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WO2025064424A9 - Tandem-cleavage linkers for antibody-drug conjugates and uses thereof - Google Patents

Tandem-cleavage linkers for antibody-drug conjugates and uses thereof Download PDF

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Publication number
WO2025064424A9
WO2025064424A9 PCT/US2024/047096 US2024047096W WO2025064424A9 WO 2025064424 A9 WO2025064424 A9 WO 2025064424A9 US 2024047096 W US2024047096 W US 2024047096W WO 2025064424 A9 WO2025064424 A9 WO 2025064424A9
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substituted
alkyl
aryl
alkenyl
heteroaryl
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WO2025064424A1 (en
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Penelope M. DRAKE
Stepan Chuprakov
Ayodele O. OGUNKOYA
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RP Scherer Technologies LLC
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RP Scherer Technologies LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • 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
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/6861—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 the tumour determinant being from kidney or bladder cancer 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
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment

Definitions

  • ADCs Antibody-drug conjugates
  • FIG. 1 the concept of an ADC can be displayed in a simple drawing (FIG. 1), the details of ADC construction and how ADC composition relates to function - including efficacy and tolerability - are highly complex.
  • practical concerns including both translatability of preclinical to clinical outcomes and compound manufacturability/stability impact which concepts are able to successfully be incorporated into therapeutic drugs. Indeed, the field’s understanding of how best to construct, produce, and use ADCs is still evolving.
  • the present disclosure provides cleavable linkers for antibody-drug conjugates (ADCs) where the linker includes a tandem-cleavage moiety.
  • ADCs antibody-drug conjugates
  • the disclosure also encompasses compounds and methods for production of such ADCs, as well as methods of using the ADCs.
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • W 2 is an antibody
  • X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
  • R 1 is H and R 2 is alkyl or substituted alkyl.
  • k1 is 2.
  • R 3 is a chemically-cleavable moiety.
  • R 3 is an enzymatically-cleavable moiety.
  • R 3 is a glycoside or a glycoside derivative.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
  • X comprises a maleimide
  • the native amino acid residue of the antibody comprises a cysteine residue
  • the conjugate is of formula (II): wherein: each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 2 is an antibody.
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • W 2 is an antibody.
  • L A comprises:
  • T 1 -V 1 a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f -
  • a, b, c, d, e and f are each independently 0 or 1
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, hctcroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH) m -,
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • L A includes the following:
  • T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , - (CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 )q-, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 - , -SO 2 NR 15 -, -NR 15 SO 2 -, and -P(O)OH-; wherein:
  • (PEG) n is , where n is an integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1 ;
  • each R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring.
  • the conjugate comprises: wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-; and b, c, d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is an amino acid analog and V 2 is -NH-;
  • T 3 is (PEG) n and V 3 is -CO-; and d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-;
  • T 2 is substituted (C 1 -C 12 )alkyl and V 2 is -CO-; and c, d, e and f are each 0.
  • one of T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 ,V 5 or V 6 is a branched group.
  • the branched group is selected from -CONR 15 - and 4AP.
  • the branched group is attached to a second linker, L B .
  • the second linker L B comprises:
  • T 7 , T 8 , T 9 , T 10 , T 11 and T 12 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, hctcroaryl, substituted hctcroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH)
  • V 7 , V 8 , V 9 , V 10 ,V 11 and V 12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • L B is attached to a compound of formula (III): wherein: each R 4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R 5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
  • R 6 comprises a cleavable moiety
  • W 1a is a drug.
  • R 4 is H and R 5 is alkyl or substituted alkyl.
  • k2 is 2.
  • R 6 is a chemically-cleavable moiety.
  • R 6 is an enzymatically-cleavable moiety.
  • R 6 is a glycoside or a glycoside derivative.
  • T 1 is (C 1 -C 12 )alkyl and V 1 is a branched group (-CONR 15 )-;
  • T 2 is (C 1 -C 12 )alkyl and V 2 is -CONH-;
  • T 3 is substituted (C 1 -C 12 )alkyl and V 3 is -CO-; d, e and f are each 0;
  • T 7 is (C 1 -C 12 )alkyl and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (PEG) n and V 3 is -CONH-;
  • T 4 is substituted (C 1 -C 12 )alkyl and V 4 is -CO-; e and f are each 0;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (C 1 -C 12 )alkyl and V 3 is absent;
  • T 4 is heteroaryl (triazole) and V 4 is absent;
  • T 5 is (C 1 -C 12 )alkyl and V 5 is -CONH-;
  • T 6 is substituted (C 1 -C 12 )alkyl and V 6 is -CO-;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0.
  • W 1 and W 1a are the same drug.
  • W 1 and W 1a are different drugs.
  • the conjugate is selected from:
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug; and X is a conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody.
  • R 1 is H and R 2 is alkyl or substituted alkyl.
  • k1 is 2.
  • R 3 is a chemically-cleavable moiety.
  • R 3 is an enzymatically-cleavable moiety.
  • R 3 is a glycoside or a glycoside derivative.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
  • X comprises a maleimide
  • the native amino acid residue of the antibody comprises a cysteine residue
  • the compound is of formula (V): wherein: each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, hctcrocyclyl, and substituted hctcrocyclyl; k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • L A comprises:
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) U , (AA) P , -(CR 13 OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • L A includes the following:
  • T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , - (CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
  • each R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring.
  • the compound comprises: wherein:
  • T 3 is (PEG) n and V 3 is -CO-; and d, e and f are each 0; or wherein:
  • T 2 is substituted (C 1 -C 12 )alkyl and V 2 is -CO-; and c, d, e and f are each 0.
  • the branched group is selected from -CONR 15 - and 4AP.
  • the branched group comprises a second linker, L B .
  • second linker L B comprises: -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -, wherein g, h, i, j, k and l are each independently 0 or 1;
  • T 7 , T 8 , T 9 , T 10 , T 11 and T 12 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH) 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to
  • V 7 , V 8 , V 9 , V 10 ,V 11 and V 12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • L B is attached to a compound of formula (VI): wherein: each R 4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R 5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
  • R 6 comprises a cleavable moiety
  • W 1a is a drug.
  • R 4 is H and R 5 is alkyl or substituted alkyl.
  • k2 is 2.
  • R 6 is a chemically-cleavable moiety.
  • R 6 is an enzymatically-cleavable moiety.
  • R 6 is a glycoside or a glycoside derivative.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • the compound comprises: wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is a branched group (-CONR 15 )-;
  • T 2 is (C 1 -C 12 )alkyl and V 2 is -CONH-;
  • T 3 is substituted (C 1 -C 12 )alkyl and V 3 is -CO-; d, e and f are each 0;
  • T 7 is (C 1 -C 12 )alkyl and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (PEG) n and V 3 is -CONH-;
  • T 4 is substituted (C 1 -C 12 )alkyl and V 4 is -CO-; e and f are each 0;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (C 1 -C 12 )alkyl and V 3 is absent;
  • T 4 is heteroaryl (triazole) and V 4 is absent;
  • T 5 is (C 1 -C 12 )alkyl and V 5 is -CONH-;
  • T 6 is substituted (C 1 -C 12 )alkyl and V 6 is -CO-;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0.
  • W 1 and W 1a are the same drug.
  • W 1 and W 1a are different drugs.
  • the conjugate is selected from:
  • aspects of the present disclosure include a method comprising administering to a subject a conjugate according to the present disclosure.
  • FIG. 5 shows a drawing of a tandem-cleavage linker requires two consecutive enzymatic steps to occur in order to release the drug after internalization of ADC.
  • FIG. 6 shows an illustration of tandem-cleavage linker that prevents release of cytotoxic payload in circulation in the event of retro-Michael deconjugation of the maleimide- thioether linkage.
  • FIG. 7 shows examples of branched maleimide linkers designs that allow to conjugate more than one molecule of payload in single step.
  • FIG. 9 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 38.
  • FIG. 13 shows a SEC assessment of Trastuzumab antibody conjugated to Compound 38.
  • FIG. 19 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 36.
  • FIG. 20 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 34.
  • FIG. 21 shows a HIC assessment of Enhertu (trastuzumab deruxtecan).
  • FIG. 22 shows Trastuzumab antibody conjugated to Compound 36 is 97% monomeric as determined by SEC.
  • FIG. 26 shows in vitro stability of trastuzumab MMAE conjugates incubated at 37° C in rat serum for up to seven days.
  • FIG. 30 shows Trastuzumab antibody conjugated to Compound 37 yields a DAR of 7.55 as determined by PLRP.
  • FIG. 38 shows a scheme illustrating that an N-terminal amine can be oxidized with N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport’s salt, RS) to afford an aldehyde or ketone tagged antibody, followed by conjugation using an oxime or hydrazone linkage, according to embodiments of the present disclosure.
  • Rapoport N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt
  • FIG. 40 shows a graph illustrating efficacy of trastuzumab MMAE conjugates against the HER2+ NCI-N87 gastric tumor xenograft model, according to embodiments of the present disclosure.
  • FIG. 41 shows a schematic depicting ELISA assay set up for analysis of total antibody (left) and total ADC concentrations (right) in rat plasma, according to embodiments of the present disclosure.
  • FIG. 42 shows a graph of rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
  • FIG. 43 shows a graph of clinical observations (average sum per animal is shown) from rat toxicity studies comparing enfortumab conjugates carrying different linker-payloads, according to embodiments of the present disclosure.
  • the vedotin conjugate (Compound 38 conjugate) induced clinical observations (mostly fur and skin related) in all of the dosed animals, reaching a maximum of 2.6 average clinical obscrvations/animals on Day 17, when an animal died.
  • no clinical observations were noted in animals dosed with enfortumab conjugated with either Compound 14 or Compound 18.
  • FIG. 46 shows a graph of alanine transaminase levels in rats dosed with enfortumab conjugates or vehicle alone, according to embodiments of the present disclosure.
  • FIG. 47 shows a graph of reticulocyte counts in rats dosed with enfortumab conjugates or vehicle alone, according to embodiments of the present disclosure.
  • FIG. 49 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
  • FIG. 50 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
  • FIG. 51 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
  • FIG. 52 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
  • FIG. 53 shows a graph illustrating the efficacy of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 moderate JIMT-1 breast tumor xenograft model, according to embodiments of the present disclosure.
  • FIG. 54 shows a graph illustrating in vitro potency of enfortumab conjugates according to embodiments of the present disclosure against HEK-293 cells overexpressing rat nectin-4.
  • FIG. 55 shows a graph illustrating in vitro potency of enfortumab conjugates according to embodiments of the present disclosure against HEK-293 cells overexpressing human nectin-4.
  • FIG. 56 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
  • FIG. 57 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line SK-BR- 3.
  • FIG. 58 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
  • FIG. 59 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
  • FIG. 61 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
  • FIG. 62 shows an HPLC trace demonstrating that Compound 14 conjugated to Enfortumab yields a DAR of 3.7 as determined by reduced PLRP..
  • FIG. 65 shows an HPLC trace demonstrating that Compound 18 conjugated to Enfortumab is 98.0% monomeric as determined by analytical SEC.
  • FIG. 66 shows an HPLC trace demonstrating that Compound 36 conjugated to Trastuzumab yields a DAR of 7.84 as determined by reduced PLRP.
  • FIG. 67 shows an HPLC trace demonstrating that Compound 36 conjugated to Trastuzumab is 96.9% monomeric as determined by analytical SEC.
  • FIG. 68 shows an HPLC trace demonstrating that Compound 37 conjugated to Trastuzumab yields a DAR of 7.81 as determined by reduced PLRP.
  • FIG. 69 shows an HPLC trace demonstrating that Compound 37 conjugated to Trastuzumab is 95.5% monomeric as determined by analytical SEC.
  • FIG. 70 shows an HPLC trace demonstrating that Compound 37 conjugated to Ibalizumab yields a DAR of 7.1 as determined by reduced PLRP.
  • FIG. 71 shows an HPLC trace demonstrating that Compound 37 conjugated to Ibalizumab is 98.0% monomeric as determined by analytical SEC.
  • FIG. 72 shows an HPLC trace demonstrating that Compound 42 conjugated to anti-FITC yields a DAR of 7.5 as determined by reduced PLRP.
  • FIG. 73 shows an HPLC trace demonstrating that Compound 42 conjugated to anti-FITC is 96.6% monomeric as determined by analytical SEC.
  • FIG. 74 shows an HPLC trace demonstrating that Compound 46 conjugated to Trastuzumab yields a DAR of 7.9 as determined by reduced PLRP.
  • FIG. 75 shows an HPLC trace demonstrating that Compound 46 conjugated to Trastuzumab is 94.9% monomeric as determined by analytical SEC.
  • FIG. 76 shows an HPLC trace demonstrating that Compound 46 conjugated to anti-FITC yields a DAR of 7.7 as determined by reduced PLRP.
  • FIG. 77 shows an HPLC trace demonstrating that Compound 46 conjugated to anti-FITC is 97.2% monomeric as determined by analytical SEC.
  • FIG. 78 shows an HPLC trace demonstrating that Compound 50 conjugated to Trastuzumab yields a DAR of 7.75 as determined by reduced PLRP.
  • FIG. 79 shows an HPLC trace demonstrating that Compound 50 conjugated to Trastuzumab is 96.0% monomeric as determined by analytical SEC.
  • FIG. 80 shows an HPLC trace demonstrating that Compound 50 conjugated to anti-FITC yields a DAR of 7.82 as determined by reduced PLRP.
  • FIG. 81 shows an HPLC trace demonstrating that Compound 50 conjugated to anti-FITC is 97.8% monomeric as determined by analytical SEC.
  • FIG. 82 shows an HPLC trace demonstrating that Compound 57 conjugated to Trastuzumab yields a DAR of 7.61 as determined by reduced PLRP.
  • FIG. 83 shows an HPLC trace demonstrating that Compound 57 conjugated to Trastuzumab is 95.9% monomeric as determined by analytical SEC.
  • FIG. 84 shows an HPLC trace demonstrating that Compound 57 conjugated to anti-FITC yields a DAR of 7.72 as determined by reduced PLRP.
  • FIG. 85 shows an HPLC trace demonstrating that Compound 57 conjugated to anti-FITC is 97.3% monomeric as determined by analytical SEC.
  • FIG. 86 shows an HPLC trace demonstrating that Compound 60 conjugated to Trastuzumab yields a DAR of 7.8 as determined by reduced PLRP.
  • FIG. 88 shows an HPLC trace demonstrating that Compound 60 conjugated to anti-FITC yields a DAR of 7.7 as determined by reduced PLRP.
  • FIG. 90 shows an HPLC trace demonstrating that Compound 61 conjugated to Trastuzumab yields a DAR of 3.9 as determined by reduced PLRP.
  • FIG. 91 shows an HPLC trace demonstrating that Compound 61 conjugated to Trastuzumab is 97.1% monomeric as determined by analytical SEC.
  • FIG. 92 shows an HPLC trace demonstrating that Compound 61 conjugated to anti-FITC yields a DAR of 3.9 as determined by reduced PLRP.
  • FIG. 95 shows an HPLC trace demonstrating that Compound 65 conjugated to Trastuzumab is 95.3% monomeric as determined by analytical SEC.
  • FIG. 96 shows an HPLC trace demonstrating that Compound 65 conjugated to Trastuzumab yields a DAR of 6.9 as determined by reduced PLRP.
  • FIG. 99 shows an HPLC trace demonstrating that Compound 62 conjugated to anti-FITC is 98.7% monomeric as determined by analytical SEC.
  • FIG. 100 shows an HPLC trace demonstrating that Compound 62 conjugated to Trastuzumab yields a DAR of 3.8 as determined by reduced PLRP.
  • FIG. 105 shows an HPLC trace demonstrating that Compound 66 conjugated to anti-FITC is 97.0% monomeric as determined by analytical SEC.
  • FIG. 108 shows an HPLC trace demonstrating that Compound 59 conjugated to Trastuzumab yields a DAR of 7.06 as determined by reduced PLRP.
  • FIG. 109 shows an HPLC trace demonstrating that Compound 59 conjugated to Trastuzumab is 95.8% monomeric as determined by analytical SEC.
  • Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms.
  • This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CFE CH ), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 )2CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -), and neopentyl ((CH 3 ) 3 CCH 2 -).
  • linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CFE CH ), n-butyl
  • Alkylene refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 - and the like.
  • This term includes, by way of example, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), iso-propylene (-CH 2 CH(CH 3 )-), (-C(CH 3 ) 2 CH 2 CH 2 -), (-C(CH 3 ) 2 CH 2 C(O)-), (-C(CH 3 ) 2 CH 2 C(O)NH-), (-CH(CH 3 )CH 2 -), and the like.
  • “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below.
  • alkane refers to alkyl group and alkylene group, as defined herein.
  • alkylaminoalkyl refers to the groups R’NHR”- where R’ is alkyl group as defined herein and R” is alkylene, alkenylene or alkynylene group as defined herein.
  • alkaryl or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.
  • Alkoxy refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec- butoxy, n-pentoxy, and the like.
  • alkoxy also refers to the groups alkenyl-O-, cycloalky l-O- , cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
  • substituted alkoxy refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
  • alkoxyamino refers to the group -NH-alkoxy, wherein alkoxy is defined herein.
  • haloalkoxy refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.
  • haloalkyl refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group.
  • Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like.
  • alkylalkoxy refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
  • alkylthioalkoxy refers to the group -alkylene-S-alkyl, alkylene-S- substitutcd alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substitutcd alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
  • Alkenyl refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi- vinyl, allyl, and but-3-en-1-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.
  • Alkynyl refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C ⁇ CH), and propargyl (-CH 2 C ⁇ CH).
  • substituted alkynyl refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, al
  • Alkynyloxy refers to the group -O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, cthynyloxy, propynyloxy, and the like.
  • Acyl refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl- C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkenyl-C(
  • “Acylamino” refers to the groups -NR 20 C(O)alkyl, -NR 20 C(O)substituted alkyl, N R 20 C(O)cycloalkyl, -NR 20 C(O)substituted cycloalkyl, - NR 20 C(O)cycloalkenyl, -NR 20 C(O) substituted cycloalkenyl, -NR 20 C(O)alkenyl, - NR 20 C(O)substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O)substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O)substituted aryl, -NR 20 C(O)heteroaryl, -NR 20 C(O) substituted heteroaryl, -NR 20 C(O)heterocyclic, and -NR 20 C(
  • Aminocarbonyl or the term “aminoacyl” refers to the group -C(O)NR 21 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
  • Aminocarbonylamino refers to the group -NR 21 C(O)NR 22 R 23 where R 21 , R 22 , and R 23 arc independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.
  • alkoxycarbonylamino refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
  • acyloxy refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
  • Aminosulfonyl refers to the group -SO 2 NR 21 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,
  • “Sulfonylamino” refers to the group -NR 21 SO 2 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 21 and R 22 are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted
  • Aryl refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl.
  • such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thi
  • Aryloxy refers to the group -O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.
  • Amino refers to the group -NH 2 .
  • substituted amino refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.
  • azido refers to the group -N 3 .
  • Carboxyl refers to -CO 2 H or salts thereof.
  • Carboxyl ester or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(C(O)O
  • (Carboxyl ester)oxy” or “carbonate” refers to the groups -O-C(O)O- alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O- C(O)O-alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O- C(O)O-cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O- substituted cycloalkenyl, -O-C(O)O-heteroaryl,
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems.
  • suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
  • substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,
  • Cycloalkenyl refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.
  • substituted cycloalkenyl refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,
  • Cycloalkynyl refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.
  • Halo or “halogen” refers to fluoro, chloro, bromo, and iodo.
  • any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent group, e.g., an alkyl group or other substituent as described herein.
  • the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N- oxide (N ⁇ O), sulfinyl, or sulfonyl moieties.
  • N ⁇ O N- oxide
  • sulfinyl sulfonyl moieties.
  • This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1 ,2,3,4- tetrahydroiso
  • heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hctcroaryl, hctcroaryloxy, hctcrocyclyl, hctcrocyclyl, hctcrocycl
  • Heterocyclyloxy refers to the group -O-heterocyclyl.
  • heterocyclylthio refers to the group heterocyclic-S-.
  • heterocyclene refers to the diradical group formed from a heterocycle, as defined herein.
  • hydroxyamino refers to the group -NHOH.
  • Niro refers to the group -NO 2 .
  • “Sulfonyloxy” refers to the group -OSO 2 -alkyl, -OSCT-substituted alkyl, -OSO 2 - alkenyl, -OSO 2 -substituted alkenyl, -OSO 2 -cycloalkyl, -OSO 2 -substituted cylcoalkyl, -OSO 2 - cycloalkenyl, -OSO 2 -substituted cylcoalkenyl, -OSO 2 -aryl, -OSO 2 -substituted aryl, -OSO 2 - heteroaryl, -OSO 2 -substituted heteroaryl, -OSO 2 -heterocyclic, and -OSO 2 -substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl
  • “Sulfate” or “sulfate ester” refers the group -O-SO 2 -OH, -O-SO 2 -O-alkyl, -O-SO 2 -O- substituted alkyl, -O-SO 2 -O-alkenyl, -O-SO 2 -O-substituted alkenyl, -O-SO 2 -O-cycloalkyl, -O- SO 2 -O- substituted cylcoalkyl, -O-SO 2 -O-cycloalkenyl, -O-SO 2 -O-substituted cylcoalkenyl, -O- SO 2 -O-aryl, -O-SO 2 -O-substituted aryl, -O-SO 2 -O-heteroaryl, -O-SO 2 -O-substituted heteroaryl, - O-SO 2 -O-
  • aminocarbonyloxy refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
  • Thiol refers to the group -SH.
  • Alkylthio or the term “thioalkoxy” refers to the group -S-alkyl, wherein alkyl is as defined herein.
  • sulfur may be oxidized to -S(O)-.
  • the sulfoxide may exist as one or more stereoisomers.
  • substituted thioalkoxy refers to the group -S-substituted alkyl.
  • thioaryloxy refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.
  • heteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.
  • heterocyclooxy refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.
  • substituted when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
  • Each M + may independently be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 )4; or an alkaline earth ion, such as [Ca 2+ ]o.s, [Mg 2+ ]o.5, or [Ba 2+ ]o.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions).
  • an alkali ion such as K + , Na + , Li +
  • an ammonium ion such as + N(R 60 )4
  • -NR 80 R 80 is meant to include -NH 2 , -NH-alkyl, A-pyrrolidinyl, /V-pipcrazinyl, 4/V-mcthyl-pipcrazin- 1 -yl and N- morpholinyl.
  • substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O-M + , -OR 70 , -SR 70 , -S M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO 2 , -N 3 , -SO 2 RTM, -SO 3 M + , -SO 3 R 70 , -OSO 2 R 70 , -OSO 3 M + , -OSO 3 R 70 , -PO 3 -2 (M + ) 2 , -P(O)(OR 70 )O M + , -P(O)(OR 70 ) 2 , -C(O)R 70 , -C(S)R 70
  • substituent groups for hydrogens on nitrogen atoms in “substituted” hctcroalkyl and cyclohetero alkyl groups are, unless otherwise specified, -R 60 , -O-M + , -ORTM, -SR 70 , -S-M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R 70 , -S(O) 2 O M + , -S(O) 2 OR 70 , -OS(O) 2 R 70 , -OS(O) 2 -O-M + .
  • a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
  • substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment.
  • substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.
  • any of the groups disclosed herein which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
  • pharmaceutically acceptable salt means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
  • “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.
  • salt thereof means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like.
  • the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient.
  • salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
  • solvent refers to a complex formed by combination of solvent molecules with molecules or ions of the solute.
  • the solvent can be an organic compound, an inorganic compound, or a mixture of both.
  • Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
  • Stereoisomers refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
  • pyrazoles imidazoles, benzimidazoles, triazoles, and tetrazoles.
  • a pharmaceutically or therapeutically effective amount refers to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder.
  • a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.
  • “Patient” refers to human and non-human subjects, especially mammalian subjects.
  • the term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.
  • polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless specifically indicated otherwise, “polypeptide,” “peptide,” and “protein” can include genetically coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, proteins which contain at least one N-terminal methionine residue (e.g., to facilitate production in a recombinant host cell); immunologically tagged proteins; and the like.
  • a polypeptide is an antibody.
  • Native amino acid sequence or “parent amino acid sequence” are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include at least one modified amino acid residue.
  • “Native amino acid” or “native amino acid residue” are used interchangeably herein to refer to natural amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Vai or V, Trp or W, Tyr or Y).
  • a native amino acid can be positioned in its naturally occurring position in a native amino acid sequence.
  • amino acid analog may be used interchangeably, and include amino acid-like compounds that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Vai or V, Trp or W, Tyr or Y).
  • Amino acid analogs also include natural amino acids with modified side chains or backbones.
  • Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs.
  • the amino acid analogs share backbone structures, and/or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule.
  • modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof.
  • amino acid analogs may include ⁇ - hydroxy acids, and ⁇ -amino acids, and the like.
  • Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.
  • amino acid side chain or “side chain of an amino acid” and the like may be used to refer to the substituent attached to the ⁇ -carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs.
  • An amino acid side chain can also include an amino acid side chain as described in the context of the modified amino acids and/or conjugates described herein.
  • carbohydrate and the like may be used to refer to monomers units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
  • sugar may be used to refer to the smaller carbohydrates, such as monosaccharides, disaccharides.
  • carbohydrate derivative includes compounds where one or more functional groups of a carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemistry) or absent (e.g., eliminated or replaced by H).
  • a variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates.
  • glycoside refers to a sugar molecule or group bound to a moiety via a glycosidic bond.
  • the moiety that the glycoside is bound to can be a cleavable linker as described herein.
  • a glycosidic bond can link the glycoside to the other moiety through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O- glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl).
  • glycosides can be cleaved from the moiety they are attached to, such as by chemically-mediated hydrolysis or enzymatically- mediated hydrolysis.
  • antibody is used in the broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), and the like.
  • An antibody is capable of binding a target antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
  • a target antigen can have one or more binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) formed by one or more variable regions of an antibody.
  • CDRs complementarity determining regions
  • natural antibody refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism.
  • Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies.
  • the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies.
  • humanized antibody or “humanized immunoglobulin” refers to a non- human (e.g., mouse or rabbit) antibody containing one or more amino acids (in a framework region, a constant region or a CDR, for example) that have been substituted with a correspondingly positioned amino acid from a human antibody.
  • humanized antibodies produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.
  • framework substitutions are identified by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No.
  • a subject rabbit antibody may be humanized according to the methods set forth in US20040086979 and US20050033031. Accordingly, the antibodies described above may be humanized using methods that are well known in the art.
  • chimeric antibodies refer to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.
  • variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3.
  • An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although domains from other mammalian species may be used.
  • An immunoglobulin polypeptide immunoglobulin light or heavy chain variable region is composed of a framework region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”.
  • the extent of the framework region and CDRs have been defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, 1991).
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • substantially purified refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated.
  • physiological conditions is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.
  • N-terminus refers to the terminal amino acid residue of a polypeptide having a free amine group, which amine group in non-N-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide.
  • one moiety is conjugated to an amino acid residue of the polypeptide.
  • two moieties may be conjugated to the same amino acid residue of the polypeptide.
  • a first moiety is conjugated to a first amino acid residue of the polypeptide and a second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example where a polypeptide is conjugated to a first moiety at a first amino acid residue and conjugated to two other moieties at a second amino acid residue.
  • two amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, three amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, four amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, five amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein.
  • the conjugation moiety includes an N-hydroxysuccinimide ester (NHS ester) or derivative thereof.
  • NHS ester N-hydroxysuccinimide ester
  • an NHS-ester conjugation moiety may be conjugated to a lysine residue of the polypeptide (e.g., antibody), such as a native lysine residue of the polypeptide (e.g., antibody), thus indirectly binding the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) together through an NHS-ester conjugation moiety.
  • the linker is a branched linker, such as a branched linker as described herein.
  • the conjugation moieties may be attached (e.g., covalently attached) to two or more linkers.
  • embodiments of the present disclosure include a conjugation moiety attached to two or more drugs or active agents each through a corresponding linker.
  • conjugates of the present disclosure may include two or more linkers, where each linker attaches a corresponding drug or active agent to the conjugation moiety.
  • the conjugation moiety and two or more linkers may be viewed overall as a “branched linker”, where the conjugation moiety is attached to two or more “branches”, where each branch includes a linker attached to a drug or active agent.
  • the reactive partner-containing drug may include a conjugation moiety as described above.
  • a drug or active agent may be modified to include a conjugation moiety.
  • the drug or active agent is attached to a conjugation moiety, such as covalently attached to conjugation moiety through a linker, such as a linker as described in detail herein.
  • the conjugate includes a polypeptide (e.g., an antibody) having at least one native amino acid residue attached to a linker as described herein, which in turn is attached to one or more drugs or active agents.
  • the conjugate may include a polypeptide (e.g., an antibody) having at least one native amino acid residue that is conjugated to the one or more moieties of interest (e.g., one or more drugs or active agents) as described above.
  • L A is a first linker
  • X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
  • R 2 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C& aryl or C 6 substituted aryl.
  • R 2 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
  • R 1 is H and R 2 is alkyl or substituted alkyl.
  • R 1 is H and R 2 is methyl; or R 1 is H and R 2 is isopropyl.
  • k1 is an integer from 1 to 10. In certain embodiments, k1 is 1. In certain embodiments, k1 is 2. In certain embodiments, k1 is 3. In certain embodiments, k1 is 4. In certain embodiments, k1 is 5. In certain embodiments, k1 is 6. In certain embodiments, k1 is 7. In certain embodiments, k1 is 8. In certain embodiments, k1 is 9. In certain embodiments, k1 is 10.
  • R 3 comprises a cleavable moiety, as described in more detail herein.
  • R 3 can be a chemically-cleavable moiety, as described in more detail herein.
  • R 3 can be an enzymatically-cleavable moiety, as described in more detail herein (e.g., a glycoside or a glycoside derivative).
  • W 1 is a drug (or active agent). Further description of drugs (or active agents) that find use in the subject conjugates is found in the disclosure herein.
  • W 2 is a polypeptide.
  • W 2 can be an antibody.
  • W 2 comprises one or more amino acid residues (e.g., native amino acid residues) that are attached to the drug, W 1 , through the linker, L, as described herein.
  • the polypeptide e.g., antibody
  • the polypeptide is attached to the rest of the conjugate through a native amino acid residue as described herein. Further description of polypeptides and antibodies that find use in the subject conjugates is found in the disclosure herein.
  • X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
  • the antibody and the drug are conjugated through the conjugation moiety, X.
  • the antibody and the drug may each be bound (e.g., covalently bonded) to the conjugation moiety, X, thus indirectly binding the antibody and the drug together through the conjugation moiety, X.
  • conjugation groups that may be used for attachment to a cysteine residue of the antibody include those exemplified in FIG. 34 herein.
  • Other conjugation groups that may be used for attachment to a cysteine residue of the antibody include P5 phosphoramidate or thiobridge conjugation moieties, as shown in FIG. 35 and FIG. 36, respectively.
  • the conjugation moiety, X includes an N-hydroxysuccinimide (NHS) or derivative thereof.
  • NHS N-hydroxysuccinimide
  • an NHS-ester conjugation moiety may react with and thus be attached (e.g., covalently attached) to a lysine residue of the antibody, such as a native lysine residue of the antibody, thus indirectly binding the antibody and the drug together through an NHS-ester conjugation moiety.
  • Examples of NHS-ester conjugation moieties are exemplified in FIG. 37 herein.
  • conjugation groups that may be used for attachment include conjugation moieties for attachment to an N-terminal amine of the antibody.
  • an N-terminal amine can be oxidized with N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport’s salt, RS) to afford an aldehyde or ketone tagged antibody, followed by conjugation using an oxime or hydrazone linkage.
  • Rapoport’s salt, RS N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt
  • An example of conjugation to an N-terminal amine is illustrated in FIG. 38 herein.
  • the conjugate of formula (I) includes a linker, L A .
  • the linker may be utilized to bind one or more moieties of interest (e.g., drug or active agent) to one or more polypeptides through the conjugation moiety, X.
  • the linker may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein) at any convenient position.
  • the linker may attach the conjugation moiety to a drug.
  • the conjugation moiety may be used to conjugate the linker (and thus the drug) to a polypeptide, such as an antibody.
  • the conjugation moiety may be used to conjugate the linker (and thus the drug) to a native amino acid residue of the antibody, as described herein.
  • L A is attached to W 2 through the conjugation moiety, X, and thus W 2 is indirectly bonded to the linker L A through the conjugation moiety.
  • W 2 is an antibody, and thus L A is attached through the conjugation moiety, X, to the antibody, e.g., the linker L A is indirectly bonded to the antibody through the conjugation moiety, X.
  • linker L A may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • the linker L A may include an alkyl or substituted alkyl group. In certain embodiments, the linker L A may include an alkenyl or substituted alkenyl group. In certain embodiments, the linker L A may include an alkynyl or substituted alkynyl group. In certain embodiments, the linker L A may include an alkoxy or substituted alkoxy group. In certain embodiments, the linker L A may include an amino or substituted amino group. In certain embodiments, the linker L A may include a carboxyl or carboxyl ester group. In certain embodiments, the linker L A may include an acyl amino group. In certain embodiments, the linker L A may include an alkylamide or substituted alkylamide group.
  • the linker L A may include an aryl or substituted aryl group. In certain embodiments, the linker L A may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the linker L A may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the linker L A may include a heterocyclyl or substituted hctcrocyclyl group.
  • the linker L A may include a polymer.
  • the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone, combinations thereof, and the like.
  • the polymer is a polyalkylene glycol.
  • the polymer is a polyethylene glycol.
  • Other linkers are also possible, as shown in the conjugates and compounds described in more detail below.
  • L A is a linker (e.g., a first linker) described by the formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, wherein L 1 , L 2 , L 3 , L 4 , L 5 and L 6 are each independently a linker subunit, and a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.
  • a linker e.g., a first linker
  • the sum of a, b, c, d, e and f is 1. In certain embodiments, the sum of a, b, c, d, e and f is 2. In certain embodiments, the sum of a, b, c, d, e and f is 3. In certain embodiments, the sum of a, b, c, d, e and f is 4. In certain embodiments, the sum of a, b, c, d, e and f is 5. In certain embodiments, the sum of a, b, c, d, e and f is 6. In certain embodiments, a, b, c, d, e and f are each 1.
  • a, b, c, d and e are each 1 and f is 0. In certain embodiments, a, b, c and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e and f are each 0. In certain embodiments, a and b are each 1 and c, d, e and f are each 0. In certain embodiments, a is 1 and b, c, d, e and f are each 0.
  • the linker subunit L 1 is attached to the conjugation moiety, X (e.g., as shown in formula (I) above).
  • the linker subunit L 2 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • the linker subunit L 3 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • the linker subunit L 4 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • the linker subunit L 5 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L 6 , if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • Linker subunits may be utilized in the linker L A .
  • Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and poly acrylates, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof.
  • each of L 1 , L 2 , L 3 , L 4 , L 5 and L 6 (if present) comprise one or more groups independently selected from a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, and a diamine (e.g., a linking group that includes an alkylene diamine).
  • L 1 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine.
  • L 1 comprises a polyethylene glycol.
  • L 1 comprises a modified polyethylene glycol.
  • L 1 comprises an amino acid residue.
  • L 1 comprises an alkyl group or a substituted alkyl.
  • L 1 comprises an aryl group or a substituted aryl group.
  • L 1 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 2 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine.
  • L 2 comprises a polyethylene glycol.
  • L 2 comprises a modified polyethylene glycol.
  • L 2 comprises an amino acid residue.
  • L 2 comprises an alkyl group or a substituted alkyl.
  • L 2 comprises an aryl group or a substituted aryl group.
  • L 2 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 3 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine.
  • L 3 comprises a polyethylene glycol.
  • L 3 comprises a modified polyethylene glycol.
  • L 3 comprises an amino acid residue.
  • Tn some embodiments, L 3 comprises an alkyl group or a substituted alkyl.
  • L 3 comprises an aryl group or a substituted aryl group.
  • L 3 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 4 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine.
  • L 4 comprises a polyethylene glycol.
  • L 4 comprises a modified polyethylene glycol.
  • L 4 comprises an amino acid residue.
  • L 4 comprises an alkyl group or a substituted alkyl.
  • L 4 comprises an aryl group or a substituted aryl group.
  • L 4 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 5 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine.
  • L 5 comprises a polyethylene glycol.
  • L 5 comprises a modified polyethylene glycol.
  • L 5 comprises an amino acid residue.
  • L 5 comprises an alkyl group or a substituted alkyl.
  • L 5 comprises an aryl group or a substituted aryl group.
  • L 5 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 6 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine.
  • L 6 comprises a polyethylene glycol.
  • L 6 comprises a modified polyethylene glycol.
  • L 6 comprises an amino acid residue.
  • L 6 comprises an alkyl group or a substituted alkyl.
  • L 6 comprises an aryl group or a substituted aryl group.
  • L 6 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L A is a linker comprising -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e - (L 6 ) f -, where:
  • -(L 1 ) a - is -(T 1 -V 1 )a-;
  • -(L 2 ) b - is -(T 2 -V 2 ) b -;
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 if present, are tether groups;
  • V 1 , V 2 , V 3 , V 4 , V 5 and V 6 are covalent bonds or linking functional groups; and a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.
  • L 1 is attached to the conjugation moiety, X (e.g., as shown in formula (I) above).
  • T 1 is attached to the conjugation moiety, X (e.g., as shown in formula (I) above).
  • V 1 is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • L 2 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • T 2 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above), or V 2 , if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • L 3 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • T 3 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above), or V 3 , if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • L 4 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • T 4 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above), or V 4 , if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • L 5 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • T 5 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above), or V 5 , if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • L 6 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • T 6 if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above), or V 6 , if present, is attached to the -NR 1 - group (e.g., as shown in formula (I) above).
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 any convenient tether groups may be utilized in the subject linkers.
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 each comprise one or more groups independently selected from a covalent bond, a (C 1 -C 12 )alkyl, a substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, hctcroaryl, substituted hctcroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) p , - (CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, where each
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and/or T 6 ) includes a (C 1 -C 12 )alkyl or a substituted (C 1 -C 12 )alkyl.
  • (C 1 -C 12 )alkyl is a straight chain or branched alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms.
  • (C 1 -C 12 )alkyl may be an alkyl or substituted alkyl, such as C 1 -C 12 alkyl, or C 1 -C 10 alkyl, or C 1 -C 6 alkyl, or C 1 -C 3 alkyl. In some instances, (C 1 -C 12 )alkyl is a C 2 -alkyl.
  • (C 1 -C 12 )alkyl may be an alkylene or substituted alkylene, such as C 1 -C 12 alkylene, or C 1 -C 10 alkylene, or C 1 -C 6 alkylene, or C 1 -C 3 alkylene.
  • (C 1 -C 12 )alkyl is a C 2 -alkylene (e.g., CH 2 CH 2 ). In some instances, (C 1 -C 12 )alkyl is a C 3 -alkylene (e.g., CH 2 CH 2 CH 2 ).
  • substituted (C 1 -C 12 )alkyl may include C 1 -C 12 alkylene (e.g., C 3 -alkylene or C 5 -alkylene) substituted with a (PEG) n group as described herein (e.g.,-CONH(PEG) 3 or -NHCO(PEG) 7 ), or may include C 1 -C 12 alkylene (e.g., C 3 - alkylene) substituted with a -CONHCH 2 CH 2 SO 3 H group, or may include C 1 -C 12 alkylene (e.g., C 5 -alkylene) substituted with a -NHCOCH 2 SO 3 H group.
  • C 1 -C 12 alkylene e.g., C 3 -alkylene or C 5 -alkylene substituted with a -NHCOCH 2 SO 3 H group.
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and/or T 6 ) includes an aryl, substituted aryl, hctcroaryl, substituted hctcroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and T 6 ) includes an aryl or substituted aryl.
  • the aryl can be phenyl.
  • the substituted aryl is a substituted phenyl.
  • the substituted phenyl can be substituted with one or more substituents selected from (C 1 -C 12 )alkyl, a substituted (C 1 - C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • the substituted aryl is a substituted phenyl, where the substituent includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and/or T 6 ) includes a heteroaryl or substituted heteroaryl. In some instances, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and T 6 ) includes a cycloalkyl or substituted cycloalkyl. In some instances, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and T 6 ) includes a heterocyclyl or substituted heterocyclyl.
  • the substituent on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclyl includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and/or T 6 ) includes an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, where the aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl is produced from a reaction, such as a click-chemistry reaction.
  • click- chemistry reactions may be used to connect two portions of a linker together during synthesis of the linker.
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and/or T 6 ) includes an ethylene diamine (EDA) moiety, e.g., an EDA containing tether group.
  • EDA ethylene diamine
  • W includes one or more EDA moieties, such as where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1 , 2, 3, 4, 5 or 6).
  • the linked ethylene diamine (EDA) moictics may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or a substituted aryl.
  • each R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl and a substituted aryl.
  • any two adjacent R 12 groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring.
  • y is 1 and the two adjacent R 12 groups are an alkyl group, cyclically linked to form a piperazinyl ring.
  • R 12 includes a polyethylene glycol moiety described by the formula: (PEG) k , which may be represented by the structure: where k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, k is 2.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and/or T 6 ) includes (AA) P , where AA is an amino acid residue.
  • Any convenient amino acids may be utilized.
  • Amino acids of interest include but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 primary alpha-amino acids and beta-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), such as a non-naturally occurring alpha-amino acid or a non-naturally occurring beta-amino acid, etc.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 and/or T 6 ) includes an amino acid analog.
  • Amino acid analogs include compounds that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Vai or V, Trp or W, Tyr or Y).
  • Naturally occurring proteins e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q,
  • Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and/or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule.
  • R 13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 13 is hydrogen.
  • R 13 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl.
  • R 13 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl.
  • R 13 is alkynyl or substituted alkynyl.
  • R 13 is alkoxy or substituted alkoxy.
  • R 13 is amino or substituted amino. In certain embodiments, R 13 is carboxyl or carboxyl ester. In certain embodiments, R 13 is acyl or acyloxy. In certain embodiments, R 13 is acyl amino or amino acyl. In certain embodiments, R 13 is alkylamide or substituted alkylamide. In certain embodiments, R 13 is sulfonyl. In certain embodiments, R 13 is thioalkoxy or substituted thioalkoxy.
  • R 13 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C 6 aryl or C 6 substituted aryl.
  • R 13 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
  • R 13 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 13 is heterocyclyl or substituted heterocyclyl, such as C 3-8 heterocyclyl or C 3-8 substituted heterocyclyl, such as a C 3-6 heterocyclyl or C 3-6 substituted heterocyclyl, or a C 3-5 heterocyclyl or C 3-5 substituted heterocyclyl.
  • R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.
  • alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 .
  • linking functional groups V 1 , V 2 , V 3 , V 4 , V 5 and V 6
  • any convenient linking functional groups may be utilized in the linker.
  • Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoraidate, and the like.
  • V 1 , V 2 , V 3 , V 4 , V 5 and V 6 are each independently selected from a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, - CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and - P(O)OH-, where q is an integer from 1 to 6.
  • q is an integer from 1 to 6 (e.g., 1 , 2, 3, 4, 5 or 6). In certain embodiments, q is 1 . In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.
  • each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 15 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl.
  • R 15 is alkynyl or substituted alkynyl. In certain embodiments, R 15 is alkoxy or substituted alkoxy. In certain embodiments, R 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15 is acyl amino or amino acyl. In certain embodiments, R 15 is alkylamide or substituted alkylamide. In certain embodiments, R 15 is sulfonyl. In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy.
  • R 15 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C 6 aryl or C 6 substituted aryl.
  • R 15 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
  • R 15 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 15 is heterocyclyl or substituted heterocyclyl, such as C 3-8 heterocyclyl or C 3-8 substituted heterocyclyl, such as a C 3-6 heterocyclyl or C 3-6 substituted heterocyclyl, or a C 3-5 heterocyclyl or C 3-5 substituted heterocyclyl.
  • each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, hctcrocyclyl, and substituted hctcrocyclyl.
  • alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R 15 .
  • the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.
  • L A is a linker comprising -(T 1 - V 1 ) a - (T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f -, where a, b, c, d, e and f are each independently 0 or 1, where the sum of a, b, c, d, e and f is 1 to 6.
  • T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from (C 1 -C 12 )alkyl, substituted (C 1 - C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH) m -, 4- amino-piperidine (4AP), an acetal group, a disulfide, a hydrazine, and an ester; and
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are each independently selected from a covalent bond, -CO-, - NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, - SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein q is an integer from 1 to 6; wherein:
  • (PEG) n is where n is an integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure:
  • AA is an amino acid residue, where p is an integer from 1 to 20; and each R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 and V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are selected from the following: wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-; and b, c, d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is an amino acid analog and V 2 is -NH-;
  • T 3 is (PEG) n and V 3 is -CO-; and d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-;
  • the conjugate may be of formula (II): wherein: each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
  • L A is a first linker
  • W 1 is a drug
  • the conjugate may be of formula (Ila): wherein: each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • W 2 is an antibody.
  • R 1 , R 2 , k1, R 3 , L A , W 1 and W 2 are as described herein (e.g., as in the description related to formula (I) herein).
  • the antibody can be linked to one drug or active agent through the conjugation moiety and linker.
  • the antibody can be linked to more than one drug or active agent through the conjugation moiety and linker.
  • the conjugation moiety can be linked to two or more drugs or active agents.
  • Each drug or active agent can be linked via a corresponding linker to the same conjugation moiety, which in turn can be attached to the antibody as described herein, thus linking the antibody to two or more drugs or active agents.
  • the linker, L A is a branched linker.
  • one of T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 ,V 5 or V 6 is a branched group.
  • the branched group is selected from -CONR 15 - and 4AP.
  • the branched group is -CONR 15 -.
  • the branched group is 4AP.
  • the branched group is attached to a second linker, L B .
  • the branched group of the first linker, L A can be attached to a first drug, W 1 , as shown in formula (I) above, and also attached to a second drug,
  • the second linker L B is a linker described by the formula: -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -, wherein L 7 , L 8 , L 9 , L 10 , L 11 and L 12 are each independently a linker subunit, and g, h, i, j, k and l are each independently 0 or 1, wherein the sum of g, h, i, j, k and l is 1 to 6.
  • the sum of g, h, i, j, k and l is I. In certain embodiments, the sum of g, h, i, j, k and l is 2. In certain embodiments, the sum of g, h, i, j, k and l is 3. In certain embodiments, the sum of g, h, i, j, k and l is 4. In certain embodiments, the sum of g, h, i, j, k and l is 5. In certain embodiments, the sum of g, h, i, j, k and l is 6. In certain embodiments, g, h, i, j, k and l are each 1.
  • g, h, i, j and k are each 1 and l is 0. In certain embodiments, g, h, i and j are each 1 and k and l are each 0. In certain embodiments, g, h, and i are each 1 and j, k and l are each 0. Tn certain embodiments, g and h are each 1 and i, j, k and l arc each 0. In certain embodiments, g is 1 and h, i, j, k and l arc each 0.
  • the linker subunit L 7 is attached to the branched group.
  • the linker subunit L 8 if present, is attached to the second drug, W 1a .
  • the linker subunit L 9 if present, is attached to the second drug, W 1a .
  • the linker subunit L 10 if present, is attached to the second drug, W 1a .
  • the linker subunit L 11 if present, is attached to the second drug, W 1a .
  • the linker subunit L 12 if present, is attached to the second drug, W 1a .
  • any convenient linker subunits may be utilized in the second linker L B .
  • any of the linker subunits described above in relation to L 1 , L 2 , L 3 , L 4 , L 5 and L 6 may be used for the linker subunits L 7 , L 8 , L 9 , L 10 , L 11 and L 12 .
  • the second linker L B is a linker comprising: -(L 7 )g-(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -, where:
  • T 7 , T 8 , T 9 , T 10 , T 11 and T 12 are tether groups;
  • V 7 , V 8 , V 9 , V 10 , V 11 and V 12 are covalent bonds or linking functional groups; and g, h, i, j, k and l are each independently 0 or 1, wherein the sum of g, h, i, j, k and 1 is 1 to 6.
  • V 7 , V 8 , V 9 , V 10 ,V 11 and V 12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • any convenient tether groups may be utilized for T 7 , T 8 , T 9 , T 10 , T 11 and T 12 .
  • any of the tether groups described above in relation to T 1 , T 2 , T 3 , T 4 , T 5 and T 6 may be used for the tether groups T 7 , T 8 , T 9 , T 10 , T 11 and T 12 .
  • each R 4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k2 is an integer from 1 to 10;
  • R 6 comprises a cleavable moiety
  • L B may be attached to a compound of formula (III).
  • L B may be attached to a compound of formula (III) at the bond indicated by the wavy line in formula (III).
  • R 4 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl. In certain embodiments, R 4 is alkynyl or substituted alkynyl.
  • each R 5 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • R 5 is hydrogen.
  • R 5 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl.
  • R 5 is methyl. In certain embodiments, R 5 is isopropyl. In certain embodiments, R 5 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl. In certain embodiments, R 5 is alkynyl or substituted alkynyl.
  • R 5 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C 6 aryl or C 6 substituted aryl.
  • R 5 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
  • R 5 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 5 is heterocyclyl or substituted heterocyclyl, such as a C 3-6 heterocyclyl or C 3-6 substituted heterocyclyl, or a C 3-5 heterocyclyl or C 3-5 substituted heterocyclyl.
  • R 4 is H and R 5 is alkyl or substituted alkyl.
  • R 4 is H and R 5 is methyl; or R 4 is H and R 5 is isopropyl.
  • k2 is an integer from 1 to 10. In certain embodiments, k2 is 1. In certain embodiments, k2 is 2. In certain embodiments, k2 is 3. In certain embodiments, k2 is 4. In certain embodiments, k2 is 5. In certain embodiments, k2 is 6. In certain embodiments, k2 is 7. In certain embodiments, k2 is 8. In certain embodiments, k2 is 9. In certain embodiments, k2 is 10.
  • k2 is 2, and each R 4 is H and each R 5 is alkyl or substituted alkyl.
  • k2 is 2, and each R 4 is H and one R 5 is methyl and the other R 5 is isopropyl.
  • R 6 comprises a cleavable moiety, as described in more detail herein.
  • R 6 can be a chemically-cleavable moiety, as described in more detail herein.
  • R 6 can be an enzymatically-cleavable moiety, as described in more detail herein (e.g., a glycoside or a glycoside derivative).
  • W 1a is a drug (or active agent).
  • the left-hand side of the linker structure is attached to the branched group, and the right-hand side of the linker structure is attached to the second drug, W 1a , as shown in formula (III). Further description of drugs (or active agents) that find use in the subject conjugates is found in the disclosure herein.
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 and V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 , and T 7 , T 8 , T 9 , T 10 , T 11 and T 12 and V 7 , V 8 , V 9 , V 10 ,V 11 and V 12 are selected from the following: wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is a branched group (-CONR 15 )-;
  • T 2 is (C 1 -C 12 )alkyl and V 2 is -CONH-;
  • T 3 is substituted (C 1 -C 12 )alkyl and V 3 is -CO-; d, e and f are each 0;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (PEG) n and V 3 is -CONH-;
  • T 4 is substituted (C 1 -C 12 )alkyl and V 4 is -CO-; e and f are each 0;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (C 1 -C 12 )alkyl and V 3 is absent;
  • T 4 is heteroaryl (tlriazole) and V 4 is absent;
  • T 5 is (C 1 -C 12 )alkyl and V 5 is -CONH-;
  • T 6 is substituted (C 1 -C 12 )alkyl and V 6 is -CO-;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0.
  • Combinations of the same or different payloads may be conjugated to the antibody through the branched linker.
  • the two payloads (e.g., drugs or active agents) attached to the branched linker are the same payload (e.g., drug or active agent).
  • a first branch of a branched linker may be attached to a payload (e.g., drug or active agent) and a second branch of the branched linker may be attached to the same payload (e.g., drug or active agent) as the first branch.
  • the two payloads (e.g., drugs or active agents) attached to the branched linker are different payloads (e.g., drugs or active agents).
  • a first branch of a branched linker may be attached to a first pay load (e.g., drug or active agent) and a second branch of the branched linker may be attached to a second payload (e.g., drug or active agent) different from the first payload (e.g., drug or active agent) attached to the first branch.
  • the branched linker is attached to a first drug, W 1 , and a second drug, W 1a .
  • W 1 and W 1a are the same drug. In other cases, W 1 and W 1a are different drugs.
  • the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect.
  • synergistic By “synergistic”, “synergism” or “synergy” is meant a therapeutic effect that is greater than the sum of the effects of the drugs or active agents taken separately.
  • the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both pay loads act, thereby increasing overall potency of the ADC.
  • the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately.
  • the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).
  • the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways.
  • payload combinations can include, but are not limited to, cytotoxic drugs, immunomodulatory molecules to activate or inhibit immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, and the like.
  • the payloads may be selected from combinations of drugs or active agents and detectable labels.
  • a first payload may be a detectable label that is used as an imaging agent or tracer to detect the location of the ADC in vivo
  • a second payload may be a drug or active agent that provides a therapeutic activity.
  • linker is a cleavable linker, such as a cleavable linker as described herein.
  • the conjugate is an antibody-drug conjugate where the antibody and the drug are linked together by a linker (e.g., L A and/or L B ), as described above.
  • the linker is a cleavable linker.
  • a cleavable linker is a linker that includes one or more cleavable moieties, where the cleavable moiety includes one or more bonds that can dissociate under certain conditions, thus separating the cleavable linker into two or more separatable portions.
  • the cleavable moiety may include one or more covalent bonds, which under certain conditions, can dissociate or break apart to separate the cleavable linker into two or more portions.
  • the cleavable linker includes a first clcavablc moiety and a second clcavablc moiety that hinders cleavage of the first cleavable moiety.
  • hinders cleavage is meant that the presence of an uncleaved second cleavable moiety reduces the likelihood or substantially inhibits the cleavage of the first cleavable moiety, thus substantially reducing the amount or preventing the cleavage of the cleavable linker.
  • the presence of uncleaved second cleavable moiety can hinder cleavage of the first cleavable moiety.
  • Cleavage of the first cleavable moiety can result in the cleavable linker dissociating or separating into two or more portions as described above to release the drug from the antibody- drug conjugate. In some instances, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety.
  • the second cleavable moiety can protect the first cleavable moiety from cleavage.
  • the presence of uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug.
  • cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thus allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, which, in turn, separates or releases the drug from the antibody at a desired target site of action for the drug as described above.
  • cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety does not in and of itself result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is still needed in order to cleave the cleavable linker).
  • the cleavable moieties included in the cleavable linker may each be an enzymatically cleavable moiety.
  • the first cleavable moiety can be a first enzymatically cleavable moiety and the second cleavable moiety can be a second enzymatically cleavable moiety.
  • An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more portions as described above through the enzymatic action of an enzyme.
  • the cleavage of an enzymatically cleavable moiety can be controlled such that substantial cleavage occurs at the desired site of action, whereas cleavage does not significantly occur in other areas (e.g., systemically) or before the antibody-drug conjugate reaches the desired site of action.
  • antibody-drug conjugates of the present disclosure can be used for the treatment of cancer, such as for the delivery of a cancer therapeutic drug to a desired site of action where the cancer cells are present.
  • enzymes such as the protease enzyme cathepsin B
  • cathepsin B can be a biomarker for cancer that is overexpressed in cancer cells.
  • the overexpression, and thus localization, of certain enzymes in cancer can be used in the context of the enzymatically cleavable moieties included in the cleavable linkers of the antibody-drug conjugates of the present disclosure to specifically release the drug at the desired site of action (i.e., the site of the cancer (and overexpressed enzyme)).
  • the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by an enzyme that is overexpressed in cancer cells.
  • the enzyme can be the protease enzyme cathepsin B.
  • the enzymatically cleavahle moiety is a clcavablc moiety (c.g., a peptide) that can be cleaved by a protease enzyme, such as cathepsin B.
  • the enzymatically cleavable moiety is a peptide.
  • the peptide can be any peptide suitable for use in the cleavable linker and that can be cleaved through the enzymatic action of an enzyme.
  • Non-limiting examples of peptides that can be used as an enzymatically cleavable moiety include, for example, Vai- Ala, Phe-Lys, and the like.
  • the first cleavable moiety described above i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety
  • uncleaved second cleavable moiety can protect the first cleavable moiety (peptide) from cleavage by a protease enzyme (e.g., cathepsin B), and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug.
  • a protease enzyme e.g., cathepsin B
  • one of the amino acid residues of the peptide that comprises the first cleavable moiety is linked to or includes a substituent, where the substituent comprises the second cleavable moiety.
  • the second cleavable moiety includes a glycoside.
  • the cleavable moiety may be a peptide, such as a peptide described by the moiety: as included in the formulae described herein (e.g., as included in formulae (I), (II), (III), (IV), (V), or (VI) described herein).
  • the enzymatically clcavablc moiety is sugar moiety, such as a glycoside (or glyosyl).
  • the glycoside can facilitate an increase in the hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include the glycoside.
  • the glycoside can be any glycoside or glycoside derivative suitable for use in the cleavable linker and that can be cleaved through the enzymatic action of an enzyme.
  • the second cleavable moiety i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage
  • the second cleavable moiety can be a glycoside or glycoside derivative.
  • the first cleavable moiety includes a peptide and the second cleavable moiety includes a glycoside or glycoside derivative.
  • the second clcavablc moiety is a glycoside or glycoside derivative selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • the second cleavable moiety is a glucuronide.
  • the second cleavable moiety is a galactoside.
  • the second cleavable moiety is a glucoside.
  • the second cleavable moiety is a mannoside. In some instances, the second cleavable moiety is a fucoside. In some instances, the second cleavable moiety is O-GlcNAc. In some instances, the second cleavable moiety is O-GalNAc.
  • the cleavable moiety may be a glycoside or glycoside derivative, such as where R 3 or R 6 is a glycoside or glycoside derivative (e.g., as shown in formulae (I), (II), (III), (IV), (V), or (VI) described herein).
  • the glycoside or glycoside derivative can be attached (covalently bonded) to the cleavable linker through a glycosidic bond.
  • the glycosidic bond can link the glycoside or glycoside derivative to the cleavable linker through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O-glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl).
  • the glycosidic bond is an O-glycosidic bond (an O-glycoside).
  • the glycoside or glycoside derivative can be cleaved from the cleavable linker it is attached to by an enzyme (e.g., through enzymatically-mediated hydrolysis of the glycosidic bond).
  • a glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme that is able to carry out the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker.
  • An example of an enzyme that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is a glucuronidase, a glycosidase, such as a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like.
  • Other suitable enzymes may also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker.
  • the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug of the antibody-drug conjugate.
  • the enzyme can be a lysosomal enzyme, such as a lysosomal glycosidase, found in cells at or near the desired site of action for the drug of the antibody-drug conjugate.
  • the enzyme is an enzyme found at or near the target site where the enzyme that mediates cleavage of the first clcavablc moiety is found.
  • Embodiments that include a linker with the two cleavable moieties as described above can also be referred to as having a “tandem-cleavage” linker.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • the glycoside or glycoside derivative can be selected from the following structures:
  • the present disclosure provides compounds useful for producing the conjugates described herein.
  • the compound may include a conjugation moiety useful for conjugation of a polypeptide (e.g., an antibody) and a drug or active agent.
  • a conjugation moiety useful for conjugation of a polypeptide e.g., an antibody
  • the compound may be bound to the polypeptide (antibody) and also bound to the drug or active agent, thus indirectly attaching the polypeptide (antibody) and the drug together.
  • the compound is a compound of formula (IV): wherein: each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • X is a conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody.
  • substituents R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , k 1 , k2, L A , L B , W 1 , W 1a , and X arc as described above in relation to the conjugates of formula (I).
  • the T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 and V 6 , and T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , V 7 , V 8 , V 9 , V 10 , V 11 and V 12 substituents are as described above in relation to the conjugates of formula (I).
  • the conjugation moiety, X includes maleimide or derivative thereof.
  • a maleimide conjugation moiety may be attached to a cysteine residue of the antibody, such as a native cysteine residue of the antibody.
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • the conjugation moiety, X includes an alpha-haloacetamide, such as an alpha-bromoacetamide.
  • a cysteine residue of the antibody such as a native cysteine residue can displace the halide to accomplish the conjugation.
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • L A is a first linker
  • W 1 is a drug
  • R 1 , R 2 , R 3 , k1, L A , and W 1 are as described herein (e.g., as in the description related to formulae (I) and (IV) herein).
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 and V 1 , V 2 , V 3 , V 4 , V 5 and V 6 are selected from the following: wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-; and b, c, d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is an amino acid analog and V 2 is -NH-;
  • T 3 is (PEG) n and V 3 is -CO-; and d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-;
  • T 2 is substituted (C 1 -C 12 )alkyl and V 2 is -CO-; and c, d, e and f are each 0.
  • the branched group is attached to a second linker, L B .
  • each R 4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k2 is an integer from 1 to 10;
  • W 1a is a drug.
  • T 1 is (C 1 -C 12 )alkyl and V 1 is a branched group (-CONR 15 )-;
  • T 2 is (C 1 -C 12 )alkyl and V 2 is -CONH-;
  • T 4 is substituted (C 1 -C 12 )alkyl and V 4 is -CO-; e and f are each 0;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 3 is (C 1 -C 12 )alkyl and V 3 is absent;
  • T 6 is substituted (C 1 -C 12 )alkyl and V 6 is -CO-;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (Ci-C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0.
  • the amino acid sequence of the polypeptide (antibody) is the native amino acid sequence of the polypeptide (antibody).
  • the conjugation moiety can be attached to the antibody at a native amino acid residue of the antibody.
  • maleimide-based conjugation can be used to attach the one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native cysteine residues of the polypeptide (e.g., antibody).
  • the native cysteine residues of the antibody can be derived from cysteine residues that are involved in disulfide bonds, such as the 8 hinge cysteine residues that typically form intrachain disulfide bonds of an antibody.
  • An antibody used in an antibody-drug conjugate of the present disclosure can have any of a variety of antigen-binding specificities, including but not limited to, c.g., an antigen present on a cancer cell; an antigen present on an autoimmune cell; an antigen present on a pathogenic microorganism; an antigen present on a virus-infected cell (e.g., a human immunodeficiency virus-infected cell); an antigen present on a diseased cell; and the like.
  • an antibody conjugate can bind an antigen, where the antigen is present on the surface of the cell.
  • a subject antibody conjugate can bind an antigen present on a cancer cell (e.g., a tumor- specific antigen; an antigen that is over-expressed on a cancer cell; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).
  • a subject antibody conjugate can be specific for an antigen on a cancer cell, where the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).
  • a subject antibody conjugate can bind an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by the virus; where the antigen is expressed on a cell type that is infected by a virus; etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor.
  • a subject antibody conjugate can bind an antigen present on a cell infected with a virus, and the conjugated moiety can be a drug, such as a viral fusion inhibitor.
  • a conjugate or a compound of the present disclosure can include as substituents W 1 and W 1a a drug or active agent.
  • Any of a number of drugs are suitable for use, or can be modified to be rendered suitable for use, as a reactive partner to conjugate to an antibody.
  • Examples of drugs include small molecule drugs and peptide drugs.
  • Small molecule drug refers to a compound, e.g., an organic compound, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa.
  • a small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom.
  • the drug or active agent can be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as a camptothecine, or an analog or derivative thereof, or a pharmaceutically active camptothecine moiety and/or a portion thereof.
  • a topoisomerase inhibitor (e.g., camptothecine, or analog or derivative thereof) conjugated to the polypeptide can be any of a variety of topoisomerase inhibitors, for example camptothecine or camptothecine moieties such as, but not limited to, camptothecine and analogs and derivatives thereof as described herein.
  • drugs that find use in the conjugates and compounds described herein include, but are not limited to, a topoisomerase inhibitor, for example camptothecine or a camptothecine derivative, such as SN-38, Belotecan, Exatecan, 9-aminocamptothecin (9-AC), topotecan, des- -Mc-topotccan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/US2022/012325, the disclosure of which is incorporated herein by reference.
  • a topoisomerase inhibitor for example camptothecine or a camptothecine derivative, such as SN-38, Belotecan, Exatecan, 9-aminocamptothecin (9-AC), topotecan, des- -Mc-topotccan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/US2022/012325, the disclosure
  • the drug or active agent can be a maytansine.
  • “Maytansine”, “maytansine moiety”, “maytansine active agent moiety” and “maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and/or portions thereof.
  • a maytansine conjugated to the polypeptide can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein (e.g., deacylmay tansine).
  • the drug or active agent can be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and/or a portion thereof.
  • An auristatin conjugated to the polypeptide can be any of a variety of auristatin moieties such as, but not limited to, an auristatin and analogs and derivatives thereof as described herein.
  • Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to an auristatin or an auristatin derivative, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), derivatives thereof, and the like.
  • the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and/or a portion thereof.
  • a duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin moieties such as, but not limited to, a duocarmycin and analogs and derivatives thereof as described herein.
  • Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to a duocarmycin or a duocarmycin derivative, such as duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof, and the like.
  • the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.
  • the drug is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulatory agent, a toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.
  • a cytotoxin can include any compound that leads to cell death (e.g., necrosis or apoptosis) or a decrease in cell viability.
  • Kinase inhibitors can include, but are not limited to, Adavosertib, Afatinib, Axitinib, Bosutinib, Cetuximab, Cobimetinib, Crizotinib, Cabozantinib, Dacomitinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ruxolitinib, Sorafenib, Sunitinib, Tucatinib, Vandetanib, Vemurafenib, and the like.
  • selective estrogen receptor modulators include, but are not limited to, Endoxifen, Tamoxifen, Afimoxifene, Toremifene, and the like.
  • Immuno stimulatory agents can include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, and the like.
  • TLR agonists include, but are not limited to, imiquimod, resiquimod, and the like.
  • Oligonucleotide dugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, volanesorsen, inotersen, tofersen, tominersen, and the like.
  • Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1,
  • Cytokines include, but are not limited to, Albinterferon Alfa-2B, Aldesleukin, ALT-801, Anakinra, Anccstim, Avotcrmin, Balugrastim, Bcmpcgaldcslcukin, Binctrakin, Cintredekin Besudotox, CTCE-0214, Darbepoetin alfa, Denileukin diftitox, Dulanermin, Edodekin alfa, Emfilermin, Epoetin delta, Erythropoietin, Human interleukin-2, Interferon alfa, Interferon alfa-2c, Interferon alfa-nl, Interferon alfa-n3, Interferon alfacon-1, Interferon beta-la, Interferon beta- lb, Interferon
  • Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.
  • Peptide drug refers to amino-acid containing polymeric compounds, and is meant to encompass naturally-occurring and non-naturally-occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics.
  • the peptide drugs may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source).
  • Peptide drugs can range in molecular weight, and can be from 200 Da to 10 kDa or greater in molecular weight.
  • Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; anti- angiogenic peptides; peptides that activate B cells; peptides that activate T cells; anti-viral peptides; peptides that inhibit viral fusion; peptides that increase production of one or more lymphocyte populations; anti-microbial peptides; growth factors; growth hormone-releasing factors; vasoactive peptides; anti- inflammatory peptides; peptides that regulate glucose metabolism; an anti-thrombotic peptide; an anti-nociceptive peptide; a vasodilator peptide; a platelet aggregation inhibitor; an analgesic; and the like.
  • drugs that find use in the conjugates and compounds described herein include, but are not limited to Tubulysin M, Calicheamicin, a STAT3 inhibitor, alpha- Amanitin, an aurora kinase inhibitor, belotecan, and an an thracy cline.
  • Other examples of drugs include small molecule drags, such as a cancer chemotherapeutic agent.
  • the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent.
  • Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents.
  • Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.
  • Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. 6,323,315.
  • dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure.
  • Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).
  • PBD pyrrolobenzodiazepine
  • Agents that act to reduce cellular proliferation are known in the art and widely used.
  • Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CytoxanTM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
  • alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazene
  • Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6- mercaptopurine (6-MP), pentostatin, 5 -fluorouracil (5-FU), methotrexate, 10-propargyl-5,8- dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinc phosphate, pcntostatinc, and gemcitabine.
  • CYTOSAR-U cytarabine
  • cytosine arabinoside including, but not limited to, fluorouracil (5-FU), floxuridine (FudR), 6-thi
  • Suitable natural products and their derivatives include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxy coformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g.
  • anthracycline daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.
  • phenoxizone biscyclopeptides e.g. dactinomycin
  • basic glycopeptides e.g.
  • anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
  • Hormone modulators and steroids that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.
  • adrenocorticosteroids e.g. prednisone, dexamethasone, etc.
  • estrogens and pregestins e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.
  • adrenocortical suppressants e.g.
  • estradiosteroids may inhibit T cell proliferation.
  • chemotherapeutic agents include metal complexes, e.g. cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc.
  • metal complexes e.g. cisplatin (cis-DDP), carboplatin, etc.
  • ureas e.g. hydroxyurea
  • hydrazines e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • a topoisomerase inhibitor e.g. N-methylhydrazine
  • procarbazine
  • mycophenolic acid mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4- morpholiny l)propoxy )quinazoline) ; etc .
  • Taxanes are suitable for use.
  • “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug.
  • “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOLTM, TAXOTERETM (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S.
  • Taxane also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No. 5,869,680; 6- thio derivatives described in WO 98/28288; sulfonamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivative described in U.S. Patent No. 5,415,869.
  • Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of scrinc/thrconinc kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-a; (7) IFN-y; (8) colony- stimulating factors; and (9) inhibitors of angiogenesis.
  • RTK tyrosine kinase
  • drugs include small molecule drugs, such as a cancer chemotherapeutic agent.
  • the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell
  • the antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agent.
  • the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid.
  • Embodiments of the present disclosure include conjugates where an antibody is conjugated to one or more drug moieties, such as two or more drug moieties, such as 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug moieties.
  • the drug moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein.
  • the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2.
  • DAR drug-to-antibody ratio
  • the conjugates have an average DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the conjugates have an average DAR from 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the conjugates have an average DAR of 1 to 10. In certain embodiments, the conjugates have an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugates have an average DAR of 5 to 10 (e.g., 8). In certain embodiments, the conjugates have an average DAR of 8 to 12 (e.g., 10). In certain embodiments, the conjugates have an average DAR of 10 to 15 (e.g., 12). In certain embodiments, the conjugates have an average DAR of 15 to 20 (e.g., 16). By average is meant the arithmetic mean.
  • the antibody-drug conjugate may include a branched linker, where two drugs or active agents arc attached to a branched linker.
  • the two drugs or active agents attached to a branched linker are the same drug or active agent.
  • a first branch of a branched linker may be attached to a drug or an active agent and a second branch of the branched linker may be attached to the same drug or the same active agent as the first branch.
  • the two drugs or active agents attached to the branched linker are different drugs or active agents.
  • a first branch of a branched linker may be attached to a first drug or a first active agent and a second branch of the branched linker may be attached to a second drug or a second active agent different from the first drug or the first active agent attached to the first branch.
  • the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect.
  • the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both payloads act, thereby increasing overall potency of the ADC.
  • the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately,
  • the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).
  • the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways.
  • pay load combinations can include, but are not limited to, cytotoxic drugs, immunomodulatory molecules to activate or inhibit immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, and the like.
  • the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an auristatin (e.g., MMAE) as described herein.
  • the two different drugs or active agents arc a topoisomerase inhibitor (e.g., bclotccan) as described herein and an iRGD peptide as described herein.
  • the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and an iRGD peptide as described herein.
  • the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein.
  • the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein.
  • a kinase inhibitor e.g., Sorafenib, Lapatinib, Gefitinib, and the like
  • the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein.
  • the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein.
  • a topoisomerase inhibitor e.g., belotecan
  • a selective estrogen receptor modulator e.g., Endoxifen
  • Drug s to be conjugated to a polypeptide may be modified to incorporate a reactive partner for reaction with the polypeptide.
  • the drug is a peptide drug
  • the reactive moiety e.g., aminooxy or hydrazide
  • an example of a method involves synthesizing a peptide drug having an aminooxy group.
  • the peptide is synthesized from a Boe-protected precursor.
  • An amino group of a peptide can react with a compound comprising a carboxylic acid group and oxy-N-Boc group.
  • the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidin-l-yloxy)propanoic acid.
  • Other variations on the compound comprising a carboxylic acid group and oxy-N-protecting group can include different number of carbons in the alkylene linker and substituents on the alkylene linker.
  • the reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and oxy-N-protecting group occurs through standard peptide coupling chemistry.
  • peptide coupling reagents examples include, but not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1- bcnzotriazolc dicthylphosphatc-l-cyclohcxyl-3-(2-morpholinylcthyl)carbodiimidc), EDC (1-(3- dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidinium hexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazol- 1 -yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazol
  • Deprotection to expose the amino-oxy functionality is performed on the peptide comprising an N-protecting group.
  • Deprotection of the N-oxysuccinimide group occurs according to standard deprotection conditions for a cyclic amide group. Deprotecting conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions include a hydrazine reagent, amino reagent, or sodium borohydride. Deprotection of a Boc protecting group can occur with TFA.
  • reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine.
  • the product and intermediates can be purified by conventional means, such as HPLC purification.
  • HPLC purification The ordinarily skilled artisan will appreciate that factors such as pH and steric hindrance (i.e., the accessibility of the amino acid residue to reaction with a reactive partner of interest) are of importance, Modifying reaction conditions to provide for optimal conjugation conditions is well within the skill of the ordinary artisan, and is routine in the art. Where conjugation is conducted with a polypeptide present in or on a living cell, the conditions are selected so as to be physiologically compatible.
  • the pH can be dropped temporarily for a time sufficient to allow for the reaction to occur but within a period tolerated by the cell (c.g., from about 30 min to 1 hour).
  • Physiological conditions for conducting modification of polypeptides on a cell surface can be similar to those used in a ketone-azide reaction in modification of cells bearing cell-surface azides (see, e.g., U.S. 6,570,040).
  • Small molecule compounds containing, or modified to contain, an ⁇ -nucleophilic group that serves as a reactive partner with a compound or conjugate disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the present disclosure.
  • conjugates of the present disclosure can be formulated in a variety of different ways.
  • the conjugate is formulated in a manner compatible with the drug, the antibody, the condition to be treated, and the route of administration to be used.
  • a pharmaceutical composition that includes any of the conjugates of the present disclosure and a pharmaceutically acceptable excipient.
  • the conjugate e.g., antibody-drug conjugate
  • the conjugate is provided as a liquid injectable (such as in those embodiments where they are administered intravenously or directly into a tissue)
  • the conjugate can be provided as a ready-to- use dosage form, or as a reconstitutable storage- stable powder or liquid composed of pharmaceutically acceptable carriers and excipients.
  • conjugates can be provided in a pharmaceutical composition comprising a therapeutically effective amount of a conjugate and a pharmaceutically acceptable carrier (e.g., saline).
  • a pharmaceutically acceptable carrier e.g., saline
  • the pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like).
  • the formulations are suitable for administration to a mammal, such as those that arc suitable for administration to a human.
  • the antibody-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug (i.e., the drug prior to conjugation to the antibody).
  • kits that include administering to a subject an amount (e.g., a therapeutically effective amount) of any of the conjugates of the present disclosure.
  • antibody-drug conjugates of the present disclosure can include a cleavable linker, such as an enzymatically cleavable linker that includes a first enzymatically cleavable moiety and a second enzymatically cleavable moiety.
  • the cleavable linker can be cleaved under appropriate conditions to separate or release the drug from the antibody at a desired target site of action for the drug.
  • the second cleavable moiety which protects the first cleavable moiety from cleavage, may be cleaved in order to allow the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker into two or more portions, thus releasing the drug from the antibody-drug conjugate at a desired site of action.
  • the first cleavable moiety can be an enzymatically cleavable moiety.
  • the enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated).
  • a first enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein.
  • the second cleavable moiety can be an enzymatically cleavable moiety.
  • the enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated).
  • a second enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein.
  • the first enzyme and the second enzyme are different enzymes.
  • the first enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated).
  • the first enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate.
  • the antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the first enzyme.
  • the first enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated.
  • the first enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated.
  • the first enzyme is present in an overabundance at the desired site of action due to localization of the first enzyme at a particular area or location.
  • the first enzyme may be associated with a certain structure within the desired site of action, such as lysosomes.
  • the first enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject.
  • the lysosomes that include the first enzyme are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug.
  • the first enzyme is a protease, such as a human protease enzyme (e.g., cathepsin B).
  • the second enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated).
  • the second enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate.
  • the antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the second enzyme.
  • the second enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated.
  • the second enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated.
  • the second enzyme is present in an overabundance at the desired site of action due to localization of the second enzyme at a particular area or location.
  • the second enzyme may be associated with a certain structure within the desired site of action, such as lysosomes.
  • the second enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject.
  • the lysosomes that include the second enzyme are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug.
  • the second enzyme is a glucuronidase, a glycosidase, such as a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like.
  • any suitable enzymes can be used for cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein.
  • Other enzymes may also be suitable for use in cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein, such as but not limited to, enzymes from other vertebrates (e.g., primates, mice, rats, cats, pigs, quails, goats, dogs, rabbits, etc.).
  • the antibody-drug conjugate is substantially stable under standard conditions.
  • substantially stable is meant that the cleavable linker of the antibody- drug conjugate does not undergo a significant amount of cleavage in the absence of a first enzyme and a second enzyme as described above.
  • the second cleavable moiety can protect the first cleavable moiety from being cleaved, and as such the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of a second enzyme as described above.
  • the cleavable linker of the antibody-drug conjugate may be substantially stable such that 25% or less of the antibody-drug conjugate is cleaved in the absence of the first enzyme and/or second enzyme, such as 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less.
  • the antibody-drug conjugate is substantially stable such that the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the first enzyme and/or second enzyme, but can be cleaved when in the presence of the first enzyme and the second enzyme.
  • the antibody-drug conjugate can be substantially stable after administration to a subject.
  • the antibody-drug conjugate is substantially stable after administration to a subject, and then, when the antibody-drug conjugate is in the presence of the second enzyme at a desired site of action, the second cleavable moiety can be cleaved from the cleavable linker, thus exposing the first clcavablc moiety to subsequent cleavage by the first enzyme, which in turn releases the drug at the desired site of action.
  • the antibody- drug conjugate after administration to a subject is stable for an extended period of time in the absence of the first enzyme and/or second enzyme, such as 1 hr or more, or 2 hrs or more, or 3 hrs or more, or 4 hrs or more, or 5 hrs or more, or 6 hrs or more, or 7 hrs or more, or 8 hrs or more, or 9 hrs or more, or 10 hrs or more, or 15 hrs or more, or 20 hrs or more, or 24 hrs (1 day) or more, or 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days (1 week) or more.
  • the first enzyme and/or second enzyme such as 1 hr or more, or 2 hrs or more, or 3 hrs or more, or 4 hrs or more, or 5 hrs or more, or 6 hrs or more, or 7 days (1 week) or more.
  • the antibody-drug conjugate is stable at a range pH values for an extended period of time in the absence of the first enzyme and/or second enzyme, such as at a pH ranging from 2 to 10, or from 3 to 9, or from 4 to 8, or from 5 to 8, or from 6 to 8, or from 7 to 8.
  • treatment is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated.
  • treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease; and/or (iii) relief, that is, causing the regression of clinical symptoms.
  • the subject to be treated can be one that is in need of therapy, where the subject to be treated is one amenable to treatment using the parent drug. Accordingly, a variety of subjects may be amenable to treatment using the antibody-drug conjugates disclosed herein. Generally, such subjects are “mammals”, with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees and monkeys).
  • domestic pets e.g., dogs and cats
  • livestock e.g., cows, pigs, goats, horses, and the like
  • rodents e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease
  • the amount of antibody-drug conjugate administered can be initially determined based on guidance of a dose and/or dosage regimen of the parent drug.
  • the antibody- drug conjugates can provide for targeted delivery and/or enhanced serum half-life of the bound drug, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen.
  • the antibody-drug conjugates can provide for reduced dose and/or reduced administration in a dosage regimen relative to the parent drug prior to being conjugated in an antibody-drug conjugate of the present disclosure.
  • an antibody-drug conjugate is administered.
  • the frequency of administration of an antibody-drug conjugate can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc.
  • an antibody-drug conjugate is administered once per month, twice per month, three times per month, every other week, once per week (qwk), twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily (qd/od), twice a day (bds/bid), or three times a day (tds/tid), etc.
  • the present disclosure provides methods that include delivering a conjugate of the present disclosure to an individual having a cancer.
  • the method may include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of the present disclosure, where the administering is effective to treat cancer in the subject.
  • the methods are useful for treating a wide variety of cancers, including, but not limited to breast, ovarian, colon, lung, stomach, and pancreatic cancer.
  • the term “treating” includes one or more (e.g., each) of: reducing growth of a solid tumor, inhibiting replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with a cancer.
  • Carcinomas that can be treated using a subject method include, but are not limited to, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, cervical carcinoma, uterine carcinoma, testicular carcinoma, and epithelial carcinoma, etc.
  • kits for treating cancer in a subject including administering to the subject a therapeutically effective amount of a pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to treat cancer in the subject.
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • W 2 is an antibody
  • X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
  • glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • W 2 is an antibody.
  • the first linker L A comprises: -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f -, wherein a, b, c, d, e and f are each independently 0 or 1;
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , - (CR 13 OH) m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 - , -SO 2 NR 15 -, -NR 15 SO 2 -, and -P(O)OH-; wherein:
  • (PEG) n is , where n is an integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1 ;
  • each R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring.
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-; and b, c, d, e and f are each 0; or wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is an amino acid analog and V 2 is -NH-;
  • T 3 is (PEG) n and V 3 is -CO-; and d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-;
  • T 2 is substituted (C 1 -C 12 )alkyl and V 2 is -CO-; and c, d, e and f are each 0.
  • T 7 , T 8 , T 9 , T 10 , T 11 and T 12 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is
  • V 7 , V 8 , V 9 , V 10 ,V 11 and V 12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • each R 4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k2 is an integer from 1 to 10;
  • R 6 comprises a cleavable moiety
  • W 1a is a drug.
  • glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 2 is (C 1 -C 12 )alkyl and V 2 is -CONH-;
  • T 3 is substituted (C 1 -C 12 )alkyl and V 3 is -CO-; d, e and f are each 0;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (PEG) n and V 3 is -CONH-;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (C 1 -C 12 )alkyl and V 3 is absent;
  • T 4 is hctcroaryl (triazoic) and V 4 is absent;
  • T 5 is (C 1 -C 12 )alkyl and V 5 is -CONH-;
  • T 6 is substituted (C 1 -C 12 )alkyl and V 6 is -CO-;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0.
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k1 is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug; and X is a conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody.
  • glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • the compound of any of Embodiments 29-35, X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
  • each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • kl is an integer from 1 to 10;
  • R 3 comprises a cleavable moiety
  • L A is a first linker
  • W 1 is a drug
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 arc each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxy
  • T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , - (CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
  • V 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 - , -SO 2 NR 15 -, -NR 15 SO 2 -, and -P(O)OH-; wherein:
  • (PEG) n is , where n is an integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: where y is an integer from 1 to 6 and r is 0 or 1 ;
  • each R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring.
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-; and b, c, d, e and f are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is an amino acid analog and V 2 is -NH-;
  • T 3 is (PEG) n and V 3 is -CO-; and
  • d, c and f arc each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-;
  • T 2 is substituted (C 1 -C 12 )alkyl and V 2 is -CO-; and c, d, e and f are each 0.
  • T 7 , T 8 , T 9 , T 10 , T 11 and T 12 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG) n , (AA) P , -(CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is
  • V 7 , V 8 , V 9 , V 10 ,V 11 and V 12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl
  • each R 4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl
  • each R 5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl
  • k2 is an integer from 1 to 10;
  • R 6 comprises a cleavable moiety
  • W 1a is a drug.
  • glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 1 is (C 1 -C 12 )alkyl and V 1 is a branched group (-CONR 15 )-;
  • T 2 is (C 1 -C 12 )alkyl and V 2 is -CONH-;
  • T 3 is substituted (C 1 -C 12 )alkyl and V 3 is -CO-; d, e and f are each 0;
  • T 7 is (C 1 -C 12 )alkyl and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (PEG) n and V 3 is -CONH-;
  • T 4 is substituted (C 1 -C 12 )alkyl and V 4 is -CO-; e and f are each 0;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and l are each 0; or wherein:
  • T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-;
  • T 2 is a branched group (4AP) and V 2 is -CO-;
  • T 3 is (C 1 -C 12 )alkyl and V 3 is absent;
  • T 4 is heteroaryl (triazole) and V 4 is absent;
  • T 5 is (C 1 -C 12 )alkyl and V 5 is -CONH-;
  • T 6 is substituted (C 1 -C 12 )alkyl and V 6 is -CO-;
  • T 7 is (PEG) n and V 7 is -CONH-;
  • T 8 is substituted (C 1 -C 12 )alkyl and V 8 is -CO-; and i, j, k and 1 are each 0.
  • the conjugate of any of Embodiments 42-53, wherein W 1 and W 1a are the same drug.
  • the conjugate of any of Embodiments 42-53, wherein W 1 and W 1a are different drugs.
  • the conjugate of any of Embodiments 29-55, wherein the conjugate is selected from:
  • a pharmaceutical composition comprising: a conjugate of any one of Embodiments 1 to 28; and a pharmaceutically acceptable excipient.
  • a method comprising : administering to a subject a conjugate of any of Embodiments 1 to 28.
  • a method of treating cancer in a subject comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of any one of Embodiments 1 to 28, wherein the administering is effective to treat cancer in the subject.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneally ); s.c., subcutaneous(ly); and the like.
  • first-generation ADCs relied on stochastic conjugation chemistries that utilized naturally occurring nucleophilic amino acid residues, predominantly cysteine and lysine sidechains (FIG. 2). For example, maleimide and NHS-ester based conjugation methods were used due to their simplicity.
  • the heterogeneous conjugation resulted from (random) incomplete ligation to available reactive groups on the protein.
  • most maleimide and NHS-ester conjugates targeted a drug-to-antibody ratio (DAR) of 4, which was less than the maximum number of conjugatable sites.
  • DAR drug-to-antibody ratio
  • ADC linker instability was manifest in two locations - the conjugation chemistry and the cleavable linker element, which could be a disulfide, an acid-labile group, or a peptide.
  • ADC heterogeneity of first-generation ADCs was characterized by both under- and over-conjugated species within the conjugate mixture.
  • the over-conjugated species tended towards hydrophobicity and aggregation and were often rapidly cleared by the liver upon injection into the body (Beck et al. Nature Reviews Drug Discovery, 16, 315-337 (2017), Sun et al. Bioconjugate Chemistry, 28, 1371-1381 (2017), Lyon et al. Nature Biotechnology , 33, 733-735 (2015)). This resulted in liver toxicity being observed as a common side effect of ADC dosing among these first-generation heterogeneous constructs.
  • HIC hydrophobic interaction chromatography
  • the ThioMab approach was generally used to make ADCs with lower overall drug-to-antibody ratios (e.g., DAR2) than were typically generated using the stochastic conjugation methods (e.g., DAR4).
  • the resulting site-specific DAR2 conjugates were shown to have improved biophysical and functional properties when compared to first-generation, stochastically-derived conjugates.
  • Other ways to make site-specifically conjugated ADCs were developed that used various methods to introduce biorthogonal reactive chemical handles at defined locations on an antibody. These included: a) incorporation of functionalized non-natural amino acids at specific locations in the sequence (Xu et al. Org Process Res Dev 20, 1034-1043 (2016), and Tian et al.
  • conjugation chemistry used to join the protein and small molecule component
  • composition of the linker that connected the conjugation handle to the payload Regarding conjugation chemistry, the commonly used maleimide-cysteine conjugation chemistry has inherent weaknesses.
  • the conjugation product can undergo a retro-Michael reaction that results in the detachment of the antibody from the drug-linker (Alley et al. Bioconjugate Chemistry 19, 759-765 (2008)), the latter of which can further react with free plasma thiols (such as those found on albumin, (e.g., Wei et al.
  • a common side-effect of vcMMAE-conjugated ADCs regardless of target antigen is myelosuppression, including neutropenia, thrombocytopenia, and anemia. This off-target toxicity is driven, at least in part, by premature cleavage of the ValCit dipeptide via extracellular enzymes encountered during circulation (Masters et al. Investigational New Drugs 36, 121-135 (2016)).
  • a highly successful approach to stabilize cleavable peptide-based linkers is the tandem-cleavage linker system that requires two consecutive enzymatic steps to occur in order to release the payload (FIG. 5), as disclosed herein.
  • the monosaccharide unit attached to the phenolic self-immolation moiety (PABA) serves as a protective element and has to be removed first by the corresponding lysosomal glycosidase to expose the peptide portion of the linker which is then readily cleaved by proteases to liberate the payload.
  • PABA phenolic self-immolation moiety
  • glucuronidase is only activated in the low pH environment of the lysosomal compartment, it is generally not active when found in serum.
  • the tandem-cleavage linker in order for the tandem-cleavage linker to be enzymatically degraded, it first must be internalized into a cell and trafficked to the lysosome. This requirement greatly reduces release of linker
  • Improved ADC comprising a malcimidc-tandcm-clcavagc conjugate
  • tandem-cleavage element in itself is sufficient to overcome many of the well-known hurdles to constructing stable and well-tolerated ADCs.
  • a linker equipped with a maleimide handle and elaborated by various types of payloads
  • safe and effective ADCs can be produced that exhibit qualities equal to or superior to the conjugates made using either generic maleimide-peptide based linkers or using a site-specific conjugation approach with the aldehyde-tag/HIPS-functionalized linkers.
  • the maleimide-tandem cleavage functionalized conjugates performed better than conjugates made with the standard vedotin linker (maleimide- Val-Cit-PABC-MMAE, Compound 38), which also contained maleimide and MMAE but lacked the tandem-cleavage element (see Table 1 and FIGS. 9-23, 30-32).
  • tandem-cleavage-containing ADCs had better reaction yields, shorter PLRP retention times, reduced evidence of precipitation, all of which point to improved hydrophilicity of the constructs, which led to improved overall biophysical properties.
  • Improved outcomes observed with linkers that incorporate the tandem-cleavage element may be driven by the effect of the tandem-cleavage element on overall conjugate hydrophilicity, resistance to protease degradation (through steric hindrance/shielding of the protease cleavage site), improved stability of the maleimide conjugation through influencing rate of exchange with surrounding thiols, and reduction in the propensity to form aggregates, to self- associate, or to interact with charged species.
  • the maleimide topoisomerase inhibitor conjugates with the tandem-cleavage linkers showed improved (more hydrophilic) HIC retention times as compared to Enhertu (an ADC made using trastuzumab conjugated to the maleimide-based deruxtecan linker, which has no tandem-cleavage element) (Table 2).
  • Enhertu an ADC made using trastuzumab conjugated to the maleimide-based deruxtecan linker, which has no tandem-cleavage element
  • tandem-cleavage element could impart an additional layer of stability and tolerability even to linkers that undergo deconjugation from the antibody and end up ligated to albumin (or other serum thiols) by serving as a “pro- drug” inactive form (FIG. 6).
  • Table 3 HIC Retention Times of MMAE Conjugated ADCs Produced Using Various Linkers
  • tandem-cleavage maleimide conjugate shows unaffected in vitro potency and ability to kill HER2+ gastric cell line
  • tandem-cleavage maleimide conjugated trastuzumab ADCs had equal potency and ability to kill the HER2+ gastric cell line, NCI-N87, as compared to ADCs made with traditional maleimide linkers (not containing tandem-cleavage) (FIGS. 24 and 25). Put another way, the improved stability imparted by the tandem-cleavage element did not reduce in vitro potency or negatively affect the ability of the ADC to kill target antigen expressing cells.
  • tandem-cleavage maleimide conjugate shows improved serum stability, improved in vivo efficacy and tolerability.
  • tandem- cleavage maleimide linkers generate conjugates with improved in vitro serum stability as compared to traditional maleimide linkers (e.g., vedotin) (see FIGS. 26-29). These linkers also appeared to demonstrate improved resistance to displacement by glutathione when incubated in vitro at 37° C for up to 6 days (Table 4).
  • tandem-cleavage containing linkers are affecting the stability of the maleimide conjugation site itself, perhaps through direct stabilization of the thioether bond, perhaps through influencing the maleimide ring opening.
  • conjugates containing tandem-cleavage linkers will show improved in vivo efficacy and tolerability as compared to non-tandcm-clcavagc containing ADCs.
  • conjugation chemistries such as click reactions, or NHS esters.
  • tandem-cleavage element Improved outcomes observed with linkers that incorporate the tandem-cleavage element likely are driven by the effect of the tandem-cleavage element on overall conjugate hydrophilicity, resistance to protease degradation (through steric hindrance/shielding of the protease cleavage site), improved stability of the maleimide conjugation through influencing rate of exchange with surrounding thiols, and reduction in the propensity to form aggregates, to self- associate, or to interact with charged species.
  • maleimide-functionalized tandem-cleavage linker is that it can be used in combination with a broad variety of acid- sensitive payloads (such as an thracy clines), unlike the ADCs formed based on the HIPS conjugation chemistry, thus widening the scope of application for use of the tandem-cleavage element.
  • acid- sensitive payloads such as an thracy clines
  • Conjugates with maleimide-functionalized tandem-cleavage linker carrying multiple payloads [00514]
  • DAR drug-to-antibody ratio
  • branched linkers which carry two or more payloads per one conjugation unit. Examples of such linkers are shown in FIG. 7.
  • Dicarboxylic acids 1 or 2 were further chemically modified to include a conjugation element (e.g. maleimide), then the carboxylic functions in 3 and 4 served as chemical handles to attach two linker-payload systems of choice to build compounds such as 5 and 6, where payload was chosen from Topoisomerase T, kinase, or PARP inhibitor, or some other biologically active compound.
  • a conjugation element e.g. maleimide
  • Dual payload ADCs in an ADC construct comprising a maleimide-functionalized tandem- cleavage linker
  • branched linkers may serve as a platform for developing dual-payload ADCs and provide a reliable access to cytotoxin combination modality. This can be achieved through the design of branched linkers with orthogonal connection elements as shown for compound 7 (FIG. 8), where carboxylic acid moiety was used to attach the first payload of choice, while the azide function - the second through azide-alkyne cycloaddition to access dual-payload linker 8.
  • the subject conjugates and compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods.
  • a variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.
  • Synthetic reagents were purchased from Sigma- Aldrich, Acros, AK Scientific, or other commercial sources and were used without purification. Anhydrous solvents were obtained from commercial sources in sealed bottles. Compounds 10 and 35 were obtained commercially from Shanghai Medicilon and used without purification. Compounds 58 and 63 were commercially available or synthesized via literature procedures. Payloads 11, 26, 39, and 67 as well as MC-VC-PAB-MMAE 38 (vedotin linker) were purchased from commercial sources. In all cases, solvent was removed under reduced pressure with a Buchi Rotovapor R-l 14 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage chromatography system.
  • HPLC purifications were performed using Waters preparative HPLC unit equipped with Phenomenex Kinetex 5 am EVO C18 150 x 21.2 mm column.
  • HPLC analyses were conducted on an Agilent 1100 Series Analytical HPLC equipped with a Model G1322A Degasser, Model G1311 A Quarternary Pump, Model G1329A Autosamplcr, Model G1314 Variable Wavelength Detector, Agilent Poroshcll 120 SB C18, 4.6 mm x 50 mm column at room temperature using a 10-100% gradient of water and acetonitrile containing 0.05% trifluoroacetic acid. HPLCs were monitored at 254 or 205 nm.
  • LRMS Low-resolution mass spectra
  • Reaction mixture was poured into 30 mL of water, and the resulting white precipitate was separated by spinning and collected, washed with 5 mL of water, and dried briefly under high vacuum to give 1.87 g of crude coupling product as an off-white solid, which was taken to the next step without purification.
  • Reaction mixture was stirred at 0 °C for 3 hours, then warmed up to ambient temperature, treated with 3 mL of 1 M aqueous lithium hydroxide and diluted with 3 mL of methanol. The resulting mixture was stirred at room temperature for 3 hours until hydrolysis was found complete (HPLC), then quenched by adding 1 M aqueous HC1 solution to pH 7. Reaction mixture was then concentrated under reduced pressure and washed with 10 mL of MTBE. Aqueous layer was purified by reversed- phase chromatography (C18 column, 0-40% acetonitrile-water with 0.05% TFA).
  • Carboxylic acid 22 (420 mg, 0.76 mmol) was combined with 421 mg (2.3 mmol) of pentafluorophenol in 5 mL of anhydrous DMF. The mixture was treated with EDCLHC1 (292 mg, 1.5 mmol) at room temperature and stirred for 48 hours until 22 was judged as completely consumed based on LCMS analysis. Reaction mixture was directly purified by reversed-phase chromatography (C18, 0-70% acetonitrile-water, 0.05% TFA). Pure fractions were combined at concentrated under vacuum and lyophilized to give 254 mg of PFP-ester 15 as a tan solid (0.36 mmol, 47% yield). LRMS (ESI neg.): m/z 715.1 [M-H]-, Calcd for C 31 H 29 F 5 N 2 O 10 S m/z 715.2.
  • Belotecan hydrochloride 26 (2.35 g, 5.0 mmol) was suspended in a mixture of 30 mL of anhydrous DMF and 1.75 mL of DIPEA (10 mmol). The resulting mixture was stirred and treated with HO At (0.68 g, 5 mmol), followed by PNP-carbonate 10 (5.1 g, 5 mmol) in a few small portions at room temperature. Reaction mixture was stirred at RT for 8 hours until starting materials were judged fully consumed based on HPLC analysis.

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Abstract

The present disclosure provides cleavable linkers for antibody-drug conjugates (ADCs) where the linker includes a tandem-cleavage moiety. The present disclosure also provides ADCs including the cleavable linkers having the tandem-cleavage moiety. In addition, the present disclosure encompasses compounds and methods for production of such ADCs, as well as methods of using the ADCs.

Description

TANDEM- CLEAVAGE LINKERS FOR ANTIBODY-DRUG CONJUGATES AND USES
THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 63/538,972, filed September 18, 2023, U.S. Provisional Application No.
63/544,310, filed October 16, 2023, and U.S. Provisional Application No. 63/605,310, filed December 1, 2023, the disclosures of each of which are incorporated herein by reference.
INTRODUCTION
[0002] Antibody-drug conjugates (ADCs) have emerged as a promising therapy format which combines the highly specific affinity of antibodies (targeting element) towards cell surface antigens together with the potency of small molecule agents (pay loads). The latter are connected to the protein through various chemical linkers using one of the available conjugation methods. Although the concept of an ADC can be displayed in a simple drawing (FIG. 1), the details of ADC construction and how ADC composition relates to function - including efficacy and tolerability - are highly complex. Furthermore, practical concerns, including both translatability of preclinical to clinical outcomes and compound manufacturability/stability impact which concepts are able to successfully be incorporated into therapeutic drugs. Indeed, the field’s understanding of how best to construct, produce, and use ADCs is still evolving.
SUMMARY
[0003] The present disclosure provides cleavable linkers for antibody-drug conjugates (ADCs) where the linker includes a tandem-cleavage moiety. In addition, the disclosure also encompasses compounds and methods for production of such ADCs, as well as methods of using the ADCs.
[0004] Aspects of the present disclosure include a conjugate of formula (I):
Figure imgf000003_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug;
W2 is an antibody; and
X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
[0005] In some embodiments, R1 is H and R2 is alkyl or substituted alkyl.
[0006] In some embodiments, k1 is 2.
[0007] In some embodiments, R3 is a chemically-cleavable moiety.
[0008] In some embodiments, R3 is an enzymatically-cleavable moiety.
[0009] In some embodiments, R3 is a glycoside or a glycoside derivative.
[0010] In some embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
[0011] In some embodiments, X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
[0012] In some embodiments, X comprises a maleimide, and the native amino acid residue of the antibody comprises a cysteine residue.
[0013] In some embodiments, the conjugate is of formula (II):
Figure imgf000004_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
W2 is an antibody.
[0014] In certain embodiments of Formula (I), the conjugate is of formula (Ila):
Figure imgf000005_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
W2 is an antibody.
[0015] In some embodiments, LA comprises:
-(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein a, b, c, d, e and f are each independently 0 or 1; T1, T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, hctcroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
[0016] In some embodiments, LA includes the following:
T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;
T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, - (CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:
(PEG)n is , where n is an integer from 1 to 30;
Figure imgf000006_0001
EDA is an ethylene diamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1 ;
Figure imgf000007_0001
4-amino-piperidine (4AP) is and
Figure imgf000007_0002
each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring.
[0017] In some embodiments, the conjugate comprises: wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-;
T3 is (PEG)n and V3 is -CO-; and d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0.
[0018] In some embodiments, one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4 ,V5 or V6 is a branched group.
[0019] In some embodiments, the branched group is selected from -CONR15- and 4AP.
[0020] In some embodiments, the branched group is attached to a second linker, LB.
[0021] In some embodiments, the second linker LB comprises:
-(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and l are each independently 0 or 1; T7, T8, T9, T10, T11 and T12 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, hctcroaryl, substituted hctcroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V7, V8, V9, V10 ,V11 and V12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
[0022] In some embodiments, wherein LB is attached to a compound of formula (III):
Figure imgf000008_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug. [0023] In some embodiments, R4 is H and R5 is alkyl or substituted alkyl.
[0024] In some embodiments, k2 is 2.
[0025] In some embodiments, R6 is a chemically-cleavable moiety.
[0026] In some embodiments, R6 is an enzymatically-cleavable moiety.
[0027] In some embodiments, R6 is a glycoside or a glycoside derivative.
[0028] In some embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [0029] In some embodiments, the conjugate comprises: wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (C1-C12)alkyl and V3 is absent;
T4 is heteroaryl (triazole) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-; T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0.
[0030] In some embodiments, W1 and W1a are the same drug.
[0031] In some embodiments, W1 and W1a are different drugs.
[0032] In some embodiments, the conjugate is selected from:
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
and
Figure imgf000013_0001
[0033] Aspects of the present disclosure include a compound of formula (IV):
Figure imgf000013_0002
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and X is a conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody.
[0034] In some embodiments, R1 is H and R2 is alkyl or substituted alkyl.
[0035] In some embodiments, k1 is 2.
[0036] In some embodiments, R3 is a chemically-cleavable moiety.
[0037] In some embodiments, R3 is an enzymatically-cleavable moiety.
[0038] In some embodiments, R3 is a glycoside or a glycoside derivative.
[0039] In some embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
[0040] In some embodiments, X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
[0041] In some embodiments, X comprises a maleimide, and the native amino acid residue of the antibody comprises a cysteine residue.
[0042] In some embodiments, the compound is of formula (V):
Figure imgf000014_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, hctcrocyclyl, and substituted hctcrocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker; and
W1 is a drug.
[0043] In some embodiments of compounds according to formula (IV), the compound is of formula (Va):
Figure imgf000015_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker; and
W1 is a drug.
[0044] In some embodiments, LA comprises:
-(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein a, b, c, d, e and f are each independently 0 or 1;
T1, T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)U, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
[0045] In some embodiments, LA includes the following:
T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;
T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, - (CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
V1, V2, V3, V4 ,V5 and V6 arc each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:
(PEG)n is
Figure imgf000016_0001
, where n is an integer from 1 to 30;
EDA is an ethylene diamine moiety having the following structure; , where y is an integer from 1 to 6 and r is 0 or 1 ;
Figure imgf000016_0002
4-amino-piperidine (4AP) is ; and
Figure imgf000016_0003
each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring.
[0046] In some embodiments, the compound comprises: wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-;
T3 is (PEG)n and V3 is -CO-; and d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0.
[0047] In some embodiments, one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4 ,V5 or V6 is a branched group.
[0048] In some embodiments, the branched group is selected from -CONR15- and 4AP.
[0049] In some embodiments, the branched group comprises a second linker, LB.
[0050] In some embodiments, second linker LB comprises: -(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and l are each independently 0 or 1;
T7, T8, T9, T10, T11 and T12 are each independently selected from a covalent bond, (C1 - C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH) 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V7, V8, V9, V10 ,V11 and V12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
[0051] In some embodiments, LB is attached to a compound of formula (VI):
Figure imgf000018_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug.
[0052] In some embodiments, R4 is H and R5 is alkyl or substituted alkyl.
[0053] In some embodiments, k2 is 2.
[0054] In some embodiments, R6 is a chemically-cleavable moiety.
[0055] In some embodiments, R6 is an enzymatically-cleavable moiety.
[0056] In some embodiments, R6 is a glycoside or a glycoside derivative.
[0057] In some embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [0058] In some embodiments, the compound comprises: wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (C1-C12)alkyl and V3 is absent;
T4 is heteroaryl (triazole) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-;
T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0.
[0059] In some embodiments, W1 and W1a are the same drug.
[0060] In some embodiments, W1 and W1a are different drugs.
[0061] In some embodiments, the conjugate is selected from:
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
and
Figure imgf000023_0001
[0062] Aspects of the present disclosure include a pharmaceutical composition comprising a conjugate according to the present disclosure, and a pharmaceutically acceptable excipient.
[0063] Aspects of the present disclosure include a method comprising administering to a subject a conjugate according to the present disclosure.
[0064] Aspects of the present disclosure include a method of treating cancer in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate according to the present disclosure, where the administering is effective to treat cancer in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1 shows a drawing illustrating that ADCs comprise an antibody joined to a linker and payload through a particular conjugation approach. The chemistry involved in the conjugation can vary and can contribute to the biophysical and functional properties of the molecule. [0066] FIG. 2 shows a schematic illustrating that NHS-ester and maleimide conjugation chemistries react with naturally occurring nucleophilic amino acid residues in proteins.
[0067] FIG. 3 shows a schematic illustrating maleimide conjugates that are reversible in plasma and can transfer linker-payload to other thiol-containing molecules, such as albumin.
[0068] FIG. 4 shows an illustration of ADC payload prematurely released into the circulation drives systemic toxicity.
[0069] FIG. 5 shows a drawing of a tandem-cleavage linker requires two consecutive enzymatic steps to occur in order to release the drug after internalization of ADC.
[0070] FIG. 6 shows an illustration of tandem-cleavage linker that prevents release of cytotoxic payload in circulation in the event of retro-Michael deconjugation of the maleimide- thioether linkage.
[0071] FIG. 7 shows examples of branched maleimide linkers designs that allow to conjugate more than one molecule of payload in single step.
[0072] FIG. 8 shows an orthogonally substituted branched linkers that allow to conjugate two types of pay loads in single step (MMAE/belotecan conjugation shown).
[0073] FIG. 9 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 38.
[0074] FIG. 10 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 14.
[0075] FIG. 11 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 18.
[0076] FIG. 12 shows a HIC assessment of Trastuzumab antibody with CHl/CT-tags conjugated to Compound 24.
[0077] FIG. 13 shows a SEC assessment of Trastuzumab antibody conjugated to Compound 38.
[0078] FIG. 14 shows Trastuzumab antibody conjugated to Compound 14 is 97.3% monomeric as determined by SEC.
[0079] FIG. 15 shows Trastuzumab antibody conjugated to Compound 18 is 97.5% monomeric as determined by SEC.
[0080] FIG. 16 shows Trastuzumab antibody conjugated to Compound 38 yields a DAR of 3.80 as determined by PLRP. [0081] FIG. 17 shows Trastuzumab antibody conjugated to Compound 14 yields a DAR of 3.80 as determined by PLRP.
[0082] FIG. 18 shows Trastuzumab antibody conjugated to Compound 18 yields a DAR of 3.85 as determined by PLRP.
[0083] FIG. 19 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 36.
[0084] FIG. 20 shows a HIC assessment of Trastuzumab antibody conjugated to Compound 34.
[0085] FIG. 21 shows a HIC assessment of Enhertu (trastuzumab deruxtecan).
[0086] FIG. 22 shows Trastuzumab antibody conjugated to Compound 36 is 97% monomeric as determined by SEC.
[0087] FIG. 23 shows Trastuzumab antibody conjugated to Compound 36 yields a DAR of 7.61 as determined by PLRP.
[0088] FIG. 24 shows in vitro potency against NCI-N87 cells of MMAE-conjugated trastuzumab-based ADCs. MMAE alone (Compound 11) is included as a reference.
[0089] FIG. 25 shows in vitro potency against NCI-N87 cells of topoisomerase I inhibitor-conjugated trastuzumab-based ADCs.
[0090] FIG. 26 shows in vitro stability of trastuzumab MMAE conjugates incubated at 37° C in rat serum for up to seven days.
[0091] FIG. 27 shows in vitro stability of trastuzumab MMAE conjugates incubated at 37° C in rat serum for up to seven days.
[0092] FIG. 28 shows in vitro stability of trastuzumab topoisomerase I inhibitor conjugates incubated at 37° C in rat serum for up to seven days.
[0093] FIG. 29 shows in vitro stability of trastuzumab topoisomerase I inhibitor conjugates incubated at 37° C in cynomolgus monkey serum for up to seven days.
[0094] FIG. 30 shows Trastuzumab antibody conjugated to Compound 37 yields a DAR of 7.55 as determined by PLRP.
[0095] FIG. 31 shows HIC assessment of Trastuzumab antibody conjugated to Compound 37.
[0096] FIG. 32 shows Trastuzumab antibody conjugated to Compound 37 is 94.9% monomeric as determined by SEC. [0097] FIG. 33 shows a scheme illustrating cysteine-maleimide conjugation, according to embodiments of the present disclosure.
[0098] FIG. 34 shows a scheme illustrating various conjugation chemistries that can be used for attachment to a cysteine residue, according to embodiments of the present disclosure. [0099] FIG. 35 shows a scheme illustrating P5 phosphoramidate conjugation to a cysteine residue, according to embodiments of the present disclosure.
[00100] FIG. 36 shows a scheme illustrating thiobridge conjugation to cysteine residues, according to embodiments of the present disclosure.
[00101] FIG. 37 shows examples of NHS-ester conjugation moieties that can be used for attachment to a lysine residue, according to embodiments of the present disclosure.
[00102] FIG. 38 shows a scheme illustrating that an N-terminal amine can be oxidized with N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport’s salt, RS) to afford an aldehyde or ketone tagged antibody, followed by conjugation using an oxime or hydrazone linkage, according to embodiments of the present disclosure.
[00103] FIG. 39 shows schemes illustrating different click-chemistry reactions that can be used to produce a linker according to the present disclosure. The click-chemistry reactions include azide-alkyne reactions, alkyne-DBCO (dibenzocyclooctyne) reactions, and TCO-Tz (trans-cycloctene tetrazine) reactions.
[00104] FIG. 40 shows a graph illustrating efficacy of trastuzumab MMAE conjugates against the HER2+ NCI-N87 gastric tumor xenograft model, according to embodiments of the present disclosure.
[00105] FIG. 41 shows a schematic depicting ELISA assay set up for analysis of total antibody (left) and total ADC concentrations (right) in rat plasma, according to embodiments of the present disclosure.
[00106] FIG. 42 shows a graph of rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
[00107] FIG. 43 shows a graph of clinical observations (average sum per animal is shown) from rat toxicity studies comparing enfortumab conjugates carrying different linker-payloads, according to embodiments of the present disclosure. The vedotin conjugate (Compound 38 conjugate) induced clinical observations (mostly fur and skin related) in all of the dosed animals, reaching a maximum of 2.6 average clinical obscrvations/animals on Day 17, when an animal died. By contrast, no clinical observations were noted in animals dosed with enfortumab conjugated with either Compound 14 or Compound 18.
[00108] FIG. 44 shows a graph of red blood cell (RBC) counts in rats dosed with enfortumab conjugates or vehicle alone, according to embodiments of the present disclosure. [00109] FIG. 45 shows a graph of neutrophil counts in rats dosed with enfortumab conjugates or vehicle alone, according to embodiments of the present disclosure.
[00110] FIG. 46 shows a graph of alanine transaminase levels in rats dosed with enfortumab conjugates or vehicle alone, according to embodiments of the present disclosure.
[00111] FIG. 47 shows a graph of reticulocyte counts in rats dosed with enfortumab conjugates or vehicle alone, according to embodiments of the present disclosure.
[00112] FIG. 48 shows a graph illustrating efficacy of trastuzumab topoisomerase I inhibitor conjugates against the HER2+ NCI-N87 gastric tumor xenograft model, according to embodiments of the present disclosure.
[00113] FIG. 49 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
[00114] FIG. 50 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
[00115] FIG. 51 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure.
[00116] FIG. 52 shows a graph illustrating rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown, according to embodiments of the present disclosure. [00117] FIG. 53 shows a graph illustrating the efficacy of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 moderate JIMT-1 breast tumor xenograft model, according to embodiments of the present disclosure.
[00118] FIG. 54 shows a graph illustrating in vitro potency of enfortumab conjugates according to embodiments of the present disclosure against HEK-293 cells overexpressing rat nectin-4.
[00119] FIG. 55 shows a graph illustrating in vitro potency of enfortumab conjugates according to embodiments of the present disclosure against HEK-293 cells overexpressing human nectin-4.
[00120] FIG. 56 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
[00121] FIG. 57 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line SK-BR- 3.
[00122] FIG. 58 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
[00123] FIG. 59 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
[00124] FIG. 60 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
[00125] FIG. 61 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.
[00126] FIG. 62 shows an HPLC trace demonstrating that Compound 14 conjugated to Enfortumab yields a DAR of 3.7 as determined by reduced PLRP..
[00127] FIG. 63 shows an HPLC trace demonstrating that Compound 14 conjugated to Enfortumab is 97.1% monomeric as determined by analytical SEC. [00128] FIG. 64 shows an HPLC trace demonstrating that Compound 18 conjugated to Enfortumab yields a DAR of 3.7 as determined by reduced PLRP.
[00129] FIG. 65 shows an HPLC trace demonstrating that Compound 18 conjugated to Enfortumab is 98.0% monomeric as determined by analytical SEC.
[00130] FIG. 66 shows an HPLC trace demonstrating that Compound 36 conjugated to Trastuzumab yields a DAR of 7.84 as determined by reduced PLRP.
[00131] FIG. 67 shows an HPLC trace demonstrating that Compound 36 conjugated to Trastuzumab is 96.9% monomeric as determined by analytical SEC.
[00132] FIG. 68 shows an HPLC trace demonstrating that Compound 37 conjugated to Trastuzumab yields a DAR of 7.81 as determined by reduced PLRP.
[00133] FIG. 69 shows an HPLC trace demonstrating that Compound 37 conjugated to Trastuzumab is 95.5% monomeric as determined by analytical SEC.
[00134] FIG. 70 shows an HPLC trace demonstrating that Compound 37 conjugated to Ibalizumab yields a DAR of 7.1 as determined by reduced PLRP.
[00135] FIG. 71 shows an HPLC trace demonstrating that Compound 37 conjugated to Ibalizumab is 98.0% monomeric as determined by analytical SEC.
[00136] FIG. 72 shows an HPLC trace demonstrating that Compound 42 conjugated to anti-FITC yields a DAR of 7.5 as determined by reduced PLRP.
[00137] FIG. 73 shows an HPLC trace demonstrating that Compound 42 conjugated to anti-FITC is 96.6% monomeric as determined by analytical SEC.
[00138] FIG. 74 shows an HPLC trace demonstrating that Compound 46 conjugated to Trastuzumab yields a DAR of 7.9 as determined by reduced PLRP.
[00139] FIG. 75 shows an HPLC trace demonstrating that Compound 46 conjugated to Trastuzumab is 94.9% monomeric as determined by analytical SEC.
[00140] FIG. 76 shows an HPLC trace demonstrating that Compound 46 conjugated to anti-FITC yields a DAR of 7.7 as determined by reduced PLRP.
[00141] FIG. 77 shows an HPLC trace demonstrating that Compound 46 conjugated to anti-FITC is 97.2% monomeric as determined by analytical SEC.
[00142] FIG. 78 shows an HPLC trace demonstrating that Compound 50 conjugated to Trastuzumab yields a DAR of 7.75 as determined by reduced PLRP. [00143] FIG. 79 shows an HPLC trace demonstrating that Compound 50 conjugated to Trastuzumab is 96.0% monomeric as determined by analytical SEC.
[00144] FIG. 80 shows an HPLC trace demonstrating that Compound 50 conjugated to anti-FITC yields a DAR of 7.82 as determined by reduced PLRP.
[00145] FIG. 81 shows an HPLC trace demonstrating that Compound 50 conjugated to anti-FITC is 97.8% monomeric as determined by analytical SEC.
[00146] FIG. 82 shows an HPLC trace demonstrating that Compound 57 conjugated to Trastuzumab yields a DAR of 7.61 as determined by reduced PLRP.
[00147] FIG. 83 shows an HPLC trace demonstrating that Compound 57 conjugated to Trastuzumab is 95.9% monomeric as determined by analytical SEC.
[00148] FIG. 84 shows an HPLC trace demonstrating that Compound 57 conjugated to anti-FITC yields a DAR of 7.72 as determined by reduced PLRP.
[00149] FIG. 85 shows an HPLC trace demonstrating that Compound 57 conjugated to anti-FITC is 97.3% monomeric as determined by analytical SEC.
[00150] FIG. 86 shows an HPLC trace demonstrating that Compound 60 conjugated to Trastuzumab yields a DAR of 7.8 as determined by reduced PLRP.
[00151] FIG. 87 shows an HPLC trace demonstrating that Compound 60 conjugated to Trastuzumab is 96.2% monomeric as determined by analytical SEC.
[00152] FIG. 88 shows an HPLC trace demonstrating that Compound 60 conjugated to anti-FITC yields a DAR of 7.7 as determined by reduced PLRP.
[00153] FIG. 89 shows an HPLC trace demonstrating that Compound 60 conjugated to anti-FITC is 97.4% monomeric as determined by analytical SEC.
[00154] FIG. 90 shows an HPLC trace demonstrating that Compound 61 conjugated to Trastuzumab yields a DAR of 3.9 as determined by reduced PLRP.
[00155] FIG. 91 shows an HPLC trace demonstrating that Compound 61 conjugated to Trastuzumab is 97.1% monomeric as determined by analytical SEC.
[00156] FIG. 92 shows an HPLC trace demonstrating that Compound 61 conjugated to anti-FITC yields a DAR of 3.9 as determined by reduced PLRP.
[00157] FIG. 93 shows an HPLC trace demonstrating that Compound 61 conjugated to anti-FITC is 98.6% monomeric as determined by analytical SEC. [00158] FIG. 94 shows an HPLC trace demonstrating that Compound 65 conjugated to Trastuzumab yields a DAR of 6.51 as determined by reduced PLRP.
[00159] FIG. 95 shows an HPLC trace demonstrating that Compound 65 conjugated to Trastuzumab is 95.3% monomeric as determined by analytical SEC.
[00160] FIG. 96 shows an HPLC trace demonstrating that Compound 65 conjugated to Trastuzumab yields a DAR of 6.9 as determined by reduced PLRP.
[00161] FIG. 97 shows an HPLC trace demonstrating that Compound 65 conjugated to Trastuzumab is 97.9% monomeric as determined by analytical SEC.
[00162] FIG. 98 shows an HPLC trace demonstrating that Compound 62 conjugated to anti-FITC yields a DAR of 3.7 as determined by reduced PLRP.
[00163] FIG. 99 shows an HPLC trace demonstrating that Compound 62 conjugated to anti-FITC is 98.7% monomeric as determined by analytical SEC.
[00164] FIG. 100 shows an HPLC trace demonstrating that Compound 62 conjugated to Trastuzumab yields a DAR of 3.8 as determined by reduced PLRP.
[00165] FIG. 101 shows an HPLC trace demonstrating that Compound 62 conjugated to Trastuzumab is 96.9% monomeric as determined by analytical SEC.
[00166] FIG. 102 shows an HPLC trace demonstrating that Compound 66 conjugated to Trastuzumab yields a DAR of 3.0 as determined by reduced PLRP.
[00167] FIG. 103 shows an HPLC trace demonstrating that Compound 66 conjugated to Trastuzumab is 95.4% monomeric as determined by analytical SEC.
[00168] FIG. 104 shows an HPLC trace demonstrating that Compound 66 conjugated to anti-FITC yields a DAR of 3.0 as determined by reduced PLRP.
[00169] FIG. 105 shows an HPLC trace demonstrating that Compound 66 conjugated to anti-FITC is 97.0% monomeric as determined by analytical SEC.
[00170] FIG. 106 shows an HPLC trace demonstrating that Compound 59 conjugated to anti-FITC yields a DAR of 5.72 as determined by reduced PLRP.
[00171] FIG. 107 shows an HPLC trace demonstrating that Compound 59 conjugated to anti-FITC is 97.2% monomeric as determined by analytical SEC.
[00172] FIG. 108 shows an HPLC trace demonstrating that Compound 59 conjugated to Trastuzumab yields a DAR of 7.06 as determined by reduced PLRP. [00173] FIG. 109 shows an HPLC trace demonstrating that Compound 59 conjugated to Trastuzumab is 95.8% monomeric as determined by analytical SEC.
DEFINITIONS
[00174] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.
[00175] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms.
This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CFE CH ), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).
[00176] The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C1 carbon atom) have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NRaRb, wherein R and R may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
[00177] “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -O-, -NR10-, -NR10C(O)-, -C(O)NR10- and the like. This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like. [00178] “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below. [00179] The term “alkane” refers to alkyl group and alkylene group, as defined herein.
[00180] The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl” refers to the groups R’NHR”- where R’ is alkyl group as defined herein and R” is alkylene, alkenylene or alkynylene group as defined herein.
[00181] The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.
[00182] “Alkoxy” refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec- butoxy, n-pentoxy, and the like. The term “alkoxy” also refers to the groups alkenyl-O-, cycloalky l-O- , cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
[00183] The term “substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.
[00184] The term “alkoxyamino” refers to the group -NH-alkoxy, wherein alkoxy is defined herein.
[00185] The term “haloalkoxy” refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.
[00186] The term “haloalkyl” refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group.
Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like.
[00187] The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. [00188] The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S- substitutcd alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substitutcd alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
[00189] “Alkenyl” refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi- vinyl, allyl, and but-3-en-1-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.
[00190] The term “substituted alkenyl” refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and - SO2-heteroaryl.
[00191] “Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH2C≡CH).
[00192] The term “substituted alkynyl” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and - SO2-heteroaryl. [00193] “Alkynyloxy” refers to the group -O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, cthynyloxy, propynyloxy, and the like.
[00194] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl- C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)-
[00195] “Acylamino” refers to the groups -NR20C(O)alkyl, -NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, -NR20C(O)substituted cycloalkyl, - NR20C(O)cycloalkenyl, -NR20C(O) substituted cycloalkenyl, -NR20C(O)alkenyl, - NR20C(O)substituted alkenyl, -NR20C(O)alkynyl, -NR20C(O)substituted alkynyl, -NR20C(O)aryl, -NR20C(O)substituted aryl, -NR20C(O)heteroaryl, -NR20C(O) substituted heteroaryl, -NR20C(O)heterocyclic, and -NR20C(O)substituted heterocyclic, wherein R20 is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[00196] “Aminocarbonyl” or the term “aminoacyl” refers to the group -C(O)NR21R22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21 and R22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [00197] “Aminocarbonylamino” refers to the group -NR21C(O)NR22R23 where R21, R22, and R23 arc independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.
[00198] The term “alkoxycarbonylamino” refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
[00199] The term “acyloxy” refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
[00200] “Aminosulfonyl” refers to the group -SO2NR21 R22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21 and R22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.
[00201] “Sulfonylamino” refers to the group -NR21SO2R22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21 and R22 are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[00202] “Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.
[00203] “Aryloxy” refers to the group -O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.
[00204] “Amino” refers to the group -NH2.
[00205] The term “substituted amino” refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.
[00206] The term “azido” refers to the group -N3.
[00207] “Carboxyl,” “carboxy” or “carboxylate” refers to -CO2H or salts thereof.
[00208] “Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkcnyl, aryl, substituted aryl, hctcroaryl, substituted hctcroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[00209] “(Carboxyl ester)oxy” or “carbonate” refers to the groups -O-C(O)O- alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O- C(O)O-alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O- C(O)O-cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O- substituted cycloalkenyl, -O-C(O)O-heteroaryl, -O-C(O)O-substituted heteroaryl, -O-C(O)O- heterocyclic, and -O-C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[00210] “Cyano” or “nitrile” refers to the group -CN.
[00211] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
[00212] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.
[00213] “Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds. [00214] The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.
[00215] “Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.
[00216] “Cycloalkoxy” refers to -O-cycloalkyl.
[00217] “Cycloalkenyloxy” refers to -O-cycloalkenyl.
[00218] “Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.
[00219] “Hydroxy” or “hydroxyl” refers to the group -OH.
[00220] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and l to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzo thienyl), wherein at least one ring within the ring system is aromatic. To satisfy valence requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent group, e.g., an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N- oxide (N→ O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, - SO-substitutcd alkyl, -SO-aryl, -SO-hctcroaryl, -SO2-alkyl, -SO2-substitutcd alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.
[00221] The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.
[00222] “Heteroaryloxy” refers to -O-heteroaryl.
[00223] “Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, where, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or - SO2- moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein.
[00224] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1 ,2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo [b] thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1- dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.
[00225] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hctcroaryl, hctcroaryloxy, hctcrocyclyl, hctcrocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, - SO2- substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.
[00226] “Heterocyclyloxy” refers to the group -O-heterocyclyl.
[00227] The term “heterocyclylthio” refers to the group heterocyclic-S-.
[00228] The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein.
[00229] The term “hydroxyamino” refers to the group -NHOH.
[00230] “Nitro” refers to the group -NO2.
[00231] “ Oxo” refers to the atom (=O).
[00232] “Sulfonyl” refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, - SO2- substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cylcoalkyl, -SO2-cycloalkenyl, -SO2- substituted cylcoalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.
[00233] “Sulfonyloxy” refers to the group -OSO2-alkyl, -OSCT-substituted alkyl, -OSO2- alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted cylcoalkyl, -OSO2- cycloalkenyl, -OSO2-substituted cylcoalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2- heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclic, and -OSO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[00234] “Sulfate” or “sulfate ester” refers the group -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2-O- substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O- SO2-O- substituted cylcoalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cylcoalkenyl, -O- SO2-O-aryl, -O-SO2-O-substituted aryl, -O-SO2-O-heteroaryl, -O-SO2-O-substituted heteroaryl, - O-SO2-O-heterocyclic, and -O-SO2-O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.
[00235] The term “aminocarbonyloxy” refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
[00236] “Thiol” refers to the group -SH.
[00237] “Thioxo” or the term “thioketo” refers to the atom (=S).
[00238] “Alkylthio” or the term “thioalkoxy” refers to the group -S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.
[00239] The term “substituted thioalkoxy” refers to the group -S-substituted alkyl.
[00240] The term “thioaryloxy” refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.
[00241] The term “thioheteroaryloxy” refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.
[00242] The term “thioheterocyclooxy” refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.
[00243] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
[00244] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2 or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O
M+, -SO2OR70, -OSO2R70, -OSO2O M+, -OSO2OR70, -P(O)(O )2(M+)2, -P(O)(OR70)O M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R™, -OC(S)R70, -0C(0)0 M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2 M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively, two R80 s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+ is a counter ion with a net single positive charge. Each M+ may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as +N(R60)4; or an alkaline earth ion, such as [Ca2+]o.s, [Mg2+]o.5, or [Ba2+]o.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80 is meant to include -NH2, -NH-alkyl, A-pyrrolidinyl, /V-pipcrazinyl, 4/V-mcthyl-pipcrazin- 1 -yl and N- morpholinyl.
[00245] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R™, -SO3 M+, -SO3R70, -OSO2R70, -OSO3 M+, -OSO3R70, -PO3 -2(M+)2, -P(O)(OR70)O M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2
M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2 M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2 M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S-M+. [00246] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” hctcroalkyl and cyclohetero alkyl groups are, unless otherwise specified, -R60, -O-M+, -OR™, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O M+, -S(O)2OR70, -OS(O)2R70, -OS(O)2 -O-M+. -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S)R7 0, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R7 0, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, - NR70C(O)NR80R80, -NR70C(NR70)R70 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined.
[00247] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
[00248] It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.
[00249] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.
[00250] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds. [00251] The term “pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.
[00252] The term “salt thereof’ means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
[00253] “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
[00254] “Stereoisomer” and “stereoisomers” refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
[00255] “Tautomer” refers to alternate forms of a molecule that differ only in electronic bonding of atoms and/or in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a -N=C(H)-NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.
[00256] It will be appreciated that the term “or a salt or solvate or stereoisomer thereof’ is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound. [00257] “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.
[00258] “Patient” refers to human and non-human subjects, especially mammalian subjects. [00259] The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.
[00260] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless specifically indicated otherwise, “polypeptide,” “peptide,” and “protein” can include genetically coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, proteins which contain at least one N-terminal methionine residue (e.g., to facilitate production in a recombinant host cell); immunologically tagged proteins; and the like. In certain embodiments, a polypeptide is an antibody.
[00261] Native amino acid sequence” or “parent amino acid sequence” are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include at least one modified amino acid residue.
[00262] “Native amino acid” or “native amino acid residue” are used interchangeably herein to refer to natural amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Vai or V, Trp or W, Tyr or Y). A native amino acid can be positioned in its naturally occurring position in a native amino acid sequence.
[00263] The terms “amino acid analog,” “unnatural amino acid,” and the like may be used interchangeably, and include amino acid-like compounds that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Vai or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and/or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include α- hydroxy acids, and α-amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.
[00264] The terms “amino acid side chain” or “side chain of an amino acid” and the like may be used to refer to the substituent attached to the α-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. An amino acid side chain can also include an amino acid side chain as described in the context of the modified amino acids and/or conjugates described herein.
[00265] The term “carbohydrate” and the like may be used to refer to monomers units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to the smaller carbohydrates, such as monosaccharides, disaccharides. The term “carbohydrate derivative” includes compounds where one or more functional groups of a carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemistry) or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates. [00266] The term “glycoside” or “glycosyl” refers to a sugar molecule or group bound to a moiety via a glycosidic bond. For example, the moiety that the glycoside is bound to can be a cleavable linker as described herein. A glycosidic bond can link the glycoside to the other moiety through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O- glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl). In some cases, glycosides can be cleaved from the moiety they are attached to, such as by chemically-mediated hydrolysis or enzymatically- mediated hydrolysis.
[00267] The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), and the like. An antibody is capable of binding a target antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen can have one or more binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) formed by one or more variable regions of an antibody.
[00268] The term “natural antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism. Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies. For example, the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies.
[00269] The term “humanized antibody” or “humanized immunoglobulin” refers to a non- human (e.g., mouse or rabbit) antibody containing one or more amino acids (in a framework region, a constant region or a CDR, for example) that have been substituted with a correspondingly positioned amino acid from a human antibody. In general, humanized antibodies produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, framework substitutions are identified by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies contemplated for use in the present invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO 98/45331 and WO 98/45332. In particular embodiments, a subject rabbit antibody may be humanized according to the methods set forth in US20040086979 and US20050033031. Accordingly, the antibodies described above may be humanized using methods that are well known in the art.
[00270] The term “chimeric antibodies” refer to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although domains from other mammalian species may be used.
[00271] An immunoglobulin polypeptide immunoglobulin light or heavy chain variable region is composed of a framework region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, 1991). The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen.
[00272] A “parent Ig polypeptide” is a polypeptide comprising an amino acid sequence which lacks an aldehyde-tagged constant region as described herein. The parent polypeptide may comprise a native sequence constant region, or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions). [00273] As used herein the term “isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.
[00274] As used herein, the term “substantially purified” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated.
[00275] The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.
[00276] By “reactive partner” is meant a molecule or molecular moiety that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include a cysteine residue or lysine residue, which can react with a cysteine-reactive moiety or a lysine-reactive moiety, respectively, to form a reaction product where a moiety of interest is attached to the cysteine residue or the lysine residue. For example, a maleimide group can be a reactive partner for a cysteine residue, or an N-hydroxysuccinimide ester (NHS ester) can be a reactive partner for a lysine residue.
[00277] “N-terminus” refers to the terminal amino acid residue of a polypeptide having a free amine group, which amine group in non-N-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide.
[00278] “C-terminus” refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, which carboxyl group in non-C-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide.
[00279] By “internal site” as used in referenced to a polypeptide or an amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or at the C-terminus.
[00280] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[00281] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[00282] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (i.e., compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.
[00283] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [00284] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[00285] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
[00286] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
[00287] The present disclosure provides cleavable linkers for antibody-drug conjugates (ADCs) where the linker includes a tandem-cleavage moiety. In addition, the disclosure also encompasses compounds and methods for production of such ADCs, as well as methods of using the ADCs.
ANTIBODY-DRUG CONJUGATES
[00288] The present disclosure provides a conjugate, e.g., an antibody-drug conjugate (ADC). By “conjugate” is meant a polypeptide (e.g., an antibody) that is covalently attached to one or more other moieties (e.g., drugs or active agents). For example, an antibody-drug conjugate according to the present disclosure includes one or more drugs or active agents covalently attached to an antibody. In certain embodiments, the polypeptide (e.g., antibody) and the one or more drugs or active agents are bound to each other through one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds can include a cleavable linker as described herein (e.g., a tandem-cleavage linker as described herein).
[00289] In certain embodiments, the conjugate is a polypeptide conjugate, which includes a polypeptide (e.g., an antibody) conjugated to one or more other moieties. In certain embodiments, the one or more moieties conjugated to the polypeptide can each independently be any of a variety of moieties of interest such as, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilization of the polypeptide to a membrane or a surface. In certain embodiments, the conjugate is a drug conjugate, where the polypeptide is an antibody, thus providing an antibody-drug conjugate. For instance, the conjugate can be a drug conjugate, where a polypeptide is conjugated to one or more drugs or active agents.
[00290] The one or more drugs or active agents can be conjugated to the polypeptide (e.g., antibody) at any desired site of the polypeptide. Thus, the present disclosure provides, for example, a polypeptide having a drug or active agent conjugated at a site at or near the C- terminus of the polypeptide. Other examples include a polypeptide having a drug or active agent conjugated at a position at or near the N-terminus of the polypeptide. Examples also include a polypeptide having a drug or active agent conjugated at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). Combinations of the above are also possible where the polypeptide is conjugated to two or more drugs or active agents.
[00291] In some embodiments, the one or more drugs or active agents are conjugated to the polypeptide (e.g., antibody) at one or more native amino acid residues of the polypeptide. For example, maleimide-based conjugation can be used to attach the one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native cysteine residues of the polypeptide (e.g., antibody). In other instances, N-hydroxy succinimide ester (NHS ester) based conjugation can be used to attach the one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native lysine residues of the polypeptide (e.g., antibody).
[00292] In certain embodiments, a conjugate of the present disclosure includes one or more drugs or active agents conjugated to an amino acid residue of a polypeptide at the α-carbon of an amino acid residue. Stated another way, a conjugate includes a polypeptide where the side chain of one or more amino acid residues in the polypeptide has been modified to be attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents through a linker as described herein). For example, a conjugate includes a polypeptide where the a-carbon of one or more amino acid residues in the polypeptide has been modified to be attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents through a linker as described herein).
[00293] Embodiments of the present disclosure include conjugates where a polypeptide is conjugated to one or more moieties, such as 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, 10 moieties, 11 moieties, 12 moieties, 13 moieties, 14 moieties, 15 moieties, 16 moieties, 17 moieties, 18 moieties, 19 moieties, or 20 or more moieties. The moieties may be conjugated to the polypeptide at one or more sites in the polypeptide. For example, one or more moieties may be conjugated to a single amino acid residue of the polypeptide. In some cases, one moiety is conjugated to an amino acid residue of the polypeptide. In other embodiments, two moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first moiety is conjugated to a first amino acid residue of the polypeptide and a second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example where a polypeptide is conjugated to a first moiety at a first amino acid residue and conjugated to two other moieties at a second amino acid residue. Other combinations are also possible, such as, but not limited to, a polypeptide conjugated to first and second moieties at a first amino acid residue and conjugated to third and fourth moieties at a second amino acid residue, etc. In some cases, two or more amino acid residues in the polypeptide are each conjugated to a pair of moieties (i.e., two moieties), where each pair of moieties is conjugated to the polypeptide through a branched linker as described herein. In some cases, one amino acid residue in the polypeptide is conjugated to a pair of moieties through a branched linker as described herein. In some cases, two amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, three amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, four amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, five amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, six amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, seven amino acid residues in the polypeptide arc each conjugated to a pair of moieties through a branched linker as described herein. In some cases, eight amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein.
[00294] The one or more amino acid residues of the polypeptide that are conjugated to the one or more moieties may be naturally occurring amino acids, unnatural amino acids, or combinations thereof. For instance, the conjugate may include one or more drugs or active agents conjugated to one or more naturally occurring amino acid residues (e.g., a native amino acid residues) of the polypeptide. In other instances, the conjugate may include one or more drugs or active agents conjugated to one or more unnatural amino acid residues of the polypeptide. One or more drugs or active agents may be conjugated to the polypeptide at a single natural (e.g., native) or unnatural amino acid residue as described above. One or more natural (e.g., native) or unnatural amino acid residues in the polypeptide may be conjugated to the moiety or moieties as described herein. For example, two (or more) amino acid residues (e.g., natural (e.g., native) or unnatural amino acid residues) in the polypeptide may each be conjugated to one or two moieties, such that multiple sites in the polypeptide are conjugated to the moieties of interest.
[00295] In certain embodiments, the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) are conjugated through a conjugation moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bonded) to the conjugation moiety, thus indirectly binding the polypeptide and the moiety of interest together through the conjugation moiety.
[00296] In some cases, the conjugation moiety includes maleimide or derivative thereof. For instance, a maleimide conjugation moiety may be conjugated to a cysteine residue of the polypeptide (e.g., antibody), such as a native cysteine residue of the polypeptide (e.g., antibody), thus indirectly binding the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) together through the maleimide conjugation moiety.
[00297] In some cases, the conjugation moiety includes an N-hydroxysuccinimide ester (NHS ester) or derivative thereof. For instance, an NHS-ester conjugation moiety may be conjugated to a lysine residue of the polypeptide (e.g., antibody), such as a native lysine residue of the polypeptide (e.g., antibody), thus indirectly binding the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) together through an NHS-ester conjugation moiety.
[00298] In some instances, a drug or active agent can be modified to include a conjugation moiety, which is then conjugated to a polypeptide (e.g., antibody) to produce a polypeptide (e.g., antibody) conjugate, thus attaching the drug or active agent to the polypeptide (e.g., antibody) through the conjugation moiety.
[00299] In certain embodiments, the conjugation moiety may be attached (e.g., covalently attached) to a linker. As such, embodiments of the present disclosure include a conjugation moiety attached to a drug or active agent through a linker. Various embodiments of the linkers that may be used to couple the conjugation moiety to the drug or active agent are described in detail herein. For example, in some instances, the linker is a cleavable linker, such as a cleavable linker as described herein.
[00300] In certain embodiments, the linker is a branched linker, such as a branched linker as described herein. In some instances, the conjugation moieties may be attached (e.g., covalently attached) to two or more linkers. As such, embodiments of the present disclosure include a conjugation moiety attached to two or more drugs or active agents each through a corresponding linker. Thus, conjugates of the present disclosure may include two or more linkers, where each linker attaches a corresponding drug or active agent to the conjugation moiety. Accordingly, the conjugation moiety and two or more linkers may be viewed overall as a “branched linker”, where the conjugation moiety is attached to two or more “branches”, where each branch includes a linker attached to a drug or active agent.
[00301] In certain embodiments, the polypeptide may be conjugated to one or more moieties of interest, where one or more amino acid residues of the polypeptide are native amino acid residues of the polypeptide. For example, the polypeptide may be conjugated to one or more moieties of interest, where one or more amino acid residues of the polypeptide are not modified before conjugation to the moiety of interest. Examples of native amino acid residues that may be conjugated to the one or more moieties of interest include, but are not limited to, cysteine, lysine, and the like. In some cases, the amino acid residue that is conjugated to the one or more moieties of interest is a native amino acid residue that does not have an aldehyde group, and the conjugation moiety is not a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. [00302] In some cases, to produce the conjugate, the polypeptide may be conjugated to the moiety of interest by reaction of an amino acid residue of the polypeptide with a compound (c.g., a compound containing a conjugation moiety, as described herein). For example, a polypeptide may be contacted with a reactive partner-containing drug under conditions suitable to provide for conjugation of the drug to the polypeptide. In some instances, the reactive partner-containing drug may include a conjugation moiety as described above. For example, a drug or active agent may be modified to include a conjugation moiety. In some cases, the drug or active agent is attached to a conjugation moiety, such as covalently attached to conjugation moiety through a linker, such as a linker as described in detail herein.
[00303] In certain embodiments, a conjugate of the present disclosure includes a polypeptide (e.g., an antibody) having at least one amino acid residue that has been attached to one or more moieties of interest (e.g., drugs or active agents). In order to make the conjugate, an amino acid residue of the polypeptide (e.g., a native amino acid residue) may be coupled to one or more drugs or active agents attached to a conjugation moiety as described above. In certain embodiments, the native amino acid residue of the polypeptide (e.g., antibody) is a cysteine or lysine residue. In certain embodiments, the amino acid residue is conjugated to a drug or active agent containing a conjugation moiety as described above to provide a conjugate of the present disclosure where the one or more drugs or active agents are conjugated to the polypeptide through the conjugation moiety.
[00304] In certain embodiments, the conjugate includes a polypeptide (e.g., an antibody) having at least one native amino acid residue attached to a linker as described herein, which in turn is attached to one or more drugs or active agents. For instance, the conjugate may include a polypeptide (e.g., an antibody) having at least one native amino acid residue that is conjugated to the one or more moieties of interest (e.g., one or more drugs or active agents) as described above. [00305] Aspects of the present disclosure include a conjugate of formula (I):
Figure imgf000057_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug;
W2 is an antibody; and
X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
[00306] The substituents related to conjugates of formula (I) are described in more detail below.
[00307] In certain embodiments, each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In certain embodiments, R1 is hydrogen. In certain embodiments, R1 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R1 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R1 is alkynyl or substituted alkynyl.
[00308] In certain embodiments, each R2 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R2 is hydrogen. In certain embodiments, R2 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R2 is methyl. In certain embodiments, R2 is isopropyl. In certain embodiments, R2 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R2 is alkynyl or substituted alkynyl. In certain embodiments, R2 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C& aryl or C6 substituted aryl. In certain embodiments, R2 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R2 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R2 is heterocyclyl or substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.
[00309] In certain embodiments, R1 is H and R2 is alkyl or substituted alkyl. For example, in some instances, R1 is H and R2 is methyl; or R1 is H and R2 is isopropyl.
[00310] In certain embodiments, k1 is an integer from 1 to 10. In certain embodiments, k1 is 1. In certain embodiments, k1 is 2. In certain embodiments, k1 is 3. In certain embodiments, k1 is 4. In certain embodiments, k1 is 5. In certain embodiments, k1 is 6. In certain embodiments, k1 is 7. In certain embodiments, k1 is 8. In certain embodiments, k1 is 9. In certain embodiments, k1 is 10.
[00311] In certain embodiments, k1 is 2, and each R1 is H and each R2 is alkyl or substituted alkyl. For example, in some instances, k1 is 2, and each R1 is H and one R2 is methyl and the other R2 is isopropyl.
[00312] In certain embodiments, R3 comprises a cleavable moiety, as described in more detail herein. For example, in some cases, R3 can be a chemically-cleavable moiety, as described in more detail herein. In some cases, R3 can be an enzymatically-cleavable moiety, as described in more detail herein (e.g., a glycoside or a glycoside derivative).
[00313] In certain embodiments, W1 is a drug (or active agent). Further description of drugs (or active agents) that find use in the subject conjugates is found in the disclosure herein. [00314] In certain embodiments, W2 is a polypeptide. For example, W2 can be an antibody. In certain embodiments, W2 comprises one or more amino acid residues (e.g., native amino acid residues) that are attached to the drug, W1, through the linker, L, as described herein. In certain embodiments, the polypeptide (e.g., antibody) is attached to the rest of the conjugate through a native amino acid residue as described herein. Further description of polypeptides and antibodies that find use in the subject conjugates is found in the disclosure herein.
[00315] In certain embodiments, LA is a linker (e.g., a first linker). The linker, LA, may be attached on one end of the linker to the drug, W1, and attached at another end of the linker to the antibody, W2. Linkers suitable for LA are described in more detail below.
[00316] In certain embodiments, X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody. In certain embodiments, the antibody and the drug are conjugated through the conjugation moiety, X. For example, the antibody and the drug may each be bound (e.g., covalently bonded) to the conjugation moiety, X, thus indirectly binding the antibody and the drug together through the conjugation moiety, X.
[00317] In some cases, the conjugation moiety, X, includes maleimide or derivative thereof. For instance, a maleimide conjugation moiety may react with and thus be attached (e.g., covalently attached) to a cysteine residue of the antibody, such as a native cysteine residue of the antibody, thus indirectly binding the antibody and the drug together through the maleimide conjugation moiety. Examples of a maleimide conjugation moiety are exemplified in FIG. 33 herein.
[00318] Other conjugation groups that may be used for attachment to a cysteine residue of the antibody include those exemplified in FIG. 34 herein. Other conjugation groups that may be used for attachment to a cysteine residue of the antibody include P5 phosphoramidate or thiobridge conjugation moieties, as shown in FIG. 35 and FIG. 36, respectively.
[00319] In some cases, the conjugation moiety, X, includes an N-hydroxysuccinimide (NHS) or derivative thereof. For instance, an NHS-ester conjugation moiety may react with and thus be attached (e.g., covalently attached) to a lysine residue of the antibody, such as a native lysine residue of the antibody, thus indirectly binding the antibody and the drug together through an NHS-ester conjugation moiety. Examples of NHS-ester conjugation moieties are exemplified in FIG. 37 herein.
[00320] Other conjugation groups that may be used for attachment include conjugation moieties for attachment to an N-terminal amine of the antibody. For example, an N-terminal amine can be oxidized with N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport’s salt, RS) to afford an aldehyde or ketone tagged antibody, followed by conjugation using an oxime or hydrazone linkage. An example of conjugation to an N-terminal amine is illustrated in FIG. 38 herein.
[00321] In certain embodiments, the conjugate of formula (I) includes a linker, LA. The linker may be utilized to bind one or more moieties of interest (e.g., drug or active agent) to one or more polypeptides through the conjugation moiety, X. The linker may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein) at any convenient position. For example, the linker may attach the conjugation moiety to a drug. The conjugation moiety may be used to conjugate the linker (and thus the drug) to a polypeptide, such as an antibody. For example, the conjugation moiety may be used to conjugate the linker (and thus the drug) to a native amino acid residue of the antibody, as described herein.
[00322] In some instances, as shown in formula (I) above, LA is attached to W2 through the conjugation moiety, X, and thus W2 is indirectly bonded to the linker LA through the conjugation moiety. As described above, W2 is an antibody, and thus LA is attached through the conjugation moiety, X, to the antibody, e.g., the linker LA is indirectly bonded to the antibody through the conjugation moiety, X.
[00323] Any convenient linker may be utilized for the linker LA in the subject conjugates and compounds. In certain embodiments, the linker LA may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the linker LA may include an alkyl or substituted alkyl group. In certain embodiments, the linker LA may include an alkenyl or substituted alkenyl group. In certain embodiments, the linker LA may include an alkynyl or substituted alkynyl group. In certain embodiments, the linker LA may include an alkoxy or substituted alkoxy group. In certain embodiments, the linker LA may include an amino or substituted amino group. In certain embodiments, the linker LA may include a carboxyl or carboxyl ester group. In certain embodiments, the linker LA may include an acyl amino group. In certain embodiments, the linker LA may include an alkylamide or substituted alkylamide group. In certain embodiments, the linker LA may include an aryl or substituted aryl group. In certain embodiments, the linker LA may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the linker LA may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the linker LA may include a heterocyclyl or substituted hctcrocyclyl group.
[00324] In certain embodiments, the linker LA may include a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is a polyethylene glycol. Other linkers are also possible, as shown in the conjugates and compounds described in more detail below.
[00325] In some embodiments, LA is a linker (e.g., a first linker) described by the formula: -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e-(L6)f-, wherein L1, L2 , L3, L4, L5 and L6 are each independently a linker subunit, and a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.
[00326] In certain embodiments, the sum of a, b, c, d, e and f is 1. In certain embodiments, the sum of a, b, c, d, e and f is 2. In certain embodiments, the sum of a, b, c, d, e and f is 3. In certain embodiments, the sum of a, b, c, d, e and f is 4. In certain embodiments, the sum of a, b, c, d, e and f is 5. In certain embodiments, the sum of a, b, c, d, e and f is 6. In certain embodiments, a, b, c, d, e and f are each 1. In certain embodiments, a, b, c, d and e are each 1 and f is 0. In certain embodiments, a, b, c and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e and f are each 0. In certain embodiments, a and b are each 1 and c, d, e and f are each 0. In certain embodiments, a is 1 and b, c, d, e and f are each 0.
[00327] In certain embodiments, the linker subunit L1 is attached to the conjugation moiety, X (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L2, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L3, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L4, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L5, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L6, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above).
[00328] Any convenient linker subunits may be utilized in the linker LA. Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and poly acrylates, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, each of L1, L2 , L3 , L4 , L5 and L6 (if present) comprise one or more groups independently selected from a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, and a diamine (e.g., a linking group that includes an alkylene diamine).
[00329] In some embodiments, L1 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L1 comprises a polyethylene glycol. In some embodiments, L1 comprises a modified polyethylene glycol. In some embodiments, L1 comprises an amino acid residue. In some embodiments, L1 comprises an alkyl group or a substituted alkyl. In some embodiments, L1 comprises an aryl group or a substituted aryl group. In some embodiments, L1 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
[00330] In some embodiments, L2 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L2 comprises a polyethylene glycol. In some embodiments, L2 comprises a modified polyethylene glycol. In some embodiments, L2 comprises an amino acid residue. In some embodiments, L2 comprises an alkyl group or a substituted alkyl. In some embodiments, L2 comprises an aryl group or a substituted aryl group. In some embodiments, L2 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
[00331] In some embodiments, L3 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L3 comprises a polyethylene glycol. In some embodiments, L3 comprises a modified polyethylene glycol. In some embodiments, L3 comprises an amino acid residue. Tn some embodiments, L3 comprises an alkyl group or a substituted alkyl. In some embodiments, L3 comprises an aryl group or a substituted aryl group. In some embodiments, L3 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
[00332] In some embodiments, L4 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L4 comprises a polyethylene glycol. In some embodiments, L4 comprises a modified polyethylene glycol. In some embodiments, L4 comprises an amino acid residue. In some embodiments, L4 comprises an alkyl group or a substituted alkyl. In some embodiments, L4 comprises an aryl group or a substituted aryl group. In some embodiments, L4 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
[00333] In some embodiments, L5 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L5 comprises a polyethylene glycol. In some embodiments, L5 comprises a modified polyethylene glycol. In some embodiments, L5 comprises an amino acid residue. In some embodiments, L5 comprises an alkyl group or a substituted alkyl. In some embodiments, L5 comprises an aryl group or a substituted aryl group. In some embodiments, L5 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
[00334] In some embodiments, L6 (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L6 comprises a polyethylene glycol. In some embodiments, L6 comprises a modified polyethylene glycol. In some embodiments, L6 comprises an amino acid residue. In some embodiments, L6 comprises an alkyl group or a substituted alkyl. In some embodiments, L6 comprises an aryl group or a substituted aryl group. In some embodiments, L6 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
[00335] In some embodiments, LA is a linker comprising -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e- (L6)f-, where:
-(L1 )a- is -(T1-V1)a-; -(L2)b- is -(T2-V2)b-;
-(L3)c- is -(T3-V3)c-;
-(L4)d- is -(T4-V4)d-;
-(L5)e- is -(T5-V5)e-; and
-(L6)f- is -(T6-V6)f-, wherein T1, T2, T3, T4, T5 and T6, if present, are tether groups;
V1, V2, V3, V4, V5 and V6, if present, are covalent bonds or linking functional groups; and a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.
[00336] As described above, in certain embodiments, L1 is attached to the conjugation moiety, X (e.g., as shown in formula (I) above). As such, in certain embodiments, T1 is attached to the conjugation moiety, X (e.g., as shown in formula (I) above). In certain embodiments, V1 is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, L2, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). As such, in certain embodiments, T2, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above), or V2, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, L3, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). As such, in certain embodiments, T3, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above), or V3, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, L4, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). As such, in certain embodiments, T4, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above), or V4, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, L5, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). As such, in certain embodiments, T5, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above), or V5, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). In certain embodiments, L6, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above). As such, in certain embodiments, T6, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above), or V6, if present, is attached to the -NR1- group (e.g., as shown in formula (I) above).
[00337] Regarding the tether groups, T1, T2, T3, T4, T5 and T6, any convenient tether groups may be utilized in the subject linkers. In some embodiments, T1, T2, T3, T4, T5 and T6 each comprise one or more groups independently selected from a covalent bond, a (C1-C12)alkyl, a substituted (C1-C12)alkyl, aryl, substituted aryl, hctcroaryl, substituted hctcroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)p, - (CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, where each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12.
[00338] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes a (C1-C12)alkyl or a substituted (C1-C12)alkyl. In certain embodiments, (C1-C12)alkyl is a straight chain or branched alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, (C1-C12)alkyl may be an alkyl or substituted alkyl, such as C1-C12 alkyl, or C1-C10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl. In some instances, (C1-C12)alkyl is a C2-alkyl. For example, (C1-C12)alkyl may be an alkylene or substituted alkylene, such as C1-C12 alkylene, or C1-C10 alkylene, or C1-C6 alkylene, or C1-C3 alkylene. In some instances, (C1-C12)alkyl is a C2-alkylene (e.g., CH2CH2). In some instances, (C1-C12)alkyl is a C3-alkylene (e.g., CH2CH2CH2).
[00339] In certain embodiments, substituted (C1-C12)alkyl is a straight chain or branched substituted alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, substituted (C1-C12)alkyl may be a substituted alkyl, such as substituted C1-C12 alkyl, or substituted C1-C10 alkyl, or substituted C1-C6 alkyl, or substitutedC1-C3 alkyl. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkyl. For example, substituted (C1-C12)alkyl may be a substituted alkylene, such as substituted C1-C12 alkylene, or substituted C1-C10 alkylene, or substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkylene. In some instances, substituted (C1-C12)alkyl is a substituted C3-alkylene. For example, substituted (C1-C12)alkyl may include C1-C12 alkylene (e.g., C3-alkylene or C5-alkylene) substituted with a (PEG)n group as described herein (e.g.,-CONH(PEG)3 or -NHCO(PEG)7), or may include C1-C12 alkylene (e.g., C3- alkylene) substituted with a -CONHCH2CH2SO3H group, or may include C1-C12 alkylene (e.g., C5-alkylene) substituted with a -NHCOCH2SO3H group. [00340] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes an aryl, substituted aryl, hctcroaryl, substituted hctcroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5 and T6) includes an aryl or substituted aryl. For example, the aryl can be phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl can be substituted with one or more substituents selected from (C1-C12)alkyl, a substituted (C1- C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some instances, the substituted aryl is a substituted phenyl, where the substituent includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).
[00341] In some instances, the tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes a heteroaryl or substituted heteroaryl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5 and T6) includes a cycloalkyl or substituted cycloalkyl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5 and T6) includes a heterocyclyl or substituted heterocyclyl. In some instances, the substituent on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclyl includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).
[00342] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, where the aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl is produced from a reaction, such as a click-chemistry reaction. For example, click- chemistry reactions may be used to connect two portions of a linker together during synthesis of the linker. Examples of click-chemistry reactions that can be used to produce an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl tether group include, but are not limited to, azide-alkyne reactions, alkyne-DBCO (dibenzocyclooctyne) reactions, TCO-Tz (trans-cycloctene tetrazine) reactions, and the like (see, e.g., FIG. 39).
[00343] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes an ethylene diamine (EDA) moiety, e.g., an EDA containing tether group. In certain embodiments, (EDA)W includes one or more EDA moieties, such as where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1 , 2, 3, 4, 5 or 6). The linked ethylene diamine (EDA) moictics may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the EDA moiety is described by the structure:
Figure imgf000068_0001
where y is an integer from 1 to 6, or is 0 or 1, and each R12 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5 or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl and a substituted aryl. In certain embodiments, any two adjacent R12 groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and the two adjacent R12 groups are an alkyl group, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R12 groups are selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH). [00344] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidin-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, a polyethylene glycol moiety, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the 4AP moiety is described by the structure:
Figure imgf000068_0002
where R12 is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., a polyethylene glycol or a modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R12 is a polyethylene glycol moiety. In certain embodiments, R12 is a carboxy modified polyethylene glycol.
[00345] In certain embodiments, R12 includes a polyethylene glycol moiety described by the formula: (PEG)k, which may be represented by the structure:
Figure imgf000069_0001
where k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, k is 2. In certain embodiments, R17 is selected from OH, OR, COOH, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R17 is COOH. In certain embodiments, R17 is OH. In certain embodiments, R17 is OR, such as OCH3.
[00346] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes (PEG)n, where (PEG)n is a polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG)n is described by the structure:
Figure imgf000069_0002
where n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, n is 2. In some instances, n is 3. In some instances, n is 6. In some instances, n is 12. [00347] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes (AA)P, where AA is an amino acid residue. Any convenient amino acids may be utilized. Amino acids of interest include but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 primary alpha-amino acids and beta-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), such as a non-naturally occurring alpha-amino acid or a non-naturally occurring beta-amino acid, etc. In certain embodiments, p is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.
[00348] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes an amino acid analog. Amino acid analogs include compounds that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Vai or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and/or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include α- hydroxy acids, and a-amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.
[00349] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5 and/or T6) includes a moiety described by the formula -(CR13OH)m-, where m is 0 or n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, R13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R13 is hydrogen. In certain embodiments, R13 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R13 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R13 is alkynyl or substituted alkynyl. In certain embodiments, R13 is alkoxy or substituted alkoxy. In certain embodiments, R13 is amino or substituted amino. In certain embodiments, R13 is carboxyl or carboxyl ester. In certain embodiments, R13 is acyl or acyloxy. In certain embodiments, R13 is acyl amino or amino acyl. In certain embodiments, R13 is alkylamide or substituted alkylamide. In certain embodiments, R13 is sulfonyl. In certain embodiments, R13 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R13 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R13 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R13 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R13 is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.
[00350] In certain embodiments, R13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13.
[00351] Regarding the linking functional groups, V1, V2, V3, V4, V5 and V6, any convenient linking functional groups may be utilized in the linker. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoraidate, and the like. In some embodiments, V1, V2, V3, V4, V5 and V6 are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, - CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and - P(O)OH-, where q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1 , 2, 3, 4, 5 or 6). In certain embodiments, q is 1 . In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.
[00352] In some embodiments, each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
[00353] In certain embodiments, R15 is hydrogen. In certain embodiments, each R15 is hydrogen. In certain embodiments, R15 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R15 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R15 is alkynyl or substituted alkynyl. In certain embodiments, R15 is alkoxy or substituted alkoxy. In certain embodiments, R15 is amino or substituted amino. In certain embodiments, R15 is carboxyl or carboxyl ester. In certain embodiments, R15 is acyl or acyloxy. In certain embodiments, R15 is acyl amino or amino acyl. In certain embodiments, R15 is alkylamide or substituted alkylamide. In certain embodiments, R15 is sulfonyl. In certain embodiments, R15 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R15 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R15 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R15 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R15 is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.
[00354] In certain embodiments, each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, hctcrocyclyl, and substituted hctcrocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R15.
[00355] In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.
[00356] As described above, in some embodiments, LA is a linker comprising -(T1- V1)a- (T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, where a, b, c, d, e and f are each independently 0 or 1, where the sum of a, b, c, d, e and f is 1 to 6.
[00357] In some embodiments, in the linker LA:
T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;
T2, T3, T4, T5 and T6 are each independently selected from (C1-C12)alkyl, substituted (C1- C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4- amino-piperidine (4AP), an acetal group, a disulfide, a hydrazine, and an ester; and
V1, V2, V3, V4 ,V5 and V6 are each independently selected from a covalent bond, -CO-, - NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, - SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; wherein:
(PEG)n is
Figure imgf000073_0001
where n is an integer from 1 to 30;
EDA is an ethylene diamine moiety having the following structure:
Figure imgf000073_0002
Figure imgf000073_0003
AA is an amino acid residue, where p is an integer from 1 to 20; and each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
[00358] In certain embodiments, T1, T2, T3, T4, T5 and T6 and V1, V2, V3, V4 ,V5 and V6 are selected from the following: wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-;
T3 is (PEG)n and V3 is -CO-; and d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0.
[00359] As described above, in some cases, the conjugation moiety, X, includes maleimide or derivative thereof. For instance, a maleimide conjugation moiety may be attached to a cysteine residue of the antibody, such as a native cysteine residue of the antibody. In these instances, the conjugate may be of formula (II):
Figure imgf000075_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
W2 is an antibody.
[00360] In some cases, the conjugation moiety, X, includes an acetamide or derivative thereof. For instance, an acetamide conjugation moiety may be attached to a cysteine residue of the antibody, such as a native cysteine residue of the antibody. In these instances, the conjugate may be of formula (Ila):
Figure imgf000075_0002
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety; LA is a first linker;
W1 is a drug; and
W2 is an antibody.
[00361] In embodiments of formulas (II) and (Ila), R1, R2, k1, R3, LA, W1 and W2 are as described herein (e.g., as in the description related to formula (I) herein).
[00362] In certain embodiments of the conjugate of formula (I), the antibody can be linked to one drug or active agent through the conjugation moiety and linker. In some instances, the antibody can be linked to more than one drug or active agent through the conjugation moiety and linker. For example, the conjugation moiety can be linked to two or more drugs or active agents. Each drug or active agent can be linked via a corresponding linker to the same conjugation moiety, which in turn can be attached to the antibody as described herein, thus linking the antibody to two or more drugs or active agents.
[00363] For example, in certain embodiments of the conjugate of formula (I), the linker, LA is a branched linker. In embodiments having a branched linker, one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4 ,V5 or V6 is a branched group. In some embodiments, the branched group is selected from -CONR15- and 4AP. In some embodiments, the branched group is -CONR15-. In some embodiments, the branched group is 4AP. In certain embodiments, the branched group is attached to a second linker, LB. As such, the branched group of the first linker, LA, can be attached to a first drug, W1, as shown in formula (I) above, and also attached to a second drug,
W1a, through the second linker, LB (e.g., as shown in formula (III) below).
[00364] In certain embodiments, the second linker LB is a linker described by the formula: -(L7)g-(L8)h-(L9)i-(L10)j-(L11)k-(L12)l-, wherein L7, L8 , L9, L10, L11 and L12 are each independently a linker subunit, and g, h, i, j, k and l are each independently 0 or 1, wherein the sum of g, h, i, j, k and l is 1 to 6.
[00365] In certain embodiments, the sum of g, h, i, j, k and l is I. In certain embodiments, the sum of g, h, i, j, k and l is 2. In certain embodiments, the sum of g, h, i, j, k and l is 3. In certain embodiments, the sum of g, h, i, j, k and l is 4. In certain embodiments, the sum of g, h, i, j, k and l is 5. In certain embodiments, the sum of g, h, i, j, k and l is 6. In certain embodiments, g, h, i, j, k and l are each 1. In certain embodiments, g, h, i, j and k are each 1 and l is 0. In certain embodiments, g, h, i and j are each 1 and k and l are each 0. In certain embodiments, g, h, and i are each 1 and j, k and l are each 0. Tn certain embodiments, g and h are each 1 and i, j, k and l arc each 0. In certain embodiments, g is 1 and h, i, j, k and l arc each 0.
[00366] In certain embodiments, the linker subunit L7 is attached to the branched group. In certain embodiments, the linker subunit L8, if present, is attached to the second drug, W1a. In certain embodiments, the linker subunit L9, if present, is attached to the second drug, W1a. In certain embodiments, the linker subunit L10, if present, is attached to the second drug, W1a. In certain embodiments, the linker subunit L11, if present, is attached to the second drug, W1a. In certain embodiments, the linker subunit L12, if present, is attached to the second drug, W1a.
[00367] Any convenient linker subunits may be utilized in the second linker LB. For example, any of the linker subunits described above in relation to L1, L2 , L3, L4, L5 and L6 may be used for the linker subunits L7, L8 , L9, L10, L11 and L12.
[00368] In certain embodiments, the second linker LB is a linker comprising: -(L7)g-(L8)h-(L9)i-(L10)j-(L11)k-(L12)l-, where:
-(L7)g- is -(T7-V7)g-;
-(L8)h- is -(T8-V8)h-;
-(L9)i- is -(T9-V9)i-;
-(L10)j- is -(T10-V10)j-;
-(L11)k- is -(T11-V11)k-; and
-(L12)l- is -(T12-V12)l-, wherein T7, T8, T9, T10, T11 and T12, if present, are tether groups;
V7, V8, V9, V10, V11 and V12, if present, are covalent bonds or linking functional groups; and g, h, i, j, k and l are each independently 0 or 1, wherein the sum of g, h, i, j, k and 1 is 1 to 6.
[00369] Accordingly, in certain embodiments, the second linker LB comprises: -(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and l are each independently 0 or 1;
T7, T8, T9, T10, T11 and T12 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V7, V8, V9, V10 ,V11 and V12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
[00370] Any convenient tether groups may be utilized for T7, T8, T9, T10, T11 and T12. For example, any of the tether groups described above in relation to T1, T2, T3, T4, T5 and T6 may be used for the tether groups T7, T8, T9, T10, T11 and T12.
[00371] Any convenient linking functional groups may be utilized for V7, V8, V9, V10 ,V11 and V12. For example, any of the linking functional groups described above in relation to V1, V2, V3, V4, V5 and V6 may be used for the linking functional groups V7, V8, V9, V10 ,V11 and V12.
[00372] In certain embodiments, each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13.
[00373] In certain embodiments, each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R15. In these embodiments, various possible substituents are as described above for R15. [00374] As described above, in certain embodiments, the branched group is attached to a second linker, LB. In addition, the second linker, LB, can be attached to the second drug, W1a, as shown in formula (III):
Figure imgf000079_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug.
[00375] Accordingly, LB may be attached to a compound of formula (III). For example, LB may be attached to a compound of formula (III) at the bond indicated by the wavy line in formula (III).
[00376] The substituents related to conjugates of formula (III) are described in more detail below.
[00377] In certain embodiments, each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In certain embodiments, R4 is hydrogen. In certain embodiments, R4 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R4 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R4 is alkynyl or substituted alkynyl.
[00378] In certain embodiments, each R5 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R5 is hydrogen. In certain embodiments, R5 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R5 is methyl. In certain embodiments, R5 is isopropyl. In certain embodiments, R5 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R5 is alkynyl or substituted alkynyl. In certain embodiments, R5 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R5 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R5 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R5 is heterocyclyl or substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.
[00379] In certain embodiments, R4 is H and R5 is alkyl or substituted alkyl. For example, in some instances, R4 is H and R5 is methyl; or R4 is H and R5 is isopropyl.
[00380] In certain embodiments, k2 is an integer from 1 to 10. In certain embodiments, k2 is 1. In certain embodiments, k2 is 2. In certain embodiments, k2 is 3. In certain embodiments, k2 is 4. In certain embodiments, k2 is 5. In certain embodiments, k2 is 6. In certain embodiments, k2 is 7. In certain embodiments, k2 is 8. In certain embodiments, k2 is 9. In certain embodiments, k2 is 10.
[00381] In certain embodiments, k2 is 2, and each R4 is H and each R5 is alkyl or substituted alkyl. For example, in some instances, k2 is 2, and each R4 is H and one R5 is methyl and the other R5 is isopropyl.
[00382] In certain embodiments, R6 comprises a cleavable moiety, as described in more detail herein. For example, in some cases, R6 can be a chemically-cleavable moiety, as described in more detail herein. In some cases, R6 can be an enzymatically-cleavable moiety, as described in more detail herein (e.g., a glycoside or a glycoside derivative). [00383] In certain embodiments, W1a is a drug (or active agent). In certain embodiments of the second linker LB, the left-hand side of the linker structure is attached to the branched group, and the right-hand side of the linker structure is attached to the second drug, W1a, as shown in formula (III). Further description of drugs (or active agents) that find use in the subject conjugates is found in the disclosure herein.
[00384] In certain embodiments of the branched linkers, T1, T2, T3, T4, T5 and T6 and V1, V2, V3, V4 ,V5 and V6, and T7, T8, T9, T10, T11 and T12 and V7, V8, V9, V10 ,V11 and V12 are selected from the following: wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (C1-C12)alkyl and V3 is absent;
T4 is heteroaryl (tlriazole) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-; T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0.
[00385] Combinations of the same or different payloads (e.g., drugs or active agents) may be conjugated to the antibody through the branched linker. In certain embodiments, the two payloads (e.g., drugs or active agents) attached to the branched linker are the same payload (e.g., drug or active agent). For example, a first branch of a branched linker may be attached to a payload (e.g., drug or active agent) and a second branch of the branched linker may be attached to the same payload (e.g., drug or active agent) as the first branch. In other embodiments, the two payloads (e.g., drugs or active agents) attached to the branched linker are different payloads (e.g., drugs or active agents). For example, a first branch of a branched linker may be attached to a first pay load (e.g., drug or active agent) and a second branch of the branched linker may be attached to a second payload (e.g., drug or active agent) different from the first payload (e.g., drug or active agent) attached to the first branch.
[00386] For example, in certain embodiments, the branched linker is attached to a first drug, W1, and a second drug, W1a. In some cases, W1 and W1a are the same drug. In other cases, W1 and W1a are different drugs.
[00387] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. By “synergistic”, “synergism” or “synergy” is meant a therapeutic effect that is greater than the sum of the effects of the drugs or active agents taken separately. For example, in some instances, the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both pay loads act, thereby increasing overall potency of the ADC.
[00388] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately. For example, the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).
[00389] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways. Examples of payload combinations can include, but are not limited to, cytotoxic drugs, immunomodulatory molecules to activate or inhibit immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, and the like. [00390] In some embodiments, where two different payloads are attached to the branched linker, the payloads may be selected from combinations of drugs or active agents and detectable labels. For example, a first payload may be a detectable label that is used as an imaging agent or tracer to detect the location of the ADC in vivo, while a second payload may be a drug or active agent that provides a therapeutic activity.
[00391] Various embodiments of the linkers that may couple the conjugation moiety to the drugs or active agents are described in detail herein. For example, in some instances, the linker is a cleavable linker, such as a cleavable linker as described herein.
[00392] In certain embodiments, the conjugate is an antibody-drug conjugate where the antibody and the drug are linked together by a linker (e.g., LA and/or LB), as described above. In some instances, the linker is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, where the cleavable moiety includes one or more bonds that can dissociate under certain conditions, thus separating the cleavable linker into two or more separatable portions. For example, the cleavable moiety may include one or more covalent bonds, which under certain conditions, can dissociate or break apart to separate the cleavable linker into two or more portions. As such a cleavable linker can be included in an antibody-drug conjugate, such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at a desired target site of action for the drug.
[00393] In some instances, the cleavable linker includes two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties can be configured such that cleavage of both cleavable moieties is needed in order to separate or release the drug from the antibody at a desired target site of action for the drug. For example, cleavage of the cleavable linker can be achieved by initially cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker includes a first clcavablc moiety and a second clcavablc moiety that hinders cleavage of the first cleavable moiety. By “hinders cleavage” is meant that the presence of an uncleaved second cleavable moiety reduces the likelihood or substantially inhibits the cleavage of the first cleavable moiety, thus substantially reducing the amount or preventing the cleavage of the cleavable linker. For instance, the presence of uncleaved second cleavable moiety can hinder cleavage of the first cleavable moiety. The hinderance of cleavage of the first cleavable moiety by the presence of the second cleavable moiety, in turn, substantially reduces the amount or prevents the release of the drug from the antibody. For example, the premature release of the drug from the antibody can be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired target site of action for the drug.
[00394] In some cases, since the second cleavable moiety hinders cleavage of the first cleavable moiety, cleavage of the cleavable linker can be achieved by initially cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the hinderance on the cleavage of the first cleavable moiety by exposing the first cleavable moiety, thus allowing the first cleavable moiety to be cleaved. Cleavage of the first cleavable moiety can result in the cleavable linker dissociating or separating into two or more portions as described above to release the drug from the antibody- drug conjugate. In some instances, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. By substantially is meant that about 10% or less cleavage of the first cleavable moiety occurs in the presence of an uncleaved second cleavable moiety, such as about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less cleavage of the first cleavable moiety occurs in the presence of an uncleaved second cleavable moiety.
[00395] Stated another way, the second cleavable moiety can protect the first cleavable moiety from cleavage. For instance, the presence of uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. As such, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thus allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, which, in turn, separates or releases the drug from the antibody at a desired target site of action for the drug as described above. In certain instances, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety does not in and of itself result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is still needed in order to cleave the cleavable linker).
[00396] The cleavable moieties included in the cleavable linker may each be an enzymatically cleavable moiety. For example, the first cleavable moiety can be a first enzymatically cleavable moiety and the second cleavable moiety can be a second enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more portions as described above through the enzymatic action of an enzyme. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved through the enzymatic action of an enzyme, such as, but not limited to, a peptide, a glycoside, and the like. In some instances, the enzyme that cleaves the enzymatically cleavable moiety is present at a desired target site of action, such as the desired target site of action of the drug that is to be released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in a significant amount in other areas, such as in whole blood, plasma or serum. As such, the cleavage of an enzymatically cleavable moiety can be controlled such that substantial cleavage occurs at the desired site of action, whereas cleavage does not significantly occur in other areas (e.g., systemically) or before the antibody-drug conjugate reaches the desired site of action.
[00397] For example, as described herein, antibody-drug conjugates of the present disclosure can be used for the treatment of cancer, such as for the delivery of a cancer therapeutic drug to a desired site of action where the cancer cells are present. In some cases, enzymes, such as the protease enzyme cathepsin B, can be a biomarker for cancer that is overexpressed in cancer cells. The overexpression, and thus localization, of certain enzymes in cancer can be used in the context of the enzymatically cleavable moieties included in the cleavable linkers of the antibody-drug conjugates of the present disclosure to specifically release the drug at the desired site of action (i.e., the site of the cancer (and overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by an enzyme that is overexpressed in cancer cells. For instance, the enzyme can be the protease enzyme cathepsin B. As such, in some instances, the enzymatically cleavahle moiety is a clcavablc moiety (c.g., a peptide) that can be cleaved by a protease enzyme, such as cathepsin B.
[00398] In certain embodiments, the enzymatically cleavable moiety is a peptide. The peptide can be any peptide suitable for use in the cleavable linker and that can be cleaved through the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as an enzymatically cleavable moiety include, for example, Vai- Ala, Phe-Lys, and the like. For example, the first cleavable moiety described above (i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety) can include a peptide. The presence of uncleaved second cleavable moiety can protect the first cleavable moiety (peptide) from cleavage by a protease enzyme (e.g., cathepsin B), and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. In some instances, one of the amino acid residues of the peptide that comprises the first cleavable moiety is linked to or includes a substituent, where the substituent comprises the second cleavable moiety. In some instances, the second cleavable moiety includes a glycoside.
[00399] For example, in the context of the formulae described herein, the cleavable moiety may be a peptide, such as a peptide described by the moiety:
Figure imgf000086_0001
as included in the formulae described herein (e.g., as included in formulae (I), (II), (III), (IV), (V), or (VI) described herein).
[00400] In some embodiments, the enzymatically clcavablc moiety is sugar moiety, such as a glycoside (or glyosyl). In some cases, the glycoside can facilitate an increase in the hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include the glycoside. The glycoside can be any glycoside or glycoside derivative suitable for use in the cleavable linker and that can be cleaved through the enzymatic action of an enzyme. For example, the second cleavable moiety (i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside or glycoside derivative. For instance, in some embodiments, the first cleavable moiety includes a peptide and the second cleavable moiety includes a glycoside or glycoside derivative. In certain embodiments, the second clcavablc moiety is a glycoside or glycoside derivative selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. In some instances, the second cleavable moiety is a glucuronide. In some instances, the second cleavable moiety is a galactoside. In some instances, the second cleavable moiety is a glucoside. In some instances, the second cleavable moiety is a mannoside. In some instances, the second cleavable moiety is a fucoside. In some instances, the second cleavable moiety is O-GlcNAc. In some instances, the second cleavable moiety is O-GalNAc.
[00401] For example, in the context of the formulae described herein, the cleavable moiety may be a glycoside or glycoside derivative, such as where R3 or R6 is a glycoside or glycoside derivative (e.g., as shown in formulae (I), (II), (III), (IV), (V), or (VI) described herein).
[00402] The glycoside or glycoside derivative can be attached (covalently bonded) to the cleavable linker through a glycosidic bond. The glycosidic bond can link the glycoside or glycoside derivative to the cleavable linker through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O-glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl). In some instances, the glycosidic bond is an O-glycosidic bond (an O-glycoside). In some cases, the glycoside or glycoside derivative can be cleaved from the cleavable linker it is attached to by an enzyme (e.g., through enzymatically-mediated hydrolysis of the glycosidic bond). A glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme that is able to carry out the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker. An example of an enzyme that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is a glucuronidase, a glycosidase, such as a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like. Other suitable enzymes may also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug of the antibody-drug conjugate. For instance, the enzyme can be a lysosomal enzyme, such as a lysosomal glycosidase, found in cells at or near the desired site of action for the drug of the antibody-drug conjugate. In some cases, the enzyme is an enzyme found at or near the target site where the enzyme that mediates cleavage of the first clcavablc moiety is found.
[00403] Embodiments that include a linker with the two cleavable moieties as described above can also be referred to as having a “tandem-cleavage” linker.
[00404] In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. For example, in some embodiments, the glycoside or glycoside derivative can be selected from the following structures:
Figure imgf000088_0001
[00405] In certain embodiments, the conjugate of formula (I) has a structure selected from the following:
Figure imgf000088_0002
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
[00406] Any of the chemical entities, linkers, conjugation moieties, or cleavable moieties set forth in the description above may be adapted for use in the subject compounds and conjugates.
[00407] Additional disclosure related to cleavable linkers is found in U.S. Application No. 17/423,796, filed January 22, 2020, and U.S. Application No. 17/531,343, filed November 19, 2021, the disclosures of each of which are incorporated herein by reference.
COMPOUNDS USEFUL FOR PRODUCING CONJUGATES
[00408] The present disclosure provides compounds useful for producing the conjugates described herein. In certain embodiments, the compound may include a conjugation moiety useful for conjugation of a polypeptide (e.g., an antibody) and a drug or active agent. For example, the compound may be bound to the polypeptide (antibody) and also bound to the drug or active agent, thus indirectly attaching the polypeptide (antibody) and the drug together.
[00409] In certain embodiments, the compound is a compound of formula (IV):
Figure imgf000095_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
X is a conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody. [00410] Regarding compounds of formula (IV), the substituents R1, R2, R3, R4, R5, R6, k 1 , k2, LA, LB, W1, W1a, and X arc as described above in relation to the conjugates of formula (I). Similarly, regarding the first linker LA and the second linker LB, the T1, T2, T3, T4, T5, T6, V1, V2, V3, V4, V5 and V6, and T7, T8, T9, T10, T11, T12, V7, V8, V9, V10, V11 and V12 substituents are as described above in relation to the conjugates of formula (I).
[00411] As described herein, in some cases, the conjugation moiety, X, includes maleimide or derivative thereof. For instance, a maleimide conjugation moiety may be attached to a cysteine residue of the antibody, such as a native cysteine residue of the antibody. In these instances, the compound may be of formula (V):
Figure imgf000096_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker; and
W1 is a drug.
[00412] In certain embodiments disclosed herein, the conjugation moiety, X, includes an alpha-haloacetamide, such as an alpha-bromoacetamide. In such embodiments, a cysteine residue of the antibody, such as a native cysteine residue can displace the halide to accomplish the conjugation. In one such embodiment, wherein X includes an alpha-haloacetamide, the compound has formula (Va):
Figure imgf000097_0001
(Va), wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker; and
W1 is a drug.
[00413] In embodiments of formula (V), R1, R2, R3, k1, LA, and W1 are as described herein (e.g., as in the description related to formulae (I) and (IV) herein).
[00414] In certain embodiments, T1, T2, T3, T4, T5 and T6 and V1, V2, V3, V4, V5 and V6 are selected from the following: wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-;
T3 is (PEG)n and V3 is -CO-; and d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0. [00415] As described herein, in certain embodiments, the branched group is attached to a second linker, LB. In addition, the second linker, LB, can be attached to the second drug, W1a, as shown in formula (VI):
Figure imgf000098_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug.
[00416] Accordingly, LB may be attached to a compound of formula (VI). For example, LB may be attached to a compound of formula (VI) at the bond indicated by the wavy line in formula (VI).
[00417] In embodiments of formula (VI), R4, R5, R6, k2, and W1a are as described herein (e.g., as in the description related to formulae (III) herein).
[00418] In certain embodiments of the branched linkers, T1, T2, T3, T4, T5 and T6 and V1, V2, V3, V4 ,V5 and V6, and T7, T8, T9, T10, T11 and T12 and V7, V8, V9, V10 ,V11 and V12 are selected from the following: wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-; T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l arc each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (C1-C12)alkyl and V3 is absent;
T4 is heteroaryl (triazole) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-;
T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (Ci-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0.
[00419] Compounds of formula (VI) can be used in conjugation reactions described herein, where a drug or active agent attached to a conjugation moiety is conjugated to a polypeptide (e.g., antibody) to form an antibody-drug conjugate.
In certain embodiments, the compound of formula (VI) has the following structure:
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
[00420] Any of the chemical entities, linkers, conjugation moieties and cleavable moieties set forth in the description above may be adapted for use in the subject compounds and conjugates.
POLYPEPTIDES AND ANTIBODIES
[00421] As noted above, a subject conjugate can comprise as substituent W2 a polypeptide (e.g., an antibody). As used herein, amino acids may be referred to by their standard name, their standard three letter abbreviation and/or their standard one letter abbreviation, such as: Alanine or Ala or A; Cysteine or Cys or C; Aspartic acid or Asp or D; Glutamic acid or Glu or E; Phenylalanine or Phe or F; Glycine or Gly or G; Histidine or His or H; Isoleucine or Ile or I; Lysine or Lys or K; Leucine or Leu or L; Methionine or Met or M; Asparagine or Asn or N; Proline or Pro or P; Glutamine or Gin or Q; Arginine or Arg or R; Serine or Ser or S; Threonine or Thr or T; Valine or Vai or V; Tryptophan or Trp or W; and Tyrosine or Tyr or Y.
[00422] In certain embodiments, the amino acid sequence of the polypeptide (antibody) is the native amino acid sequence of the polypeptide (antibody). In these embodiments, the conjugation moiety can be attached to the antibody at a native amino acid residue of the antibody. For example, maleimide-based conjugation can be used to attach the one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native cysteine residues of the polypeptide (e.g., antibody). The native cysteine residues of the antibody can be derived from cysteine residues that are involved in disulfide bonds, such as the 8 hinge cysteine residues that typically form intrachain disulfide bonds of an antibody. In some instances, the disulfide bonds can be reduced to provide cysteine residues where the sulfhydryl groups of the cysteine residues are available to react with and be attached (e.g., covalently attached) to a conjugation moiety (e.g., maleimide conjugation moiety) as described herein.
[00423] In other instances, N-hydroxy succinimide ester (NHS ester) based conjugation can be used to attach the one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native lysine residues of the polypeptide (e.g., antibody). The native lysine residues of the antibody can include an amine group, which is available to react with and be attached (e.g., covalently attached) to a conjugation moiety (e.g., NHS-ester conjugation moiety) as described herein. [00424] An antibody used in an antibody-drug conjugate of the present disclosure can have any of a variety of antigen-binding specificities, including but not limited to, c.g., an antigen present on a cancer cell; an antigen present on an autoimmune cell; an antigen present on a pathogenic microorganism; an antigen present on a virus-infected cell (e.g., a human immunodeficiency virus-infected cell); an antigen present on a diseased cell; and the like. For example, an antibody conjugate can bind an antigen, where the antigen is present on the surface of the cell. An antibody conjugate of the present disclosure can bind antigen with a suitable binding affinity, e.g., from 5 x 10-6 M to 10-7 M, from 10-7 M to 5 x 10-7 M, from 5 x 10-7 M to IO-8 M, from 10-8 M to 5 x 10-8 M, from 5 x 10-8 M to 10-9 M, or a binding affinity greater than 10-9 M.
[00425] As non-limiting examples, a subject antibody conjugate can bind an antigen present on a cancer cell (e.g., a tumor- specific antigen; an antigen that is over-expressed on a cancer cell; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, a subject antibody conjugate can be specific for an antigen on a cancer cell, where the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).
[00426] As further non-limiting examples, a subject antibody conjugate can bind an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by the virus; where the antigen is expressed on a cell type that is infected by a virus; etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor. For example, a subject antibody conjugate can bind an antigen present on a cell infected with a virus, and the conjugated moiety can be a drug, such as a viral fusion inhibitor.
DRUGS FOR CONJUGATION TO A POLYPEPTIDE
[00427] As noted above, a conjugate or a compound of the present disclosure can include as substituents W1 and W1a a drug or active agent. Any of a number of drugs are suitable for use, or can be modified to be rendered suitable for use, as a reactive partner to conjugate to an antibody. Examples of drugs include small molecule drugs and peptide drugs.
[00428] “Small molecule drug” as used herein refers to a compound, e.g., an organic compound, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa. A small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom.
[00429] For example, the drug or active agent can be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as a camptothecine, or an analog or derivative thereof, or a pharmaceutically active camptothecine moiety and/or a portion thereof. A topoisomerase inhibitor (e.g., camptothecine, or analog or derivative thereof) conjugated to the polypeptide can be any of a variety of topoisomerase inhibitors, for example camptothecine or camptothecine moieties such as, but not limited to, camptothecine and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to, a topoisomerase inhibitor, for example camptothecine or a camptothecine derivative, such as SN-38, Belotecan, Exatecan, 9-aminocamptothecin (9-AC), topotecan, des- -Mc-topotccan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/US2022/012325, the disclosure of which is incorporated herein by reference.
[00430] In other embodiments, the drug or active agent can be a maytansine. “Maytansine”, “maytansine moiety”, “maytansine active agent moiety” and “maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and/or portions thereof. A maytansine conjugated to the polypeptide can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein (e.g., deacylmay tansine).
[00431] In other instances, the drug or active agent can be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and/or a portion thereof. An auristatin conjugated to the polypeptide can be any of a variety of auristatin moieties such as, but not limited to, an auristatin and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to an auristatin or an auristatin derivative, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), derivatives thereof, and the like. [00432] In other cases, the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and/or a portion thereof. A duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin moieties such as, but not limited to, a duocarmycin and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to a duocarmycin or a duocarmycin derivative, such as duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof, and the like. In some embodiments, the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.
[00433] In certain embodiments, the drug is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulatory agent, a toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.
[00434] For example, a cytotoxin can include any compound that leads to cell death (e.g., necrosis or apoptosis) or a decrease in cell viability.
[00435] Kinase inhibitors can include, but are not limited to, Adavosertib, Afatinib, Axitinib, Bosutinib, Cetuximab, Cobimetinib, Crizotinib, Cabozantinib, Dacomitinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ruxolitinib, Sorafenib, Sunitinib, Tucatinib, Vandetanib, Vemurafenib, and the like.
[00436] For example, selective estrogen receptor modulators include, but are not limited to, Endoxifen, Tamoxifen, Afimoxifene, Toremifene, and the like.
[00437] Immuno stimulatory agents can include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, and the like. TLR agonists include, but are not limited to, imiquimod, resiquimod, and the like.
[00438] Oligonucleotide dugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, volanesorsen, inotersen, tofersen, tominersen, and the like.
[00439] Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1,
ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, and the like. [00440] Cytokines include, but are not limited to, Albinterferon Alfa-2B, Aldesleukin, ALT-801, Anakinra, Anccstim, Avotcrmin, Balugrastim, Bcmpcgaldcslcukin, Binctrakin, Cintredekin Besudotox, CTCE-0214, Darbepoetin alfa, Denileukin diftitox, Dulanermin, Edodekin alfa, Emfilermin, Epoetin delta, Erythropoietin, Human interleukin-2, Interferon alfa, Interferon alfa-2c, Interferon alfa-nl, Interferon alfa-n3, Interferon alfacon-1, Interferon beta-la, Interferon beta- lb, Interferon gamma- lb, Interferon Kappa, Interleukin- 1 alpha, Interleukin- 10, Interleukin-7, Lenograstim, Leridistim, Lipegfilgrastim, Lorukafusp alfa, Maxy-G34, Methoxy polyethylene glycol-epoetin beta, Molgramostim, Muplestim, Nagrestipen, Oprelvekin, Pegfilgrastim, Pegilodecakin, Peginterferon alfa-2a, Peginterferon alfa- 2b, Peginterferon beta- la, Peginterferon lambda- la, Recombinant CD40-ligand, Regramostim, Romiplostim, Sargramostim, Thrombopoietin, Tucotuzumab celmoleukin, Viral Macrophage-Inflammatory Protein, and the like.
[00441] Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.
[00442] “Peptide drug” as used herein refers to amino-acid containing polymeric compounds, and is meant to encompass naturally-occurring and non-naturally-occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics. The peptide drugs may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source). Peptide drugs can range in molecular weight, and can be from 200 Da to 10 kDa or greater in molecular weight. Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; anti- angiogenic peptides; peptides that activate B cells; peptides that activate T cells; anti-viral peptides; peptides that inhibit viral fusion; peptides that increase production of one or more lymphocyte populations; anti-microbial peptides; growth factors; growth hormone-releasing factors; vasoactive peptides; anti- inflammatory peptides; peptides that regulate glucose metabolism; an anti-thrombotic peptide; an anti-nociceptive peptide; a vasodilator peptide; a platelet aggregation inhibitor; an analgesic; and the like.
[00443] Additional examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to Tubulysin M, Calicheamicin, a STAT3 inhibitor, alpha- Amanitin, an aurora kinase inhibitor, belotecan, and an an thracy cline. [00444] Other examples of drugs include small molecule drags, such as a cancer chemotherapeutic agent. For example, where the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.
[00445] Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. 6,323,315. For example, dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).
[00446] Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
[00447] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6- mercaptopurine (6-MP), pentostatin, 5 -fluorouracil (5-FU), methotrexate, 10-propargyl-5,8- dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinc phosphate, pcntostatinc, and gemcitabine.
[00448] Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxy coformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.
[00449] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
[00450] Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
[00451] Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g. aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation; therefore compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation.
[00452] Other suitable chemotherapeutic agents include metal complexes, e.g. cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, e.g. mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4- morpholiny l)propoxy )quinazoline) ; etc .
[00453] Taxanes are suitable for use. “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis).
[00454] Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).
[00455] Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No. 5,869,680; 6- thio derivatives described in WO 98/28288; sulfonamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivative described in U.S. Patent No. 5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No. 5,824,701. [00456] Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of scrinc/thrconinc kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-a; (7) IFN-y; (8) colony- stimulating factors; and (9) inhibitors of angiogenesis.
[00457] Examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent. For example, where the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agent. In certain embodiments, the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid.
[00458] Embodiments of the present disclosure include conjugates where an antibody is conjugated to one or more drug moieties, such as two or more drug moieties, such as 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug moieties. The drug moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In certain embodiments, the conjugates have an average DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugates have an average DAR from 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the conjugates have an average DAR of 1 to 10. In certain embodiments, the conjugates have an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugates have an average DAR of 5 to 10 (e.g., 8). In certain embodiments, the conjugates have an average DAR of 8 to 12 (e.g., 10). In certain embodiments, the conjugates have an average DAR of 10 to 15 (e.g., 12). In certain embodiments, the conjugates have an average DAR of 15 to 20 (e.g., 16). By average is meant the arithmetic mean. [00459] As described herein, in some instances, the antibody-drug conjugate may include a branched linker, where two drugs or active agents arc attached to a branched linker. In certain embodiments, the two drugs or active agents attached to a branched linker are the same drug or active agent. For example, a first branch of a branched linker may be attached to a drug or an active agent and a second branch of the branched linker may be attached to the same drug or the same active agent as the first branch. In other embodiments, the two drugs or active agents attached to the branched linker are different drugs or active agents. For example, a first branch of a branched linker may be attached to a first drug or a first active agent and a second branch of the branched linker may be attached to a second drug or a second active agent different from the first drug or the first active agent attached to the first branch.
[00460] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. For example, in some instances, the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both payloads act, thereby increasing overall potency of the ADC.
[00461] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately, For example, the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).
[00462] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways. Examples of pay load combinations can include, but are not limited to, cytotoxic drugs, immunomodulatory molecules to activate or inhibit immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, and the like. [00463] In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an auristatin (e.g., MMAE) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents arc a topoisomerase inhibitor (e.g., bclotccan) as described herein and an iRGD peptide as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and an iRGD peptide as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein.
[00464] Drug s to be conjugated to a polypeptide may be modified to incorporate a reactive partner for reaction with the polypeptide. Where the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide) can be positioned at an N-terminal region, the N-terminus, a C- terminal region, the C-terminus, or at a position internal to the peptide. For example, an example of a method involves synthesizing a peptide drug having an aminooxy group. In this example, the peptide is synthesized from a Boe-protected precursor. An amino group of a peptide can react with a compound comprising a carboxylic acid group and oxy-N-Boc group. As an example, the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidin-l-yloxy)propanoic acid. Other variations on the compound comprising a carboxylic acid group and oxy-N-protecting group can include different number of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and oxy-N-protecting group occurs through standard peptide coupling chemistry. Examples of peptide coupling reagents that can be used include, but not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1- bcnzotriazolc dicthylphosphatc-l-cyclohcxyl-3-(2-morpholinylcthyl)carbodiimidc), EDC (1-(3- dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidinium hexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazol- 1 -yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazol-l-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol- 1 -yl-N,N,N’ ,N’-tetramethyluronium tetrafluoroborate), TSTU (O-(N-succinimidyl)-N,N,N’ ,N’ -tetramethyluronium tetrafluoroborate), HATU (N-[(dimethylamino)-l-H-l,2,3-triazolo[4,5,6]-pyridin-l- ylmethylene]- -N-methylmethanaminium hexafluorophosphate N-oxide), BOP-CI (bis(2-oxo-3- oxazolidinyl)phosphinic chloride), PyBOP ((l-H-l,2,3-benzotriazol-l-yloxy)- tris(pyrrolidino)phosphonium tetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy )- 1 ,2,3-benzotriazin-4(3H)-one) PyBrOP (bromotris(pyrrolidino)phosphonium hexafluorophosphate). As a non-limiting example, HOBt and DIC can be used as peptide coupling reagents.
[00465] Deprotection to expose the amino-oxy functionality is performed on the peptide comprising an N-protecting group. Deprotection of the N-oxysuccinimide group, for example, occurs according to standard deprotection conditions for a cyclic amide group. Deprotecting conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions include a hydrazine reagent, amino reagent, or sodium borohydride. Deprotection of a Boc protecting group can occur with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. The product and intermediates can be purified by conventional means, such as HPLC purification. [00466] The ordinarily skilled artisan will appreciate that factors such as pH and steric hindrance (i.e., the accessibility of the amino acid residue to reaction with a reactive partner of interest) are of importance, Modifying reaction conditions to provide for optimal conjugation conditions is well within the skill of the ordinary artisan, and is routine in the art. Where conjugation is conducted with a polypeptide present in or on a living cell, the conditions are selected so as to be physiologically compatible. For example, the pH can be dropped temporarily for a time sufficient to allow for the reaction to occur but within a period tolerated by the cell (c.g., from about 30 min to 1 hour). Physiological conditions for conducting modification of polypeptides on a cell surface can be similar to those used in a ketone-azide reaction in modification of cells bearing cell-surface azides (see, e.g., U.S. 6,570,040).
[00467] Small molecule compounds containing, or modified to contain, an α-nucleophilic group that serves as a reactive partner with a compound or conjugate disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the present disclosure.
General methods are known in the art for chemical synthetic schemes and conditions useful for synthesizing a compound of interest (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).
FORMULATIONS
[00468] The conjugates of the present disclosure can be formulated in a variety of different ways. In general, where the conjugate is an antibody-drug conjugate, the conjugate is formulated in a manner compatible with the drug, the antibody, the condition to be treated, and the route of administration to be used.
[00469] In some embodiments, provided is a pharmaceutical composition that includes any of the conjugates of the present disclosure and a pharmaceutically acceptable excipient. [00470] The conjugate (e.g., antibody-drug conjugate) can be provided in any suitable form, e.g., in the form of a pharmaceutically acceptable salt, and can be formulated for any suitable route of administration, e.g., oral, topical or parenteral administration. Where the conjugate is provided as a liquid injectable (such as in those embodiments where they are administered intravenously or directly into a tissue), the conjugate can be provided as a ready-to- use dosage form, or as a reconstitutable storage- stable powder or liquid composed of pharmaceutically acceptable carriers and excipients.
[00471] Methods for formulating conjugates can be adapted from those readily available. For example, conjugates can be provided in a pharmaceutical composition comprising a therapeutically effective amount of a conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like). In some embodiments, the formulations are suitable for administration to a mammal, such as those that arc suitable for administration to a human.
METHODS OF TREATMENT
[00472] The antibody-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug (i.e., the drug prior to conjugation to the antibody).
[00473] In some embodiments, provided are methods that include administering to a subject an amount (e.g., a therapeutically effective amount) of any of the conjugates of the present disclosure.
[00474] In certain aspects, provided are methods of delivering a drug to a target site in a subject, the method including administering to the subject a pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject. For example, as described herein, antibody-drug conjugates of the present disclosure can include a cleavable linker, such as an enzymatically cleavable linker that includes a first enzymatically cleavable moiety and a second enzymatically cleavable moiety. In some instances, the cleavable linker can be cleaved under appropriate conditions to separate or release the drug from the antibody at a desired target site of action for the drug. For example, the second cleavable moiety, which protects the first cleavable moiety from cleavage, may be cleaved in order to allow the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker into two or more portions, thus releasing the drug from the antibody-drug conjugate at a desired site of action.
[00475] In certain embodiments, the first cleavable moiety can be an enzymatically cleavable moiety. In some instances, the enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated). For example, a first enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein.
[00476] In certain embodiments, the second cleavable moiety can be an enzymatically cleavable moiety. In some instances, the enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated). For example, a second enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein. In certain embodiments, the first enzyme and the second enzyme are different enzymes.
[00477] In other instances, the first enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated). For instance, the first enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the first enzyme. In some instances, the first enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated. For example, the first enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated. In some instances, the first enzyme is present in an overabundance at the desired site of action due to localization of the first enzyme at a particular area or location. For instance, the first enzyme may be associated with a certain structure within the desired site of action, such as lysosomes. In some cases, the first enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject. In some embodiments, the lysosomes that include the first enzyme, are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug. In certain embodiments, the first enzyme is a protease, such as a human protease enzyme (e.g., cathepsin B).
[00478] In certain embodiments, the second enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated). For instance, the second enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the second enzyme. In some instances, the second enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated. For example, the second enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated. In some instances, the second enzyme is present in an overabundance at the desired site of action due to localization of the second enzyme at a particular area or location. For instance, the second enzyme may be associated with a certain structure within the desired site of action, such as lysosomes. In some cases, the second enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject. In some embodiments, the lysosomes that include the second enzyme, are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug. In certain embodiments, the second enzyme is a glucuronidase, a glycosidase, such as a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like.
[00479] Any suitable enzymes can be used for cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein. Other enzymes may also be suitable for use in cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein, such as but not limited to, enzymes from other vertebrates (e.g., primates, mice, rats, cats, pigs, quails, goats, dogs, rabbits, etc.). [00480] In certain embodiments, the antibody-drug conjugate is substantially stable under standard conditions. By substantially stable is meant that the cleavable linker of the antibody- drug conjugate does not undergo a significant amount of cleavage in the absence of a first enzyme and a second enzyme as described above. For example, as described above, the second cleavable moiety can protect the first cleavable moiety from being cleaved, and as such the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of a second enzyme as described above. For instance, the cleavable linker of the antibody-drug conjugate may be substantially stable such that 25% or less of the antibody-drug conjugate is cleaved in the absence of the first enzyme and/or second enzyme, such as 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less. In some cases, the antibody-drug conjugate is substantially stable such that the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the first enzyme and/or second enzyme, but can be cleaved when in the presence of the first enzyme and the second enzyme. For example, the antibody-drug conjugate can be substantially stable after administration to a subject. In some cases, the antibody-drug conjugate is substantially stable after administration to a subject, and then, when the antibody-drug conjugate is in the presence of the second enzyme at a desired site of action, the second cleavable moiety can be cleaved from the cleavable linker, thus exposing the first clcavablc moiety to subsequent cleavage by the first enzyme, which in turn releases the drug at the desired site of action. In certain embodiments, after administration to a subject the antibody- drug conjugate is stable for an extended period of time in the absence of the first enzyme and/or second enzyme, such as 1 hr or more, or 2 hrs or more, or 3 hrs or more, or 4 hrs or more, or 5 hrs or more, or 6 hrs or more, or 7 hrs or more, or 8 hrs or more, or 9 hrs or more, or 10 hrs or more, or 15 hrs or more, or 20 hrs or more, or 24 hrs (1 day) or more, or 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days (1 week) or more. In certain embodiments, the antibody-drug conjugate is stable at a range pH values for an extended period of time in the absence of the first enzyme and/or second enzyme, such as at a pH ranging from 2 to 10, or from 3 to 9, or from 4 to 8, or from 5 to 8, or from 6 to 8, or from 7 to 8. [00481] As described above, antibody-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug. By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease; and/or (iii) relief, that is, causing the regression of clinical symptoms.
[00482] The subject to be treated can be one that is in need of therapy, where the subject to be treated is one amenable to treatment using the parent drug. Accordingly, a variety of subjects may be amenable to treatment using the antibody-drug conjugates disclosed herein. Generally, such subjects are “mammals”, with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees and monkeys).
[00483] The amount of antibody-drug conjugate administered can be initially determined based on guidance of a dose and/or dosage regimen of the parent drug. In general, the antibody- drug conjugates can provide for targeted delivery and/or enhanced serum half-life of the bound drug, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen. Thus, the antibody-drug conjugates can provide for reduced dose and/or reduced administration in a dosage regimen relative to the parent drug prior to being conjugated in an antibody-drug conjugate of the present disclosure.
[00484] Furthermore, as noted above, because the antibody-drug conjugates can provide for controlled stoichiometry of drug delivery, dosages of antibody-drug conjugates can be calculated based on the number of drug molecules provided on a per antibody-drug conjugate basis.
[00485] In some embodiments, multiple doses of an antibody-drug conjugate are administered. The frequency of administration of an antibody-drug conjugate can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc. For example, in some embodiments, an antibody-drug conjugate is administered once per month, twice per month, three times per month, every other week, once per week (qwk), twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily (qd/od), twice a day (bds/bid), or three times a day (tds/tid), etc.
Methods of treating cancer
[00486] The present disclosure provides methods that include delivering a conjugate of the present disclosure to an individual having a cancer. For example, the method may include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of the present disclosure, where the administering is effective to treat cancer in the subject. The methods are useful for treating a wide variety of cancers, including, but not limited to breast, ovarian, colon, lung, stomach, and pancreatic cancer. In the context of cancer, the term “treating” includes one or more (e.g., each) of: reducing growth of a solid tumor, inhibiting replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with a cancer. [00487] Carcinomas that can be treated using a subject method include, but are not limited to, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, cervical carcinoma, uterine carcinoma, testicular carcinoma, and epithelial carcinoma, etc.
[00488] In certain aspects, provided are methods of treating cancer in a subject, such methods including administering to the subject a therapeutically effective amount of a pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to treat cancer in the subject.
Certain Embodiments
[00489] The present disclosure contemplates, among other things, the following numbered embodiments:
1. A conjugate of formula (I):
Figure imgf000124_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; W2 is an antibody; and
X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
2. The conjugate of Embodiment 1, wherein R1 is H and R2 is alkyl or substituted alkyl.
3. The conjugate of any of Embodiments 1-2, wherein k1 is 2.
4. The conjugate of any of Embodiments 1-3, wherein R3 is a chemically-cleavable moiety.
5. The conjugate of any of Embodiments 1-3, wherein R3 is an enzymatically-cleavable moiety.
6. The conjugate of Embodiment 5, wherein R3 is a glycoside or a glycoside derivative.
7. The conjugate of Embodiment 6, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
8. The conjugate of any of Embodiments 1-7, wherein X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
9. The conjugate of any of Embodiments 1-7, wherein X comprises a maleimide, and the native amino acid residue of the antibody comprises a cysteine residue.
10. The conjugate of Embodiment 8, wherein the conjugate is of formula (II):
Figure imgf000125_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
W2 is an antibody.
11. The conjugate of any of Embodiments 1-10, wherein the first linker LA comprises: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein a, b, c, d, e and f are each independently 0 or 1;
T1, T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
12. The conjugate of Embodiment 11, wherein:
T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;
T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, - (CR13OH) m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:
(PEG)n is , where n is an integer from 1 to 30;
Figure imgf000127_0001
EDA is an ethylene diamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1 ;
Figure imgf000127_0002
4-amino-piperidine (4AP) is
Figure imgf000127_0003
; and each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring.
13. The conjugate of any of Embodiments 11-12, wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-;
T3 is (PEG)n and V3 is -CO-; and d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0.
14. The conjugate of any of Embodiments 11-12, wherein one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4 ,V5 or V6 is a branched group.
15. The conjugate of Embodiment 14, wherein the branched group is selected from - CONR15- and 4AP.
16. The conjugate of any of Embodiments 14-15, wherein the branched group is attached to a second linker, LB.
17. The conjugate of Embodiment 16, wherein the second linker LB comprises:
-(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and l are each independently 0 or 1;
T7, T8, T9, T10, T11 and T12 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V7, V8, V9, V10 ,V11 and V12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
18. The conjugate of any of Embodiments 16-17, wherein LB is attached to a compound of formula (III):
Figure imgf000129_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug.
19. The conjugate of Embodiment 18, wherein R4 is H and R5 is alkyl or substituted alkyl.
20. The conjugate of any of Embodiments 18-19, wherein k2 is 2.
21. The conjugate of any of Embodiments 18-20, wherein R6 is a chemically-cleavable moiety. 22. The conjugate of any of Embodiments 18-20, wherein R6 is an cnzymatically-clcavablc moiety.
23. The conjugate of Embodiment 22, wherein R6 is a glycoside or a glycoside derivative.
24. The conjugate of Embodiment 23, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
25. The conjugate of any of Embodiments 11-24, wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-; T3 is (C1-C12)alkyl and V3 is absent;
T4 is hctcroaryl (triazoic) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-;
T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0. The conjugate of any of Embodiments 14-25, wherein W1 and W1a are the same drug. The conjugate of any of Embodiments 14-25, wherein W1 and W1a are different drugs. The conjugate of any of Embodiments 1-27, wherein the conjugate is selected from:
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
and
Figure imgf000134_0001
29. A compound of formula (IV):
Figure imgf000134_0002
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and X is a conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody.
30. The compound of Embodiment 29, wherein R1 is H and R2 is alkyl or substituted alkyl.
31. The compound of any of Embodiments 29-30, wherein k1 is 2.
32. The compound of any of Embodiments 29-31, wherein R3 is a chemically-cleavable moiety.
33. The compound of any of Embodiments 29-31, wherein R3 is an enzymatically-cleavable moiety.
34. The compound of Embodiment 33, wherein R3 is a glycoside or a glycoside derivative.
35. The compound of Embodiment 34, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
36. The compound of any of Embodiments 29-35, X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
37. The compound of any of Embodiments 29-35, wherein X comprises a maleimide, and the native amino acid residue of the antibody comprises a cysteine residue.
38. The compound of Embodiment 36, wherein the compound is of formula (V):
Figure imgf000135_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; kl is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker; and
W1 is a drug.
39. The compound of any of Embodiments 29-38, wherein the first linker LA comprises: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein a, b, c, d, e and f are each independently 0 or 1;
T1, T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V1, V2, V3, V4 ,V5 and V6 arc each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 40. The compound of Embodiment 39, wherein:
T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;
T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, - (CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:
(PEG)n is
Figure imgf000137_0001
, where n is an integer from 1 to 30;
EDA is an ethylene diamine moiety having the following structure: where y is an integer from 1 to 6 and r is 0 or 1 ;
Figure imgf000137_0002
4-amino-piperidine (4AP) is
Figure imgf000137_0003
; and each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring.
41. The compound of any of Embodiments 39-40, wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-; T3 is (PEG)n and V3 is -CO-; and d, c and f arc each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0.
42. The compound of any of Embodiments 39-40, wherein one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4 ,V5 or V6 is a branched group.
43. The compound of Embodiment 42, wherein the branched group is selected from - CONR15- and 4AP.
44. The compound of any one of Embodiments 42-43, wherein the branched group comprises a second linker, LB.
45. The compound of Embodiment 44, wherein the second linker LB comprises:
-(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and l are each independently 0 or 1;
T7, T8, T9, T10, T11 and T12 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V7, V8, V9, V10 ,V11 and V12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
46. The compound of any of Embodiments 44-45, wherein LB is attached to a compound of formula (VI):
Figure imgf000139_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug.
47. The compound of Embodiment 46, wherein R4 is H and R5 is alkyl or substituted alkyl.
48. The compound of any of Embodiments 46-47, wherein k2 is 2.
49. The compound of any of Embodiments 46-48, wherein R6 is a chemically-cleavable moiety. 50. The compound of any of Embodiments 46-48, wherein R6 is an enzymatically-cleavable moiety.
51. The compound of Embodiment 50, wherein R6 is a glycoside or a glycoside derivative.
52. The compound of Embodiment 51, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
53. The compound of any of Embodiments 39-52, wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (C1-C12)alkyl and V3 is absent; T4 is heteroaryl (triazole) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-;
T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and 1 are each 0. The conjugate of any of Embodiments 42-53, wherein W1 and W1a are the same drug. The conjugate of any of Embodiments 42-53, wherein W1 and W1a are different drugs. The conjugate of any of Embodiments 29-55, wherein the conjugate is selected from:
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
and
Figure imgf000144_0001
57. A pharmaceutical composition comprising: a conjugate of any one of Embodiments 1 to 28; and a pharmaceutically acceptable excipient.
58. A method comprising : administering to a subject a conjugate of any of Embodiments 1 to 28.
59. A method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of any one of Embodiments 1 to 28, wherein the administering is effective to treat cancer in the subject. EXAMPLES
[00490] The following examples arc put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. By “average” is meant the arithmetic mean. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneally ); s.c., subcutaneous(ly); and the like.
EXAMPLE 1
Summary
[00491] Historically, first-generation ADCs relied on stochastic conjugation chemistries that utilized naturally occurring nucleophilic amino acid residues, predominantly cysteine and lysine sidechains (FIG. 2). For example, maleimide and NHS-ester based conjugation methods were used due to their simplicity.
[00492] However, in spite of initial optimism that saw a rush of heterogeneously conjugated ADCs proceed into the clinic, it was eventually realized that these general conjugation approaches were not an optimal way to build complex molecular systems designed to be used as therapeutic agents. Not only were the heterogeneous preparations challenging to generate reproducibly batch-to-batch, but they were also very difficult to characterize due to their complexity. Clinically, poor tolerability and mediocre efficacy were the impediments that needed to be resolved for successful clinical trials. Multiple efforts for innovating, improving and stabilizing linker-payload designs were made (Jain et al. Pharm Res, 32, 3526-3540 (2015), Su and Zhang Frontiers in Pharmacology Vol. 12, 2021)).
[00493] In addition to these, various approaches to making more uniform conjugates ligated at the antibody intrachain disulfides were introduced, where thiol cross-linking reagents (modified maleimide reagents) were used to form bridges between adjacent cysteine resides to yield more homogenously constructed DAR4 molecules (Behrens ct al. Mol Pharm 12, 3986- 3998 (2015), Badescu et al. Bioconjugate Chemistry 25, 1124-1136 (2014), Schumacher et al. Org Biomol Chem 12, 7261-7269 (2014)). As the cause and effect of ADC design became better understood, the exaggerated toxicity and poor efficacy observed in patients treated with early ADCs came to be associated with two features that were related to the conjugation and linker chemistry (Drake and Rabuka Curr Opin Chemical Biology, 28, 174-180 (2015), Su and Zhang Frontiers in Pharmacology Vol. 12, 2021)). Specifically, these were; 1) a heterogeneous conjugate mixture and, 2) linker instability.
[00494] The heterogeneous conjugation resulted from (random) incomplete ligation to available reactive groups on the protein. For example, most maleimide and NHS-ester conjugates targeted a drug-to-antibody ratio (DAR) of 4, which was less than the maximum number of conjugatable sites. ADC linker instability was manifest in two locations - the conjugation chemistry and the cleavable linker element, which could be a disulfide, an acid-labile group, or a peptide.
[00495] ADC heterogeneity of first-generation ADCs was characterized by both under- and over-conjugated species within the conjugate mixture. In particular, the over-conjugated species tended towards hydrophobicity and aggregation and were often rapidly cleared by the liver upon injection into the body (Beck et al. Nature Reviews Drug Discovery, 16, 315-337 (2017), Sun et al. Bioconjugate Chemistry, 28, 1371-1381 (2017), Lyon et al. Nature Biotechnology , 33, 733-735 (2015)). This resulted in liver toxicity being observed as a common side effect of ADC dosing among these first-generation heterogeneous constructs.
[00496] One common way to characterize ADCs with respect to hydrophobicity is to use hydrophobic interaction chromatography, or HIC. Conjugated antibody has a longer retention time on a HIC column as compared to unconjugated antibody. For this reason, HIC is often used as a tool to assess DAR. It can also be used as a tool to compare the relative hydrophobicity of different conjugates or antibodies during optimization or lead selection (Lyon et al. Nature Biotechnology, 33, 733-735 (2015)). (Similar information can be gained from analyzing reduced ADCs on a PLRP column, which also retains more hydrophobic compounds longer than hydrophilic compounds.) In general, the more hydrophilic a conjugate (the shorter the HIC retention time [RT]), the better the in vivo outcomes with respect to pharmacokinetics and efficacy. A more hydrophilic molecule will typically enjoy longer in vivo exposure after dosing and - in turn, and all other things being equal - will exhibit stronger in vivo anti-tumor efficacy. The rationale for this outcome is that an ADC that stays in the circulation longer (has longer exposure) will be able to deliver more payload over time to the tumor and thus will show higher efficacy. This reasoning presupposes that the conjugate is stable, and that the payload remains on the antibody to be available for delivery to the tumor.
[00497] To address the issues of conjugate heterogeneity and overt hydrophobicity, site- specific conjugation methods have been developed. One of the most prominent, and commonly used site-specific conjugation approaches has been to genetically engineer an exogenous cysteine residue into the antibody constant region and conjugate a linker-payload to that cysteine using standard maleimide chemistry. This approach has been referred to as ThioMab. (Junutula et al. Nature Biotechnology 26, 925-932 (2008), Junutula et al. Clinical Cancer Research 16, 4769- 4778 (2010)). The ThioMab approach was generally used to make ADCs with lower overall drug-to-antibody ratios (e.g., DAR2) than were typically generated using the stochastic conjugation methods (e.g., DAR4). The resulting site-specific DAR2 conjugates were shown to have improved biophysical and functional properties when compared to first-generation, stochastically-derived conjugates. Other ways to make site-specifically conjugated ADCs were developed that used various methods to introduce biorthogonal reactive chemical handles at defined locations on an antibody. These included: a) incorporation of functionalized non-natural amino acids at specific locations in the sequence (Xu et al. Org Process Res Dev 20, 1034-1043 (2016), and Tian et al. PNAS 111, 1766-1771 (2014), b) exploiting the specificity of enzymes to derivatize engineered consensus peptide sequences to introduce reactive functional groups (Lee et al. Angewandte Chemie 54, 12020-12024 (2015), Strop et al. Chemistry & Biology 20, 161- 167 (2013), and Drake et al. Bioconjugate Chemistry 25, 1331-1341 (2014), and c) remodeling of the native N- linked glycans to incorporate a reactive handle via a non-natural monosaccharide group (van Geel et al. Bioconjugate Chemistry 26, 2233-2242 (2015)). In addition to these, various approaches to making more uniform conjugates ligated at the antibody intrachain disulfides were introduced, where thiol cross-linking reagents (modified maleimide reagents) were used to form bridges between adjacent cysteine resides to yield more homogenously constructed DAR4 molecules (Behrens et al. Mol Pharm 12, 3986-3998 (2015), Badescu et al. Bioconjugate Chemistry 25, 1124-1136 (2014), Schumacher et al. Org Biomol Chem 12, 7261- 7269 (2014)).
[00498] Among the benefits of these varied site-specific conjugation methods is the predictability of the structural outcome (conjugate uniformity). In addition, some site-specific approaches - such as engineered cysteines, and the aldehyde tag when used in combination with HIPS conjugation chemistry - allow for variation in linker-payload placement on the antibody. This flexibility of linker-payload placement is useful for structure-activity relationship mapping. Studies across conjugation platforms have shown that the location of the linker-payload placement on the antibody affects the biophysical properties of the ADC and the in vivo functional outcomes (e.g., efficacy, pharmacokinetics, and tolerability) of the molecule (Strop et al. Chemistry & Biology 20, 161-167 (2013), Drake et al. Bioconjugate Chemistry 25, 1331-1341 (2014), Shen et al. Nature Biotechnology 30, 184-189 (2012), Su and Zhang Frontiers in Pharmacology Vol. 12, 2021)).
[00499] Second, because the majority of the clinically tested ADC linkers are peptide- based, they are susceptible to cleavage by incidental plasma proteases or other enzymes to release the highly potent ADC payload into the circulation. This has led to systemic toxicity and poor tolerability of the ADC. Site-specific conjugation methods (and the resulting improvements in product homogeneity) do not in of themselves address the second major design problem that was identified in first-generation conjugates - that element of conjugate instability that is driven by the linker itself. Most cleavable linkers are unstable in the circulation and premature release of cytotoxic pay load into the patient’s bloodstream represents a prominent source of systemic toxicity and poor tolerability of ADCs (FIG. 4).
[00500] To address these issues, two points of instability had to be considered: the conjugation chemistry used to join the protein and small molecule component, and the composition of the linker that connected the conjugation handle to the payload. Regarding conjugation chemistry, the commonly used maleimide-cysteine conjugation chemistry has inherent weaknesses. The conjugation product can undergo a retro-Michael reaction that results in the detachment of the antibody from the drug-linker (Alley et al. Bioconjugate Chemistry 19, 759-765 (2008)), the latter of which can further react with free plasma thiols (such as those found on albumin, (e.g., Wei et al. Analytical Chemistry 88, 4979-4986 (2016)) and can continue to circulate as a secondary cytotoxic conjugate (FIG. 3). [00501] Historically, it has been very difficult to design cleavable linkers that remain stable while the ADC is in circulation. Typical release mechanisms employed for clcavablc linkers have included acid-mediated hydrolysis (Laguzza et al. J Med Chem 32, 548-555 (1989)), reduction of a disulfide bond (Saito et al. Advanced Drug Delivery Reviews 55, 199-215 (2003), Kellogg et al. Bioconjugate Chemistry 22, 717-727 (2011)), and proteolytic cleavage of a peptide motif. The latter is most notably exemplified by the Val-Cit-PABC linker with the MMAE payload, also called vedotin (Doronina et al. Nature Biotechnology 21, 778-784 (2003)).
[00502] A common side-effect of vcMMAE-conjugated ADCs regardless of target antigen is myelosuppression, including neutropenia, thrombocytopenia, and anemia. This off-target toxicity is driven, at least in part, by premature cleavage of the ValCit dipeptide via extracellular enzymes encountered during circulation (Masters et al. Investigational New Drugs 36, 121-135 (2018)).
[00503] A highly successful approach to stabilize cleavable peptide-based linkers is the tandem-cleavage linker system that requires two consecutive enzymatic steps to occur in order to release the payload (FIG. 5), as disclosed herein. The monosaccharide unit attached to the phenolic self-immolation moiety (PABA) serves as a protective element and has to be removed first by the corresponding lysosomal glycosidase to expose the peptide portion of the linker which is then readily cleaved by proteases to liberate the payload. Because glucuronidase is only activated in the low pH environment of the lysosomal compartment, it is generally not active when found in serum. Thus, in order for the tandem-cleavage linker to be enzymatically degraded, it first must be internalized into a cell and trafficked to the lysosome. This requirement greatly reduces release of linker during ADC circulation.
[00504] Most site-specific conjugation methods require extensive protein engineering - either of the protein sequence itself or of the attached carbohydrate groups - which can significantly lengthen development times and/or involve complicated manufacturing approaches with low yields. Also, most site-specific approaches are not compatible with wild-type antibody formats and so cannot be applied to existing antibodies (that sometimes represent considerable investment of both time and money). In addition, some site-specific methods appear to be incompatible for use with a number of desirable payloads. EXAMPLE 2
Improved ADC comprising a malcimidc-tandcm-clcavagc conjugate
[00505] Surprisingly, it has now been found that the tandem-cleavage element in itself is sufficient to overcome many of the well-known hurdles to constructing stable and well-tolerated ADCs. In one embodiment, it was shown that by adding the tandem-cleavage element into a linker equipped with a maleimide handle (and elaborated by various types of payloads), safe and effective ADCs can be produced that exhibit qualities equal to or superior to the conjugates made using either generic maleimide-peptide based linkers or using a site-specific conjugation approach with the aldehyde-tag/HIPS-functionalized linkers. For example, regarding elements that are typically assessed during after conjugation - including yield, conjugate retention time, DAR, % aggregate (high-molecular weight species, HMW), and evidence of precipitation in the conjugation mixture, the maleimide-tandem cleavage functionalized conjugates performed better than conjugates made with the standard vedotin linker (maleimide- Val-Cit-PABC-MMAE, Compound 38), which also contained maleimide and MMAE but lacked the tandem-cleavage element (see Table 1 and FIGS. 9-23, 30-32). The tandem-cleavage-containing ADCs had better reaction yields, shorter PLRP retention times, reduced evidence of precipitation, all of which point to improved hydrophilicity of the constructs, which led to improved overall biophysical properties. Improved outcomes observed with linkers that incorporate the tandem-cleavage element may be driven by the effect of the tandem-cleavage element on overall conjugate hydrophilicity, resistance to protease degradation (through steric hindrance/shielding of the protease cleavage site), improved stability of the maleimide conjugation through influencing rate of exchange with surrounding thiols, and reduction in the propensity to form aggregates, to self- associate, or to interact with charged species.
Table 1. Characterization of Maleimide-MMAE Conjugate Preparations
Figure imgf000150_0001
EXAMPLE 3
Improved hydrophilicity of the maleimide-tandem-cleavage conjugates [00506] A similar, but even more dramatic effect was observed when comparing the HIC retention times of malcimide-tandcm-clcavagc conjugates to sitc-spccific (aldehyde tag/HIPS) conjugates employing the tandem-cleavage element. As shown in Table 2, DAR8 topoisomerase I inhibitor conjugates made using maleimide-tandem-cleavage were eluted nearly two minutes earlier from a HIC column as compared to site-specific HIPS tandem-cleavage conjugates using the same payload. This stark improvement in hydrophilicity of the maleimide-tandem-cleavage conjugates over optimized site-specific conjugates was unexpected, and furthermore was a welcome surprise considering the potential advantages to be gained by conjugation to native cysteines rather than specific antibody engineering for a unique conjugation approach.
Table 2. HIC Retention Times of Topoisomerase I Inhibitor Conjugated ADCs Produced Using Various Linkers
Figure imgf000151_0001
EXAMPLE 4
Improved hydrophilicity of the maleimide-tandem-cleavage conjugates
[00507] At the same time, the maleimide topoisomerase inhibitor conjugates with the tandem-cleavage linkers (Compounds 36 and 37) showed improved (more hydrophilic) HIC retention times as compared to Enhertu (an ADC made using trastuzumab conjugated to the maleimide-based deruxtecan linker, which has no tandem-cleavage element) (Table 2). The latter observation again points to the improvements that are broadly offered when using the tandem- cleavage element in a linker regardless of the context of conjugation chemistry or payload.
[00508] In the context of a maleimide conjugate, the tandem-cleavage element could impart an additional layer of stability and tolerability even to linkers that undergo deconjugation from the antibody and end up ligated to albumin (or other serum thiols) by serving as a “pro- drug” inactive form (FIG. 6). Table 3. HIC Retention Times of MMAE Conjugated ADCs Produced Using Various Linkers
Figure imgf000152_0001
EXAMPLE 5
The tandem-cleavage maleimide conjugate shows unaffected in vitro potency and ability to kill HER2+ gastric cell line
[00509] With respect to potency and ability kill target cells, the tandem-cleavage maleimide conjugated trastuzumab ADCs had equal potency and ability to kill the HER2+ gastric cell line, NCI-N87, as compared to ADCs made with traditional maleimide linkers (not containing tandem-cleavage) (FIGS. 24 and 25). Put another way, the improved stability imparted by the tandem-cleavage element did not reduce in vitro potency or negatively affect the ability of the ADC to kill target antigen expressing cells.
EXAMPLE 6
[00510] The tandem-cleavage maleimide conjugate shows improved serum stability, improved in vivo efficacy and tolerability. In some embodiments, it is shown that tandem- cleavage maleimide linkers generate conjugates with improved in vitro serum stability as compared to traditional maleimide linkers (e.g., vedotin) (see FIGS. 26-29). These linkers also appeared to demonstrate improved resistance to displacement by glutathione when incubated in vitro at 37° C for up to 6 days (Table 4).
Table 4. ADC Stability to Glutathione in Buffer at 37° C
Figure imgf000152_0002
Figure imgf000153_0001
[00511] This observation indicated that the tandem-cleavage containing linkers are affecting the stability of the maleimide conjugation site itself, perhaps through direct stabilization of the thioether bond, perhaps through influencing the maleimide ring opening. These unexpected observations further suggest that conjugates containing tandem-cleavage linkers will show improved in vivo efficacy and tolerability as compared to non-tandcm-clcavagc containing ADCs. These anticipated benefits may be observed for tandem-cleavage-containing ADCs conjugated with maleimide or with other conjugation chemistries, such as click reactions, or NHS esters.
[00512] Improved outcomes observed with linkers that incorporate the tandem-cleavage element likely are driven by the effect of the tandem-cleavage element on overall conjugate hydrophilicity, resistance to protease degradation (through steric hindrance/shielding of the protease cleavage site), improved stability of the maleimide conjugation through influencing rate of exchange with surrounding thiols, and reduction in the propensity to form aggregates, to self- associate, or to interact with charged species.
[00513] Another advantage of the maleimide-functionalized tandem-cleavage linker is that it can be used in combination with a broad variety of acid- sensitive payloads (such as an thracy clines), unlike the ADCs formed based on the HIPS conjugation chemistry, thus widening the scope of application for use of the tandem-cleavage element.
EXAMPLE 7
Conjugates with maleimide-functionalized tandem-cleavage linker carrying multiple payloads [00514] In one embodiment, it was demonstrated that the modularity of the disclosed method allowed creation of conjugates with high drug-to-antibody ratio (DAR) through the use of branched linkers which carry two or more payloads per one conjugation unit. Examples of such linkers are shown in FIG. 7. Dicarboxylic acids 1 or 2 were further chemically modified to include a conjugation element (e.g. maleimide), then the carboxylic functions in 3 and 4 served as chemical handles to attach two linker-payload systems of choice to build compounds such as 5 and 6, where payload was chosen from Topoisomerase T, kinase, or PARP inhibitor, or some other biologically active compound.
EXAMPLE 8
Dual payload ADCs in an ADC construct comprising a maleimide-functionalized tandem- cleavage linker
[00515] In further embodiments, it has been shown that branched linkers may serve as a platform for developing dual-payload ADCs and provide a reliable access to cytotoxin combination modality. This can be achieved through the design of branched linkers with orthogonal connection elements as shown for compound 7 (FIG. 8), where carboxylic acid moiety was used to attach the first payload of choice, while the azide function - the second through azide-alkyne cycloaddition to access dual-payload linker 8.
EXAMPLE 9
Materials and Methods
General
[00516] The subject conjugates and compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. A variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.
[00517] Synthetic reagents were purchased from Sigma- Aldrich, Acros, AK Scientific, or other commercial sources and were used without purification. Anhydrous solvents were obtained from commercial sources in sealed bottles. Compounds 10 and 35 were obtained commercially from Shanghai Medicilon and used without purification. Compounds 58 and 63 were commercially available or synthesized via literature procedures. Payloads 11, 26, 39, and 67 as well as MC-VC-PAB-MMAE 38 (vedotin linker) were purchased from commercial sources. In all cases, solvent was removed under reduced pressure with a Buchi Rotovapor R-l 14 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage chromatography system. Preparative HPLC purifications were performed using Waters preparative HPLC unit equipped with Phenomenex Kinetex 5 am EVO C18 150 x 21.2 mm column. HPLC analyses were conducted on an Agilent 1100 Series Analytical HPLC equipped with a Model G1322A Degasser, Model G1311 A Quarternary Pump, Model G1329A Autosamplcr, Model G1314 Variable Wavelength Detector, Agilent Poroshcll 120 SB C18, 4.6 mm x 50 mm column at room temperature using a 10-100% gradient of water and acetonitrile containing 0.05% trifluoroacetic acid. HPLCs were monitored at 254 or 205 nm. Low-resolution mass spectra (LRMS) were acquired on Agilent Technology 6120 Quadrupole LC/MS, equipped with Agilent 1260 Infinity HPLC system, G1314 variable wavelength detector, and Agilent Poroshell 120 SB C18, 4.6 mm x 50 mm column at room temperature using 10-100% gradient of water and acetonitrile containing 0.1% formic acid.
Synthesis of Drug-Linkers
EXAMPLE 10
[00518] Preparation of(2S,3S,4S,5R,6S)-6-(5-((5S,8S,llS,12R)-ll-((S)-sec-butyl)-12-(2-
((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3- oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa- 4, 7, 10-triazatetradecyl )-2-( (S)-2-( (S)-2-( 6-( 2,5 -dioxo-2,5-dihydro- IH-pyrrol- 1 -yl )hexanamido)- 3-methylbutanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (14)
Figure imgf000156_0001
[00519] In a 20 mL glass vial were combined monomethyl auristatin A 11 (MMAE, 720 mg, 1.0 mmol), 5 mL of anhydrous DMF, and 0.35 mL of DIPEA (2.0 mmol) at room temperature. The resulting mixture was stirred and treated with PNP carbonate 10 (1014 mg, 1.0 mmol) as a solid in a few small portions, followed by the addition of HO At (136 mg, 1.0 mmol) in one portion at room temperature. Reaction mixture was stirred for 6 h until reaction was judged complete (HPLC). Reaction mixture was poured into 30 mL of water, and the resulting white precipitate was separated by spinning and collected, washed with 5 mL of water, and dried briefly under high vacuum to give 1.87 g of crude coupling product as an off-white solid, which was taken to the next step without purification.
[00520] A solution of crude intermediate (1.87 g) in 15 mL of THF was cooled down to 0 °C in an ice bath and treated slowly with 1 M aqueous lithium hydroxide solution (3 mL).
Reaction mixture was stirred at 0 °C for 3 hours, then warmed up to ambient temperature, treated with 3 mL of 1 M aqueous lithium hydroxide and diluted with 3 mL of methanol. The resulting mixture was stirred at room temperature for 3 hours until hydrolysis was found complete (HPLC), then quenched by adding 1 M aqueous HC1 solution to pH 7. Reaction mixture was then concentrated under reduced pressure and washed with 10 mL of MTBE. Aqueous layer was purified by reversed- phase chromatography (C18 column, 0-40% acetonitrile-water with 0.05% TFA). Pure product fractions were combined, concentrated under reduced pressure, and lyophilized to give compound 12 as a white powder (735 mg, 0.60 mmol, 60% yield over 2 steps). LRMS (ESI): m/z 1229.7 [M+H]+, Calcd for C61H96N8O18 m/z 1229.7.
[00521] In an oven dried 20 mL glass vial were combined compound 12 (50 mg, 40μmol), HOAt (50 (tmol, 50 μL of 1 M in DMA), and DIPEA (21 μL, 150 μmol) in 3 mL of DMF at RT. To this mixture were added PFP-ester 13 in one portion. After 10 minutes, reaction was judged complete by LCMS analysis. The mixture was directly purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 46 mg of product 14 as a white powder (32 μmol, 80% yield). LRMS (ESI): m/z 1422.7 [M+H]+, Calcd for C71H107N9O21 m/z 1422.8.
Synthesis of compound 13.
[00522] To a mixture of 6-maleimidohexanoic acid (2.11 g, 10 mmol) and pentafluorophenol (2.2 g, 12 mmol) in 25 mL of anhydrous THF were added DCC (2.06 g, 10 mmol) in one portion at room temperature. The resulting mixture was stirred overnight, then solids were filtered off and washed with THF. Combined filtrates were concentrated under vacuum and then purified by silica gel chromatography using (0-15% EtOAc-hexanes) to give 3.22 g of product 13 as a white solid (8.5 mmol, 85% yield). LRMS (ESI): m/z 400.1 [M+Na]+, Calcd for C16H12F5NO4 m/z 400.1.
EXAMPLE 11
Preparation of (2S,3S,4S,5R,6S)-6-(5-((5S,8S,llS,12R)-ll-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)- 3-(((lS,2R)-l -hydroxy- l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-
1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-
2-((2S,5S,18R)-22-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-5-isopropyl-2-methyl-4,7,17,20- tetraoxo- 18-(sulfomethyl)- 10,13-dioxa-3, 6,16, 19-tetraazadocosanamido)phenoxy)-3, 4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid (18) [00523] Synthesis of linker 15.
Figure imgf000158_0001
[00524] To a mixture of Fmoc-L-cysteic acid 19 (0.39 g, 1 mmol) and amine 20 (233 mg, 1 mmol) in 4 mL of DMF were added DIPEA (0.52 mL, 3 mmol), followed by HATU (380 mg, 1 mmol) in one portion at RT. After 30 minutes, reaction was judged complete by LCMS analysis. Reaction mixture was directly purified by reversed-phase chromatography (C18, 0- 50% acetonitrile-water, 0.05% TFA). Pure fractions were combined, solvents were removed in vacuum to give 485 mg of product 21 as a white foaming solid (0.8 mmol, 80% yield). LRMS (ESI neg.): m/z 605.2 [M-H]-, Calcd for C29H38N2O10S m/z 605.2.
[00525] Compound 21 (485 mg, 0.8 mmol) was dissolved in a DCM-TFA mixture (1:1, 6 mL). The resulting solution was allowed to stand at RT for 10 minutes, then solvents were removed under vacuum and the residue was purified by reversed-phase chromatography (C18 column, 0-50% acetonitrile-water, 0.05% TFA). Pure fractions were combined, solvents were removed in vacuum to give 420 mg of compound 22 as a white foaming solid (0.76 mmol, 95% yield). LRMS (ESI neg.): m/z 549.1 [M-H]-, Calcd for C25H30N2O10S m/z 549.2.
[00526] Carboxylic acid 22 (420 mg, 0.76 mmol) was combined with 421 mg (2.3 mmol) of pentafluorophenol in 5 mL of anhydrous DMF. The mixture was treated with EDCLHC1 (292 mg, 1.5 mmol) at room temperature and stirred for 48 hours until 22 was judged as completely consumed based on LCMS analysis. Reaction mixture was directly purified by reversed-phase chromatography (C18, 0-70% acetonitrile-water, 0.05% TFA). Pure fractions were combined at concentrated under vacuum and lyophilized to give 254 mg of PFP-ester 15 as a tan solid (0.36 mmol, 47% yield). LRMS (ESI neg.): m/z 715.1 [M-H]-, Calcd for C31H29F5N2O10S m/z 715.2.
Figure imgf000159_0001
[00527] In an oven dried 20 mL glass vial were mixed 60 mg of compound 12 (49 μmol), 26 μL of DIPEA (147 μmol), and HOAt solution (50 μL of 1M in DMA) in 3 mL of DMF. The mixture was stirred and treated with PFP ester 15 (35 mg, 49 μmol) at room temperature in one portion. After 30 minutes, reaction was found complete by LCMS analysis. Reaction mixture was directly treated with 96 μL of piperidine (0.98 mmol) at room temperature. After 20 minutes, reaction mixture was directly purified by reversed-phase prep HPLC (C18, 0-50% acetonitrile- water, 0.05% TFA). Pure fractions were combined and lyophilized to give 47 mg of compound 16 as a white solid (31 μmol, 63 % yield). LRMS (ESI): m/z 1539.7 [M+H]+, Calcd for C71H114N10O25S m/z 1539.8.
[00528] In a glass vial were mixed compound 16 (47 mg, 31 μmol) 2 mL of DMF and 16 μL of DIPEA (93 μmol). The resulting mixture was stirred and combined with 10 mg of PFP ester 17 (31 μmol) at room temperature. After 2 h, reaction mixture was directly purified by reversed-phase prep HPLC (C18, 0-50% acetonitrile-water, 0.05% TFA). Pure fractions were combined and freeze-dried to give 28 mg of compound 18 as a white solid (17 μmol, 55 % yield). LRMS (ESI): m/z 1690.7 [M+H]+, Calcd for C78H119N11O28S m/z 1690.8.
[00529] Synthesis of compound 17. [00530] 3-Maleimidopropanoic acid (169 mg, 1 mmol) was combined with pcntafluorophcnol (368 mg, 2 mmol) in 4 mL anhydrous THF. To this mixture were added DCC (206 mg, 1 mmol) at room temperature in one portion. The resulting mixture was stirred at RT overnight. Solids were filtered off and washed with THF, combined filtrates were concentrated under vacuum and purified by silica gel chromatography (0-20% EtOAc-Hexanes) to give 250 mg of compound 17 as an off-white solid. LRMS (ESI): m/z 358.1[M+Na]+, Calcd for C13H6F5NO4 m/z 358.0.
EXAMPLE 12
Preparation of(2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-ll-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)- 3-(((lS,2R)-l -hydroxy- 1 -phenylpropan-2-yl )amino )-l -methoxy-2 -methyl-3 -oxopropyl )pyrrolidin-
1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-
2-((2S,5S,18R)-22-(2-((l,2-dimethylhydrazineyl)methyl)-lH-pyrrolo[2,3-b]pyridin-l-yl)-5- isopropyl-2-methyl-4, 7, 17,20-tetraoxo-18-( sulfomethyl)- 10, 13 -dioxa-3, 6, 16,19- tetraazadocosanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (24)
Figure imgf000160_0001
[00531] To a stirred solution of compound 12 (735 mg, 0.60 mmol) in 3 mL of anhydrous DMA were added DIPEA (0.21 mL, 1.2 mmol) and a solution of PFP-ester 23 (575 mg, 0.60 mmol) in 2 mL of DMA at room temperature, followed by the addition of HO At (84 mg, 0.60 mmol). The resulting mixture was stirred for 30 minutes until coupling was judged complete (HPLC analysis), then treated directly with 1.2 mL of piperidine at room temperature. After 15 minutes, reaction mixture was purified by reversed- phase chromatography (C18 column, 0-40% gradient of acetonitrile-water, no acid additive). Pure fractions were combined, concentrated under reduced pressure to ~50 mL final volume, and lyophilized to give compound 24 (808 mg, 0.45 mmol, 75% yield) as a white fluffy powder. LRMS (ESI): m/z 1783.9 [M+H]+, Calcd for C84H130N14O26S m/z 1783.9.
[00532] Synthesis of compound 23.
Figure imgf000161_0001
[00533] Carboxylic acid 25 (1.33 g, 1.67 mmol) was combined with pentafluorophenol (1.23 g, 6.68 mmol) in 6.5 mL of anhydrous DMF. This mixture was treated with EDC1-HC1 (0.64 g, 3.34 mmol) in one portion at room temperature and stirred for 20 h until 25 was fully consumed as judged by HPLC analysis. Reaction mixture was loaded onto a C18 column and eluted with 0-80% gradient of acetonitrile-water with 0.05% TFA additive. Pure fractions were combined, concentrated under reduced pressure until slightly murky, and lyophilized to give PFP-ester product 23 (1.40 g, 1.46 mmol, 87% yield) as a tan powder. LRMS (ESI): m/z 961.2 [M+H]+, Calcd for C44H45F5N6O11S m/z 961.3.
EXAMPLE 13
Preparation of (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(3-(5-((S)-28-(((S)-l-(((S)-l-((2- ((( 2S,3R,4S,5S,6S )-6-carboxy-3,4,5-lrihydroxylelrahydro-2H-pyran-2-yl)oxy)-4-( (((2-((S )-4- ethyl-4-hydroxy-3, 14-dioxo-3,4,12, 14-tetrahydro-lH-pyrano[3 ',4 ':6, 7 ]indolizino[1,2-b ]quinolin- 11 -yl )ethyl)( isopropyl)carbamoyl )oxy )methyl )phenyl )amino )-l -oxopropan-2-yl )amino)-3-methyl- l-oxobutan-2-yl)carbamoyl)-26,34-dioxo-2,5,8,ll,14,17,20,23-octaoxa-27,33- diazaheptatriacontan-37-amido)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-l- yl)propanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,ll,14,17,20,23-octaoxa- 27,30,33-triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14- tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-ll- yl )ethyl)( isopropyl )carbamoyl )oxy )methyl )phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (34)
Figure imgf000162_0001
[00534] Belotecan hydrochloride 26 (2.35 g, 5.0 mmol) was suspended in a mixture of 30 mL of anhydrous DMF and 1.75 mL of DIPEA (10 mmol). The resulting mixture was stirred and treated with HO At (0.68 g, 5 mmol), followed by PNP-carbonate 10 (5.1 g, 5 mmol) in a few small portions at room temperature. Reaction mixture was stirred at RT for 8 hours until starting materials were judged fully consumed based on HPLC analysis. The mixture was poured onto 300 mL of ice with vigorous stirring, the resulting yellowish precipitate was collected by filtration, washed with 30 mL of cold water twice, dried on air overnight to give 6.7 g of crude coupling product as a light-yellow powder which was taken to the next step without purification. [00535] A solution of crude coupling intermediate (6.7 g) in 30 mL of THF was cooled down to 0 °C in an ice bath and treated slowly with 2 M aqueous lithium hydroxide solution (10 mL). Reaction mixture was stirred at 0 °C for 1 hour, then another 10 mL of 2 M LiOH solution was added and stirring continued for 15 minutes before warming the reaction mixture to room temperature and adding another 10 mL of 2 M lithium hydroxide. The resulting mixture was stirred for 1 hour at room temperature, then quenched by adding 2 M aqueous HC1 solution to pH ~2-3 and let stir for 30 minutes. The mixture was transferred to a separatory funnel and washed with MTBE (2 x 50 mL). Aqueous layer was separated, then directly loaded on a C18 column, and eluted with 0-40% CH3CN-H2O with 0.05% TFA. Pure fractions were combined, concentrated under reduced pressure to ~70 mL volume, and lyophilized to give 3.4 g of product 27 (3.6 mmol, 72% yield over 2 steps) as a bright-yellow fluffy powder. LRMS (ESI): m/z 945.4 [M+H]+, Calcd for C47H56N6O15 m/z 945.4.
[00536] To a stirred mixture of compound 27 (945 mg, 1.0 mmol) and DIPEA (0.35 mL, 2 mmol) in 4 mL DMF were added Boc-Lys(Fmoc)-OPfp 28 (635 mg, 1 mmol) in one portion at room temperature. The resulting turbid mixture was stirred for 30 minutes until it became a clear yellow solution, while the reaction was judged complete by LCMS analysis. Reaction mixture was poured into 50 mL of 10% aq. citric acid solution and extracted with ethyl acetate (2x100 mL). Organic layer was washed once with brine, dried over sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using 5-10% MeOH in DCM as an eluent. Solvents removed under vacuum to give 1.15 g (0.82 mmol, 82% yield) of product 29 as a bright yellow solid. LRMS (ESI): m/z 1395.6 [M+H]+, Calcd for C73H86N8O20 m/z 1395.6.
[00537] To a stirred solution of compound 29 (1.15 g, 0.82 mmol) in 5 mL of anhydrous DCM were added 5 mL of TFA at RT. Reaction mixture was stirred for 15 minutes, monitored for completion by LCMS. Solvents were immediately removed under reduced pressure, the dark oily residue was briefly dried under high vacuum and triturated with 25 mL MTBE. The resulting light-yellow precipitate was collected by filtration, transferred to a round-bottom flask, and dried under high vacuum overnight to give 1.2 g of crude product 30 as light-yellow powder (TFA salt). LRMS (ESI): m/z 1295.5 [M+H]+, Calcd for C68H78N8O18m/z 1295.6.
[00538] To a solution of mPEG8 acid (340 mg, 0.82 mmol) in 5 mL of anhydrous THF were added pentafluorophenol (180 mg, 0.98 mmol), followed by DCC (170 mg, 0.82 mmol) at room temperature. The resulting mixture was stirred overnight, solids were filtered off, and washed with THF on filter. Combined filtrate was concentrated under reduced pressure, to give crude compound 31 (clear oil), which was re-dissolved in 2 mL of DMF and combined with a stirred mixture of crude compound 30 (0.82 mmol) and DIPEA (0.43 mL, 2.5 mmol) in 4 mL of DMF. Reaction mixture was stirred for 1 hour at room temperature until judged complete by HPLC analysis based on consumption of 30. Reaction mixture was directly treated with 3 mL of triethylamine and stirring continued at room temperature for 7 hours until Fmoc -deprotection was complete based on HPLC analysis. Triethylamine and partially DMF were removed under reduced pressure, and the residual mixture was purified by reversed-phase chromatography (C18 column 0-40% CH3CN-H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure to ~70 mL final volume, and lyophilized to give 932 mg of product 32 as a bright-yellow light powder (0.64 mmol, 78% yield over 3 steps starting from 29). LRMS (ESI): m/z 1467.7 [M+H]+, Calcd for C71H102N8O25 m/z 1467.7.
[00539] To a stirred solution of compound 32 (3.5 g, 2.4 mmol) in 16 mL of anhydrous DMA were added DIPEA (0.84 mL, 4.8 mmol) and HOAt (0.33 g, 2.4 mmol) at room temperature. The resulting mixture was treated with a separately prepared solution of bis-PFP- ester 33 (1.0 g, 1.1 mmol in 4 mL of DMA) in small portions (2 mL, 1 mL, 0.75 mL, and 0.5 mL) with 10 minutes intervals between additions. After the addition was complete, reaction mixture was stirred for 15 minutes at room temperature and treated with 2.1 mL of piperidine (22 mmol). After 20 minutes, reaction mixture was directly purified by reversed-phase chromatography (C18 column, 0-40% CH3CN-H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure to -100 mL final volume, and lyophilized to obtain 3.2 g of compound 34 as a bright-yellow fluffy powder (0.98 mmol, 89% yield for two steps based on 33). LRMS (ESI): m/z 1638.3 [M+H]2+, Calcd for C160H224N20O53 m/z 1638.8.
[00540] Synthesis of Boc-Lys(Fmoc)-OPfp 28.
[00541] To a stirred solution of Boc-Lys(Fmoc)-OH (4.7 g, 10 mmol) in 40 mL of anhydrous THF were added pentafluorophenol (2.2 g, 12 mmol), followed by DCC (2.1 g, 10 mmol) in small portions at room temperature. The resulting mixture was stirred overnight, solids were filtered off, washed with THF on filter. Combined filtrate was concentrated under vacuum, and the white solid residue was triturated with DCM-EtOAc mixture, collected by filtration, and dried under vacuum to give 4.9 g (7.8 mmol, 78% yield) of Boc-Lys(Fmoc)-OPfp 28 as a white powder. LRMS (ESI): m/z 657.3 [M+Na]+, Calcd for C32H31F5N2O6 m/z 657.2.
[00542] Synthesis of bis-PFP ester 33.
Figure imgf000165_0001
[00543] A solution of bis-acid 35 (2.0 g, 3.34 mmol) in 40 mL of anhydrous THF was combined with 6.2 g (33.4 mmol) of pentafluorophenol. The mixture was stirred and treated with DCC (2.0 g, 10 mmol) in a few small portions at room temperature. Reaction mixture was stirred for 48 hours at RT, then all solids were removed by filtration and washed with THF on filter. Combined filtrates were concentrated under vacuum and purified by silica gel chromatography (0-25-35% gradient of EtOAc-Hexanes) to give 2.4 g of bis-PFP ester 33 (2.6 mmol, 77% yield) as a white foaming solid. LRMS (ESI): m/z 931.2 [M+H]+, Calcd for C45H32F10N4O7 m/z 931.2.
EXAMPLE 14
Preparation of (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(6-(2,5-dioxo-2,5-dihydro-lH- pyrrol- 1 -yl )hexanamido )butyl )-31 -isopropyl-34-methyl-26,29,32-trioxo-2, 5,8,11,14,17,20,23- octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-
3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11- yl )ethyl)( isopropyl )carbamoyl )oxy )methyl )phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (36)
Figure imgf000165_0002
[00544] Compound 32 (20 mg, 14 μmol) was dissolved in 2 mL of anhydrous DMF and combined with HOAt (14 μmol,14 μL of IM solution in DMA) and DIPEA (6 μL). To this mixture were added compound 13 (5 mg, 14 μmol) at room temperature. Reaction mixture was stirred for 10 minutes until judged complete by LCMS analysis and purified directly by reversed- phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were lyophilized to give 20 mg of compound 36 as a yellow solid (12 μmol, 86% yield). LRMS (ESI): m/z 1660.8 [M+H]+, Calcd C81H113N9O28 m/z 1660.8.
EXAMPLE 15
Preparation of (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl )hexanamido )-3 -methylbutanamido )propanamido)-5-( (((2-((S )-4-ethyl-4-hydroxy-3, 14-dioxo-
3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-ll- yl )ethyl)( isopropyl )carbamoyl )oxy )methyl )phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (37)
Figure imgf000166_0001
[005451 To a solution of compound 21 (25 mg, 26 μmol) in anhydrous DMF (2 mL) were added compound (12 mg, 32 μmol). The resulting mixture was stirred and treated with DIPEA (30 μL) and HOAt (20 μL of IM solution in DMA, 20 μmol). After 20 minutes, reaction mixture was directly purified by reversed-phase prep HPLC (C18, 0-50% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 23 mg of compound 37 as a yellow solid (20 μmol, 77% yield). LRMS (ESI): m/z 1138.4 [M+H]+, Calcd C57H67N7O18 m/z 1138.5.
EXAMPLE 16
Preparation of (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl )hexanamido)-3-methylbutanamido)propanamido)-5-( ((((1S,9S )-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10, 13 -dioxo -2, 3, 9,10,13, 15 -hexahydro- 1H, 12H- benzo[ de ]pyrano[3 ',4 ':6, 7 ]indolizino[ 1 ,2-b ]quinolin-l -yl)carbamoyl)oxy)methyl )phenoxy)-
3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (42)
Figure imgf000167_0001
[00546] Exatecan 39 (mesylate, 266 mg, 0.5 mmol) was suspended in 4 mL of anhydrous DMF and treated with DIPEA (0.26 mL, 1.5 mmol). The resulting clear dark solution was treated with HOAT (0.5 mL of IM in DMA), followed by compound 10 (507 mg, 0.5 mmol) in a few small portions at RT with stirring. The mixture was stirred for 5 hours at RT, until reaction was judged complete by LCMS analysis. Reaction mixture was poured into 20 mL of water, the resulting precipitate was separated by spinning and washed with water to obtain 650 mg of crude compound 40 as a light-yellow solid. LRMS (ESI): m/z 1309.4 [M+H]+, Calcd for
C68H69FN6O20 m/z 1309.5
[00547] Crude compound 40 (0.5 mmol) was dissolved in 15 mL of THF. The solution was cooled down to 0 °C, treated dropwise with 7 mL of LiOH solution (aq. IM) and stirred at 0 °C for 3 h, monitored for completion by HPLC. Reaction mixture was quenched by adding HC1 (aq. IM solution) to adjust the pH to 2-3, and allowed to stir for 15 minutes, then washed with MTBE (15 mL). Aqueous layer was separated, loaded onto C18 column, and eluted with 0- 40% acetonitrile- water with 0.05% TFA. Pure fractions were combined and lyophilized to give 130 mg of compound 41 (0.14 mmol, 28% yield over two steps) as a yellow solid. LRMS (ESI): m/z 947.3 [M+H]+, Calcd for C46H51FN6O15 m/z 947.3
[00548] Compound 41 (25 mg, 26 μmol) was dissolved in anhydrous DMF (2 mL) and combined with DIPEA (30 μL, 172 μmol) and Pfp-ester 13 (15 mg, 40 μmol) at RT. Reaction mixture was stirred for 20 minutes and purified by reversed-phase prep HPLC (C18, 0-50% acetonitrile- water, 0.05% TFA). Pure fractions were lyophilized to give 14 mg of compound 42 (12 μmol, 46% yield) as a yellow solid. LRMS (ESI): m/z 1140.4 [M+H]+, Calcd for C56H62FN7O18 m/z 1140.4 EXAMPLE 17
Preparation of (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(6-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)hexanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,ll,14,17,20,23- octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-( ((( (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10, 13 -dioxo -2, 3, 9,10,13, 15 -hexahydro- 1H, 12H- benzo[ de ]pyrano[3 ',4 ':6, 7 ]indolizino[ 1 ,2-b ] quinolin- 1 -yl)carbamoyl)oxy)methyl )phenoxy)- 3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (46)
Figure imgf000168_0001
[00549] Compound 41 (110 mg, 0.12 mmol) was dissolved in 3 mL of anhydrous DMF treated with DIPEA (60 μL, 0.34 mmol) at RT and stirred. To this mixture were added Pfp- ester 28 (74 mg, 0.12 mmol) as a solid, followed by HO At (0.12 mL of IM in DMA, 0.12 mmol). After 30 minutes, reaction was judged complete by LCMS analysis. Reaction mixture was quenched by pouring into 20 mL of 10% aq. citric acid, extracted with ethyl acetate (2 X 20 mL). Combined organic layer was washed with brine and dried over sodium sulfate. Solvents were removed in vacuum and the residue was purified on silica gel with 2-12% MeOH-DCM to give 125 mg of compound 43 (90 μmol, 75% yield) as a yellow solid. LRMS (ESI): m/z 1397.5 [M+H]+, Calcd for C72H8IFN802O m/z 1397.6.
[00550] Compound 43 (125 mg, 90 μmol) was dissolved in a mixture of DCM-TFA (1:1 v/v, 4 mL) at RT and let stand for 10 minutes. Solvents were removed in vacuum and the dark oily residue was triturated with MTBE to solidify and collect crude compound 44 as a light- yellow powder which was dried under high vacuum overnight and used in the next step without any purification. LRMS (ESI): m/z 1297.4 [M+H]+, Calcd for C67H73FN8O18 m/z 1297.5.
[00551] To a solution of mPEG8 acid (37 mg, 90 μmol) in 2 mL of anhydrous THF were added pentafluorophenol (20 mg, 108 μmol), followed by DCC (19 mg, 90 μmol) at room temperature. The resulting mixture was stirred overnight, solids were filtered off and the resulting filtrate was concentrated under reduced pressure, to give crude compound 31 (clear oil), which was re-dissolved in 1 mL of DMF and combined at room temperature with a stirred mixture of crude compound 44 (90 μmol), DIPEA (46 μL, 0.27 mmol), and HOAt (90 μL of IM in DMA, 90 μmol) in 2 mL of DMF. Reaction mixture was stirred for 30 minutes at room temperature until judged complete by HPLC analysis based on consumption of 44. Reaction mixture was then directly treated with 100 μL of piperidine, stirred for 20 minutes, and purified by reversed-phase prep HPLC (C18 column, 0-50% acetonitrile-water with 0.05% TFA). Pure fractions were combined and lyophilized to give 60 mg of compound 45 as a yellow powder (41 μmol, 46% yield over 3 steps starting from 43). LRMS (ESI): m/z 1469.6 [M+H]+, Calcd for C70H97FN8O25 m/z 1469.7.
[00552] Compound 45 (20 mg, 21 μmol) was dissolved in anhydrous DMF (2 mL) and combined with DIPEA (15 μL, 86 μmol) and Pfp-ester 13 (7 mg, 20 μmol) at RT. Reaction mixture was stirred for 1 h and purified by reversed-phase prep HPLC (C18, 0-50% acetonitrile- water, 0.05% TFA). Pure fractions were lyophilized to give 19 mg of compound 46 (11 μmol, 52 % yield) as a yellow solid. LRMS (ESI): m/z 1662.6 [M+H]+, Calcd for C80H108FN9O28 m/z 1662.7.
EXAMPLE 18
Preparation of (2S,3S,4S,5R,6S)-6-(2-((29S,32S,35S)-29-(4-(6-(2,5-dioxo-2,5-dihydro-lH- pyrrol-1 -yl)hexanamido)butyl)-32-isopropyl-35-methyl-27, 30, 33-trioxo-2, 5, 8,11,14,17,20,23,26- nonaoxa-28,31 ,34-triazahexatriacontan-36-amido)-5-((((2-((S)-4-ethyl-44rydroxy-3, 14-dioxo-
3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-ll- yl )ethyl)( isopropyl )carbamoyl )oxy )methyl )phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (50).
Figure imgf000170_0001
[00553] To a stirred solution of compound 47 (907 mg, 1.16 mmol) in 2 mL of anhydrous DMF were added DIPEA (0.60 mL, 3.4 mmol), followed by HATU (443 mg, 1.16 mmol) at room temperature. The resulting mixture was stirred for 30 min and then combined with a solution od compound 27 (1.0 g, 1.06 mmol) in 4 mL of DMF. After 1 h, reaction mixture was treated with 1.2 mL of triethylamine, stirred at RT for 5 h, until reaction was judged complete by LCMS analysis. Reaction mixture was concentrated under reduced pressure to remove triethylamine and partly DMF, and the residue was purified by reversed-phase chromatography (C18, 0-40% acetonitrile- water/0.05% TFA). Pure fractions were combined and lyophilized to give 800 mg of compound 48 (0.54 mmol, 51% yield over two steps) as a light-yellow powder. LRMS (ESI): m/z 1483.6 [M+H]+, Calcd for C71H102N8O26 m/z 1483.7.
[00554] To a stirred solution of compound 48 (287 mg, 0.19 mmol) in 2 mL of anhydrous DMA were added DIPEA (0.10 mL, 0.57 mmol), followed by PFP-ester 13 (73 mg, 0.19 mmol) in one portion at room temperature. Reaction mixture was stirred for 30 min, then directly purified by reversed-phase column chromatography (C18, 0-40% acetonitrile-water/0.05% TFA). Pure fractions were combined, concentrated in vacuum, and lyophilized to give 220 mg of compound 50 (0.13 mmol, 68% yield) as a light-yellow powder. LRMS (ESI): m/z 1676.7 [M+H]+, Calcd for C81H113N9O29 m/z 1676.8. [00555] Synthesis of mP8c-Lys(Fmoc)-OH (47)
Figure imgf000171_0001
[00556] To a stirred solution of mPEG8-alcohol 51 (385 mg, 1.0 mmol) in 4 mL of anhydrous DCM were added DIPEA (0.35 mL, 2 mmol), followed by bis-(4-nitrophenyl) carbonate (335 mg, 1.1 mmol) at room temperature. Reaction mixture was stirred overnight and then directly purified by silica gel chromatography using 0-5% MeOH-DCM gradient to obtain 507 mg of compound 52 (0.92 mmol, 92% yield) as a light-yellow clear oil. LRMS (ESI): m/z 550.3 [M+H]+, Calcd for C24H39NO13 m/z 550.2.
[00557] Amino acid H-Lys(Fmoc)-OH (53) (184 mg, 0.5 mmol) was suspended in 2 mL of anhydrous DMF, treated with DIPEA (0.17 mL, I mmol) at RT, and stirred for 5 min. To this mixture were added HOAt (0.6 mL of IM in DMA, 0.6 mmol), followed by PNP-carbonate 52 (330 mg, 0.6 mmol) as a solution in 1 mL of DMF. The resulting mixture was stirred for 2 h at RT and then directly purified by reversed-phase chromatography (C18, 0-70% acetonitrile- water/0.05% TFA). Pure fractions were combined and lyophilized to give 220 mg of compound 47 (0.28 mmol, 56% yield) as a clear colorless oil. LRMS (ESI): m/z. 779.4 [M+H]+, Calcd for C39H58N2O14 m/z 779.4.
EXAMPLE 19
Preparation of (2S,3S,4S,5R,6S)-6-(2-((29S,32S,35S)-29-(4-(2-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)acetamido)butyl)-32-isopropyl-35-methyl-27,30,33-trioxo-2,5,8,ll,14,17,20,23,26- nonaoxa-28,31 ,34-triazahexatriacontan-36-amido )-5-( (((2-(( S)-4-ethyl-4-hydroxy-3, 14-dioxo- 3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11- yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (57)
Figure imgf000172_0001
[005581 Carboxylic acid 55 (388 mg, 2.5 mmol) was combined with pentafluorophenol (1.38 g, 7.5 mmol) in 10 mL of anhydrous THF. The resulting solution was treated with DCC (1.55 g, 7.5 mmol) at room temperature. Reaction mixture was stirred overnight, filtered, concentrated in vacuum, and purified by silica gel chromatography to give 505 mg of PFP ester 56 (1.6 mmol, 64% yield). LRMS (ESI): m/z 322.0 [M+H]+, Calcd for C12H4F5NO4 m/z 322.0. [00559] To a stirred solution of compound 48 (21 mg, 14 μmol) in 2 mL of anhydrous DMF were added DIPEA (6 μL, 35 μmol) and HOAt (14 μL, IM in DMA, 14 μmol) at room temperature. The resulting mixture was treated with PFP ester 56 (4.6 mg, 14 μmol). After 30 min, reaction mixture was directly purified by reversed-phase prep HPLC (C18, 0-70 acetonitrile- water/0.05% TFA). Pure fractions were combined, concentrated, and lyophilized to obtain 20 mg of compound 57 (12 μmol, 86% yield) as a bright-yellow fluffy powder. LRMS (ESI): m/z 1620.6 [M+H]+- Calcd for C77H105N9O29 m/z 1620.7.
EXAMPLE 20
Preparation of (2S,3S,4S,5R,6S)-6-(2-((29S,32S,35S)-29-(4-(2-bromoacetamido)butyl)-32- isopropyl-35-methyl-27,30,33-trioxo-2,5,8,11,14,17,20,23,26-nonaoxa-28,31,34- triazahexatriacontan-36-amido)-5-( (((2-((S )-4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro- 1H-pyrano[ 3', 4 ':6, 7]indolizino[ 1,2-b ]quinolin-11 - yl )ethyl)( isopropyl )carbamoyl )oxy )methyl )phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (59)
Figure imgf000172_0002
[00560] To a stirred solution of compound 48 (30 mg, 20 μmol) in 1 mF of anhydrous DMF were added DIPEA (7 pF, 40 μmol), followed by Pfp-ester 58 (6 mg, 20 μmol) in one portion at ambient temperature. After 15 minutes, reaction mixture was directly purified by reversed-phase prep HPLC (C18, 0-50% ACN-water/0.05% TFA). Pure fractions were combined and lyophilized to give 19 mg (12 μmol, 60% yield) of compound 59 as a yellow powder. LRMS (ESI): m/z 1605.5 [M+H]+- Calcd for C73H103BrN8O27 m/z 1620.7.
EXAMPLE 21
Preparation of ( 2S,3S,4S, 5R, 6S)-6-(2-((S)-2-((S)-2-(2-(2, 5-dioxo-2, 5 -dihydro- 1H-pyrrol-1 - yl)acetamido)-3-methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-
3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-11- yl )ethyl)( isopropyl )carbamoyl )oxy )methyl )phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (60)
Figure imgf000173_0001
[00561] In an oven dried 20 mL glass vial were combined compound 27 (25 mg, 26 μmol), HOAt (33 μmol, 33 μL of 1 M in DMA), and DIPEA (17 μL, 99 μmol) in 3 mL of DMF at RT. To this mixture were added PFP-ester 56 (9 mg, 27 μmol) in one portion. After 10 minutes, reaction was judged complete by LCMS analysis. The mixture was directly purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 6 mg of compound 60 (6 μmol, 23% yield) as a yellow powder. LRMS (ESI): m/z 1082.4 [M+H]+, Calcd for C53H60N7O18 m/z 1082.4. EXAMPLE 22
Preparation of(2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)- 3-(((1S,2R)-1 -hydroxy- 1 -phenylpropan-2-yl )amino )-l -methoxy-2 -methyl-3 -oxopropyl )pyrrolidin-
1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-
2-((S)-2-((S)-2-(2-(2, 5-dioxo-2, 5-dihydro-l H-pyrrol-1 -yl)acetamido )-3- methylbutanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (61)
Figure imgf000174_0001
[00562] In an oven dried 20 mL glass vial were combined compound 12 (36 mg, 29 (imol), HOAt (36 pmol, 36 μL of 1 M in DMA), and DIPEA (19 μL, 108 μmol) in 3 mL of DMF at RT. To this mixture were added PFP-ester 56 (10 mg, 30 μmol) in one portion. After 10 minutes, reaction was judged complete by LCMS analysis. The mixture was directly purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 27 mg of compound 61 as a white powder (20 μmol, 69% yield). LRMS (ESI): m/z 1366.6 [M+H]+, Calcd for C67H100N9O21 m/z 1366.7.
EXAMPLE 23
Preparation of (2S,3S,4S,5R,6S)-6-(5-((5S,8S,HS,12R)-ll-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)-
3 -(((1S,2R)-1 -hydroxy- l-phenylpropan-2-yl)amino)-1 -methoxy-2 -methyl-3 -oxopropyl)pyrrolidin-
1-yl)-2-oxoeth.yl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-
2-((4R,17S,20S)-l-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-isopropyl-20-methyl-2,5,15,18- tetraoxo-4-( sulfomethyl )-9, 12-dioxa-3,6, 16, 19-tetraazahenicosan-21 -amido)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid (62)
Figure imgf000175_0001
[00563] In an oven dried 20 mL glass vial were combined compound 16 (5 mg, 3.2 μmol), HO At (3.3 μmol, 3.3 μL of 1 M in DMA), and DIPEA (1.7 μL, 9.9 μmol) in 1 mL of anhydrous DMF at RT. To this mixture were added PFP-ester 56 (1.1 mg, 3.2 pmol) in one portion. After 10 minutes, reaction was judged complete by LCMS analysis. The mixture was directly purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 2 mg of compound 62 as a yellow powder (1.2 μmol, 38% yield). LRMS (ESI): m/z 1676.7 [M+H]+, Calcd for C77H118N11O28S m/z 1676.8.
EXAMPLE 24
Preparation of (2S,3S,4S,5R,6S)-6-(5-((5S,8S,l 1S,12R)-1 l-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)- 3-(((1S,2R)-l -hydroxy- l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-
1-yl)-2-oxoeth.yl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-
2-((2S,5S)-15-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-5-isopropyl-2-methyl-4,7-dioxo-10,13- dioxa-3, 6-diazapentadecanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (64)
Figure imgf000176_0001
[00564] In an oven dried 20 mL glass vial were combined compound 12 (14 mg, 11 p.mol), HOAt (14 μmol, 14 ofμ 1L M in DMA), and DIPEA (7.5 , 42 μmol) in 3 mL of DMF at RT. To this mixture were added PFP-ester 63 (5 mg, 12 μmol) in one portion. After 10 minutes, reaction was judged complete by LCMS analysis. The mixture was directly purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 8 mg of compound 64 as a white powder (5.5 pmol, 50% yield). LRMS (ESI): m/z 1468.7 [M+H]+, Calcd for C72H110N9O23 m/z 1468.8.
EXAMPLE 25
Preparation of(2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-(2-bromoacetamido)-3- methylbutanamido)propanamido)-5-( (((2-(( S)-4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14- tetrahydro-1H-pyrano[3 ',4':6, 7]indolizino[l,2-b ] quinolin- 11 - yl )ethyl)( isopropyl )carbamoyl )oxy )methyl )phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-
Figure imgf000176_0002
[00565] To a stirred solution of compound 27 (13 mg, 14 μmol) in 3 mL of anhydrous DMF were added DIPEA (20 μL) and Pfp-ester 58 (4.2 mg, 14 μmol) at RT. After 10 min, reaction mixture was analyzed by LCMS and directly purified by reversed-phase prep HPLC (C18, acetonitrile-water, 0.05% TFA). Pure fractions were lyophilized to give 5 mg of compound 65 (4.7 μmol, 34% yield) as a yellow solid. LRMS (ESI): m/z. 1067.3 [M+H]+, Calcd for C49H57BrN6O16 m/z 1067.3.
EXAMPLE 26
Preparation of ( 2S,3S,4S,5R,6S)-6-(2-( ( S )-2-( ( S )-2-( 2-bromoacetamido)-3- methylbutanamido)propanamido)-5-((5S,8S,llS,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((lR,2R)- 3 -(((1S,2R)-1 -hydroxy- 1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-
1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10- triaz.atetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (66)
Figure imgf000177_0001
[00566] To a solution of compound 12 (20 mg, 16 μmol) in 3 mL of DMF were added DIPEA (30 μL) and Pfp-ester 58 (5 mg, 16 μmol) at RT. Reaction mixture was stirred for 10 min and directly purified by reversed-phase prep HPLC (C18 column, 0-50% acetonitrile-water, 0.05% TFA). Pure fractions were collected and lyophilized to give 17.5 mg of compound 66 (13 μmol) as a white solid. LRMS (ESI): m/z 1351.6 [M+H]+, Calcd for C63H97BrN8O19 m/z 1351.6. EXAMPLE 27
Preparation of ( 2S,3S,4S,5R,6S)-6-( 5-( ((( 4-( ( 4-amino-2-butyl-lH-imidazo[4,5-c ]quinolin-1 - yl)methyl)benzyl)carbamoyl)oxy)methyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-1- yl)hexanamido)-3miethylbutanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-carboxylic acid (70)
Figure imgf000178_0001
[00567] To a stirred solution of 1MDQ (67, 51 mg, 0.14 mmol) in anhydrous DMF (2 mL) were added compound 10 (141 mg, 0.14 mmol), followed by DIPEA (75 p.L, 0.78 mmol)) and HOAt (140 μL of IM in DMA, 0.14 mmol). The resulting mixture was stirred for 3 h and quenched by pouring in 5 mL of water. The resulting precipitate was separated on centrifuge, washed with water, and dried on air to give 150 mg of crude compound 68 as a yellowish solid, which was subjected to the next step without purification. LRMS (ESI): m/z 1233.5 [M+H]+, Calcd for C66H72N8O16 m/z 1233.5.
[00568] Crude compound 68 (150 mg, -0.14 mmol) was dissolved in THF (3 mL) and treated with aqueous lithium hydroxide solution (0.65 mL of 2M, 1.3 mmol) at 0 °C. Reaction mixture was stirred at 0 °C for 2 h, then warmed up to RT and stirred for 1 h. Reaction mixture was quenched by adding 10% aq. TFA to adjust pH to 7 and purified by reversed-phase prep HPLC (C18, 0-50% acetonitrile-water, 0.05% TFA). Puere fractions were combined and lyophilized to give 71 mg of compound 69 (82 μmol, 59% yield) as a white solid. LRMS (ESI): m/z 871.4 [M+H]+, Calcd for C44H54N8O11 m/z 871.4.
[00569] Compound 69 (22 mg, 25 μmol) was combined with Pfp-ester 13 (13 mg, 35 μmol) in 2 mL of anhydrous DMF. The mixture was treated with DIPEA (20 μL, 115 μmol) and stirred for 1 h at RT. Reaction mixture was directly purified by reversed-phase prep HPLC (Cl 8, 0-50% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 18 mg of compound 70 (17 μmol, 68% yield) as a white solid. LRMS (ESI): m/z 1064.4 [M+H]+, Calcd for C54H65N9O14 w/z 1064.5.
EXAMPLE 28
HIPS conjugation of aldehyde-tagged antibodies.
[00570] Antibodies (15 mg/mL) bearing one or two aldehyde tags (single or double- tagged constructs) were conjugated to linker-payloads at 1.1 or 1.7 mM, respectively. Reactions proceeded for 72 h at 37 °C in 20 mM sodium citrate, 50 mM NaCl pH 5.5 (20/50 buffer) containing 0.85-2.5% DMA. After conjugation, free drug was removed using a 30 kD MWCO 0.5 mL Amicon spin concentrator. Samples were added to the spin concentrator, centrifuged at 15,000 x g for 7 min, then diluted with 450 μL 20 mM sodium citrate, 50 mM NaCl pH 5.5 and centrifuged again. The process was repeated 10 times. To determine the DAR of the final product, ADCs were examined by analytical chromatography using HIC (Tosoh #14947) or PLRP-RP (Agilent PL1912-1802 1000A, 8 um, 50 x 2.1 mm) columns. HIC analysis used mobile phase A: 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0, and mobile phase B: 25% isopropanol, 18.75 mM sodium phosphate pH 7.0. PLRP analysis used mobile phase A: 0.1% trifluoroacetic acid in water, and mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. Prior to PLRP analysis, sample was denatured with the addition of 50 mM DTT, 4 M guanidine HC1 (final concentrations) and heating at 37 °C for 30 min. To determine aggregation, samples were analyzed using analytical size exclusion chromatography (SEC; Tosoh #08541) with a mobile phase of 300 mM NaCl, 25 mM sodium phosphate pH 6.8 with 5% isopropanol.
Maleimide conjugation of untagged (wild type) antibodies
[00571] Maleimide conjugation of untagged (wild type) antibodies. Antibodies (5 mg/mL) were reduced using 2.5 mol. equivalents of TCEP for 90 min at 37° C in in PBS, pH 8.0, 1 mM DTPA. TCEP was removed and the protein was exchanged into PBS, pH 7.4, 1 mM DTPA using tangential flow filtration. Reduced antibody (3 mg/mL) was conjugated with 10 mol. equiv of maleimide-functionalized linker-payloads for 60 min on ice. Free drug was removed, and final ADC was exchanged into PBS, pH 7.4 using tangential flow filtration. Hinge region cysteine conjugation using bromoacetamide (BrAc) linker-payloads
[00572] An antibody (final concentration = 5 mg/mL) in pH 8 DPBS buffer with 1 mM DTPA was reduced with 1.55 mM TCEP (stock was prepared as follows: 10 mM TCEP in pH 7.4 PBS with 1 mM DTPA) for 90 min at 37 °C. After 90 min, a Zeba protein desalting column was used to remove TCEP and exchange the reduced antibody back into pH 8 DPBS with 1 mM DTPA. The antibody was then conjugated at 3 mg/mL in pH 8 DPBS with 1 mM DTPA and 0.24 mM of either compound 59 or compound 65 (12 eq). The drug-linker stock solution was 50-100 mM in DMA. The conjugation mixture was incubated at 37 °C for 24 h, and the free drug was removed subsequently using a 30 kDa MWCO 0.5 mL Amicon spin concentrator exchanging into pH 5.5 histidine-trehalose buffer (20 mM Histidine, 6 % trehalose). The resulting conjugate was analyzed via PLRP and HIC to determine DAR and via SEC to determine aggregation.
Table 5. Cysteine conjugates of tandem-cleavage linkers included with present disclosure
Figure imgf000180_0001
Figure imgf000181_0001
EXAMPLE 29
In vitro cytotoxicity assays.
[00573] Cell lines were plated in 96- well plates (Costar 3610) at a density of 5 x 104 cells/well in 100 μL of growth media. The next day cells were treated with 20 μL of test articles serially diluted in media. After incubation at 37 °C with 5% CO2 for 5 days, viability was measured using the Promega CellTiter Gio® reagent according to the manufacturer’ s recommendations. GI50 curves were calculated in GraphPad Prism normalized to the pay load concentration.
EXAMPLE 30
Glutathione stability screen.
[00574] ADCs were prepared at 2-3 mg/mL in phosphate buffered saline with a final concentration of 500 pM glutathione. Replicate samples were prepared, incubated for the indicated times (0, 3 and 7 days) and analyzed by PLRP as described, using TCEP instead of DTT to reduce the samples prior to analysis. PLRP analysis indicated the DAR of the sample, and changes in DAR over time relative to starting point were interpreted as a sign of deconjugation.
EXAMPLE 31
In vitro stability.
[00575] ADCs were spiked into rat or cynomolgus monkey serum at 40 pg/mL. Samples were aliquoted and stored at -80 °C until use. Aliquots were placed at 37 °C under 5% CO2 for the indicated times (e.g., 0, 3, and 6 days), and then were analyzed by ELISA. A freshly thawed aliquot was used as the Day 0 time point to represent the starting conditions for the study. A standard curve was prepared in serum, and the standards and samples were diluted 1:1000 into casein blocking buffer (Thermo Scientific), so that the concentrations would be in the linear range of the ELISA assays. Analytes were captured on plates coated with human HER2/ERBB2 recombinant protein (ACROBiosystems HE2-H5225). Then, conjugated payload was detected with either a mouse anti-MMAE antibody or a mouse anti-belotecan/anti-Dxd antibody (generated internally) followed by an HRP-conjugated anti-mouse secondary antibody. Total antibody was detected with an HRP-conjugated anti-human Fc-specific secondary antibody (Jackson Immunoresearch). Bound secondary antibody was visualized using Ultra TMB One- Step ELISA substrate (Thermo Fisher). The colorimetric reaction was stopped with H2SO4, and absorbance at 450 nm was determined using a BioTek Synergy Neo2 plate reader. The OD 450 nm signal for the standard curve was plotted against the log of the known concentrations and fit using a 4-parameter logistic model. Stability samples were quantified with reference to the standard curve fit using a 4-parameter regression analysis.
EXAMPLE 32
Xenograft studies
Methods:
[00576] For the NCI-N87 study, female BALB/c nude mice (8/group) were inoculated subcutaneously with 10 million NCLN87 cells in a 1:1 solution of PBS:Matrigel. Treatment began when the tumors reached an average of 219 mm3. [00577] Animals were dosed one time intravenously with vehicle alone, or with 3 mg/kg of ADCs comprising trastuzumab conjugated to cither Compound 14, Compound 18, or Compound 38 with DARs of 4. Alternatively, animals were dosed with 3 mg/kg of Enhertu or with 3 mg/kg of an ADC comprising trastuzumab conjugated to Compound 36 with a DAR of 8. The animals were monitored twice weekly for body weight and tumor size. Animals were euthanized when tumors reached 2000 mm3.
Results:
NCI-N87 study
[00578] A single, 3 mg/kg dose of each of the three ADC compounds (ADCs of Compound 14, Compound 18, or Compound 38) resulted in tumor regressions that were sustained for more than 3 weeks post-dose (FIG. 40).
[00579] A single, 3 mg/kg dose of the ADC compound (ADC of Compound 36) resulted in tumor regressions that were sustained for more than 3 weeks post-dose (FIG. 48).
JIMT-1 study
[00580] For the JIMT-1 study, female NOD/SCID mice (10/group) were inoculated subcutaneously with 5 million JIMT-1 cells in 0.1 ml of PBS for tumor development. Treatment began when the tumors reached an average of 190 mm3.
[00581] Animals were treated with 10 mg /kg of human IgG on the day of randomization (day 0). One day later (day 1), animals were dosed one time intravenously with vehicle alone or with 3 mg/kg of ADCs comprising trastuzumab conjugated to either Compound 36 or Compound 37 with DARs of approximately 8. The animals were monitored twice weekly for body weight and tumor size. Animals were euthanized when tumors reached 2000 mm3.
Results:
[00582] A single, 3 mg/kg dose of each of the two ADC compounds (ADCs of Compound 36 or Compound 37) resulted in tumor growth inhibition that was sustained for more than 3 weeks post-dose (FIG. 53). EXAMPLE 33
Rat Pharmacokinetic Studies
[00583] Study Design: Sprague-Dawley rats (3/group) were given a single i.v. bolus dose of 3 mg/kg of ADCs comprising trastuzumab conjugated to either Compound 14, Compound 18, or Compound 38 with DARs of 4. Alternatively, animals were dosed with 10 mg/kg of Enhertu or with 10 mg/kg of ADCs comprising trastuzumab conjugated to either Compound 36 or Compound 37 with DARs of 8. Plasma samples were collected at the designated times and were analyzed for total antibody, total conjugate, and total ADC concentrations.
[00584] Pharmacokinetic sample analysis: The concentrations of total antibody and total ADC (DAR-sensitive), were quantified by ELISA as shown in the schematic (FIG. 41). For total antibody, conjugates were captured with an anti-human IgG-specific antibody and detected with an HRP-conjugated anti-human Fc-specific antibody. For total ADC, conjugates were captured with an anti-human Fab-specific antibody and detected with a mouse anti-MMAE primary antibody, followed by an HRP-conjugated anti-mouse IgG-subclass 1-specific secondary antibody. Bound secondary antibody was detected using Ultra TMB One-Step ELISA substrate (Thermo Fisher). After quenching the reaction with sulfuric acid, signals were read by taking the absorbance at 450 nm on a BioTek Synergy Neo2 plate reader.
[00585] Results for ADCs of Compound 14, Compound 18, and Compound 38 are shown in FIG. 42, which shows a graph of rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown.
[00586] Results for ADCs of Compound 36 and Compound 37 are shown in FIG. 49, which shows a graph of rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown.
[00587] Study Design: Sprague-Dawley rats (3/group) were given a single i.v. bolus dose of 3 mg/kg of ADCs comprising trastuzumab conjugated to either Compound 61 or Compound 65 with DARs of approximately 4 or 7, respectively. Alternatively, animals were dosed with 10 mg/kg of ADCs comprising trastuzumab conjugated to either Compound 57 or Compound 60 with DARs of approximately 8. Plasma samples were collected at the designated times and were analyzed for total antibody and total ADC concentrations. [00588] Pharmacokinetic sample analysis: The concentrations of total antibody and total ADC (DAR-scnsitivc), were quantified by ELISA as shown in the schematic (FIG. 41). For the total antibody determination, conjugates were captured with an anti-human IgG-specific antibody and detected with an HRP-conjugated anti-human Fc-specific antibody. For total ADC, conjugates were captured with an anti-human Fab-specific antibody and detected with a mouse anti-MMAE or a mouse anti-belotecan primary antibody, followed by an HRP-conjugated anti- mouse IgG- subclass 1- specific secondary antibody. Bound secondary antibody was detected using Ultra TMB One-Step ELISA substrate (Thermo Fisher). After quenching the reaction with sulfuric acid, signals were read by taking the absorbance at 450 nm on a BioTek Synergy Neo2 plate reader.
[00589] Results for ADC of Compound 61 are shown in FIG. 50, which shows a graph of rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by EEISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown.
[00590] Results for ADCs of Compound 57 and Compound 60 are shown in FIG. 51 which shows a graph of rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown.
[00591] Results for ADC of Compound 65 are shown in FIG. 52, which shows a graph of rat pharmacokinetic data showing total antibody and total ADC concentrations as measured by EEISA in plasma samples from rats dosed with the indicated compounds and sampled at the times shown.
EXAMPLE 34
Multi-dose non-GLP rat toxicology study
[00592] Male Sprague-Dawley rats (8-9 wk old at study start, 5 animals/group) were dosed intravenously with either vehicle alone or with nectin-4-targeted conjugates made using antibodies carrying the variable regions of the rat cross -reactive antibody, enfortumab. The tested ADCs were enfortumab vedotin (Compound 38), enfortumab conjugated to Compound 14, and enfortumab conjugated to Compound 18. Dosing at 10 mg/kg occurred weekly for a total of 4 doses (days 1, 8, 15, and 22). Animals were observed for 7 days post last dose. Body weights were recorded four times/week. Blood was collected from all animals for clinical pathology on days 5, 12, 19, and 26, and for toxicokinctic analysis at 8 h and days 4 and 7 post-dose (for all doses). Clinical observations were conducted daily. The clinical observation scoring system scale ranged from 0 (normal) to 3 (severe) is shown in Table 6.
Table 6. Clinical Observation Scoring System
Figure imgf000186_0001
Results:
[00593] Enfortumab ADCs conjugated to either vedotin (Compound 38), Compound 14, or to Compound 18 were compared for tolerability at equal payload/equal antibody dosing levels in a multi-dose rat study. One of the most prominent observations from this study was the numerous clinical observations noted in the vedotin dosing group. Most of the observations were related to skin lesions. By contrast, there were no clinical observations noted in the Compound 14 or Compound 18 dosing groups (FIG. 43). Considering that all of the ADCs release the same payload (free MMAE), share similar pharmacokinetic profiles, and display similar levels of efficacy against tumor xenografts, the striking difference in tolerability in this physiologically - relevant (antigen cross-reactive) rat model was unexpected and demonstrates that the tandem- cleavage linker element confers unique properties on conjugates when used in any context - including maleimide conjugation to the antibody. This finding was novel, unexpected, and of potential therapeutic utility.
[00594] FIG. 44 shows a graph of red blood cell (RBC) counts in rats dosed with enfortumab conjugates or vehicle alone.
[00595] FIG. 45 shows a graph of neutrophil counts in rats dosed with enfortumab conjugates or vehicle alone.
[00596] FIG. 46 shows a graph of alanine transaminase levels in rats dosed with enfortumab conjugates or vehicle alone.
[00597] FIG. 47 shows a graph of reticulocyte counts in rats dosed with enfortumab conjugates or vehicle alone.
[00598] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:
1. A conjugate of formula (I):
Figure imgf000188_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug;
W2 is an antibody; and
X is a conjugation moiety attached to the antibody at a native amino acid residue of the antibody.
2. The conjugate of Claim 1, wherein R1 is H and R2 is alkyl or substituted alkyl.
3. The conjugate of any of Claims 1-2, wherein k1 is 2.
4. The conjugate of any of Claims 1-3, wherein R3 is a chemically-cleavable moiety.
5. The conjugate of any of Claims 1-3, wherein R3 is an enzymatically-cleavable moiety.
6. The conjugate of Claim 5, wherein R3 is a glycoside or a glycoside derivative.
7. The conjugate of Claim 6, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannosidc, a fucosidc, O-GlcNAc, and O-GalNAc.
8. The conjugate of any of Claims 1-7, wherein X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
9. The conjugate of any of Claims 1-7, wherein X comprises a maleimide, and the native amino acid residue of the antibody comprises a cysteine residue.
10. The conjugate of Claim 8, wherein the conjugate is of formula (II):
Figure imgf000189_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
W2 is an antibody.
11. The conjugate of claim 8, wherein the conjugate is of formula (Ila):
Figure imgf000189_0002
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
W2 is an antibody.
12. The conjugate of any of Claims 1-11, wherein the first linker LA comprises: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein a, b, c, d, e and f are each independently 0 or 1;
T1, T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m- 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
13. The conjugate of Claim 12, wherein:
T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;
T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, - (CR13OH) m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:
(PEG)n is
Figure imgf000191_0001
, where n is an integer from 1 to 30;
EDA is an ethylene diamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1 ;
Figure imgf000191_0002
4-amino-piperidine (4AP) is
Figure imgf000191_0003
; and each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring.
14. The conjugate of any of Claims 12-13, wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-;
T3 is (PEG)n and V3 is -CO-; and d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0.
15. The conjugate of any of Claims 12-13, wherein one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4 ,V5 or V6 is a branched group.
16. The conjugate of Claim 15, wherein the branched group is selected from -CONR15- and 4AP.
17. The conjugate of any of Claims 15-16, wherein the branched group is attached to a second linker, LB.
18. The conjugate of Claim 17, wherein the second linker LB comprises:
-(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and l are each independently 0 or 1;
T7, T8, T9, T10, T11 and T12 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V7, V8, V9, V10 ,V11 and V12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
19. The conjugate of any of Claims 17-18, wherein LB is attached to a compound of formula (III):
Figure imgf000193_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug.
20. The conjugate of Claim 19, wherein R4 is H and R5 is alkyl or substituted alkyl.
21. The conjugate of any of Claims 19-20, wherein k2 is 2.
22. The conjugate of any of Claims 19-21, wherein R6 is a chemically-cleavable moiety.
23. The conjugate of any of Claims 19-21 , wherein R6 is an enzymatically-cleavable moiety.
24. The conjugate of Claim 23, wherein R6 is a glycoside or a glycoside derivative.
25. The conjugate of Claim 24, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
26. The conjugate of any of Claims 12-25, wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (C1-C12)alkyl and V3 is absent;
T4 is heteroaryl (triazole) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-; T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0.
27. The conjugate of any of Claims 15-26, wherein W1 and W1a are the same drug.
28. The conjugate of any of Claims 15-26, wherein W1 and W1a are different drugs.
29. The conjugate of any of Claims 1-28, wherein the conjugate is selected from:
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
and
Figure imgf000201_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker;
W1 is a drug; and
X is a conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody.
31. The compound of Claim 30, wherein R1 is H and R2 is alkyl or substituted alkyl.
32. The compound of any of Claims 30-31, wherein k1 is 2.
33. The compound of any of Claims 30-32, wherein R3 is a chemically-cleavable moiety.
34. The compound of any of Claims 30-32, wherein R3 is an cnzymatically-clcavablc moiety.
35. The compound of Claim 34, wherein R3 is a glycoside or a glycoside derivative.
36. The compound of Claim 35, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O- GalNAc.
37. The compound of any of Claims 30-36, X comprises a conjugation moiety configured to attach to a cysteine residue or a lysine residue of the antibody.
38. The compound of any of Claims 30-36, wherein X comprises a maleimide, and the native amino acid residue of the antibody comprises a cysteine residue.
39. The compound of Claim 37, wherein the compound is of formula (V):
Figure imgf000202_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker; and
W1 is a drug.
40. The compound of Claim 37, wherein the compound is of formula (Va):
Figure imgf000203_0001
wherein: each R1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10;
R3 comprises a cleavable moiety;
LA is a first linker; and
W1 is a drug.
41. The compound of any of Claims 30-40, wherein the first linker LA comprises: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein a, b, c, d, e and f are each independently 0 or 1;
T1, T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-pipcridinc (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
42. The compound of Claim 41, wherein:
T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;
T2, T3, T4, T5 and T6 are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, - (CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and
V1, V2, V3, V4 ,V5 and V6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:
(PEG)n is , where n is an integer from 1 to 30;
Figure imgf000204_0001
EDA is an ethylene diamine moiety having the following structure:
, where y is an integer from 1 to 6 and r is 0 or 1 ;
Figure imgf000204_0002
4-amino-piperidine (4AP) is
Figure imgf000204_0003
; and each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring.
43. The compound of any of Claims 41-42, wherein:
T1 is (C1-C12)alkyl and V1 is -CO-; and b, c, d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is an amino acid analog and V2 is -NH-;
T3 is (PEG)n and V3 is -CO-; and d, e and f are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CONH-;
T2 is substituted (C1-C12)alkyl and V2 is -CO-; and c, d, e and f are each 0.
44. The compound of any of Claims 41-42, wherein one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4 ,V5 or V6 is a branched group.
45. The compound of Claim 44, wherein the branched group is selected from -CONR15- and 4AP.
46. The compound of any one of Claims 44-45, wherein the branched group comprises a second linker, LB.
47. The compound of Claim 46, wherein the second linker LB comprises:
-(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and l are each independently 0 or 1;
T7, T8, T9, T10, T11 and T12 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)W, (PEG)n, (AA)P, -(CR13OH)m-, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;
V7, V8, V9, V10 ,V11 and V12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
48. The compound of any of Claims 46-47, wherein LB is attached to a compound of formula (VI):
Figure imgf000206_0001
wherein: each R4 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R5 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k2 is an integer from 1 to 10;
R6 comprises a cleavable moiety; and
W1a is a drug.
49. The compound of Claim 48, wherein R4 is H and R5 is alkyl or substituted alkyl.
50. The compound of any of Claims 48-49, wherein k2 is 2.
51. The compound of any of Claims 48-50, wherein R6 is a chemically-cleavable moiety.
52. The compound of any of Claims 48-50, wherein R6 is an enzymatically-cleavable moiety.
53. The compound of Claim 52, wherein R6 is a glycoside or a glycoside derivative.
54. The compound of Claim 53, wherein the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O- GalNAc.
55. The compound of any of Claims 41-54, wherein:
T1 is (C1-C12)alkyl and V1 is a branched group (-CONR15)-;
T2 is (C1-C12)alkyl and V2 is -CONH-;
T3 is substituted (C1-C12)alkyl and V3 is -CO-; d, e and f are each 0;
T7 is (C1-C12)alkyl and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein:
T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (PEG)n and V3 is -CONH-;
T4 is substituted (C1-C12)alkyl and V4 is -CO-; e and f are each 0;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CO-;
T2 is a branched group (4AP) and V2 is -CO-;
T3 is (C1-C12)alkyl and V3 is absent;
T4 is heteroaryl (triazole) and V4 is absent;
T5 is (C1-C12)alkyl and V5 is -CONH-;
T6 is substituted (C1-C12)alkyl and V6 is -CO-;
T7 is (PEG)n and V7 is -CONH-;
T8 is substituted (C1-C12)alkyl and V8 is -CO-; and i, j, k and l are each 0.
56. The compound of any of Claims 44-55, wherein W1 and W1a are the same drug.
57. The compound of any of Claims 44-55, wherein W1 and W1a are different drugs.
58. The compound of any of Claims 30-55, wherein the compound is selected from:
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
59. A pharmaceutical composition comprising: a conjugate of any one of Claims 1 to 29; and a pharmaceutically acceptable excipient.
60. A method comprising: administering to a subject a conjugate of any of Claims 1 to 29.
61. A method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of any one of Claims 1 to 29, wherein the administering is effective to treat cancer in the subject.
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