WO2024054821A2 - Conjugués anticorps-médicament de facteur tissulaire et leurs utilisations - Google Patents

Conjugués anticorps-médicament de facteur tissulaire et leurs utilisations Download PDF

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WO2024054821A2
WO2024054821A2 PCT/US2023/073516 US2023073516W WO2024054821A2 WO 2024054821 A2 WO2024054821 A2 WO 2024054821A2 US 2023073516 W US2023073516 W US 2023073516W WO 2024054821 A2 WO2024054821 A2 WO 2024054821A2
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substituted
alkyl
amino acid
aryl
amino
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WO2024054821A3 (fr
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Robyn M. BARFIELD
Maxine Bauzon
Penelope M. DRAKE
Seema Kantak
Brian Alan MENDELSOHN
Tiffany UNSULANGI
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Exelixis, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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
    • A61K47/6801—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
    • A61K47/6889—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present disclosure relates generally to antibody-drug conjugates (ADCs) that bind to tissue factor (TF, e.g., human TF) and methods of their use.
  • ADCs antibody-drug conjugates
  • TF Tissue factor
  • FVIIa serine protease factor Vila
  • the TF/FVIIa complex catalyzes conversion of the inactive protease factor X (FX) into the active protease factor Xa (FXa).
  • FXa and its co-factor FVa form the prothrombinase complex, which generates thrombin from prothrombin.
  • Thrombin converts soluble fibrinogen into insoluble strands of fibrin and catalyzes many other coagulation-related processes.
  • TF is over-expressed on multiple types of solid tumors. In cancer, TF/FVIIa signaling can support angiogenesis, tumor progression, and metastasis.
  • ADCs Antibody-drug conjugates
  • a typical ADC includes an antibody -based targeting element attached to a highly potent pharmaceutical agent (payload) via a chemical linker using an available bioconjugation method.
  • the molar ratio of targeting element (e.g., antibody) to attached payload can vary, and is referred to as the drug-to-antibody ratio (DAR).
  • DAR drug-to-antibody ratio
  • bioconjugation methods either exploit endogenous amino acid residues of a protein (z.e., lysine and cysteine), or rely on selective engagement of a bioorthogonal functional group that has been intentionally introduced into the protein.
  • the Hydrazino-Ao-Pictet-Spengler (HIPS) conjugation method takes an advantage of an aldehyde functional group (an “aldehyde tag”), which can be introduced into a protein, such as an antibody, through various means (e.g., by the action of formyl generating enzyme (FGE)), serving as the conjugation handle.
  • FGE formyl generating enzyme
  • the aldehyde group cleanly reacts with the HIPS indole moiety to form a stable carbon-carbon bond that permanently attaches the payload of choice to the protein in a single chemical step.
  • ADCs that can target TF to treat, prevent, or alleviate TF-mediated diseases, disorders, or conditions, such as those involving tumor cells expressing TF.
  • ADCs comprising an antibody that binds to tissue factor (“TF-ADC”).
  • TF-ADCs bind to the same epitope of human TF as an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) described herein.
  • VH heavy chain variable region
  • VL light chain variable region
  • compositions comprising a TF- ADC that comprises an antibody or fragment thereof that binds to TF (“TF antibody”) and a drug conjugated (directly or indirectly) thereto.
  • TF antibody an antibody or fragment thereof that binds to TF
  • Such pharmaceutical compositions include TF-ADCs comprising an antibody or fragment thereof that binds to essentially the same epitope of human TF as an antibody comprising a VH and a VL described herein.
  • the present disclosure also provides methods of treating, preventing, or alleviating a TF-mediated disease, disorder, or condition, such as alleviating one or more symptoms of the TF-mediated disease, disorder, or condition with a TF-ADC.
  • a TF-ADC comprising (a) a TF antibody and (b) one or more pyridazine-pyrrolo coupling moieties comprising a drug conjugated to the pyridazine-pyrrolo coupling moiety through a linker, for example, using the Hydrazino-Ao-Pictet-Spengler (HIPS) conjugation method.
  • HIPS Hydrazino-Ao-Pictet-Spengler
  • a TF-ADC as disclosed herein comprises branched HIPS linkers that carry two (or more) molecules of the same or different payload per one HIPS moiety and are therefore capable of conjugating two (or more) small molecule payloads per one aldehyde group in a protein in a single conjugation step (FIG. 2).
  • the present disclosure provides TF-ADC structures, which comprises (a) a TF antibody, (b) a branched HIPS linker, and (c) a drug.
  • the disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the conjugates.
  • TF-ADC comprising (a) a TF antibody; and (b) one or more pyridazine-pyrrolo coupling moieties comprising one or more drugs conjugated to the pyridazine-pyrrolo coupling moiety via one or more linkers.
  • a TF-ADC is represented by Formula (I), the TF-ADC comprising: a. an antibody that binds to tissue factor (TF); and b. two or more drugs conjugated to a pyridazine-pyrrolo coupling moiety, each via a linker wherein:
  • Ab represents the antibody that binds to TF
  • Z 1 , Z 2 , and Z 4 are each independently CR 4 ;
  • Z 3 is C-L B -W 2 ;
  • R 1 , R 2 , R 3 , and R 4 are each selected from hydrogen and alkyl
  • L A is a first linker comprising:
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (Ci- Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA) P , -(CR 13 OH) X -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-
  • 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)-, -SO2-, -SO2NR 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, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carb
  • L B is a second linker comprising:
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each independently selected from a covalent bond, (Ci-Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA) P , -(CR 13 OH) X -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para- amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy
  • V 7 , V 8 , V 9 , V 10 ,V n , V 12 and V 13 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2)q-, -NR 15 (CeH4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl,
  • W 1 is a first drug
  • W 2 is a second drug.
  • Z 1 is CR 4 .
  • Z 3 is C-L B -W 2 .
  • W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • 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, provided at least one of a, b, c, d, e, and f is 1;
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (Ci- Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA) P , -(CR 13 OH) X -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-
  • 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)-, -SO2-, -SO2NR 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, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carb
  • T 1 is selected from a (Ci-Ci2)alkyl and a substituted (Ci-Ci2)alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (Ci- Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA)p, -(CR 13 OH)X-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PAB A, PAP, PHP, 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)-, -SO2- , -SO2NR 15 -, -NR 15 SO 2 -, and -P(O)OH-; wherein: integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: 4-amino-piperidine 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;
  • a, b, c, and d are each 1; and e and f are 0.
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L A is a linker wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is substituted (Ci-Ci2)alkyl and V 2 is -CO-;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PABC and V 4 is absent; p is an integer from 1 to 10; a, b, c, and d are each 1; and e and f are each 0.
  • the PABC is substituted with a glycoside, for example, a hydrogen of PABC is replaced with a glycoside, such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • a glycoside such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L B comprises:
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each independently selected from a covalent bond, (Ci-Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA) P , -(CR 13 OH) X -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para- amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy
  • V 7 , V 8 , V 9 , V 10 ,V n , V 12 and V 13 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2)q-, -NR 15 (CeH4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R 13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl,
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 7 is a covalent bond
  • T 8 , T 9 , T 10 , T 11 and T 12 are each independently selected from a covalent bond, (Ci- Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA)p, -(CR 13 OH)X-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and
  • V 7 , V 8 , V 9 , V 10 ,V n and V 12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2)q-, -NR 15 (CeH4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR 15 -, -NR 15 SO2-, and -P(O)OH-; wherein: integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: 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; g, h, i, j, and k are each 1; and
  • T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L B is a linker wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is substituted (Ci-Ci2)alkyl and V 9 is -CO-;
  • T 10 is (AA) P and V 10 is absent;
  • T 11 is PABC and V 11 is absent;
  • p is an integer from 1 to 10;
  • g, h, i, j, and k are each 1;
  • the PABC is substituted with a glycoside, for example, a hydrogen of PABC is replaced with a glycoside, such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • a glycoside such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • a TF-ADC is represented by Formula (I): wherein:
  • Ab represents the antibody that binds to TF
  • Z 1 , Z 2 , and Z 4 are each independently CR 4 ;
  • Z 3 is C-L B -W 2 ;
  • R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (Ci-Ci2)alkyl;
  • L A is a first linker wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -C0NH-;
  • T 2 is substituted (Ci-Ci2)alkyl and V 2 is -CO-;
  • T 3 is (AA) P where p is an integer from 1-20 and V 3 is a covalent bond;
  • T 4 is PABC and V 4 is a covalent bond; a, b, c, and d are each 1; e and f are each 0; and
  • L B is a second linker
  • T 7 is a covalent bond and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is substituted (Ci-Ci2)alkyl and V 9 is -CO-;
  • T 10 is (AA) P where p is an integer from 1-20 and V 10 is a covalent bond;
  • T 11 is PABC and V 11 is a covalent bond; and g, h, i, j, and k are each 1; and
  • 1 and m are each 0; s is an integer from 1 to 10;
  • W 1 is a first drug
  • W 2 is a second drug.
  • W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • a TF-ADC is represented by Formula (I): wherein:
  • Ab represents the antibody that binds to TF
  • Z 1 , Z 2 , and Z 4 are each independently CR 4 ;
  • Z 3 is C-L B -W 2 ;
  • R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (Ci-Ci2)alkyl;
  • L A is a linker wherein:
  • T 1 is (Ci-Ce)alkyl and V 1 is -CONH-;
  • T 2 is (Ci-Ce)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10 and V 2 is -CO-;
  • T 3 is (AA)2 and V 3 is a covalent bond
  • T 4 is PABC substituted with a glycoside and V 4 is a covalent bond; a, b, c, and d are each 1; and e and f are each 0; and
  • L B is a linker
  • T 7 is a covalent bond and V 7 is -NHCO-;
  • T 8 is (Ci-Ce)alkyl and V 8 is -CONH-;
  • T 9 is (Ci-Ce)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10 and V 9 is -CO-;
  • T 10 is (AA)2 and V 10 is a covalent bond
  • T 11 is PABC substituted with a glycoside and V 11 is a covalent bond; g, h, i, j, and k are each 1; and
  • 1 and m are each 0; s is an integer from 1 to 10;
  • W 1 is a first drug
  • W 2 is a second drug.
  • the PABC of one or both of T 4 and T 11 is substituted with a glucuronide.
  • one or both of T 1 and T 8 is ethyl.
  • one or both of T 2 and T 9 is Cs alkylene substituted with -NHCO(PEG)k, where k is an integer from 5-10.
  • one or both of W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • a TF-ADC is represented by Formula (II): wherein:
  • Ab represents the antibody that binds to TF; and s is an integer from 1 to 10.
  • s is an integer from 1 to 4.
  • Formula (II) may be prepared by conjugating one or more linker-payloads of Formula (Ila), shown below, with a TF antibody:
  • a TF-ADC can be represented by Formula (I) or (II), wherein Ab comprises: a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26.
  • a TF-ADC can be represented by Formula (I) or (II), wherein Ab comprises: a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:41 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:42.
  • a TF-ADC can be represented by Formula (I) or (II), wherein Ab comprises: (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NOs: l, 7, 8, 15, 21, 27, 31, 32, 35, or 39, a VH CDR2 comprising the amino acid sequence of SEQ ID NOs:2, 9, 14, 16, or 22, and a VH CDR3 comprising the amino acid sequence of SEQ ID NOs:3, 10, 17, or 23; and (ii) a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NOs:4, 11, 18, 24, 28, 33, 36, or 40, a VL CDR2 comprising the amino acid sequence of SEQ ID NOs:5, 12, 19, 29, or 37, and a VL CDR3 comprising the amino acid sequence of SEQ ID NOs:6, 13, 20, 30, 34, or 38.
  • Ab comprises: (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of
  • a TF-ADC can be represented by Formula (I) or (II), wherein the Ab competes with any one of the TF antibodies as disclosed herein in binding to TF.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and/or a framework 4 (FR4) sequence as set forth in any one of SEQ ID NOs:25, 26, 41, and 42.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises human framework sequences.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL comprising the amino acid sequence of SEQ ID NO:26.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:82.
  • the Ab comprises a VL comprising the amino acid sequence of SEQ ID NO:91.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:82 and a VL comprising the amino acid sequence of SEQ ID NO:91.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:79.
  • the Ab comprises a VL comprising the amino acid sequence of SEQ ID NO:91.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:79 and a VL comprising the amino acid sequence of SEQ ID NO:91.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO:41 and a VL comprising the amino acid sequence of SEQ ID NO:42.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:85.
  • the Ab comprises a VL comprising the amino acid sequence of SEQ ID NO:92.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:85 and a VL comprising the amino acid sequence of SEQ ID NO:92.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88.
  • the Ab comprises a VL comprising the amino acid sequence of SEQ ID NO:92.
  • a TF-ADC can be represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a VL comprising the amino acid sequence of SEQ ID NO:92.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a TF- ADC, wherein the TF-ADC can be represented by Formula (I) or Formula (II) and a pharmaceutically acceptable excipient, wherein the TF antibody (TF Ab or Ab) is as described in any embodiment described herein.
  • such a pharmaceutical composition can have a drug-to-antibody ratio (DAR) of the TF-ADC of about 1 to about 20, for example, a DAR of about 2 to about 8, about 1 to about 4, about 2 to about 4, about 3 to about 4, about 4, about 1 to about 8, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 6.5 to about 8, about 6 to about 7, about 6.5 to about 7.5, about 7 to about 8, about 6.5, about 7, about 7.5, or about 8.
  • DAR drug-to-antibody ratio
  • the present disclosure also provides a method for treating a cancer or a tumor in a subject comprising administering to the subject the TF-ADC, wherein the TF-ADC can be represented by Formula (I) or (II) or the pharmaceutical composition comprising a TF-ADC of Formula (I) or (II) and a pharmaceutically acceptable excipient, wherein the TF antibody is as described in any embodiment herein.
  • the TF-ADC can be represented by Formula (I) or (II) or the pharmaceutical composition comprising a TF-ADC of Formula (I) or (II) and a pharmaceutically acceptable excipient, wherein the TF antibody is as described in any embodiment herein.
  • kits comprising the antibody-drug conjugate as disclosed herein or the pharmaceutical composition as disclosed herein, and instructions for use.
  • FIG. 1 shows a schematic drawing of the HIPS ligation for the synthesis of ADCs.
  • Antibodies carrying aldehyde moieties are reacted with a Hydrazino-Ao-Pictet-Spengler (HIPS) linker and payload to generate a site-specifically conjugated ADC with a stable azacarboline linkage.
  • HIPS Hydrazino-Ao-Pictet-Spengler
  • FIG. 2 shows a schematic representation of branched HIPS ligation for the synthesis of ADCs.
  • Antibodies carrying four aldehyde moieties are reacted with a branched HIPS linker to generate ADCs with drug-to-antibody (DAR) value of up to 8, according to embodiments of the present disclosure.
  • DAR drug-to-antibody
  • FIG. 3A shows a graph of total antibody and ADC concentrations for pharmacokinetic (PK) sample analysis.
  • Square TF-ADC 6-8 - total mAb measurement
  • triangle TF-ADC 6-8 - total ADC measurement.
  • FIG. 3B shows a graph of total antibody and ADC concentrations for pharmacokinetic (PK) sample analysis.
  • Diamond TF-ADC 6-4 - total mAb measurement; star: TF-ADC 6-4 - total ADC measurement.
  • FIG. 3C shows a graph of total antibody and ADC concentrations for pharmacokinetic (PK) sample analysis. Inverted triangle: TF-ADC 7-8 - total mAh measurement; cross: TF-ADC 7-8 - total ADC measurement.
  • FIG. 3D shows a graph of total antibody and ADC concentrations for pharmacokinetic (PK) sample analysis.
  • FIG. 4 shows graphs of in vitro cytotoxicity assays of free drugs, z.e., Monomethyl auristatin E (MMAE), Belotecan or an Exatecan derivative DxD, in various TF positive cancer cell lines, including RF/6A, A431, HCT-116, BxPC3, AU-565, HPAF-II, MDA-MB- 231, AsPC-1, HCC1954, and SKOV-3.
  • MMAE Monomethyl auristatin E
  • Belotecan Belotecan or an Exatecan derivative DxD
  • FIG. 5 shows graphs of in vitro cytotoxicity assays of the TF-ADC 6-8 TF antibody conjugated to belotecan (grey circle), the TF-ADC 7-8 TF antibody conjugated to a belotecan (square), a corresponding isotype antibody conjugated to belotecan (triangle), MMAE (diamond), or the free belotecan (black circle), in various TF positive cancer cell lines, including RF/6A, A431, HCT-116, BxPC3, AU-565, HPAF-II, MDA-MB-231, AsPC-1, HCC1954, and SKOV-3.
  • FIG. 6 shows graphs of in vitro cytotoxicity assays of FITC-ADC 8 (top panel, circle as marked with FITC), TF-ADC 6-8 (top panel, square without any mark), TF-ADC 7-8 (top panel, triangle), Belotecan (top panel, square as marked with B), FITC-ADC 4 (bottom panel, circle as marked with FITC), TF-ADC 6-4 (bottom panel, square), TF-ADC 7-4 (bottom panel, triangle), MMAE (bottom panel, diamond), or Belotecan (bottom panel, circle as marked with B), in three selected TF positive cancer cell lines, including A431, HPAF-II, and BxPC-3.
  • FIG. 7 shows graphs of in vitro cytotoxicity assays of TF-ADC 6-8 having various DAR levels in three selected TF positive cancer cell lines, including A431, HPAF-II, and BxPC-3.
  • FIG. 8 shows graphs of in vitro cytotoxicity assays of TF-ADC 6-4 having different DAR levels in three selected TF positive cancer cell lines, including A431, HPAF-II, and BxPC-3.
  • FIG. 9A shows a graph of mean tumor volume (mm 3 ) vs. days, which indicates in vivo efficacy against a BxPC3 xenograft of TF -targeted ADCs.
  • FIG. 9B shows body weights of the tested mice.
  • FIG. 9C plots the in vivo efficacy data of TF-ADC 6-8 and TF-ADC 7-8.
  • FIG. 9D plots the in vivo efficacy data of TF-ADC 6-4 and TF-ADC 7-4.
  • FIG. 9E plots the in vivo efficacy data of TF-ADC 6-4 and TF-ADC 6-8.
  • FIG. 9F plots the in vivo efficacy data of TF-ADC 7-4 and TF-ADC 7-8.
  • a single i.v. dose was delivered on Day 0.
  • FIG. 10A shows a graph of in vivo efficacy against an HPAF-II xenograft of TF- targeted ADCs.
  • FIG. 10B shows body weights of the tested mice.
  • FIG. 10C plots the in vivo efficacy data of TF-ADC 6-8 and TF-ADC 7-8.
  • FIG. 10D plots the in vivo efficacy data of TF-ADC 6-4 and TF-ADC 7-4.
  • FIG. 10E plots the in vivo efficacy data of TF-ADC 6-4 and TF-ADC 6-8.
  • FIG. 10F plots the in vivo efficacy data of TF-ADC 7-4 and TF-ADC 7-8.
  • a single i.v. dose was delivered on Day 0.
  • FIGs. 11A and 11B show graphs of concentrations of indicated ADC (tADC) and antibody thereof (tAb) in plasma of mice dosed with various ADCs.
  • FIG. 11A plots data from mice dosed with 3 mg/kg or 10 mg/kg of TF-ADC 6-4 or TF-ADC 6-8.
  • FIG. 11B plots data from mice dosed with 1 mg/kg TF-ADC 6-4 or TF-ADC 6-8.
  • EXMA-006 served as a negative control in both graphs, while a benchmark ADC served as a comparator as shown in FIG. 11B
  • FIGs. 12A and 12B show graphs of tumor volumes measured in HPAF-II xenograft mice treated with various dosages of TF-ADC 6-4 (FIG. 12A) or TF-ADC 6-8 (FIG. 12B).
  • FIGs. 13A and 13B provide the concentrations of indicated ADC (ADC) and its antibody (tAb) in plasma of non-human primates (NHP) treated with various dosages of TF- ADC 6-4 (FIG. 13A) or TF-ADC 6-8 (FIG. 13B)
  • FIGs. 14A and 14B provide the concentrations of payload in plasma of NHP treated with various dosages of TF-ADC 6-4 (FIG. 14A) or TF-ADC 6-8 (FIG. 14B).
  • FIGs. 15A-15C provides exemplary ATP release results of treated tumor cells (FIGs. 15A-15B, A431; FIG. 15C, SKOV3).
  • FIGs. 15B-15C provide areas under the curve (AUCs) of tumor cells treated at various concentrations of TF-ADC 6-8, isotype control (an isotype IgGl antibody conjugated to belotecan), or belotecan, while FIG. 15A plots the AUCs in the treatment groups of control (an isotype IgGl antibody conjugated to belotecan), 33 nM TF- ADC 6-8, 33 nM belotecan, 100 nM MMAE, or 100 nM benchmark ADC.
  • FIG. 16 plots HMGB1 released by A431 tumor cells treated with control, 33 nM TF- ADC 6-8, 33 nM belotecan, 100 nM MMAE, or 100 nM benchmark ADC.
  • FIGs. 17A and 17B plot fFNy released by PBMCs co-cultured for 24 hours (FIG. 17A) or 96 hours (FIG. 17B) with tumor cells pre-treated with isotype ADC, TF-ADC 6-8, free belotecan, or free MMAE plus an anti-PD-1 antibody (referred to herein as MMAE + aPDl). Each bar represents data from one donor.
  • FIGs. 18A and 18B plot TNFa released by PBMCs co-cultured for 24 hours (FIG. 18A) or 96 hours (FIG. 18B) with tumor cells pre-treated with isotype ADC, TF-ADC 6-8, belotecan, or MMAE plus an anti-PD-1 antibody (referred to herein as MMAE + aPDl). Each bar represents data from one donor.
  • FIGs. 19A and 19B plot IP- 10 released by PBMCs co-cultured for 24 hours (FIG. 19A) or 96 hours (FIG. 19B) with tumor cells pre-treated with isotype ADC, TF-ADC 6-8, belotecan, or MMAE plus an anti-PD-1 antibody (referred to herein as MMAE + aPDl). Each bar represents data from one donor.
  • FIGs. 20A and 20B plot MIP-1 a released by PBMCs co-cultured for 24 hours (FIG. 20A) or 96 hours (FIG. 20B) with tumor cells pre-treated with isotype ADC, TF-ADC 6-8, belotecan, or MMAE plus an anti-PD-1 antibody (referred to herein as MMAE + aPDl). Each bar represents data from one donor.
  • the present disclosure provides antibody-drug conjugates (ADCs) that bind to TF and a drug conjugated (directly or indirectly) thereto.
  • ADCs antibody-drug conjugates
  • Such TF-ADCs are useful in compositions and in methods of treating, preventing, or alleviating a TF-mediated disease, disorder, or condition, including one or more symptoms of the disease, disorder, or condition.
  • TF- mediated diseases, disorders, and conditions include a variety of cancers, including, but not limited to, any cancer wherein the tumor cells express or overexpress TF.
  • TF- ADCs are useful for the killing and/or removal of tumor cells.
  • TF-ADCs described herein are useful in compositions and in methods for treating cancer.
  • 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 (CEE-), ethyl (CEECEE-), n-propyl (CEECEECEE-), isopropyl ((CEE)2CH-), n-butyl (CEECH2CH2CH2-), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH2CH 2 CH 2 CH2-), and neopentyl ((CH 3 ) 3 CCH 2 -).
  • substituted alkyl refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the Ci 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, thiohetero
  • 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-,
  • This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH 2 CH(CH 3 )-), (-C(CH 3 )2CH 2 CH2-), (-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-, cycloalkyl-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.
  • 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- substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted 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-l-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.
  • 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, alkoxy
  • 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, ethynyloxy, 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, -
  • 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 are 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 -SChNR 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, substituted
  • “Sulfonylamino” refers to the group -NR 21 SO2R 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 -NH2.
  • 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.
  • Carboxyl refers to -CO2H 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 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, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamin
  • 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.
  • Cycloalkoxy refers to -O-cycloalkyl.
  • Cycloalkenyloxy refers to -O-cycloalkenyl.
  • Halo or “halogen” refers to fluoro, chloro, bromo, and iodo.
  • Heteroaryl refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 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 benzothienyl), wherein at least one ring within the ring system is aromatic.
  • 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— >0), sulfinyl, or sulfonyl moieties.
  • This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • 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, thio
  • heteroarylkyl 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.
  • Heteroaryloxy refers to -O-heteroaryl.
  • Heterocycle refers 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.
  • 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.
  • 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.
  • 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,
  • 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, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,
  • 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 -NO2.
  • “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, wherein al
  • “Sulfonyloxy” refers to the group -OSCh-alkyl, -OSCh-substituted alkyl, -OSO2- alkenyl, -OSCh-substituted alkenyl, -OSCh-cycloalkyl, -OSCh-substituted cylcoalkyl, -OSO2- cycloalkenyl, -OSCh-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, wherein alky
  • “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
  • 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.
  • heteroaryl oxy 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.
  • substituent groups for substituting for one or more hydrogens are, unless otherwise specified, -R 60 , halo,
  • 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.5, [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 -NH2, -NH-alkyl, 7V-pyrrolidinyl, 7V-piperazinyl, 47V-methyl-piperazin-l-yl and A-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, -NO2, -N3, -SO2R 70 , -SO 3 M + , -SO3R 70 , -OSO2R 70 , -OSO3-M+ -OSO3R 70 , -PO3' 2 (M + )2, -P(O)(OR 70 )O-M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70
  • substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O M + , -OR 70 , -SR 70 , -S'M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -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 + , -OS(O) 2 OR 70 , -P(O)(O-) 2 (M + )2, -P(O)(OR 70 )O'M + , -P(O)(OR 70 )(OR 70 ), -C(O) R 70 , -C
  • 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.
  • 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.
  • 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, -di methyl form am ide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
  • 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.
  • pyrazoles imidazoles, benzimidazoles, triazoles, and tetrazoles.
  • “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.
  • 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 noncoded 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.
  • 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, He 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 a-hydroxy acids, and a-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 a-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, di saccharides.
  • 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 nonhuman (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 nonhumanized 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.
  • 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.
  • 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 which 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.
  • 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 preexisting amino acid sequence modifications (such as additions, deletions and/or substitutions).
  • 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.
  • 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.
  • 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 or serine of a sulfatase motif and Formylglycine Generating Enzyme (FGE), which react to form a reaction product of a converted aldehyde tag containing a formylglycine (fGly) in lieu of cysteine or serine in the motif.
  • FGE Formylglycine Generating Enzyme
  • exemplary reactive partners include an aldehyde of an fGly residue of a converted aldehyde tag (e.g., a reactive aldehyde group) and an “aldehyde-reactive reactive partner”, which comprises an aldehyde-reactive group and a moiety of interest, and which reacts to form a reaction product of a polypeptide having the moiety of interest conjugated to the polypeptide through the fGly residue.
  • a converted aldehyde tag e.g., a reactive aldehyde group
  • aldehyde-reactive reactive partner which comprises an aldehyde-reactive group and a moiety of interest
  • 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.
  • 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.
  • 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.
  • subject refers to human and non-human subjects, especially mammalian subjects.
  • treating means the treating or treatment of a disease or medical condition in a subject, 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 subject; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a subject; or (d) alleviating a symptom of the disease or medical condition in a subject.
  • the term “treating,” or “treatment” excludes a prophylactic treatment.
  • amino acid sequence refers to the amino acid sequence of a polypeptide prior to modification to include a modified amino acid residue.
  • amino acid analog 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, He 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, He 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
  • 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 embodiments, 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 can 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, and the like) or an atom (such as Cl or Br, and the like), deletion of a group, substitution of a covalent bond (single bond for double bond, and the like), or combinations thereof.
  • amino acid analogs can include a-hydroxy acids, and a- amino acids, and the like.
  • amino acid side chain is used to refer to the substituent attached to the a-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 is used to refer to monomer units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
  • sugar is 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 can 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.
  • tissue Factor tissue Factor
  • platelet tissue factor factor III
  • thromboplastin thromboplastin
  • CD 142 tissue Factor
  • the TF protein is a TF protein naturally expressed by a primate (e.g., a monkey or a human), a rodent (e.g, a mouse or a rat), a dog, a camel, a cat, a cow, a goat, a horse, a pig or a sheep.
  • a primate e.g., a monkey or a human
  • rodent e.g., a mouse or a rat
  • TF encompasses “full-length” TF, as well as any form of TF or any fragment thereof, for example those resulted from processing in a cell.
  • the TF comprises a signal sequence. In some embodiments, the TF does not include a signal sequence.
  • the term TF refers to a fragment of the full-length TF, which comprises an TF extracellular domain (ECD).
  • ECD TF extracellular domain
  • the term TF also encompasses naturally occurring variants of TF, such as SNP variants, splice variants and allelic variants.
  • the TF protein is human TF (hTF;
  • the human TF does not comprise a signal peptide, for example amino acid (aa) 33 to aa 295 of SEQ ID NO: 175.
  • the human TF as used herein refers to the extracellular domain (ECD) of the human TF, for example, aa 33 to aa 251 of SEQ ID NO: 175.
  • the TF protein is cynomolgus TF (cTF;
  • the cynomolgus TF as used herein refers to its ECD, for example aa 1 to aa 220 of SEQ ID NO: 176.
  • the TF protein is mouse TF (mTF;
  • the mouse TF as used herein refers to its ECD, for example aa 1 to aa 223 of SEQ ID NO: 177.
  • the TF protein is pig TF (pTF;
  • the pig TF as used herein refers to its ECD, for example aa to aa 216 of SEQ ID NO: 178.
  • TF is a cell surface receptor for the serine protease factor Vila. It is often times constitutively expressed by certain cells surrounding blood vessels and in some disease settings.
  • the term TF as used herein refers to a TF epitope.
  • antibody immunoglobulin
  • immunoglobulin or “Ig” are used interchangeably herein and are used in the broadest sense and specifically covers, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full length heavy and/or light chains.
  • Antibodies also include single antibody domains as well as antibody fragments (and/or polypeptides that comprise antibody fragments) that retain TF binding characteristics.
  • Non-limiting examples of antibody fragments include antigenbinding regions and/or effector regions of the antibody, e.g., Fab, Fab’, F(ab’)2, Fv, scFv, (SCFV)2, single-chain antibody molecule, dual variable domain antibody, single variable domain antibody, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fvs (dsFv), single-domain antibody (e.g., nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled).
  • variable (V) region can be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) variable regions.
  • VH immunoglobulin heavy
  • VL light
  • antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, and an antibody heavy chain monomer.
  • the V region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers that bind TF.
  • a VH region and a VL region can be covalently coupled either directly or through a linker to form a single-chain Fv (scFv).
  • scFv proteins are referred to herein as included in the category “antibody fragments.”
  • Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody.
  • CDRs also termed “minimal recognition units” or “hypervariable regions” can be obtained by constructing polynucleotides that encode one or more CDRs of interest.
  • Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al.
  • Antibody fragments can be incorporated, for example, into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable regions of new antigen receptors (v-NAR), and bis-single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9): 1126-1136, 2005).
  • antibodies comprising a VH and/or VL contain a light chain and/or a heavy chain constant region, such as one or more constant regions, including one or more IgGl, IgG2, IgG3 and/or IgG4 constant regions.
  • antibodies can include epitope-binding fragments of any of the above.
  • the antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.
  • the term “monospecific,” as used herein denotes an antibody that has one or more binding sites each of which binds to the same epitope of the same antigen.
  • bispecific means that the antibody can specifically bind to at least two distinct antigenic determinants, for example two binding sites each formed by a pair of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) binding to different antigens or to different epitopes on the same antigen.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • Such a bispecific antibody can have a 1+1 format (comprising one binding site for a first antigen or epitope and one binding site for a second antigen or epitope).
  • bispecific antibody formats can be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope).
  • a bispecific antibody comprises two antigen binding sites, each can bind to a different antigenic determinant.
  • Such a bispecific antibody can bind to two different epitopes on the same antigen (e.g., epitopes on TF).
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well known in the art.
  • nucleic acids or polypeptides are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the amino acid sequences that is at least about ten residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the full-length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about ten bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a side chain with similar chemical characteristics.
  • Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site.
  • Methods of identifying amino acid conservative substitutions that do not eliminate binding are well known in the art.
  • polypeptide refers to a polymer of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can include (e.g., be substituted with) non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage to or conjugation with (directly or indirectly) a moiety such as a labeling component or a drug (e.g., toxin).
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • polypeptides of this disclosure can be based upon antibodies or other members of the immunoglobulin superfamily, in some embodiments, the polypeptides can occur as single chains or dimers of single chains.
  • an “antigen” is a moiety or molecule that contains an epitope to which an antibody can bind.
  • an antigen can be bound by an antibody.
  • the antigen to which an antibody described herein binds is TF (e.g., human TF), or a fragment thereof, such as a fragment that comprises one or more regions of TF.
  • an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can bind.
  • An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope.
  • an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope), e.g., human TF.
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • an antibody binds to a group of amino acids regardless of whether they are folded in a natural three-dimensional protein structure.
  • an antibody requires amino acid residues making up the epitope to exhibit a particular conformation (c.g, bend, twist, turn or fold) in order to recognize and bind the epitope.
  • An antibody binds “an epitope,” “essentially the same epitope,” or “the same epitope” as a reference antibody.
  • the most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping, or adjacent epitopes in a three- dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody.
  • the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent or enzyme labels.
  • Epitope binning is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, using competition assays combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct binding specificities. Additional details regarding methods for epitope binning and determining epitope binding of antibodies are described herein, as shown in Example 5.
  • the terms “specifically binds,” “specifically recognizes,” “immunospecifically binds,” “selectively binds,” “immunospecifically recognizes” and “immunospecific” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope) as understood by one skilled in the art.
  • “specifically binds” means, for instance, that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins.
  • a molecule that specifically binds to an antigen can bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIACORETM, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), the OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art.
  • an antibody or antigen binding region binds to or specifically binds to an antigen when it binds to the antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIAs) and enzyme linked immunosorbent assays (ELISAs).
  • RIAs radioimmunoassays
  • ELISAs enzyme linked immunosorbent assays
  • a specific or selective reaction will be at least twice background signal or noise and can be more than ten times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity.
  • the extent of binding of an antibody or antigen-binding region to a “nontarget” protein is less than about 10% of the binding of the antibody or antigen-binding region to its target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIAs.
  • FACS fluorescence activated cell sorting
  • molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the KA when the molecules bind to another antigen.
  • molecules that specifically bind to an antigen do not cross react with other proteins.
  • molecules that specifically bind to an antigen do not cross react with other non- TF proteins.
  • “specifically binds” means, for instance, that a polypeptide or molecule binds a protein or target with a KD of about 0.1 mM or less, but more usually less than about 1 pM. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KD of at least about 0.1 pM or less, at least about 0.01 pM or less, or at least about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species.
  • specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a polypeptide or molecule that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, e.g. , binding to a single target. Thus, in some embodiments, a polypeptide or molecule can specifically bind more than one target. In some embodiments, multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule.
  • an antibody can comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins.
  • an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities.
  • binding affinity generally refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., an antigen such as TF).
  • binding affinity refers to intrinsic binding affinity that reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD).
  • KD dissociation constant
  • Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity is known in the art, any of which can be used for purposes of the present disclosure.
  • the “KD” or “KD value” can be measured by biolayer interferometry (BLI) using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA).
  • the KD may also be measured in a radiolabeled antigen binding assay (RIA), for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865- 881) or using surface plasmon resonance (SPR) assays by BIACORETM, using, for example, a BIACORETM-2000 or a BIACORETM-3000 (BIAcore, Inc., Piscataway, NJ).
  • RIA radiolabeled antigen binding assay
  • SPR surface plasmon resonance
  • an “on-rate” or “rate of association” or “association rate” or “k O n,” as well as an “off-rate” or “rate of dissociation” or “dissociation rate” or “koff,” can also be determined with the same SPR or BLI techniques described herein using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) or a BIACORETM-2000 or a BIACORETM-3000 (BIACORETM, Inc., Piscataway, NJ), respectively.
  • Compet when used in the context of a TF antibody, describes a binding agent that, in the presence of another binding agent, is at least partially inhibited from binding to an epitope or binding site due to binding of the other binding agent.
  • Competition can be determined by an assay in which the binding agent under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., TF).
  • a reference molecule e.g., a reference ligand, or reference antigen binding protein, such as a reference antibody
  • TF common antigen
  • Numerous types of competitive binding assays can be used to determine if a test binding agent competes with a reference molecule for binding to TF (e.g., human TF).
  • assays examples include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competition assay see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253
  • solid phase direct biotin-avidin EIA see, e.g., Kirkland et al., (1986) J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); and direct labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82).
  • such an assay involves the use of a purified antigen (e.g., TF, such as human TF) bound to a solid surface or cells bearing either of an unlabeled test antigen binding protein (e.g., test TF antibody or ADC) or a labeled reference antigen binding protein (e.g., reference TF antibody or ADC).
  • a purified antigen e.g., TF, such as human TF
  • test antigen binding protein e.g., test TF antibody or ADC
  • a labeled reference antigen binding protein e.g., reference TF antibody or ADC
  • Antibodies identified by competition assay include antibodies binding to the same epitope as the reference antibody and/or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur (e.g., similar epitope or overlapping epitope).
  • a competing antibody when it is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 20%, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.
  • binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.
  • constant region and “constant domain” are used interchangeably herein, are well-known antibody terms of art, and refer to an antibody portion, for example, a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to an antigen, but which can exhibit various effector functions, such as interaction with an Fc receptor.
  • the term includes the portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable region.
  • Antibody effector functions refer to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody and which vary with the antibody isotype.
  • Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226 (of the EU numbering system) or from Pro230 (of the EU numbering system) to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 of the EU numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like.
  • Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.
  • a “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and/or changed (e.g., isolated, purified, selected, including, or combining with other sequences such as variable region sequences) by a human.
  • Native sequence human Fc regions include a native sequence human IgGl Fc region (non- A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region; as well as naturally occurring variants thereof.
  • a “variant Fc region” comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, (e.g., substituting, addition, or deletion) preferably one or more amino acid substitution(s).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide.
  • the variant Fc region described herein can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • the variant Fc region herein described herein can have a loss of effector function (e.g., silent Fc).
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes one or more constant regions.
  • the “heavy chain” can refer to any distinct types, e.g., for example, alpha (a), delta (5), epsilon (a), gamma (y) and mu (p), based on the amino acid sequence of the constant region, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGl, IgG2, IgG3 and IgG4.
  • the term “light chain” when used in reference to an antibody can refer to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • Light chain amino acid sequences are well known in the art.
  • antigen binding fragment refers to a portion of an antibody that comprises amino acid residues that interact with an antigen and confer on the binding fragment or region its specificity and affinity for the antigen (e.g., the CDRs).
  • Antigen binding fragment as used herein includes “antibody fragment,” which comprises a portion of an antibody including one or more CDRs, such as the antigen binding or variable region of the antibody.
  • Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, and the like), camelized antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fvs (sdFv), anti -idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • synthetic antibodies e.g., monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, and the like), camelized antibodies, Fab fragments, F(ab’)
  • antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain one or more antigen binding sites that bind to TF.
  • a TF antibody as described herein, can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule.
  • a TF antibody, as described herein is an IgG antibody (e.g., human IgG), or a class (e.g., human IgGl, IgG2, IgG3, or IgG4) or a subclass thereof.
  • a TF antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs, wherein the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical.
  • the H and/or L chains comprise constant regions, for example, human constant regions.
  • the L chain constant region of a TF antibody is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region.
  • the H chain constant region of a TF antibody comprises a gamma heavy chain constant region, for example, a human gamma heavy chain constant region.
  • a TF antibody comprise an IgG constant region, for example, human IgG constant regions (e.g., IgGl, IgG2, IgG3, and/or IgG4 constant regions).
  • TF antibody and “antibody that binds to TF” are used interchangeably and refer to an antibody that preferentially binds to TF.
  • An antibody or fragment thereof can preferentially bind to TF, such as human TF, which means that the antibody or fragment thereof binds to TF, such as human TF, with greater affinity than it binds to an unrelated control protein.
  • the antibody or fragment thereof can specifically recognize and bind to TF or a portion thereof.
  • Specific binding means that the TF antibody or fragment thereof binds to TF with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme).
  • the TF antibody or fragment thereof can bind TF substantially exclusively (e.g., is able to distinguish TF from other known polypeptides, for example, by virtue of measurable differences in binding affinity).
  • a TF antibody can react with TF sequences other than human TF sequences (e.g., cynomolgus TF sequences).
  • variable region and “variable domain” are used interchangeably to refer to a portion of the light and heavy chains of an antibody that are generally located at the amino-terminal of the light and heavy chain, has a length of about 120 to 130 amino acids in the heavy chain, about 100 to 110 amino acids in the light chain, and is used in the binding and specificity of each antibody for its antigen.
  • the variable region of the heavy chain is referred to herein as “VH.”
  • the variable region of the light chain is referred to herein as “VL.”
  • variable refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its antigen.
  • variable regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” or alternatively called “complementarity determining regions.”
  • the variable regions of heavy and light chains each comprise four frameworks (FR1, FR2, FR3 and FR4), largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P sheet structure.
  • the hypervariable regions in each chain are held together in proximity by the frameworks and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)).
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • the variable regions differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable region are referred to as framework regions (FR).
  • the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen.
  • the variable region is a human variable region.
  • hypervariable region refers to the regions of an antibody variable region that are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six hypervariable regions: three in the VH (Hl or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3), and three in the VL (LI or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3).
  • the Kabat CDRs are based on sequence variability and are the most used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag).
  • the “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.
  • IMGT® ImMunoGeneTics
  • IG immunoglobulins
  • TR T cell receptors
  • MHC major histocompatibility complex
  • Hypervariable regions can comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26- 35A (Hl), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH.
  • hypervariable region As used herein, the terms “hypervariable region,” “HVR,” “HV,” “complementarity determining region,” or “CDR” are used interchangeably.
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, for example, to introduce a nucleic acid sequence into a host cell.
  • vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome.
  • a vector can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
  • Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL region) both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • PCR polymerase chain reaction
  • suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., a TF antibody), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • TF-mediated disease includes a cancer including, but not limited to, cancers that express or overexpress TF.
  • tumor in any embodiment herein, refers to any neoplastic cell growth or proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • ADC refers to an antibody-drug conjugate, which in the context of the present invention refers to a TF antibody, which is coupled to another moiety which includes a drug, as described herein.
  • drug refers to a compound that has biological activity, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, and the like).
  • a cytotoxic compound e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, and the like.
  • drugs include small molecule drugs, such as a cancer chemotherapeutic agent.
  • a cancer chemotherapeutic agent such as an antibody (or fragment thereof) that has specificity for a tumor cell
  • the antibody can be modified 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 (z.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 a TF-ADC 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. Set. 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., pyrrol Whyzodiazepine (PBD)).
  • PBD pyrroleauzodiazepine
  • 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 (CYNOTANTM), 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 triazenes,
  • 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, fludarabine phosphate, pentostatin, and gemcitabine.
  • CYTOSAR-U® cytosine arabinoside
  • fluorouracil (5-FU) floxuridine
  • 6-MP 6-mercaptopurine
  • pentostatin 5 -fluorouraci
  • Suitable natural products and their derivatives include, but are not limited to, Ara-C, paclitaxel (TAXOL®), docetaxel (TAXOTERE®), deoxycoformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, e.g. etoposide, teniposide, and the like; antibiotics, e.g.
  • anthracycline daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, and the like; 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.
  • 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, and the like; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; and the like; adrenocortical suppressants, e.g.
  • estradiosteroids aminoglutethimide; 17a-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (DROGENIL®), toremifene (FARESTON®), and goserelin (ZOLADEX®), and the like.
  • Estrogens stimulate proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.
  • chemotherapeutic agents include metal complexes, e.g. cisplatin (cis- DDP), carboplatin, and the like; ureas, e.g. hydroxyurea; hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; and the like
  • metal complexes e.g. cisplatin (cis- DDP), carboplatin, and the like
  • ureas e.g. hydroxyurea
  • hydrazines e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • a topoisomerase inhibitor e.g. N-methylhydrazine
  • procarbazine mitoxantrone
  • leucovorin tegafur
  • mycophenolic acid mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); gefitinib (IRESSA®, ZD 1839, 4-(3-chloro-4-fluorophenylamino)- 7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); and the like.
  • 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) can 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.
  • 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 herein) and paclitaxel conjugates (e.g., paclitaxel -PEG, paclitaxel-dextran, or paclitaxel -xylose).
  • analogs and derivatives e.g., TAXOTERE® docetaxel, as noted herein
  • paclitaxel conjugates e.g., paclitaxel -PEG, paclitaxel-dextran, or paclitaxel -xylose.
  • Taxane also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but are 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; sulfenamide 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.
  • Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine 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) colonystimulating factors; and (9) inhibitors of angiogenesis.
  • RTK tyrosine kinase
  • an “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with a disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount.
  • therapeutically effective amount refers to the amount of an antibody or ADC described herein that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder, or condition, and/or a symptom related thereto.
  • a therapeutically effective amount of an agent, including a therapeutic agent can be an amount necessary for (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and/or (iii) to improve or enhance the therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or ADC described herein).
  • another therapy e.g., a therapy other than the administration of an antibody or ADC described herein.
  • a “therapeutically effective amount” of a substance/molecule/agent of the present disclosure can vary based on a number of factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent, to elicit a desired response in the individual.
  • a therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule/agent are outweighed by the therapeutically beneficial effects.
  • the term “therapeutically effective amount” refers to an amount of an antibody or other agent (e.g., or drug) effective to “treat” a disease, disorder, or condition, in a subject or mammal.
  • the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid.
  • the drug is an antimitotic agent, such as an auristatin or an active auristatin analog or derivative thereof.
  • the drug is a DNA alkylating agent.
  • pharmaceutically acceptable means being approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • Excipients include carriers, excipients, preservatives, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed and can be included, for example, to affect stability, bulk up formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form (e.g., facilitating absorption, reducing viscosity, enhancing solubility).
  • An “excipient” can be an organic or inorganic ingredient, natural or synthetic with which the active ingredient is combined to facilitate the use of the active ingredient, e.g., the administration of the active ingredient to a subject.
  • excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (e.g., less than about ten amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants such as ascorbic acid
  • excipient can also refer to a diluent, adjuvant (e.g., Freund’s adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered.
  • excipients can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition in any embodiment, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • Oral compositions can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • suitable excipients are described in Remington: The Science and Practice of Pharmacy (2020) (Elsevier Science, Amsterdam, Netherlands), including pharmaceutical compounds, can contain an effective amount or therapeutically effective amount of a TF-ADC, for example, in isolated or purified form, together with a suitable amount of excipient to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • TF antibody also referred to herein as “TF antibody,” “anti-TF antibody,” “TF Ab,” “Ab” or “antibody”
  • a drug can be linked directly or indirectly to each other via a pyridazine-pyrrolo coupling moiety to form a TF-ADC as described herein.
  • the TF antibody and the two or more drugs or active agents are bound to each other through one or more functional groups and covalent bonds.
  • the one or more functional groups and covalent bonds can include a branched linker as described herein.
  • Moi eties of interest can be conjugated to the TF antibody at any desired site of the antibody.
  • the present disclosure provides, for example, a TF antibody having moieties conjugated at two or more sites on the antibody, such as a site at or near the C-terminus of the antibody, a position at or near the N-terminus of the antibody, and a position between the C-terminus and the N-terminus of the antibody (e.g., at an internal site of the antibody). Combinations of the above conjugation sites are also possible.
  • a conjugate of the present disclosure includes two (or more) drugs or active agents conjugated to an amino acid residue of a TF antibody at the a- carbon of an amino acid residue.
  • a conjugate includes a TF antibody where the side chain of an amino acid residue in the antibody has been modified and attached to two (or more) drugs or active agents (e.g., attached to two drugs or active agents through a branched linker as described herein).
  • a conjugate includes a TF antibody where the a-carbon of an amino acid residue in the antibody has been modified and attached to two drugs or active agents (e.g. , attached to two drugs or active agents through a branched linker as described herein).
  • Embodiments of the present disclosure include conjugates where a TF antibody is conjugated to two or more moieties, such as 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 TF antibody at multiple sites in the antibody. In some embodiments, two moieties may be conjugated to a single amino acid residue of the TF antibody.
  • two moieties may be conjugated to the same amino acid residue of the TF antibody.
  • two moieties are conjugated to a first amino acid residue of the TF antibody and two other moieties are conjugated to a second amino acid residue of the TF antibody.
  • a TF antibody can be 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.
  • two or more amino acid residues in the TF antibody are each conjugated to a pair of moieties (z.e., two moieties), where each pair of moieties is conjugated to the TF antibody through a branched linker as described herein.
  • 1 amino acid residue in the TF antibody is conjugated to a pair of moieties through a branched linker as described herein.
  • 2 or more amino acid residues, such as 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid residues in the TF antibody are each conjugated to a pair of moieties through a branched linker as described herein.
  • the one or more amino acid residues of the TF antibody that are conjugated to the moieties of interest may be naturally occurring amino acids, unnatural amino acids, or combinations thereof.
  • the conjugate may include moieties of interest (e.g., drugs or active agents) conjugated to a naturally occurring amino acid residue of the TF antibody.
  • the conjugate may include moieties of interest conjugated to an unnatural amino acid residue of the TF antibody.
  • the moieties of interest may be conjugated to the TF antibody at a single natural or unnatural amino acid residue as described above.
  • One or more natural or unnatural amino acid residues in the TF antibody may be conjugated to the moieties of interest as described herein.
  • two (or more) amino acid residues e.g., natural or unnatural amino acid residues) in the TF antibody may each be conjugated to two moieties through a branched linker, such that multiple sites in the TF antibody are conjugated to the moieties of interest.
  • a TF antibody may be conjugated to two or more moieties of interest.
  • the moiety of interest is a payload, for instance, a chemical entity, such as a drug, an active agent, or a detectable label.
  • drugs or active agents, such as cytokines
  • detectable labels may be conjugated to the TF antibody.
  • combinations of different payloads may be conjugated to the TF antibody.
  • embodiments of the present disclosure include, but are not limited to, the following: a conjugate of a TF antibody and two or more drugs; a conjugate of a TF antibody and two or more active agents, such as cytokines; a conjugate of a TF antibody and two or more detectable labels; and combinations thereof.
  • the TF antibody and the moi eties of interest are conjugated through a conjugation moiety.
  • the TF antibody and the moi eties of interest may each be bound (e.g., covalently bonded) to the conjugation moiety, thus indirectly binding the TF antibody and the moieties of interest together through the conjugation moiety.
  • the conjugation moiety includes a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound.
  • a general scheme for coupling moieties of interest to a TF antibody through a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety is shown in the general reaction scheme below.
  • Hydrazinyl- indolyl and hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also referred to herein as a hydrazino-/.w-Pictet-Spengler (HIPS) conjugation moiety and an aza-hydrazino-/.w-Pictet- Spengler (azaHIPS) conjugation moiety, respectively.
  • HIPS hydrazino-/.w-Pictet-Spengler
  • azaHIPS aza-hydrazino-/.w-Pictet- Spengler
  • the carbon which is denoted in a formula as disclosed herein, such as Formula (I) or (II), as being immediately adjacent to Ab was originally a part of the antibody prior to the conjugation.
  • this carbon is conjugated to a (fGly’) residue, thus conjugating the antibody and the linker-payload.
  • this carbon is interpreted as a part of a (fGly’) residue conjugating the antibody and the linker-payload.
  • each R independently includes a moiety of interest (e.g., drug or active agent) that is conjugated to the TF antibody (e.g., conjugated to the TF antibody through a linker as described herein), where n is an integer from 1 to 4.
  • a conjugation moiety e.g., a hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety
  • R is attached to two or more drugs or active agents, R.
  • a TF antibody that includes a 2-formylglycine residue (fGly) is reacted with the conjugation moiety to produce a TF antibody conjugate, thus attaching the two or more drugs or active agents to the TF antibody through the conjugation moiety.
  • the moieties can be any of a variety of moieties such as, but not limited to, chemical entities, such as detectable labels, or a drugs or active agents.
  • R’ and R may each independently be any desired substituent, such as, but not limited to, 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.
  • Z may be CR 21 , NR 22 , N, O or S, where R 21 and R 22 are each independently selected from any of the substituents described for R’ and R” above.
  • hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also possible, as shown in the conjugates and compounds described herein.
  • the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties may be attached (e.g., covalently attached) to two or more linkers.
  • embodiments of the present disclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl 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 hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety.
  • the hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety and two or more linkers may be viewed overall as a “branched linker”, where the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is attached to two of more “branches”, where each branch includes a linker attached to a drug or active agent.
  • Combinations of the same or different payloads may be conjugated to the TF antibody through the branched linker.
  • the two payloads (e.g., drugs, active agents or detectable labels) attached to the branched linker are the same payload (e.g., drug, active agent or detectable label).
  • a first branch of a branched linker may be attached to a payload (e.g., drug, active agent or detectable label) and a second branch of the branched linker may be attached to the same payload (e.g., drug, active agent or detectable label) as the first branch.
  • the two payloads (e.g., drugs, active agents or detectable labels) attached to the branched linker are different payloads (e.g., drugs, active agents or detectable labels).
  • a first branch of a branched linker may be attached to a first payload (e.g., a first drug, active agent or detectable label) and a second branch of the branched linker may be attached to a second payload (e.g., a second drug, active agent or detectable label) different from the first payload (e.g., the first drug, active agent or detectable label) attached to the first branch.
  • 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 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.
  • 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 TF antibody may be conjugated to two or more moieties of interest, where one or more amino acids of the TF antibody are modified before conjugation to the moieties of interest. Modification of one or more amino acids of the TF antibody may produce a TF antibody that contains one or more reactive groups suitable for conjugation to the moieties of interest.
  • the TF antibody may include one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to the moieties of interest (e.g., where two or more moieties are attached to a conjugation moiety, such as a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above).
  • an amino acid of the TF antibody may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde).
  • a reactive aldehyde may be included in an “aldehyde tag” or “aid-tag”, which, as used herein, refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C/S)TPSR, SEQ ID NO:99) that has been converted by action of a formylglycine generating enzyme (FGE) to contain a 2-formylglycine residue (referred to herein as “fGly”).
  • FGE formylglycine generating enzyme
  • the fGly residue generated by an FGE may also be referred to as a “formylglycine”.
  • aldehyde tag is used herein to refer to an amino acid sequence that includes a “converted” sulfatase motif (z.e., a sulfatase motif in which a cysteine or serine residue has been converted to fGly by action of an FGE, e.g., L(fGly)TPSR, SEQ ID NO: 123).
  • FGE e.g., L(fGly)TPSR, SEQ ID NO: 123
  • a converted sulfatase motif may be produced from an amino acid sequence that includes an “unconverted” sulfatase motif (i.e., a sulfatase motif in which the cysteine or serine residue has not been converted to fGly by an FGE, but is capable of being converted, e.g, an unconverted sulfatase motif with the sequence: LCTPSR, SEQ ID NO: 100).
  • an “unconverted” sulfatase motif i.e., a sulfatase motif in which the cysteine or serine residue has not been converted to fGly by an FGE, but is capable of being converted, e.g, an unconverted sulfatase motif with the sequence: LCTPSR, SEQ ID NO: 100).
  • conversion as used in the context of action of a formylglycine generating enzyme (FGE) on a sulfatase motif refers to biochemical modification of a cysteine or serine residue in a sulfatase motif to a formylglycine (fGly) residue (e.g, Cys to fGly, or Ser to fGly). Additional aspects of aldehyde tags and uses thereof in site-specific protein modification are described in U.S. Patent No. 7,985,783 and U.S. Patent No. 8,729,232, the disclosures of each of which are incorporated herein by reference.
  • the TF antibody containing the fGly residue may be conjugated to the moieties of interest by reaction of the fGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety, as described above).
  • a compound e.g., a compound containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety, as described above.
  • an fGly-containing TF antibody may be contacted with a reactive partner under conditions suitable to provide for conjugation of two or more drugs to the TF antibody.
  • the reactive partner may include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above.
  • two or more drugs or active agents may be attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • the drugs or active agents are attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, such as covalently attached to a hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl, where each drug or active agent is attached through a corresponding linker to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • a conjugate of the present disclosure includes a TF antibody having at least one amino acid residue that has been attached to two or more moi eties of interest (e.g., drugs or active agents).
  • an amino acid residue of the TF antibody may be modified and then coupled to two or more drugs or active agents attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above.
  • an amino acid residue of the TF antibody is a cysteine or serine residue that is modified to an fGly residue, as described above.
  • the modified amino acid residue (e.g., fGly residue) is conjugated to two or more drugs or active agents containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety as described above to provide a conjugate of the present disclosure where the two or more drugs or active agents are conjugated to the TF antibody through the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • the term fGly refers to the amino acid residue of the TF antibody that is coupled to the moi eties of interest (e.g., drugs or active agents).
  • the conjugate includes a TF antibody having at least one amino acid residue attached to a branched linker as described herein, which in turn is attached to two or more drugs or active agents.
  • the conjugate may include a TF antibody having at least one amino acid residue (fGly’) that is conjugated to the moieties of interest (e.g., drugs or active agents) as described above.
  • Ab represents the antibody that binds to TF
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from CR 4 , N and C-L B -W 2 , wherein at least one Z 1 , Z 2 , Z 3 and Z 4 is C-L B -W 2 ;
  • R 1 is 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 2 and R 3 are each 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, or R 2 and R 3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl; each R 4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl
  • L A is a first linker
  • L B is a second linker; s is an integer from 1 to 10;
  • W 1 is a first drug
  • W 2 is a second drug.
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from CR 4 , N and C-L B -W 2 , wherein at least one Z 1 , Z 2 , Z 3 and Z 4 is C-L B -W 2 .
  • Z 1 is CR 4 .
  • Z 1 is N.
  • Z 1 is C- L B -W 2 .
  • Z 2 is CR 4 .
  • Z 2 is N.
  • Z 2 is C-L B -W 2 .
  • Z 3 is CR 4 .
  • Z 3 is N.
  • Z 3 is C-L B -W 2 .
  • Z 4 is CR 4 .
  • Z 4 is N.
  • Z 4 is C-L B -W 2 .
  • each of Z 1 , Z 3 , and Z 4 is CR 4 .
  • Z 3 is C-L B -W 2 .
  • Z 1 is C-L B -W 2
  • Z 2 is CR 4
  • Z 3 is CR 4
  • Z 4 is CR 4
  • Z 1 is CR 4
  • Z 2 is C-L B -W 2
  • Z 3 is CR 4
  • Z 4 is CR 4
  • Z 1 is CR 4
  • Z 2 is CR 4
  • Z 3 is C-L B - W 2
  • Z 4 is CR 4 .
  • Z 1 is CR 4
  • Z 2 is CR 4
  • Z 3 is CR 4
  • Z 4 is C-L B -W 2 .
  • R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl.
  • R 1 is hydrogen.
  • R 1 is alkyl or substituted alkyl, such as Ci-6 alkyl or Ci-6 substituted alkyl, or Ci-4 alkyl or Ci-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl.
  • R 1 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.
  • R 1 is alkynyl or substituted alkynyl, 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.
  • R 1 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 Ce aryl or Ce substituted aryl.
  • R 1 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 Ce heteroaryl or Ce substituted heteroaryl.
  • R 1 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.
  • R 1 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.
  • R 2 and R 3 are each 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, or R 2 and R 3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl.
  • R 2 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.
  • R 2 is hydrogen. In certain embodiments, R 2 is alkyl or substituted alkyl, such as Ci-6 alkyl or Ci-6 substituted alkyl, or Ci-4 alkyl or Ci-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R 2 is methyl. In certain embodiments, R 2 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, R 2 is alkynyl or substituted alkynyl.
  • R 2 is alkoxy or substituted alkoxy. In certain embodiments, R 2 is amino or substituted amino. In certain embodiments, R 2 is carboxyl or carboxyl ester. In certain embodiments, R 2 is acyl or acyloxy. In certain embodiments, R 2 is acyl amino or amino acyl. In certain embodiments, R 2 is alkylamide or substituted alkylamide. In certain embodiments, R 2 is sulfonyl. In certain embodiments, R 2 is thioalkoxy or substituted thioalkoxy.
  • R 2 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 Ce aryl or Ce substituted aryl.
  • R 2 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 Ce heteroaryl or Ce substituted heteroaryl.
  • R 2 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.
  • R 2 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.
  • R 3 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.
  • R 3 is hydrogen. In certain embodiments, R 3 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or Ci-4 alkyl or Ci-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R 3 is methyl. In certain embodiments, R 3 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, R 3 is alkynyl or substituted alkynyl.
  • R 3 is alkoxy or substituted alkoxy. In certain embodiments, R 3 is amino or substituted amino. In certain embodiments, R 3 is carboxyl or carboxyl ester. In certain embodiments, R 3 is acyl or acyloxy. In certain embodiments, R 3 is acyl amino or amino acyl. In certain embodiments, R 3 is alkylamide or substituted alkylamide. In certain embodiments, R 3 is sulfonyl. In certain embodiments, R 3 is thioalkoxy or substituted thioalkoxy.
  • R 3 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 Ce aryl or Ce substituted aryl.
  • R 3 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 Ce heteroaryl or Ce substituted heteroaryl.
  • R 3 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.
  • R 3 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.
  • both R 2 and R 3 are methyl.
  • R 2 and R 3 are optionally cyclically linked to form a 5 or 6- membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5 or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 6-membered heterocyclyl.
  • each R 4 is independently selected from hydrogen, halogen, 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 4 is hydrogen. In certain embodiments, each R 4 is hydrogen. In certain embodiments, R 4 is halogen, such as F, Cl, Br or I. In certain embodiments, R 4 is F. In certain embodiments, R 4 is Cl. In certain embodiments, R 4 is Br. In certain embodiments, R 4 is I. In certain embodiments, R 4 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or Ci-4 alkyl or Ci-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R 4 is methyl.
  • R 4 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.
  • R 4 is alkynyl or substituted alkynyl.
  • R 4 is alkoxy or substituted alkoxy.
  • R 4 is amino or substituted amino.
  • R 4 is carboxyl or carboxyl ester.
  • R 4 is acyl or acyloxy.
  • R 4 is acyl amino or amino acyl.
  • R 4 is alkylamide or substituted alkylamide. In certain embodiments, R 4 is sulfonyl. In certain embodiments, R 4 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 4 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 Ce aryl or Ce substituted aryl (e.g., phenyl or substituted phenyl).
  • R 4 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 Ce heteroaryl or Ce substituted heteroaryl.
  • R 4 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.
  • R 4 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.
  • L A is a first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.
  • L B is a second linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.
  • W 1 is a first drug (or a first active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.
  • W 2 is a second drug (or a second active agent).
  • a second drug or a second active agent. Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.
  • Ab represents an antibody that binds to tissue factor (“TF antibody”).
  • TF antibody tissue factor
  • Ab comprises one or more fGly’ residues as described herein.
  • the TF antibody is attached to the rest of the conjugate through an fGly’ residue as described herein. Examples of TF antibodies that can be used in the conjugates of the present disclosure are described in more detail below.
  • the conjugate of Formula (I) includes a first linker, L A .
  • the first linker, L A may be utilized to bind a first moiety of interest (e.g., a first drug or active agent) to a TF antibody through a conjugation moiety.
  • the first linker, L A may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein).
  • the first linker, L A may attach a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a first drug.
  • the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety may be used to conjugate the first linker, L A , (and thus the first drug) to a TF antibody.
  • L A is attached to Ab through a conjugation moiety, and thus Ab is indirectly bonded to the linker L A through the hydrazinyl- indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • Ab is a TF antibody, and thus L A is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety to the TF antibody, e.g, the linker L A is indirectly bonded to the TF antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • the first 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 first linker L A may include an alkyl or substituted alkyl group. In certain embodiments, the first linker L A may include an alkenyl or substituted alkenyl group. In certain embodiments, the first linker L A may include an alkynyl or substituted alkynyl group. In certain embodiments, the first linker L A may include an alkoxy or substituted alkoxy group. In certain embodiments, the first linker L A may include an amino or substituted amino group. In certain embodiments, the first linker L A may include a carboxyl or carboxyl ester group. In certain embodiments, the first linker L A may include an acyl amino group.
  • the first linker L A may include an alkylamide or substituted alkylamide group. In certain embodiments, the first linker L A may include an aryl or substituted aryl group. In certain embodiments, the first linker L A may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker L A may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker L A may include a heterocyclyl or substituted heterocyclyl group.
  • the first 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 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, provided that at least one of a, b, c, d, e and f is 1. [00301] In certain embodiments, the sum of a, b, c, d, e and f is 1 to 6.
  • 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 hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • the linker subunit L 2 if present, is attached to the first drug or active agent W 1 .
  • the linker subunit L 3 if present, is attached to the first drug or active agent W 1 .
  • the linker subunit L 4 if present, is attached to the first drug or active agent W 1 .
  • linker subunit L 5 if present, is attached to the first drug or active agent W 1 .
  • linker subunit L 6 if present, is attached to the first drug or active agent W 1 .
  • Any convenient linker subunits may be utilized in the first linker L A .
  • Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and polyacrylates, 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 comprises 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.
  • 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 first linker comprising -(L 1 )a-(L 2 )b-(L 3 )c-(L 4 )d-(L 5 ) e -(L 6 )f-, where:
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are tether groups;
  • V 1 , V 2 , V 3 , V 4 , V 5 and V 6 if present, are covalent bonds or linking functional groups;
  • a, b, c, d, e and f are each independently 0 or 1, provided that at least one of a, b, c, d, e and f is 1.
  • the sum of a, b, c, d, e and f is 1 to 6. 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.
  • the sum of a, b, c, d, e and f is 6.
  • a, b, c, d, e and f are each 1.
  • a, b, c, d and e are each 1 and f is 0.
  • a, b, c and d are each 1 and e and f are each 0.
  • a, b, and c are each 1 and d, e and f are each 0.
  • a and b are each 1 and c, d, e and f are each 0.
  • a is 1 and b, c, d, e and f are each 0.
  • L 1 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • T 1 is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • V 1 is attached to the first drug or active agent.
  • L 2 if present, is attached to the first drug or active agent.
  • T 2 is attached to the first drug or active agent, or V 2 , if present, is attached to the first drug or active agent.
  • L 3 if present, is attached to the first drug or active agent.
  • T 3 if present, is attached to the first drug or active agent, or V 3 , if present, is attached to the first drug or active agent.
  • L 4 if present, is attached to the first drug or active agent.
  • T 4 if present, is attached to the first drug or active agent, or V 4 , if present, is attached to the first drug or active agent.
  • L 5 if present, is attached to the first drug or active agent.
  • T 5 if present, is attached to the first drug or active agent, or V 5 , if present, is attached to the first drug or active agent.
  • L 6 if present, is attached to the first drug or active agent.
  • T 6 if present, is attached to the first drug or active agent, or V 6 , if present, is attached to the first drug or active agent.
  • the conjugate of Formula (I) includes a second linker, L B .
  • the second linker, L B may be utilized to bind a second moiety of interest e.g., a second drug or active agent) to a TF antibody through a conjugation moiety.
  • the second linker, L B may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein).
  • the second linker, L B may attach a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety to a second drug.
  • the hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety may be used to conjugate the second linker, L B , (and thus the second drug) to a TF antibody.
  • L B is attached to Ab through a conjugation moiety, and thus Ab is indirectly bonded to the second linker L B through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • Ab is a TF antibody, and thus L B is attached through the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety to the TF antibody, e.g., the linker L B is indirectly bonded to the TF antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • the second linker L B 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 second linker L B may include an alkyl or substituted alkyl group. In certain embodiments, the second linker L B may include an alkenyl or substituted alkenyl group. In certain embodiments, the second linker L B may include an alkynyl or substituted alkynyl group. In certain embodiments, the second linker L B may include an alkoxy or substituted alkoxy group. In certain embodiments, the second linker L B may include an amino or substituted amino group. In certain embodiments, the second linker L B may include a carboxyl or carboxyl ester group. In certain embodiments, the second linker L B may include an acyl amino group.
  • the second linker L B may include an alkylamide or substituted alkylamide group. In certain embodiments, the second linker L B may include an aryl or substituted aryl group. In certain embodiments, the second linker L B may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the second linker L B may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the second linker L B may include a heterocyclyl or substituted heterocyclyl group.
  • the second linker L B 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 B is a second linker described by the formula: -(L 7 ) g -(L 8 )h-(L 9 )i-(L 10 )j-(L 11 )k-(L 12 )i-(L 13 )m, wherein L 7 , L 8 , L 9 , L 10 , L 11 , L 12 and L 13 are each independently a linker subunit, and g, h, i, j, k, 1 and m are each independently 0 or 1, provided at least one of g, h, i, j, k, 1 and m is 1.
  • the sum of g, h, i, j, k, 1 and m is 1 to 7. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 1. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 2. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 3. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 4. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 5.
  • the sum of g, h, i, j, k, 1 and m is 6. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 7. In certain embodiments, g, h, i, j, k, 1 and m are each 1. In certain embodiments, g, h, i, j, k and 1 are each 1 and m is 0. In certain embodiments, g, h, i, j and k are each 1 and 1 and m are each 0. In certain embodiments, g, h, i and j are each 1 and k, 1 and m are each 0.
  • g, h, and i are each 1 and j, k, 1 and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, 1 and m are each 0. In certain embodiments, g is 1 and h, i, j, k, 1 and m are each 0. In certain embodiments, g, h, i, j, k, 1 and m are each 0.
  • the linker subunit L 7 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • the linker subunit L 8 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 9 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 10 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 11 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 12 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 13 if present, is attached to the second drug or active agent W 2 .
  • Any convenient linker subunits may be utilized in the second linker L B .
  • Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and polyacrylates, 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 7 , L 8 , L 9 , L 10 , L 11 , L 12 and L 13 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 7 (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 7 comprises a polyethylene glycol.
  • L 7 comprises a modified polyethylene glycol.
  • L 7 comprises an amino acid residue.
  • L 7 comprises an alkyl group or a substituted alkyl.
  • L 7 comprises an aryl group or a substituted aryl group.
  • L 7 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 8 (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 8 comprises a polyethylene glycol.
  • L 8 comprises a modified polyethylene glycol.
  • L 8 comprises an amino acid residue.
  • L 8 comprises an alkyl group or a substituted alkyl.
  • L 8 comprises an aryl group or a substituted aryl group.
  • L 8 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 9 (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 9 comprises a polyethylene glycol.
  • L 9 comprises a modified polyethylene glycol.
  • L 9 comprises an amino acid residue.
  • L 9 comprises an alkyl group or a substituted alkyl.
  • L 9 comprises an aryl group or a substituted aryl group.
  • L 9 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 10 (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 10 comprises a polyethylene glycol.
  • L 10 comprises a modified polyethylene glycol.
  • L 10 comprises an amino acid residue.
  • L 10 comprises an alkyl group or a substituted alkyl.
  • L 10 comprises an aryl group or a substituted aryl group.
  • L 10 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 11 (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 11 comprises a polyethylene glycol.
  • L 11 comprises a modified polyethylene glycol.
  • L 11 comprises an amino acid residue.
  • L 11 comprises an alkyl group or a substituted alkyl.
  • L 11 comprises an aryl group or a substituted aryl group.
  • L 11 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 12 (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 12 comprises a polyethylene glycol.
  • L 12 comprises a modified polyethylene glycol.
  • L 12 comprises an amino acid residue.
  • L 12 comprises an alkyl group or a substituted alkyl.
  • L 12 comprises an aryl group or a substituted aryl group.
  • L 12 comprises a diamine e.g., a linking group comprising an alkylene diamine).
  • L 13 (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 13 comprises a polyethylene glycol.
  • L 13 comprises a modified polyethylene glycol.
  • L 13 comprises an amino acid residue.
  • L 13 comprises an alkyl group or a substituted alkyl.
  • L 13 comprises an aryl group or a substituted aryl group.
  • L 13 comprises a diamine e.g., a linking group comprising an alkylene diamine).
  • L B is a second linker comprising -(L 7 ) g -(L 8 )h-(L 9 )i-(L 10 )j-(L 11 )k-(L 12 )i-(L 13 )m-, where: -(L 7 ) g - is -(T 7 -V 7 ) g -;
  • -(L 13 )m- is -(T 13 -V 13 )m-, wherein T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 , if present, are tether groups;
  • V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 are covalent bonds or linking functional groups; and g, h, i, j, k, 1 and m are each independently 0 or 1, provided at least one of g, h, i, j, k, 1, and m is 1.
  • the sum of g, h, i, j, k, 1 and m is 1 to 7. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 1. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 2. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 3. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 4. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 5.
  • the sum of g, h, i, j, k, 1 and m is 6. In certain embodiments, the sum of g, h, i, j, k, 1 and m is 7. In certain embodiments, g, h, i, j, k, 1 and m are each 1. In certain embodiments, g, h, i, j, k and 1 are each 1 and m is 0. In certain embodiments, g, h, i, j and k are each 1 and 1 and m are each 0. In certain embodiments, g, h, i and j are each 1 and k, 1 and m are each 0.
  • g, h, and i are each 1 and j, k, 1 and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, 1 and m are each 0. In certain embodiments, g is 1 and h, i, j, k, 1 and m are each 0. In certain embodiments, g, h, i, j, k, 1 and m are each 0.
  • L 7 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • T 7 is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • V 7 is attached to the second drug or active agent.
  • L 8 if present, is attached to the second drug or active agent.
  • T 8 is attached to the second drug or active agent, or V 8 , if present, is attached to the second drug or active agent.
  • L 9 if present, is attached to the second drug or active agent.
  • T 9 if present, is attached to the second drug or active agent, or V 9 , if present, is attached to the second drug or active agent.
  • L 10 if present, is attached to the second drug or active agent.
  • T 10 if present, is attached to the second drug or active agent, or V10 4 , if present, is attached to the second drug or active agent.
  • L 11 if present, is attached to the second drug or active agent.
  • T 11 if present, is attached to the second drug or active agent, or V 11 , if present, is attached to the second drug or active agent.
  • L 12 if present, is attached to the second drug or active agent.
  • T 12 if present, is attached to the second drug or active agent, or V 12 , if present, is attached to the second drug or active agent.
  • L 13 if present, is attached to the second drug or active agent.
  • T 13 if present, is attached to the second drug or active agent, or V 13 , if present, is attached to the second drug or active agent.
  • any convenient tether groups may be utilized in the subject linkers.
  • T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 each comprise one or more groups independently selected from a covalent bond, a (Ci-Ci2)alkyl, a substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA) P , -(CR 13 OH) X -, 4-amino- piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), paraamino
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes a (Ci-Ci2)alkyl or a substituted (Ci-Ci2)alkyl.
  • (Ci-Ci2)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.
  • (Ci-Ci2)alkyl may be an alkyl or substituted alkyl, such as C1-C12 alkyl, or C1-C10 alkyl, or Ci-Ce alkyl, or C1-C3 alkyl.
  • (Ci-Ci2)alkyl is a C2-alkyl.
  • (Ci-Ci2)alkyl may be an alkylene or substituted alkylene, such as C1-C12 alkylene, or C1-C10 alkylene, or Ci-Ce alkylene, or C1-C3 alkylene.
  • (Ci-Ci2)alkyl is a Ci-alkylene (e.g, CH2).
  • (Ci-Ci2)alkyl is a C2-alkylene (e.g, CH2CH2).
  • (Ci-Ci2)alkyl is a C3-alkylene (e.g., CH2CH2CH2).
  • substituted (Ci-Ci2)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.
  • substituted (Ci-Ci2)alkyl may be a substituted alkyl, such as substituted C1-C12 alkyl, or substituted C1-C10 alkyl, or substituted Ci-Ce alkyl, or substituted C1-C3 alkyl.
  • substituted (Ci-Ci2)alkyl is a substituted C2-alkyl.
  • substituted (Ci-Ci2)alkyl may be a substituted alkylene, such as substituted C1-C12 alkylene, or substituted C1-C10 alkylene, or substituted Ci-Ce alkylene, or substituted C1-C3 alkylene.
  • substituted (Ci-Ci2)alkyl is a substituted Ci-alkylene (e.g., Ci-alkylene substituted with -SO3H).
  • substituted (Ci-Ci2)alkyl is a substituted C2-alkylene.
  • substituted (Ci- Ci2)alkyl is a substituted C3-alkylene.
  • substituted (Ci-Ci2)alkyl may include Ci- C12 alkylene (e.g., C3-alkylene or Cs-alkylene) substituted with a (PEG)k group as described herein (e.g.,-CONH(PEG)k, such as -CONH(PEG) 3 or -CONH(PEG) 5 ; or -NHCO(PEG)k, such as -NHCO(PEG)?), or may include C1-C12 alkylene (e.g., C3-alkylene) substituted with a -CONHCH2CH2SO3H group, or may include C1-C12 alkylene (e.g., Cs-alkylene) substituted with a -NHCOCH2SO3H group.
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) 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 (Ci-Ci2)alkyl, a substituted (Ci-Ci2)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 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes a heteroaryl or substituted heteroaryl, such triazolyl (e.g., 1,2,3- triazolyl).
  • triazolyl e.g., 1,2,3- triazolyl
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes a cycloalkyl or substituted cycloalkyl. In some instances, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) 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 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13
  • EDA ethylene diamine
  • (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) moieties 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.
  • the EDA moiety is described by the structure: where y is an integer from 1 to 6, r is 0 or 1, and each R 12 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.
  • 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.
  • y is 1 and the adjacent R 12 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).
  • an alkyl e.g, methyl
  • a substituted alkyl e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH.
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) 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.
  • the 4AP moiety is described by the structure: where R 12 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.
  • R 12 is a polyethylene glycol moiety.
  • R 12 is a carboxy modified polyethylene glycol.
  • 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.
  • R 17 is selected from OH, COOH, OR, 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.
  • R 17 is COOH.
  • R 17 is OH.
  • R 17 is OCH3.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes (PEG)n, where (PEG)n is a polyethylene glycol or a modified polyethylene glycol linking unit.
  • (PEG)n is described by the structure: 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 I 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.
  • n is 2.
  • n is 3.
  • n is 6.
  • n is 12.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) 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.
  • 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.
  • p is 1.
  • p is 2.
  • a tether group (e.g, T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) 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, He 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 a-hydroxy acids, and a- amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes a moiety described by the formula -(CR 13 OH) X -, where x is 0 or x 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, x is 1. In certain embodiments, x is 2.
  • 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 Ci-6 alkyl or Ci-6 substituted alkyl, or Ci-4 alkyl or Ci-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl.
  • R 13 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.
  • 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 C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a Ce aryl or Ce substituted aryl.
  • R 13 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 Ce heteroaryl or Ce substituted heteroaryl.
  • R 13 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.
  • R 13 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.
  • 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 .
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes an acetal group, a disulfide, a hydrazine, or an ester.
  • the tether group includes an acetal group.
  • the tether group includes a hydrazine.
  • the tether group includes a disulfide.
  • the tether group includes an ester.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 ) includes a meta-amino-benzyloxy (MABO), meta-amino- benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), or para-hydroxy -phenyl (PHP).
  • MABO meta-amino-benzyloxy
  • MABC meta-amino- benzyloxycarbonyl
  • PABO para-amino-benzyloxycarbonyl
  • PABC para-amino-benzyloxycarbonyl
  • a tether group includes a MABO group described by the following structure:
  • a tether group includes a MABC group described by the following structure:
  • a tether group includes a PABO group described by the following structure:
  • a tether group includes a PABC group described by the following structure:
  • a tether group includes a PAB group described by the following structure:
  • a tether group includes a PABA group described by the following structure:
  • a tether group includes a PAP group described by the following structure:
  • a tether group includes a PHP group described by the following structure:
  • each R 14 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 14 is hydrogen. In certain embodiments, each R 14 is hydrogen. In certain embodiments, R 14 is alkyl or substituted alkyl, such as Ci-6 alkyl or Ci-6 substituted alkyl, or Ci-4 alkyl or Ci-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R 14 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, R 14 is alkynyl or substituted alkynyl.
  • R 14 is alkoxy or substituted alkoxy. In certain embodiments, R 14 is amino or substituted amino. In certain embodiments, R 14 is carboxyl or carboxyl ester. In certain embodiments, R 14 is acyl or acyloxy. In certain embodiments, R 14 is acyl amino or amino acyl. In certain embodiments, R 14 is alkylamide or substituted alkylamide. In certain embodiments, R 14 is sulfonyl. In certain embodiments, R 14 is thioalkoxy or substituted thioalkoxy.
  • R 14 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 Ce aryl or Ce substituted aryl.
  • R 14 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5- 8 substituted heteroaryl, such as a Cs heteroaryl or Cs substituted heteroaryl, or a Ce heteroaryl or Ce substituted heteroaryl.
  • R 14 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.
  • R 14 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.
  • the phenyl ring may be substituted with one or more additional groups selected from halogen, 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.
  • one or more of the tether groups T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and/or T 13 is each optionally substituted with a glycoside or glycoside derivative.
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each optionally substituted with a glycoside.
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each optionally substituted with a glycoside.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative.
  • the phenyl ring may be substituted with one or more additional groups selected from a glycoside and 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 PABC is substituted with a glycoside, for example, a hydrogen of PABC is replaced with a glycoside, such as 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:
  • linking functional groups V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 any convenient linking functional groups may be utilized in the subject linkers.
  • 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 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 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)-, -SO2-, -SO2NR 15 -, -NR 15 SO2- 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 C1-6 alkyl or C1-6 substituted alkyl, or Ci-4 alkyl or Ci-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R 15 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, R 15 is alkynyl or substituted alkynyl.
  • 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 C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a Ce aryl or Ce substituted aryl.
  • R 15 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 Ce heteroaryl or Ce substituted heteroaryl.
  • R 15 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.
  • R 15 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.
  • 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.
  • 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 .
  • L A is a first linker comprising -(TkV ⁇ 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, provided at least one of a, b, c, d, e, and f is 1.
  • T 1 is selected from a (Ci-Ci2)alkyl and a substituted (Ci-Ci2)alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from (Ci-Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA) P , -(CR 13 OH) X -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, 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)-, -SO2-, -SO2NR 15 -, -NR 15 SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; wherein: integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: integer from 1 to 6 and r is 0 or 1;
  • 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.
  • L A comprises:
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (Ci- Cnjalkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA) P , -(CR 13 OH) X -, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy
  • 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)-, -SO2-, -SO2NR 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, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carb
  • T 1 is selected from a (Ci-Ci2)alkyl and a substituted (Ci-Ci2)alkyl;
  • T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (Ci- Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA)p, -(CR 13 OH)X-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PAB A, PAP, PHP, 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)-, -SO2- , -SO2NR 15 -, -NR 15 SO 2 -, and -P(O)OH-; wherein: integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: 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; a, b, c, and d are each 1; and e and f are 0.
  • T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • 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 (Ci-Ci 2 )alkyl and V 1 is -CO-;
  • T 2 is (AA) P and V 2 is absent;
  • T 3 is PABC and V 3 is absent; p is an integer from 1 to 10; and d, e and f are each 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is (PEG)n and V 2 is -CO-;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PABC and V 4 is absent; p is an integer from 1 to 10; and e and f are each 0; or wherein:
  • T 1 is (Ci-Ci 2 )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-;
  • T 4 is (AA) P and V 4 is absent;
  • T 5 is PABC and V 5 is absent; p is an integer from 1 to 10; and f is 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is (PEG)n and V 2 is -CO-;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PABC and V 4 is absent; p is an integer from 1 to 10; and e and f are each 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is substituted (Ci-Ci2)alkyl and V 2 is -CO-;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PABC and V 4 is absent; p is an integer from 1 to 10; and e and f are each 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is (PEG)n and V 2 is -CO-;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PABA and V 4 is -CO-;
  • T 5 is (Ci-Ci2)alkyl and V 5 is absent; p is an integer from 1 to 10; and f is 0; or wherein:
  • T 1 is (Ci-Ci 2 )alkyl and V 1 is -CO-;
  • T 2 is 4AP and V 2 is -CO-;
  • T 3 is (Ci-Ci 2 )alkyl and V 3 is -CO-;
  • T 4 is (AA) P and V 4 is absent;
  • T 5 is PABC and V 5 is absent; p is an integer from 1 to 10; and f is 0; or wherein:
  • T 1 is (Ci-Ci 2 )alkyl and V 1 is -CO-;
  • T 2 is 4AP and V 2 is -CO-;
  • T 3 is (Ci-Ci2)alkyl and V 3 is -O-;
  • T 4 is (Ci-Ci 2 )alkyl and V 4 is -CO-;
  • T 5 is (AA) P and V 5 is absent; p is an integer from 1 to 10; and
  • T 6 is PABC and V 6 is absent; or wherein:
  • T 1 is (Ci-Ci 2 )alkyl and V 1 is -CO-;
  • T 2 is an amino acid analog and V 2 is absent;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PABC and V 4 is absent; p is an integer from 1 to 10; and e and f are each 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is (PEG)n and V 2 is -CONH-;
  • T 3 is substituted (Ci-Ci2)alkyl and V 3 is -CO-;
  • T 4 is (AA) P and V 4 is absent;
  • T 5 is PABC and V 5 is absent; p is an integer from 1 to 10; and f is 0; or wherein:
  • T 1 is (Ci-Ci 2 )alkyl and V 1 is -CO-;
  • T 2 is an (AA) P and V 2 is -NH-;
  • T 3 is (PEG)n and V 3 is -CO-;
  • T 4 is (AA) P and V 4 is absent;
  • T 5 is PABC and V 5 is absent; p is an integer from 1 to 10; and f is 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is (PEG)n and V 2 is -CO-;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PAP and V 4 is -C(O)O-; p is an integer from 1 to 10; and e and f are each 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is substituted (Ci-Ci2)alkyl and V 2 is -CO-;
  • T 3 is (AA) P and V 3 is absent;
  • T 4 is PABC and V 4 is absent; p is an integer from 1 to 10; and e and f are each 0; or wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -CONH-;
  • T 2 is substituted (Ci-Ci2)alkyl and V 2 is -CO-;
  • T 3 is PABC and V 3 is absent; and d, e and f are each 0.
  • the left-hand side of the above linker structure for the first linker L A is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and the right-hand side of the above linker structure for the first linker L A is attached to the first drug or active agent.
  • L B is a second linker comprising -(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 )i-(T 13 -V 13 )m-, where g, h, i, j, k, 1 and m are each independently 0 or 1, provided that at least one of g, h, i, j, k, 1 and m is 1.
  • T 7 is selected from a (Ci-Ci2)alkyl and a substituted (Ci-Ci2)alkyl;
  • T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each independently selected from (Ci-Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA) P , -(CR 13 OH)X-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a disulfide, a hydrazine, and an ester; and
  • V 7 , V 8 , V 9 , V 10 ,V n , V 12 and V 13 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)-, -SO2-, -SO2NR 15 -, -NR 15 SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; wherein: 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.
  • any convenient tether groups may be utilized for T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 .
  • 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 , T 12 and T 13 .
  • Any convenient linking functional groups may be utilized for V 7 , V 8 , V 9 , V 10 ,V n , V 12 and V 13 .
  • any of the linking functional groups described above in relation to V 1 , V 2 , V 3 , V 4 , V 5 and V 6 may be used for the linking functional groups V 7 , V 8 , V 9 , V 10 ,V n , V 12 and V 13 .
  • each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.
  • alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 .
  • 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.
  • 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 .
  • various possible substituents are as described above for R 15 .
  • one or more of the tether groups T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 is each optionally substituted with a glycoside or 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 MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative.
  • the phenyl ring may be substituted with one or more additional groups selected from a glycoside and 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.
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L B In some embodiments of L B : g, h, i, j, and k are each 1;
  • T 7 is a covalent bond
  • T 8 , T 9 , T 10 , T 11 and T 12 are each independently selected from a covalent bond, (Ci- Ci2)alkyl, substituted (Ci-Ci2)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) W , (PEG)n, (AA)p, -(CR 13 OH)X-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and
  • V 7 , V 8 , V 9 , V 10 ,V n and V 12 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2)q-, -NR 15 (CeH4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2- , -SO2NR 15 -, -NR 15 SO2-, and -P(O)OH-; wherein: integer from 1 to 30;
  • EDA is an ethylene diamine moiety having the following structure: 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 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 and V 7 , V 8 , V 9 , V 10 ,V n , V 12 and V 13 are selected from the following: wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CO-;
  • T 9 is (AA) P and V 9 is absent;
  • T 10 is PABC and V 10 is absent; and k, 1 and m are each 0; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is (PEG)n and V 9 is -CO-;
  • T 10 is (AA) P and V 10 is absent;
  • T 11 is PABC and V 11 is absent;
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CO-;
  • T 9 is an amino acid analog and V 9 is -NH-;
  • T 10 is (PEG)n and V 10 is -CO-;
  • T 11 is (AA) P and V 11 is absent;
  • T 12 is PABC and V 12 is absent; and m is 0; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is (PEG)n and V 9 is -CO-;
  • T 10 is (AA) P and V 10 is absent;
  • T 11 is PABC and V 11 is absent;
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is substituted (Ci-Ci2)alkyl and V 9 is -CO-;
  • T 10 is (AA) P and V 10 is absent;
  • T 11 is PABC and V 11 is absent;
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is (PEG)n and V 9 is -CO-;
  • T 10 is (AA) P and V 10 is absent;
  • T 11 is PABA and V 11 is -CO-;
  • T 12 is (Ci-Ci2)alkyl and V 12 is absent; and m is 0; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CO-;
  • T 9 is 4AP and V 9 is -CO-;
  • T 10 is (Ci-Ci 2 )alkyl and V 10 is -CO-; T 11 is (AA) P and V 11 is absent;
  • T 12 is PABC and V 12 is absent; and m is 0; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CO-;
  • T 9 is 4AP and V 9 is -CO-;
  • T 10 is (Ci-Ci 2 )alkyl and V 10 is -O-;
  • T 11 is (Ci-Ci 2 )alkyl and V 11 is -CO-;
  • T 12 is (AA) P and V 12 is absent;
  • T 13 PABC and V 13 is absent; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CO-;
  • T 9 is an amino acid analog and V 9 is absent;
  • T 10 is (AA) P and V 10 is absent;
  • T 11 is PABC and V 11 is absent;
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is (PEG)n and V 9 is -CONH-;
  • T 10 is substituted (Ci-Ci2)alkyl and V 10 is -CO-;
  • T 11 is (AA) P and V 11 is absent;
  • T 12 is PABC and V 12 is absent; and m is 0; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CO-;
  • T 9 is (AA) P and V 9 is -NH-;
  • T 10 is (PEG)n and V 10 is -CO-;
  • T 11 is (AA) P and V 11 is absent;
  • T 12 is PABC and V 12 is absent; and m is 0; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is (PEG)n and V 9 is -CO-;
  • T 10 is (AA) P and V 10 is absent;
  • T 11 is PAP and V 11 is -C(O)O-;
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CO-;
  • T 9 is (AA) P and V 9 is absent;
  • T 10 is PABC and V 10 is absent;
  • T 11 is PAP and V 11 is -C(O)O-;
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is substituted (Ci-Ci2)alkyl and V 9 is -CO-;
  • T 10 is PABC and V 10 is absent; and k, 1 and m are each 0; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci2)alkyl and V 8 is absent;
  • T 9 is heteroaryl and V 9 is absent;
  • T 10 is (Ci-Ci 2 )alkyl and V 10 is -CONH-;
  • T 11 is (PEG)n and V 11 is -CO-;
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci2)alkyl and V 8 is absent;
  • T 9 is heteroaryl and V 9 is absent;
  • T 10 is (Ci-Ci 2 )alkyl and V 10 is -CONH-;
  • T 11 is substituted (Ci-Ci2)alkyl and V 11 is -CO-;
  • T 12 is (AA) P and V 12 is absent; and T 13 PAB and V 13 is absent; or wherein:
  • T 7 is absent and V 7 is -NHCO-;
  • T 8 is (Ci-Ci2)alkyl and V 8 is absent;
  • T 9 is heteroaryl and V 9 is absent;
  • T 10 is (Ci-Ci 2 )alkyl and V 10 is -C0NH-;
  • T 11 is substituted (Ci-Ci2)alkyl and V 11 is -CO-;
  • T 12 is (AA) P and V 12 is absent;
  • T 13 PABC and V 13 is absent.
  • the left-hand side of the above linker structure for the second linker L B is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and the right-hand side of the above linker structure for the second linker L B is attached to the second drug or active agent.
  • the conjugate is an antibody-drug conjugate where the TF antibody and the drugs are linked together by linkers as described above.
  • the linker m(e.g., L A and/or L B ) 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 separable 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.
  • linkers that are included in an antibody-drug conjugate can be cleavable linkers, 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.
  • a 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 TF antibody at a desired target site of action for the drug.
  • cleavage of a cleavable linker can be achieved by initially cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties.
  • a cleavable linker includes a first cleavable moiety and a second cleavable 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.
  • the hinderance of cleavage of the first cleavable moiety by the presence of the second cleavable moiety substantially reduces the amount or prevents the release of the drug from the antibody.
  • 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.
  • 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, 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.
  • substantially means 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.
  • 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
  • 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 (z.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 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, an ester, a peptide, a glycoside, and the like.
  • 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.
  • 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.
  • 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 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 an esterase that cleaves ester bonds or a glycosidase that cleaves glycosidic bonds, 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 (z.e., the site of the cancer (and overexpressed enzyme)).
  • the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester or a glycoside) that can be cleaved by an enzyme that is overexpressed in cancer cells.
  • the enzyme can be an esterase.
  • the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase enzyme.
  • the enzyme can be a glycosidase.
  • the enzymatically cleavable moiety is a cleavable moiety (e.g., a glycoside or glycoside derivative) that can be cleaved by a glycosidase enzyme.
  • the enzymatically cleavable moiety is an ester bond.
  • the first cleavable moiety described above i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety
  • the presence of uncleaved second cleavable moiety can protect the first cleavable moiety (ester) from cleavage by an esterase enzyme, 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 portion of the linker adjacent to 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 or glycoside derivative.
  • the enzymatically cleavable moiety is sugar moiety, such as a glycoside (or glyosyl) or glycoside derivative.
  • the glycoside or glycoside derivative can facilitate an increase in the hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include the glycoside or glycoside derivative.
  • the glycoside or glycoside derivative 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 (z.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside or glycoside derivative.
  • the first cleavable moiety includes an ester and the second cleavable moiety includes a glycoside or glycoside derivative.
  • the second cleavable 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. 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.
  • 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 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 glycosidase, such as a glucuronidase, a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like.
  • a glycosidase such as a glucuronidase, 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 cleavable moiety is found.
  • a TF-ADC is represented by Formula (I): wherein:
  • Ab represents the antibody that binds to TF
  • Z 1 , Z 2 , and Z 4 are each independently CR 4 ;
  • Z 3 is C-L B -W 2 ;
  • R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (Ci-Ci2)alkyl;
  • L A is a first linker wherein:
  • T 1 is (Ci-Ci2)alkyl and V 1 is -C0NH-;
  • T 2 is substituted (Ci-Ci2)alkyl and V 2 is -CO-;
  • T 3 is (AA) P where p is an integer from 1-20 and V 3 is a covalent bond;
  • T 4 is PABC and V 4 is a covalent bond; a, b, c, and d are each 1; e and f are each 0; and
  • L B is a second linker wherein T 7 is a covalent bond and V 7 is -NHCO-;
  • T 8 is (Ci-Ci 2 )alkyl and V 8 is -CONH-;
  • T 9 is substituted (Ci-Ci2)alkyl and V 9 is -CO-;
  • T 10 is (AA) P where p is an integer from 1-20 and V 10 is a covalent bond;
  • T 11 is PABC and V 11 is a covalent bond; and h, i, j, and k are each 1; and
  • 1 and m are each 0; s is an integer from 1 to 10;
  • W 1 is a first drug
  • W 2 is a second drug.
  • W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • a TF-ADC is represented by Formula (I): wherein:
  • Ab represents the antibody that binds to TF
  • Z 1 , Z 2 , and Z 4 are each independently CR 4 ;
  • Z 3 is C-L B -W 2 ;
  • R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (Ci-Ci2)alkyl;
  • L A is a linker wherein:
  • T 1 is (Ci-Ce)alkyl and V 1 is -CONH-;
  • T 2 is (Ci-Ce)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10 and V 2 is -CO-;
  • T 3 is (AA)2 and V 3 is a covalent bond
  • T 4 is PABC substituted with a glycoside and V 4 is a covalent bond; a, b, c, and d are each 1; and e and f are each 0; and
  • L B is a linker wherein T 7 is a covalent bond and V 7 is -NHCO-;
  • T 8 is (Ci-Ce)alkyl and V 8 is -CONH-;
  • T 9 is (Ci-Ce)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10 and V 9 is -CO-;
  • T 10 is (AA)2 and V 10 is a covalent bond
  • T 11 is PABC substituted with a glycoside and V 11 is a covalent bond; h, i, j, and k are each 1; and
  • 1 and m are each 0; s is an integer from 1 to 10;
  • W 1 is a first drug
  • W 2 is a second drug.
  • the PABC of one or both of T 4 and T 11 is substituted with a glucuronide.
  • one or both of T 1 and T 8 is ethyl.
  • one or both of T 2 and T 9 is Cs alkylene substituted with -NHCO(PEG)k, where k is an integer from 5 to 10.
  • one or both of W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • a TF-ADC as disclosed herein comprises any payload, any linker, or any linker-payload as disclosed in US Patent Application No. 2022-0241423 and International Publication No. WO 2022187370, each of which is incorporated herein by reference in its entirety.
  • the TF-ADC is represented by Formula (II): wherein:
  • Ab represents the antibody that binds to TF; and s is an integer from 1 to 10.
  • s is an integer from 1 to 4.
  • a subject conjugate comprises an antibody (Ab) that binds to TF.
  • 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;
  • I l l Phenylalanine or Phe or F Glycine or Gly or G; Histidine or His or H; Isoleucine or He 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.
  • TF-ADCs described herein comprise a drug and TF antibody conjugated thereto.
  • a TF antibody refers to an antibody, specifically binding to TF, such as a TF protein, a TF polypeptide, a TF polypeptide fragment, a TF peptide, or a TF epitope.
  • the TF antibody is a human or humanized antibody (e.g., comprising human constant regions) that binds to TF.
  • a TF antibody can bind to TF which is expressed on the surface of a mammalian (e.g., human) cell, including a TF- expressing tumor cell.
  • a TF antibody binds a TF extracellular epitope expressed on a cell such as a tumor cell (e.g., an extracellular TF epitope).
  • TF is a human TF.
  • An exemplary amino acid sequence of human TF is described herein (SEQ ID NO: 175).
  • the TF antibody competes for binding to TF with a reference TF antibody that comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the antibodies described herein, such as an amino acid sequence of a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 as described in any one of Tables 1-2.
  • the TF antibody competes for binding to TF with a reference TF antibody that comprises one, two, and/or three VH CDRs and/or one, two, and/or three VL CDRs from: (a) the antibody designated as EXMA-006; or (b) the antibody designated as EXMA-007.
  • a TF-ADC comprises a drug conjugated (directly or indirectly) to a TF antibody that competes for binding to TF with a reference TF antibody that comprises one, two, and/or three VH CDRs and one, two, and/or three VL CDRs from: (a) the antibody designated as EXMA-006; or (b) the antibody designated as EXMA-007.
  • the TF antibody competes for binding to TF with a reference TF antibody that comprises a VH region and VL region from: (a) the antibody designated as EXMA-006; or (b) the antibody designated as EXMA-007.
  • the TF antibody competes for binding to TF with a reference TF antibody that comprises: (a) a VH region comprising the amino acid sequence of SEQ ID NO:25 and a VL region comprising the amino acid sequence of SEQ ID NO:26; or (b) a VH region comprising the amino acid sequence of SEQ ID NO:41 and a VL region comprising the amino acid sequence of SEQ ID NO:42.
  • other suitable TF antibodies can be used, see, for example, International Publication Nos. WO2019136309 and W02021003399, each of which in incorporated herein by reference in its entirety.
  • the TF antibody comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the antibodies described herein, such as an amino acid sequence of a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 as described in any one of Tables 1-2.
  • the TF antibody comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from: (a) the antibody designated as EXMA-006; or (b) the antibody designated as EXMA-007.
  • the TF antibody comprises one, two, and/or three heavy chain CDRs and one, two, and/or three light chain CDRs from: (a) the antibody designated as EXMA-006; or (b) the antibody designated as EXMA-007.
  • the TF antibody comprises a VH region, which comprises one or more (such as one, two, or three) of VH CDR1, VH CDR2, and VH CDR3 as described herein, such as in any one of Tables 1-2; and/or a VL region, which comprises one or more (such as one, two, or three) of VL CDR1, VL CDR2, and VL CDR3 as described herein, such as in any one of Tables 1-2.
  • the TF antibody is bispecific and comprises a first binding region that comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs as described in any one of Tables 1-2 and a second region that comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from a binding agent that binds to a second target antigen that is not TF.
  • the TF antibody is bispecific and comprises a first binding domain that comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs as described in any one of Tables 1-2 and a second binding domain that comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from a binding agent that binds to a second TF epitope.
  • the antibody designated as EXMA-006 comprises a VH amino acid sequence of SEQ ID NO:25 and a VL amino acid sequence of SEQ ID NO:26.
  • the antibody designated as EXMA-007 comprises a VH amino acid sequence of SEQ ID NO:41 and a VL amino acid sequence of SEQ ID NO:42.
  • Table 1 Antibody EXMA-006-CDR Sequences
  • the TF antibody comprises a VH region. In some embodiments, the TF antibody comprises a VL region. In some embodiments, the TF antibody has a combination of (i) a VH region; and (ii) a VL region.
  • the TF antibody comprises a heavy chain having a combination of (i) a VH as described herein, such as in any one of Tables 1-2, and (ii) one or more heavy chain constant regions (e.g., CHI, hinge, CH2, and CH3).
  • a VH as described herein, such as in any one of Tables 1-2
  • one or more heavy chain constant regions e.g., CHI, hinge, CH2, and CH3
  • An exemplary IgG heavy chain can comprise any VH amino acid sequence as described herein and the following CHI, hinge, CH2, and CH3 amino acid sequence:
  • the TF antibody comprises a heavy chain having a combination of (i) a VH as described herein, such as in any one of Tables 1-2, and (ii) one or more heavy chain constant regions (e.g., CHI, hinge, CH2, and CH3).
  • a VH as described herein, such as in any one of Tables 1-2
  • one or more heavy chain constant regions e.g., CHI, hinge, CH2, and CH3
  • An exemplary IgG heavy chain can comprise any VH amino acid sequence as described herein and the following CHI, hinge, CH2, and CH3 amino acid sequence:
  • a TF antibody comprises a light chain having a combination of (i) a VL region as described herein, such as in any one of Tables 1-2; and (ii) a light chain constant region (CL).
  • An exemplary light chain e.g., for pairing with an IgG heavy chain
  • a TF antibody comprises a light chain having a combination of (i) a VL region as described herein, such as in any one of Tables 1-2; and (ii) a light chain constant region (CL).
  • An exemplary light chain (e.g., for pairing with an IgG heavy chain) can comprise any VL amino acid sequence described herein and the following CL amino acid sequence:
  • the TF antibody comprises (a) a heavy chain having a combination of (i) a VH as described herein, such as in any one of Tables 1-2, and (ii) one or more heavy chain constant regions e.g., CHI, hinge, CH2, and CH3); and (b) a light chain having a combination of (i) a VL as described herein, such as in any one of Tables 1-2, and (ii) a light chain constant region in an IgG format (CL1).
  • the TF antibody comprises a VH having an amino acid sequence of:
  • the TF antibody comprises a VH having an amino acid sequence of:
  • the antibody that binds to TF comprises a heavy chain having an amino acid sequence of:
  • one or both of the heavy chains of the TF antibody further comprises a signal peptide, for example at the N terminus of the chain.
  • one or both of the light chains of the TF antibody further comprises a signal peptide, for example at the N terminus of the chain.
  • the signal peptide comprises an amino acid sequence of MMSFVSLLLVGILFHATQA (SEQ ID NO:97).
  • the signal peptide comprises an amino acid sequence of MGWSLILLFLVAVATRVHS (SEQ ID NO:98).
  • the TF antibody comprises a heavy chain with a signal peptide and therefore have an amino acid sequence of: MMSFVSLLLVGILFHATQAOVQLVQSGAEVKKPGASVKVSCKASGYTFDVYGISWV RQAPGQGLEWMGWIAPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTA VYYCARDAGTYSPFGYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKD YFPEP VT VSWNSGALTSGVHTFP AVLQ SSGL YSLS S VVTVPS S SLGTQT YIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVI ⁇ FNWYVDGVEVHNAI ⁇ TI ⁇ PREEQYNSTYRVVSVLTVLHQD WLNGKEYKCK
  • the TF antibody comprises a heavy chain with a signal peptide and therefore have an amino acid sequence of:
  • a TF antibody comprises one or more CDRs (e.g., one, two, three, four, five, or six CDRs), for example, a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 as described in Table 1.
  • a TF antibody comprises one or more CDRs, (e.g., one, two, three, four, five, or six CDRs), for example, a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 as described in Table 2
  • a TF antibody comprises one or more CDRs, (e.g., one, two, or three VH CDRs), for example, a VH CDR1, VH CDR2, VH CDR3, as described in Table 1.
  • a TF antibody comprises one or more CDRs, (e.g., one, two, or three VL CDRs), for example, a VL CDR1, VL CDR2, and/or VL CDR3, as described in Table 1.
  • a TF antibody comprises one or more CDRs, (e.g., one, two, or three VH CDRs), for example, a VH CDR1, VH CDR2, VH CDR3, as described in Table 1 and one or more CDRs, (e.g., one, two, or three VL CDRs), for example, a VL CDR1, VL CDR2, and/or VL CDR3, as described in Table 1.
  • CDRs e.g., one, two, or three VH CDRs
  • CDRs e.g., one, two, or three VL CDRs
  • a TF antibody comprises one or more CDRs, (e.g., one, two, or three VH CDRs), for example, a VH CDR1, VH CDR2, VH CDR3, as described in Table 2.
  • a TF antibody comprises one or more CDRs, (e.g., one, two, or three VL CDRs), for example, a VL CDR1, VL CDR2, and/or VL CDR3, as described in Table 2.
  • a TF antibody comprises one or more CDRs, (e.g., one, two, or three VH CDRs), for example, a VH CDR1, VH CDR2, VH CDR3, as described in Table
  • VL CDRs e.g., one, two, or three VL CDRs
  • a TF antibody comprises one or more (e.g., one, two, or three) VH CDRs as described herein, such as in Tables 1-2. In other embodiments, a TF antibody comprises one or more (e.g., one, two, or three) VL CDRs as described herein, such as in Tables 1-2. In some embodiments, a TF antibody comprises one or more (e.g., one, two, or three) VH CDRs as described herein, such as in Tables 1-2 and one or more (e.g., one, two, or three) VL CDRs as described herein, such as in Tables 1-2.
  • a TF antibody comprises a VH CDR1 comprising an amino acid sequence comprising any one of SEQ ID NOs: l, 7, 8, 15, 21, 27, 31, 32, 35, and 39.
  • a TF antibody comprises a VH CDR2 comprising an amino acid sequence comprising any one of SEQ ID NOs:2, 9, 14, 16, and 22.
  • a TF antibody comprises a VH CDR3 comprising an amino acid sequence of any one of SEQ ID NOs:3, 10, 17, and 23.
  • a TF antibody comprises a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from a VH CDR1, VH CDR2, VH CDR3 as described in any one of Tables 1-2.
  • a TF antibody comprises a VL CDR1 comprising an amino acid sequence of any one of SEQ ID NOs:4, 11, 18, 24, 28, 33, 36, and 40.
  • a TF antibody comprises a VL CDR2 comprising an amino acid sequence of any one of SEQ ID NOs:5, 12, 19, 29, and 37.
  • a TF antibody comprises a VL CDR3 comprising an amino acid sequence of any one of SEQ ID NOs:6, 13, 20, 30, 34, and 38.
  • a TF antibody comprises a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from a VL CDR1, VL CDR2, VL CDR3 as described herein, such as in any one of Tables 1-2.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises all three heavy chain CDRs and/or all three light chain CDRs from: (a) the antibody designated as EXMA-006 that comprises a VH amino acid sequence of SEQ ID NO:25 and a VL amino acid sequence of SEQ ID NO:26; or (b) the antibody designated as EXMA-007 that comprises a VH amino acid sequence of SEQ ID NO:41 and a VL amino acid sequence of SEQ ID NO:42.
  • a TF-ADC comprises a TF antibody, wherein the TF antibody comprises all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated as EXMA-006. In some embodiments, a TF- ADC comprises a TF antibody, wherein the TF antibody comprises all three heavy chain CDRs and/or all three light chain CDRs from the antibody designated as EXMA-006.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises: (a) a VH region comprising a VH CDR1, a VH CDR2, and a VH CDR3 amino acid sequence as described herein, such as in Tables 1-2; and/or (b) a VL region comprising a VL CDR1, a VL CDR2, and a VL CDR3 amino acid sequence as described herein, such as in Tables 1-2.
  • a TF-ADC comprises a TF antibody wherein the antibody comprises: a VH region comprising a VH CDR1, a VH CDR2, and a VH CDR3 amino acid sequence as described herein, such as in Tables 1-2.
  • a TF- ADC comprises a TF antibody, wherein the antibody comprises a VL region comprising a VL CDR1, a VL CDR2, and a VL CDR3 amino acid sequence as described herein, such as in Tables 1-2
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:8, a VH CDR2 comprising the amino acid sequence of SEQ ID NOV, a VH CDR3 comprising the amino acid sequence of SEQ ID NOTO, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 13.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3, a VL CDR1 comprising the amino acid sequence of SEQ ID NON, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:21, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:22, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:23, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:24, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:30.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NON 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NON, a VH CDR3 comprising the amino acid sequence of SEQ ID NON, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:30.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:32, a VH CDR2 comprising the amino acid sequence of SEQ ID N0:9, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:33, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:34.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 14, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:28, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:29, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:30.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:35, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:36, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:37, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:38.
  • the TF antibody comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:22, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:23, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:40, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:30.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises a VH region and/or VL region described herein, wherein an VH and/or VL comprises human framework sequences.
  • an VH region and/or VL region comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and/or a framework 4 (FR4) sequence, such as a human FR1, a human FR2, a human FR3 and/or a human FR4.
  • an CDRs of a TF antibody can be determined by the Kabat system (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • CDRs of a TF antibody can be determined by the Chothia system, which will be referred to herein as the “Chothia CDRs” (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A. et al., 1990, J. Mol. Biol. 215(1): 175-82; and U.S. Patent No. 7,709,226).
  • CDRs of a TF antibody can be determined by the ImMunoGeneTics (IMGT®) system, for example, as described in Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-212 (“IMGT® CDRs”).
  • IMGT® CDRs ImMunoGeneTics
  • CDRs of a TF antibody can be determined by the AbM system, which will be referred to herein as the “AbM CDRs,” for example as described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. See also, e.g., Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).
  • CDRs of a TF antibody can be determined by the Contact system, which will be referred to herein as the “Contact CDRs” (see, e.g., MacCallum RM et al., 1996, J Mol Biol 5:732-745).
  • the Contact CDRs are based on an analysis of the available complex crystal structures.
  • a TF antibody comprises a heavy chain variable region (VH) comprising: (1) a VH CDR1 comprising an amino acid sequence of any one of: (i) SEQ ID NO: 1, (ii) SEQ ID NO:7, (iii) SEQ ID NO:8, (iv) SEQ ID NO: 15, and (v) SEQ ID NO:21; (2) a VH CDR2 comprising an amino acid sequence of any one of: (i) SEQ ID NO:2, (ii) SEQ ID NOV, (iii) SEQ ID NO: 14, (iv) SEQ ID NO: 16, and (v) SEQ ID NO:22; and (3) a VH CDR3 comprising an amino acid sequence of any one of: (i) SEQ ID NO:3, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 17, and (iv) SEQ ID NO:23; and/or a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of any one of: (i
  • a TF antibody comprises a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of any one of: (i) SEQ ID NO: 1, (ii) SEQ ID NOV, (iii) SEQ ID NO:8, (iv) SEQ ID NO: 15, and (v) SEQ ID NO:21; (2) a VH CDR2 comprising an amino acid sequence of any one of: (i) SEQ ID NO:2, (ii) SEQ ID NOV, (iii) SEQ ID NO: 14, (iv) SEQ ID NO: 16, and (v) SEQ ID NO:22; and (3) a VH CDR3 comprising an amino acid sequence of any one of: (i) SEQ ID NO:3, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 17, and (iv) SEQ ID NO:23.
  • a TF antibody comprises a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of any one of: (i) SEQ ID NON, (ii) SEQ ID NO: 11, (iii) SEQ ID NO: 18, and (iv) SEQ ID NO:24; (2) a VL CDR2 comprising an amino acid sequence of any one of: (i) SEQ ID NO:5, (ii) SEQ ID NO: 12, and (iii) SEQ ID NO: 19; and (3) a VL CDR3 comprising an amino acid sequence of any one of: (i) SEQ ID NO:6, (ii) SEQ ID NO: 13, and (iii) SEQ ID NO:20.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises: (1) a VL CDR1 comprising an amino acid sequence of any one of: SEQ ID NOs:4, 11, 18, and 24; (2) a VL CDR2 comprising an amino acid sequence of any one of: SEQ ID NOs:5, 12, and 19; and (3) a VL CDR3 comprising an amino acid sequence of any one of: SEQ ID NOs:6, 13, and 20.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NON; (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:6.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO:1; and (b) a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of any one of SEQ ID NOsN, 11, 18, and 24; (2) a VL CDR2 comprising an amino acid sequence of any one of SEQ ID NOs:5, 12, and 19; and (3) a VL CDR3 comprising an amino acid sequence of any one of SEQ ID NOs:6, 13, and 20.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO:1; and (b) a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NON; (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:5; and (3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO:6.
  • a TF-ADC is prepared from a TF antibody, wherein the antibody comprises a VH comprising an amino acid sequence of SEQ ID NO:25, and/or a VL comprising an amino acid sequence of SEQ ID NO:26.
  • a TF-ADC is prepared from a TF antibody, wherein the antibody comprises a heavy chain that is inserted with one or more fGly site(s) and comprises an amino acid sequence of SEQ ID NO:78 or 81 and/or a light chain comprising an amino acid sequence of SEQ ID NO:26.
  • a TF-ADC is prepared from a TF antibody, wherein the antibody comprises a heavy chain that is inserted with one or more fGly site(s) and comprises an amino acid sequence of SEQ ID NO:78 or 81 and/or a light chain comprising an amino acid sequence of SEQ ID NO:91.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises a heavy chain that is inserted with one or more fGly site(s) conjugated to a linkerdrug construct as disclosed herein and comprises an amino acid sequence of SEQ ID NO:84 or 87, and/or a light chain comprising an amino acid sequence of SEQ ID NO:26.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises a heavy chain that is inserted with one or more fGly site(s) conjugated to a linker-drug construct as disclosed herein and comprises an amino acid sequence of SEQ ID NO:84 or 87, and/or a light chain comprising an amino acid sequence of SEQ ID NO:91.
  • a TF antibody comprises a heavy chain variable region (VH) comprising: (1) a VH CDR1 comprising an amino acid sequence of any one of: (i) SEQ ID NO:27, (ii) SEQ ID NO:31, (iii) SEQ ID NO:32, (iv) SEQ ID NO:35, and (v) SEQ ID NO:39; (2) a VH CDR2 comprising an amino acid sequence of any one of: (i) SEQ ID NO:2, (ii) SEQ ID NOV, (iii) SEQ ID NO: 14, (iv) SEQ ID NO: 16, and (v) SEQ ID NO:22; and (3) a VH CDR3 comprising an amino acid sequence of any one of: (i) SEQ ID NO:3, (ii) SEQ ID NO: 10, (iii) SEQ ID NO: 17, and (iv) SEQ ID NO:23; and/or a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of any one of
  • a TF antibody comprises a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of any one of: (i) SEQ ID NO:27, (ii) SEQ ID NO:31, (iii) SEQ ID NO:32, (iv) SEQ ID NO:35, and (v) SEQ ID NO:39; (2) a VH CDR2 comprising an amino acid sequence of any one of: (i) SEQ ID NO:2, (ii) SEQ ID NOV, (iii) SEQ ID NO: 14, (iv) SEQ ID NO: 16, and (v) SEQ ID NO:22; and (3) a VH CDR3 comprising an amino acid sequence of any one of: (i) SEQ ID NOV, (ii) SEQ ID NOVO, (iii) SEQ ID NO: 17, and (iv) SEQ ID NO:23.
  • a TF antibody comprises a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of any one of: (i) SEQ ID NO:28, (ii) SEQ ID NO:33, (iii) SEQ ID NO:36, and (iv) SEQ ID NO:40; (2) a VL CDR2 comprising an amino acid sequence of any one of: (i) SEQ ID NO: 12, (ii) SEQ ID NO:29, and (iii) SEQ ID NO:37; and (3) a VL CDR3 comprising an amino acid sequence of any one of: (i) SEQ ID NO:30, (ii) SEQ ID NO:34, and (iii) SEQ ID NO:38.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises: a VL CDR1 comprising an amino acid sequence of any one of SEQ ID NOs:28, 33, 36, and 40; (2) a VL CDR2 comprising an amino acid sequence of any one of SEQ ID SEQ ID NOs: 12, 29, and 37; and (3) a VL CDR3 comprising an amino acid sequence of any one of SEQ ID SEQ ID NOs:30, 34, and 38.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NO:28; (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 12; and (3) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 13.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO:27; and (b) a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of any one of SEQ ID NOs:28, 33, 36, and 40; (2) a VL CDR2 comprising an amino acid sequence of any one of SEQ ID NOs: 12, 29, and 37; and (3) a VL CDR3 comprising an amino acid sequence of any one of SEQ ID SEQ ID NOs:30, 34, and 38.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises: (a) a VH region comprising: (1) a VH CDR1 comprising an amino acid sequence of SEQ ID NO:27; and (b) a VL region comprising: (1) a VL CDR1 comprising an amino acid sequence of SEQ ID NO:28; and (2) a VL CDR2 comprising an amino acid sequence of SEQ ID NO:29; and (3) a VL CDR3 comprising an amino acid sequence of SEQ ID SEQ ID NO:30.
  • a TF-ADC is prepared from a TF antibody, wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO:41, and/or a light chain comprising an amino acid sequence of SEQ ID NO:42.
  • a TF-ADC is prepared from a TF antibody, wherein the antibody comprises a heavy chain that is inserted with one or more fGly site(s) and comprises an amino acid sequence of SEQ ID NO:78 or 81 and/or a light chain comprising an amino acid sequence of SEQ ID NO:42.
  • a TF-ADC is prepared from a TF antibody, wherein the antibody comprises a heavy chain that is inserted with one or more fGly site(s) and comprises an amino acid sequence of SEQ ID NO:78 or 81 and/or a light chain comprising an amino acid sequence of SEQ ID NO:92.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises a heavy chain that is conjugated to one or more fGly site(s) conjugated to a linkerdrug construct as disclosed herein and comprises an amino acid sequence of SEQ ID NO:84 or 87, and/or a light chain comprising an amino acid sequence of SEQ ID NO:42.
  • a TF-ADC comprises a TF antibody, wherein the antibody comprises a heavy chain that is conjugated to one or more fGly site(s) conjugated to a linker-drug construct as disclosed herein and comprises an amino acid sequence of SEQ ID NO:84 or 87, and/or a light chain comprising an amino acid sequence of SEQ ID NO:92.
  • the position of one or more (e.g., one, two, three) CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of a TF antibody can vary by one, two, three, four, five, or six amino acid positions provided that binding to TF (e.g., human TF) is maintained e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the position defining a CDR of any of Table 1 or Table 2 can vary by shifting the N-terminal and/or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the current CDR position, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the length of one or more (e.g., one, two, three) CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and/or VL (e.g., CDR1, CDR2, or CDR3) region of a TF antibody (e.g., a human TF antibody) described herein can vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • a VH and/or VL CDR1, CDR2, and/or CDR3 can be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NOs: 1-24 and 27-40, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • a VH and/or VL CDR1, CDR2, and/or CDR3 can be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NOs: 1-24 and 27-40, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the amino terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24 and 27-40, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the carboxy terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 can be extended by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24 and 27-40, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the amino terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24 and 27-40, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the carboxy terminus of a VH and/or VL CDR1, CDR2, and/or CDR3 can be shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOs: 1-24 and 27-40, provided that binding to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Any method known in the art can be used to ascertain whether binding to TF (e.g., human TF) is maintained, for example, the binding assays and conditions described in the “Examples” section described herein.
  • TF e.g., human TF
  • a TF-ADC comprises a TF antibody that comprises one or more (e.g., one, two, three, four, or more) conservative sequence modifications.
  • conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families are disclosed herein. Thus, in some embodiments, a predicted nonessential amino acid residue in a TF antibody can be replaced with another amino acid residue from the same side chain family.
  • the conservative sequence modifications described herein modify the amino acid sequences of a TF antibody (e.g., a human TF antibody) by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.
  • the amino acid sequence modifications refer to at most one, two, three, four, five, or six amino acid substitutions to the CDRs, such as those described in any one of Tables 1-2.
  • each such CDR can contain up to five conservative amino acid substitutions, for example up to (not more than) four conservative amino acid substitutions, for example up to (not more than) three conservative amino acid substitutions, for example up to (not more than) two conservative amino acid substitutions, or no more than one conservative amino acid substitution.
  • a TF-ADC comprises a TF antibody that contains one or more, (e.g., one, two, three, four, five, or six CDRs) having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of EXMA006 or EXMA007 (see, e.g., Tables 1-2).
  • a TF-ADC comprises a TF antibody that contains a VH and a VL comprising CDRs identical to those of EXMA006 or EXMA007 (see, e.g., Tables 1-2).
  • the amino acid sequence modifications do not include any modification within a specificity determining residue (SDR). In some embodiments, the amino acid sequence modifications do not include any modification within a CDR (such as CDR1, CDR2, CDR3, or any combination thereof). In further embodiments, the amino acid sequence modifications are in the framework or constant region.
  • SDR specificity determining residue
  • CDR CDR1, CDR2, CDR3, or any combination thereof.
  • the antibody in a TF-ADC comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:25 and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:26, and the binding of the antibody to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the antibody in a TF-ADC comprises a VH comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:41 and a VL comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:42, and the binding of the antibody to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the antibody in a TF-ADC comprises a heavy chain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs:77-90 and a light chain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:91 or 92, and the binding of the antibody to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the antibody in a TF-ADC comprises a heavy chain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:93 and a light chain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:91, and the binding of the antibody to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the antibody in a TF-ADC comprises a heavy chain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:94 and a light chain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:92, and the binding of the antibody to TF (e.g., human TF) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).
  • TF e.g., human TF
  • the antibody in the TF-ADC competes with the antibody designated as EXMA-006. Additionally or alternatively, the antibody in the TF-ADC competes with the antibody designated EXMA-007. In some embodiments, the antibody in the TF-ADC competes with any TF antibody as disclosed herein.
  • a nucleic acid encoding a TF binding agent (e.g., antibody or antibody fragment), a nucleic acid complementary thereto, a vector comprising a nucleic acid as disclosed herein, a cell comprising a nucleic acid or a vector as disclosed herein.
  • the cell expresses the TF binding agent.
  • the cell replicates the nucleic acid or the vector.
  • materials for generating TF antibodies, including human TF antibodies, useful for the preparation of ADCs e.g., an isolated cell
  • a cell e.g., an isolated cell
  • a polynucleotide can comprise one or more nucleic acid sequences encoding a TF antibody or antibody fragment.
  • the polynucleotide is an isolated and/or recombinant polynucleotide.
  • the isolated polynucleotide comprises a nucleotide sequence that encodes an antibody heavy chain variable region (VH) and/or an antibody light chain variable region (VL), wherein the VH and the VL comprise CDRs identical to CDRs as described herein.
  • one or more vectors can comprise one or more polynucleotides for expression of the one or more polynucleotides in a suitable host cell.
  • Such vectors are useful, for example, for amplifying the polynucleotides in host cells to create useful quantities thereof, and for expressing binding agents, such as antibodies or antibody fragments, using recombinant techniques.
  • one or more vectors are expression vectors wherein one or more polynucleotides are operatively linked to one or more polynucleotides comprising expression control sequences.
  • Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating one or more polynucleotides encoding antibody sequences that bind TF are specifically contemplated.
  • Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression systems in which the expression construct is to be utilized. Promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.
  • Expression constructs can also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct.
  • Expression constructs can also include sequences that facilitate, e.g., promote, homologous recombination in a host cell.
  • expression constructs also include sequences necessary for replication in a host cell.
  • an expression control sequence may include a promoter/enhancer sequence, e.g., cytomegalovirus promoter/enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41 :521-530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4: 151, 1993); Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); simian virus 40 promoter; DRA (downregulated in adenoma; Alrefai et al., Am. J.
  • a promoter/enhancer sequence e.g., cytomegalovirus promoter/enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41 :5
  • a promoter can be an epithelial-specific promoter or endothelial-specific promoter.
  • a polynucleotide can also optionally include a suitable polyadenylation sequence (e.g, the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (e.g, 3’) of the polypeptide coding sequence.
  • a suitable polyadenylation sequence e.g, the SV40 or human growth hormone gene polyadenylation sequence
  • operably linked downstream e.g, 3’
  • one or more polynucleotides can optionally additionally comprise one or more nucleotide sequences encoding one or more secretory signal peptides fused in frame with the polypeptide sequences.
  • the one or more secretory signal peptides can direct secretion of the antibody polypeptides by the cells that express the one or more polynucleotides and can be cleaved by the cell from the secreted polypeptide.
  • one or more polynucleotides can further optionally comprise one or more sequences whose only intended function is to facilitate large scale production of the vector.
  • Expression vectors can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).
  • viral vectors are rendered replication-deficient by, e.g., deleting or disrupting select genes required for viral replication.
  • a cell can comprise one or more polynucleotides and/or one or more vectors.
  • a cell can be transformed or transfected with one or more polynucleotides encoding a TF antibody (e.g., a human TF antibody) or one or more vectors comprising the one or more polynucleotides encoding a TF antibody (e.g., a human TF antibody).
  • a cell can express a TF antibody (e.g., a human TF antibody) containing one or more, (e.g., one, two, three, four, five, or six) CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to CDRs as described herein, such as EXMA006 or EXMA007 (see, e.g., Tables 1-2).
  • a TF antibody e.g., a human TF antibody
  • CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to CDRs as described herein, such as EXMA006 or EXMA007 (see, e.g., Tables 1-2).
  • a cell can express a TF antibody (e.g., a human TF antibody) containing VH regions and VL regions comprising CDRs identical to those as described herein, such as of EXMA006 or EXMA007 (see, e.g., Tables 1-2).
  • a TF antibody e.g., a human TF antibody
  • VH regions and VL regions comprising CDRs identical to those as described herein, such as of EXMA006 or EXMA007 (see, e.g., Tables 1-2).
  • Suitable cells for generating a TF antibody as described herein include prokaryotic cells, such as Escherichia coli (see, e.g., Pliickthun et al., Methods Enzymol., 178:497-515, 1989), and eukaryotic cells, such as an animal cell (e.g., a myeloma cell, Chinese Hamster Ovary (CHO) cell, or hybridoma cell), yeast (e.g., Saccharomyces cerevisiae), or a plant cell (e.g., a tobacco, com, soybean, or rice cell).
  • prokaryotic cells such as Escherichia coli (see, e.g., Pliickthun et al., Methods Enzymol., 178:497-515, 1989
  • eukaryotic cells such as an animal cell (e.g., a myeloma cell, Chinese Hamster Ovary (CHO) cell, or hybridoma
  • mammalian host cells can provide for translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) that can be desirable to confer optimal biological activity on recombinant expression products.
  • an antibody e.g., TF antibody, including a human TF antibody
  • TF antibody can be glycosylated or non-glycosylated and/or have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
  • Methods for introducing DNA or RNA into a host cell include, but are not limited to, transformation, transfection, electroporation, nuclear injection, and fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.
  • host cells are useful for amplifying polynucleotides and for expressing polypeptides (e.g., antibodies) encoded by the polynucleotides.
  • a process to produce a TF antibody can comprise introducing RNA or DNA that encodes for a TF antibody, as described herein, into a host cell, culturing the host cell, and isolating the TF antibody thus produced.
  • a variety of methods for producing an antibody from a polynucleotide is generally well known. For example, basic molecular biology procedures are described by Maniatis et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989 (see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory, New York, 2001).

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Abstract

La présente divulgation concerne des structures conjuguées anticorps-médicament d'anti-facteur tissulaire. Les structures conjuguées anticorps-médicament comprennent un lieur ramifié, au moins deux charges utiles par lieur ramifié étant fixées à un anticorps. La divulgation concerne également des composés et des méthodes de production de tels conjugués. La divulgation concerne, en outre, des méthodes d'utilisation des conjugués.
PCT/US2023/073516 2022-09-07 2023-09-06 Conjugués anticorps-médicament de facteur tissulaire et leurs utilisations WO2024054821A2 (fr)

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