KR20200107837A (ko) * 2019-03-06 2020-09-16 주식회사 레고켐 바이오사이언스 인간 dlk1에 대한 항체를 포함하는 항체 약물 접합체 및 이의 용도
KR20210068591A (ko) * 2018-10-31 2021-06-09 주식회사 인투셀 융합된 헤테로시클릭 벤조디아제핀 유도체 및 그의 용도

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WO1997044000A2 (en) * 1996-05-23 1997-11-27 Panorama Research, Inc. Dna-binding indole derivatives, their prodrugs and immunoconjugates as anticancer agents
WO2014080251A1 (en) * 2012-11-24 2014-05-30 Hangzhou Dac Biotech Co., Ltd. Hydrophilic linkers and their uses for conjugation of drugs to cell binding molecules
KR20190064619A (ko) * 2016-10-14 2019-06-10 메디뮨 리미티드 피롤로벤조디아제핀 컨쥬게이트
KR20210068591A (ko) * 2018-10-31 2021-06-09 주식회사 인투셀 융합된 헤테로시클릭 벤조디아제핀 유도체 및 그의 용도
KR20200107837A (ko) * 2019-03-06 2020-09-16 주식회사 레고켐 바이오사이언스 인간 dlk1에 대한 항체를 포함하는 항체 약물 접합체 및 이의 용도

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12274898B2 (en) 2018-10-31 2025-04-15 Intocell, Inc. Substituted benzodiazepines as antibody-drug conjugates
WO2025155901A1 (en) 2024-01-19 2025-07-24 24M Technologies, Inc. Systems and methods for preparing semi-solid electrodes
WO2025214466A1 (en) * 2024-04-12 2025-10-16 BeiGene Guangzhou Biologics Manufacturing Co., Ltd. Targeted pyrrolobenzodiazapine conjugates
WO2026059501A1 (en) * 2024-09-13 2026-03-19 Hummingbird Bioscience Pte. Ltd. Linker moieties

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