Classifications

• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
  • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• 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 (i.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.
• the HIPS conjugation method has been used to produce conjugates carrying one payload per HIPS moiety per aldehyde tag, which produces antibody conjugates with DAR values of up to 4.
• the present disclosure provides the use of 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 antibody-drug conjugate (ADC) structures, which include a branched HIPS linker.
• ADC antibody-drug conjugate
• the disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the conjugates.
• 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
• Z 1 is CR 4 .
• Z 1 is N.
• Z 3 is C-L B -W 2 .
• L A comprises: -(T 1 -V 1 )a-(T 2 -V 2 )b-(T 3 -V 3 )c-(T 4 -V 4 )d-(T 5 -V 5 )e-(T 6 -V 6 )f-, wherein a, b, c, d, e and f are each independently 0 or 1; T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, hetero
• T 1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl
• T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C1-C12)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 1 , V 2 , V 3 , V 4 ,V 5 and V 6 are
• 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 (C 1 -C 12 )alkyl and V 1 is -CO-; T 2 is AA and V 2 is absent; T 3 is PABC and V 3 is absent; and d, e and f are each 0; or wherein: T 1 is (C1-C12)alkyl and V 1 is -CONH-; T 2 is (PEG) n and V 2 is -CO-; T 3 is AA and V 3 is absent; T 4 is PABC and V 4 is absent; and e and f are each 0; or wherein: T 1 is (C1-C12)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 and V 4 is absent; T 5 is PABC and V 5 is absent; and f is 0; or wherein: T 1 is (C 1 -C 12 )al
• L B comprises: -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -(T 13 -V 13 ) m -, wherein g, h, i, j, k, 1 and m are each independently 0 or 1;
• 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
• 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 (CH 2 ) q -, -NR 15 (C 6 H 4 )-, -CONR 15 -, -NR 15 CO-, -C(0)0-, -OC(O)-, -0-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 S0 2 - and -P(0)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 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 PAB
• PABA PABA
• PAP PAP
• 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 -CO-;
• T 9 is AA and V 9 is absent; T 10 is PABC and V 10 is absent; and k, l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)alkyl and V 8 is -CONH-; T 9 is (PEG)n and V 9 is -CO-; T 10 is AA and V 10 is absent; and T 11 is PABC and V 11 is absent; and l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)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 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 (C
• the conjugate is selected from:
• 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 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
• W 1 is a first drug
• W 2 is a second drug
• Z 1 is CR 4 .
• Z 1 is N.
• Z 3 is C-L B -W 2 .
• 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- 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
• 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(0)0-, - OC(O)-, -0-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 S0 2 - and -P(0)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, 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, PABA, 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(0)0-, - OC(O)-, -0-, -S-, -S(O)-, -SO2- , -SO2NR 15 -, -NR 15 S0 2 -, and -P(0)0H-; 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.
• 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 (C 1 -C 12 )alkyl and V 1 is -CO-; T 2 is AA and V 2 is absent; T 3 is PABC and V 3 is absent; and d, e and f are each 0; or wherein: T 1 is (C1-C12)alkyl and V 1 is -CONH-; T 2 is (PEG) n and V 2 is -CO-; T 3 is AA and V 3 is absent; T 4 is PABC and V 4 is absent; and e and f are each 0; or wherein: T 1 is (C1-C12)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 and V 4 is absent; T 5 is PABC and V 5 is absent; and f is 0; or wherein: T 1 is (C 1 -C 12 )al
• L B comprises: -(T 7 -V 7 )g-(T 8 -V 8 )h-(T 9 -V 9 )i-(T 10 -V 10 )j-(T 11 -V 11 )k-(T 12 -V 12 )l-(T 13 -V 13 )m-, wherein g, h, i, j, k and 1 are each independently 0 or 1; T 7 , T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each independently selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA)p, -
• 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 is a linker wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C 1 -C 12 )alkyl and V 8 is -CO-; T 9 is AA and V 9 is absent; T 10 is PABC and V 10 is absent; and k, l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)alkyl and V 8 is -CONH-; T 9 is (PEG) n and V 9 is -CO-; T 10 is AA and V 10 is absent; and T 11 is PABC and V 11 is absent; and l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C 1 -C 12 )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 9
• the compound is selected from:
• aspects of the present disclosure include a pharmaceutical composition comprising a conjugate as described herein, and a pharmaceutically-acceptable excipient.
• aspects of the present disclosure include a method of administering a conjugate to a subject, where the method includes administering to a subject a conjugate as described herein.
• aspects of the present disclosure include a method of treating cancer, where the method includes administering to a subject a therapeutically effective amount of a conjugate as described herein, where the administering is effective to treat the cancer in the subject.
• 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 dmg-to-antibody (DAR) value of up to 8, according to embodiments of the present disclosure.
• DAR dmg-to-antibody
• FIG. 3 shows a graph indicating Construct 15 CT-tagged polatuzumab conjugate yielded a DAR of 3.57 as determined by hydrophobic interaction chromatography (HIC), according to embodiments of the present disclosure.
• HIC hydrophobic interaction chromatography
• FIG. 4 shows a graph indicating Construct 15 CT-tagged polatuzumab conjugate was 97.5% monomeric as determined by analytical size-exclusion chromatography (SEC), according to embodiments of the present disclosure.
• FIG. 5 shows a graph indicating Construct 15 CHl-3/CT-tagged polatuzumab conjugate yielded a DAR of 7.19 as determined by polymeric reversed phase (PLRP), according to embodiments of the present disclosure.
• FIG. 6 shows a graph indicating Construct 15 CHl-3/CT-tagged polatuzumab conjugate was 97.7% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 7 shows a graph indicating Construct 15 LC-9/CT double-tagged polatuzumab conjugate yielded a DAR of 7.06 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 8 shows a graph indicating Construct 15 LC-9/CT double-tagged polatuzumab conjugate was 99.1% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 9 shows a graph indicating Construct 15 LC-9/CH2-3 double-tagged polatuzumab conjugate yielded a DAR of 7.36 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 10 shows a graph indicating Construct 15 LC-9/CH2-3 double-tagged polatuzumab conjugate was 87.3% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 11 shows a graph indicating Construct 15 CH1-5/CT double-tagged polatuzumab conjugate yielded a DAR of 5.78 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 12 shows a graph indicating Construct 15 CH1-5/CT double-tagged polatuzumab conjugate was 99.7% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 13 shows a graph indicating Construct 15 CH1-6/CT double-tagged polatuzumab conjugate yielded a DAR of 6.86 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 14 shows a graph indicating Construct 15 CH1-6/CT double-tagged polatuzumab conjugate was 99.7% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 15 shows a graph indicating Construct 15 CH1-5/CH2-3 double-tagged polatuzumab conjugate yielded a DAR of 7.06 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 16 shows a graph indicating Construct 15 CH1-5/CH2-3 double-tagged polatuzumab conjugate was 92.8% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 17 shows a graph indicating Construct 15 CH1-6/CH2-3 double-tagged polatuzumab conjugate yielded a DAR of 7.14 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 18 shows a graph indicating Construct 15 CH1-6/CH2-3 double-tagged polatuzumab conjugate was 98.5% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 19 shows a graph indicating Construct 22 CH1-3/CT double-tagged polatuzumab conjugate yielded a DAR of 3.87 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 20 shows a graph indicating Construct 22 CH1-3/CT double-tagged polatuzumab conjugate was 93.6% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 21 shows a graph indicating Construct 22 CT-tagged polatuzumab conjugate yielded a DAR of 1.91 as determined by HIC, according to embodiments of the present disclosure.
• FIG. 22 shows a graph indicating Construct 22 CT-tagged polatuzumab conjugate is 95.0% monomeric was determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 23 shows a graph indicating Construct 25 CHl-3/CT-tagged polatuzumab conjugate yielded a DAR of 4.66 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 24 shows a graph indicating Construct 25 CHl-3/CT-tagged polatuzumab conjugate was 96.9% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 25 shows a graph indicating Construct 25 CT-tagged polatuzumab conjugate yielded a DAR of 2.27 as determined by HIC, according to embodiments of the present disclosure.
• FIG. 26 shows a graph indicating Construct 25 CT-tagged polatuzumab conjugate was 96.7% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 27 shows a graph indicating Construct 18 CT-tagged polatuzumab conjugate yielded a DAR of 3.58 as determined by HIC, according to embodiments of the present disclosure.
• FIG. 28 shows a graph indicating Construct 18 CT-tagged polatuzumab conjugate was 96.7% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 29 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 7.0 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 30 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-FITC conjugate was 98.1% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 31 shows a graph indicating Construct 15 CHl-3/CT-tagged daclizumab conjugate yielded a DAR of 7.07 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 32 shows a graph indicating Construct 15 CHl-3/CT-tagged daclizumab conjugate was 98.6% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 33 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-GPC-3 conjugate yielded a DAR of 6.86 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 34 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-GPC-3 conjugate was 98.2% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 35 shows a graph indicating Construct 15 CH2-3/CT-tagged anti-FITC conjugate yielded a DAR of 4.60 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 36 shows a graph indicating Construct 15 CH2-3/CT-tagged anti-FITC conjugate was 96.7% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 37 shows a graph indicating Construct 15 CHl-3/CT-tagged alemtuzumab conjugate yielded a DAR of 7.07 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 38 shows a graph indicating Construct 15 CHl-3/CT-tagged alemtuzumab conjugate was 98.0% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 39 shows a graph indicating Construct 15 CHl-3/CT-tagged daclizumab conjugate yielded a DAR of 7.24 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 40 shows a graph indicating Construct 15 CHl-3/CT-tagged daclizumab conjugate was 98.9% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 41 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-CD70 conjugate yielded a DAR of 6.97 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 42 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-CD70 conjugate was 99.4% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 43 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-nectin-4 conjugate yielded a DAR of 7.56 as determined by HIC, according to embodiments of the present disclosure.
• FIG. 44 shows a graph indicating Construct 15 CHl-3/CT-tagged anti-nectin-4 conjugate was 99.5% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 45 shows a graph indicating Construct 18 CHl-3/CT-tagged polatuzumab conjugate yielded a DAR of 6.57 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 46 shows a graph indicating Construct 18 CHl-3/CT-tagged polatuzumab conjugate was 98.1% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 47 shows a graph indicating Construct 18 CHl-3/CT-tagged anti-nectin-4 conjugate yielded a DAR of 6.1 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 48 shows a graph indicating Construct 18 CHl-3/CT-tagged anti-nectin-4 conjugate was 96.6% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 49 shows a graph indicating Construct 18 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 6.16 as determined by PLRP, according to embodiments of the present disclosure.
• FIG. 50 shows a graph indicating Construct 18 CHl-3/CT-tagged anti-FITC conjugate was 93.5% monomeric as determined by analytical SEC, according to embodiments of the present disclosure.
• FIG. 51 shows a graph of in vitro cytotoxicity assays of free topoisomerase inhibitors in NCI-N87 gastric cancer cells.
• FIG. 52 shows a graph of in vitro cytotoxicity assays of free topoisomerase inhibitors in Sk-Br-3 breast cancer cells.
• FIG. 53 shows a graph of in vitro cytotoxicity assays of free topoisomerase inhibitors in Granta NHL cells.
• FIG. 54 shows a graph of in vitro cytotoxicity assays of free topoisomerase inhibitors in MDA-MB-468 breast cancer cells.
• FIG. 55 shows a graph of in vitro cytotoxicity assays of free topoisomerase inhibitors in MDA-PCa-2b prostate cancer cells.
• FIG. 56 shows a graph of in vitro cytotoxicity assays in MDA-MB-468 breast cancer cells of a TROP-2 targeted ADC made using Compound 32, according to embodiments of the present disclosure.
• FIG. 57 shows a graph of in vitro cytotoxicity assays in NCI-N87 gastric cancer cells of a HER2 targeted ADC made using Compound 32, according to embodiments of the present disclosure.
• FIG. 58 shows a graph of in vitro cytotoxicity assays in SU-DHL-1 ALCL cells of a CD25 targeted ADC made using Compound 32, according to embodiments of the present disclosure.
• FIG. 59 shows a graph of in vitro cytotoxicity assays in BxPC-3 pancreatic cancer cells of a TROP-2 targeted ADC made using Compound 32, according to embodiments of the present disclosure.
• FIG. 60 shows a graph of in vitro cytotoxicity assays in NCI-N87 gastric cancer cells of a HER2 targeted ADC made using Compound 36, according to embodiments of the present disclosure.
• FIG. 61 shows a graph of in vitro cytotoxicity assays in Sk-Br-3 breast cancer cells of a HER2 targeted ADC made using Compound 36, according to embodiments of the present disclosure.
• FIG. 62 shows a schematic drawing of ELISA assays used to determine total antibody and ADC concentrations for pharmacokinetic (PK) sample analysis.
• FIG. 63 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of trastuzumab antibody.
• FIG. 64 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of polatuzumab antibody.
• FIG. 65 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of a conventional HER2 topoisomerase inhibitor conjugated ADC bearing a protease-cleavable linker, according to embodiments of the present disclosure.
• FIG. 66 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of CHl-3/CT-tagged trastuzumab conjugated to construct 32, according to embodiments of the present disclosure, according to embodiments of the present disclosure.
• FIG. 67 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of CHl-3/CT-tagged trastuzumab conjugated to construct 36, according to embodiments of the present disclosure.
• FIG. 68 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of CT-tagged polatuzumab conjugated to construct 15, according to embodiments of the present disclosure.
• FIG. 69 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of CH2-3-tagged polatuzumab conjugated to construct 15, according to embodiments of the present disclosure.
• FIG. 70 shows a graph of concentration (pg/mL) vs. days post dose following a 0.9 mg/kg dose of CHl-2-tagged polatuzumab conjugated to construct 15, according to embodiments of the present disclosure.
• FIG. 72 shows structures of dual-payload constructs carrying a MMAE payload and a second payload with an alternative mechanism of action (MOA), according to embodiments of the present disclosure.
• FIG. 73 shows structures of dual-payload constructs carrying a belotecan payload and a second payload with an alternative MOA, according to embodiments of the present disclosure.
• FIG. 76 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (36) or (175) as compared to free payload against NCI-N87 cells, according to embodiments of the present disclosure.
• FIG. 77 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (36) or (175) as compared to free payloads against SK-BR-3 cells, according to embodiments of the present disclosure.
• FIG. 78 shows a graph of in vitro potency of TROP-2 targeted or isotype control ADCs carrying (36) or (80) as compared to (16) against MDA-MB-468 cells, according to embodiments of the present disclosure.
• FIG. 79 shows a graph of in vitro potency of TROP-2 targeted or isotype control ADCs carrying (36) or (80) as compared to (16) against BxPC-3 cells, according to embodiments of the present disclosure.
• FIG. 80 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (99), (103), or (110) as compared to (16) against NCI-N87 cells, according to embodiments of the present disclosure.
• FIG. 81 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (36), (86), or (92) as compared to (16) against SK-BR-3 cells, according to embodiments of the present disclosure.
• FIG. 82 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (36), (86), or (92) as compared to (16) against NCI-N87 cells, according to embodiments of the present disclosure.
• FIG. 83 shows a graph of in vitro potency of TROP-2 targeted or isotype control ADCs carrying (65), Trodelvy, or a CL2A-SN38 isotype control conjugate as compared to (1) or (2) against SK-BR-3 cells, according to embodiments of the present disclosure.
• FIG. 84 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (65), Enhertu, or a MC-GGFG-Dxd isotype control conjugate as compared to (2) against NCI-N87 cells, according to embodiments of the present disclosure.
• FIG. 85 shows a graph of in vitro potency of TROP-2 targeted or isotype control ADCs carrying (36) or (113) as compared to (16) against MDA-MB-468 cells, according to embodiments of the present disclosure.
• FIG. 86 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (36), (136), or (142) as compared to (16) against NCI-N87 cells, according to embodiments of the present disclosure.
• FIG. 87 shows a graph of in vitro potency of TROP-2 targeted or isotype control ADCs carrying (36), (127), or (131) as compared to (16) against MDA-MB-468 cells, according to embodiments of the present disclosure.
• FIG. 88 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (36), (127), or (131) as compared to (16) against NCI-N87 cells, according to embodiments of the present disclosure.
• FIG. 89 shows a graph of in vitro potency of TROP-2 targeted or isotype control ADCs carrying (151), (147), or (131) as compared to (16) against NCI-N87 cells, according to embodiments of the present disclosure.
• FIG. 90 shows a graph of in vitro potency of HER2 targeted or isotype control ADCs carrying (151), (147), or (131) as compared to (16) against SK-BR-3 cells, according to embodiments of the present disclosure.
• FIG. 91 shows a graph indicating Compound 127 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 7.15 as determined by PLRP.
• FIG. 92 shows a graph indicating Compound 131 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 6.80 as determined by PLRP.
• FIG. 93 shows a graph indicating Compound 127 CHl-3/CT-tagged trastuzumab conjugate was 94.4% monomeric as determined by analytical SEC.
• FIG. 94 shows a graph indicating Compound 131 CHl-3/CT-tagged trastuzumab conjugate was 93.6% monomeric as determined by analytical SEC.
• FIG. 95 shows a graph indicating Compound 80 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 5.86 as determined by PLRP.
• FIG. 96 shows a graph indicating Compound 80 CHl-3/CT-tagged trastuzumab conjugate was 97.4% monomeric as determined by analytical SEC.
• FIG. 97 shows a graph indicating Compound 80 CHl-3/CT-tagged sacituzumab conjugate yielded a DAR of 6.19 as determined by PLRP.
• FIG. 98 shows a graph indicating Compound 80 CHl-3/CT-tagged sacituzumab conjugate was 97.1% monomeric as determined by analytical SEC.
• FIG. 99 shows a graph indicating Compound 86 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 5.46 as determined by PLRP.
• FIG. 100 shows a graph indicating Compound 86 CHl-3/CT-tagged anti-FITC conjugate was 98.0% monomeric as determined by analytical SEC.
• FIG. 101 shows a graph indicating Compound 92 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 1.58 as determined by PLRP.
• FIG. 102 shows a graph indicating Compound 92 CHl-3/CT-tagged anti-FITC conjugate was 96.1% monomeric as determined by analytical SEC.
• FIG. 103 shows a graph indicating Compound 99 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 3.07 as determined by PLRP.
• FIG. 104 shows a graph indicating Compound 99 CHl-3/CT-tagged anti-FITC conjugate was 97.9% monomeric as determined by analytical SEC.
• FIG. 105 shows a graph indicating Compound 103 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 6.56 as determined by PLRP.
• FIG. 106 shows a graph indicating Compound 103 CHl-3/CT-tagged trastuzumab conjugate was 97.3% monomeric as determined by analytical SEC.
• FIG. 107 shows a graph indicating Compound 110 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 5.66 as determined by PLRP.
• FIG. 108 shows a graph indicating Compound 110 CHl-3/CT-tagged anti-FITC conjugate was 98.5% monomeric as determined by analytical SEC.
• FIG. 109 shows a graph indicating Compound 113 CHl-3/CT-tagged sacituzumab conjugate yielded a DAR of 6.41 as determined by PLRP.
• FIG. 110 shows a graph indicating Compound 113 CHl-3/CT-tagged sacituzumab conjugate was 97.4% monomeric as determined by analytical SEC.
• FIG. Ill shows a graph indicating Compound 123 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 5.56 as determined by PLRP.
• FIG. 112 shows a graph indicating Compound 123 CHl-3/CT-tagged anti-FITC conjugate was 95.5% monomeric as determined by analytical SEC.
• FIG. 113 shows a graph indicating Compound 123 CHl-3/CT-tagged sacituzumab conjugate yielded a DAR of 6.27 as determined by PLRP.
• FIG. 114 shows a graph indicating Compound 123 CHl-3/CT-tagged sacituzumab conjugate was 98.0% monomeric as determined by analytical SEC.
• FIG. 115 shows a graph indicating Compound 151 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 5.67 as determined by PLRP.
• FIG. 116 shows a graph indicating Compound 151 CHl-3/CT-tagged anti-FITC conjugate was 97.8% monomeric as determined by analytical SEC.
• FIG. 117 shows a graph indicating Compound 147 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 6.47 as determined by PLRP.
• FIG. 118 shows a graph indicating Compound 147 CHl-3/CT-tagged anti-FITC conjugate was 96.4% monomeric as determined by analytical SEC.
• FIG. 119 shows a graph indicating Compound 73 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 5.41 as determined by PLRP.
• FIG. 120 shows a graph indicating Compound 67 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 4.02 as determined by PLRP.
• FIG. 121 shows a graph indicating Compound 136 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 7.26 as determined by PLRP.
• FIG. 122 shows a graph indicating Compound 136 CHl-3/CT-tagged trastuzumab conjugate was 98.9% monomeric as determined by analytical SEC.
• FIG. 123 shows a graph indicating Compound 142 CHl-3/CT-tagged trastuzumab conjugate yielded a DAR of 6.9 as determined by PLRP.
• FIG. 124 shows a graph indicating Compound 175 CHl-3/CT-tagged anti-FITC conjugate yielded a DAR of 5.08 as determined by PLRP.
• FIG. 125 shows a graph indicating Compound 175 CHl-3/CT-tagged anti-FITC conjugate was 93.0% monomeric as determined by analytical SEC.
• FIG. 126 shows a graph indicating Compound 181 CT-tagged trastuzumab conjugate yielded a DAR of 3.45 as determined by HIC.
• FIG. 127 shows a graph indicating Compound 181 CT-tagged trastuzumab conjugate was 97.6% monomeric as determined by analytical SEC.
• FIG. 128 shows a graph indicating Compound 181 CT-tagged anti-MUCl conjugate yielded a DAR of 3.59 as determined by HIC.
• FIG. 129 shows a graph indicating Compound 181 CT-tagged anti-MUCl conjugate was 99.0% monomeric as determined by analytical SEC.
• FIG. 130 shows a graph indicating Compound 181 CHl/CT-tagged anti-MUCl conjugate yielded a DAR of 2.13 as determined by PLRP.
• FIG. 131 shows a graph indicating Compound 181 CHl/CT-tagged anti-MUCl conjugate was 96.9% monomeric as determined by analytical SEC.
• FIG. 132 shows a graph indicating Compound 181 CHl/CT-tagged trastuzumab conjugate yielded a DAR of 3.28 as determined by PLRP.
• FIG. 133 shows a graph indicating Compound 181 CHl/CT-tagged trastuzumab conjugate was 92.2% monomeric as determined by analytical SEC.
• FIG. 134 shows a graph indicating Compound 194 CT-tagged trastuzumab conjugate yielded a DAR of 3.04 as determined by HIC.
• FIG. 135 shows a graph indicating Compound 194 CT-tagged trastuzumab conjugate was 97.2% monomeric as determined by analytical SEC.
• FIG. 136 shows a graph indicating Compound 194 CT-tagged anti-MUCl conjugate yielded a DAR of 3.01 as determined by HIC
• FIG. 137 shows a graph indicating Compound 194 CT-tagged anti-MUCl conjugate was 99.3% monomeric as determined by analytical SEC.
• FIG. 138 shows a graph indicating Compound 194 CHl/CT-tagged anti-MUCl conjugate yielded a DAR of 5.07 as determined by PLRP.
• FIG. 139 shows a graph indicating Compound 194 CHl/CT-tagged anti-MUCl conjugate was 97.0% monomeric as determined by analytical SEC.
• FIG. 140 shows a graph indicating Compound 194 CHl/CT-tagged trastuzumab conjugate yielded a DAR of 6.77 as determined by PLRP.
• FIG. 141 shows a graph indicating Compound 194 CHl/CT-tagged trastuzumab conjugate was 96.6% monomeric as determined by analytical SEC.
• FIG. 142 shows a graph indicating Compound 200 CHl/CT-tagged trastuzumab conjugate yielded a DAR of 6.49 as determined by PLRP.
• FIG. 143 shows a graph indicating Compound 200 CHl/CT-tagged trastuzumab conjugate was 94.2% monomeric as determined by analytical SEC.
• FIG. 144 shows a graph indicating Compound 200 CHl/CT-tagged sacituzumab conjugate yielded a DAR of 6.19 as determined by PLRP.
• FIG. 145 shows a graph indicating Compound 200 CHl/CT-tagged sacituzumab conjugate was 96.8% monomeric as determined by analytical SEC.
• FIG. 146 shows a graph indicating Compound 200 CHl/CT-tagged anti-FITC conjugate yielded a DAR of 6.5 as determined by PLRP.
• FIG. 147 shows a graph indicating Compound 200 CHl/CT-tagged anti-FITC conjugate is 94.4% monomeric as determined by analytical SEC.
• Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms.
• This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH 3 )2CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH 3 ) 3 CCH2-).
• linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH3CH2CH2CH
• 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 -0-, -N-, -S-, -S(0) 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, thioheteroary
• 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 -0-, -NR 10 -, -NR 10 C(O)-, -C(0)NR 10 - and the like.
• 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 CH 2 -), (-C(CH 3 ) 2 CH 2 C(0)-), (-C(CH 3 ) 2 CH 2 C(0)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
• Alkynyl refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-CoCH), and propargyl (-CH 2 CoCH).
• 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
• 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(0)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 cycl
• Aminocarbonylamino refers to the group -NR 21 C(0)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(0)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(0)0-, substituted alkyl-C(0)0-, cycloalkyl-C(0)0-, substituted cycloalkyl-C(0)0-, aryl-C(0)0-, heteroaryl-C(0)0-, and heterocyclyl-C(0)0- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
• Amino sulfonyl refers to the group -S0 2 NR 21 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted cycloalken
• “Sulfonylamino” refers to the group -NR 21 S0 2 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 21 and R 22 are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted
• Aryl refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl.
• such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thi
• Aryloxy refers to the group -O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.
• amino refers to the group -Nth.
• 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(0)0-alkyl, -C(0)0-substituted alkyl, -C(0)0-alkenyl, -C(0)0-substituted alkenyl, -C(0)0-alkynyl, -C(0)0-substituted alkynyl, -C(0)0-aryl, -C(0)0-substituted aryl, -C(0)0-cycloalkyl, -C(0)0-substituted cycloalkyl, -C(0)0-cycloalkenyl, -C(0)0-substituted cycloalkenyl, -C(0)0-heteroaryl, -C(0)0-substituted heteroaryl, -C(0)0-heterocyclic, and -C(0)0-substituted heterocyclic, wherein
• (Carboxyl ester)oxy refers to the groups -0-C(0)0- alkyl, -0-C(0)0-substituted alkyl, -0-C(0)0-alkenyl, -0-C(0)0-substituted alkenyl, -O- C(0)0-alkynyl, -0-C(0)0-substituted alkynyl, -0-C(0)0-aryl, -0-C(0)0-substituted aryl, -O- C(0)0-cycloalkyl, -0-C(0)0-substituted cycloalkyl, -0-C(0)0-cycloalkenyl, -0-C(0)0- substituted cycloalkenyl, -0-C(0)0-heteroaryl, -0-C(0)0-substituted heteroaryl, -0-C(0)0- heterocyclic, and -0-C(0)0-substituted heterocyclic, wherein alkyl, substituted alkyl
• 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, substituted thioalkoxy,
• Cycloalkenyl refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.
• substituted cycloalkenyl refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy
• Cycloalkynyl refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.
• Cycloalkoxy refers to –O-cycloalkyl.
• Cycloalkenyloxy refers to –O-cycloalkenyl.
• Halo or “halogen” refers to fluoro, chloro, bromo, and iodo.
• “Hydroxy” or “hydroxyl” refers to the group –OH.
• 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 benzo thienyl), 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.
• N 0 N- oxide
• sulfinyl 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- tetrahydrois
• 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,
• Heterocyclyloxy refers to the group -O-heterocyclyl.
• heterocyclylthio refers to the group heterocyclic-S-.
• heterocyclene refers to the diradical group formed from a heterocycle, as defined herein.
• hydroxyamino refers to the group -NHOH.
• Niro refers to the group -NO2.
• “Sulfonyl” refers to the group -S0 2 -alkyl, -S0 2 -substituted alkyl, -S0 2 -alkenyl, - SO2- substituted alkenyl, -S02-cycloalkyl, -SO2- substituted cylcoalkyl, -S 02-cycloalkenyl, -SO2- substituted cylcoalkenyl, -S0 2 -aryl, -S0 2 -substituted aryl, -S0 2 -heteroaryl, -SO2- substituted heteroaryl, -S0 2 -heterocyclic, and -S0 2 -substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, wherein alky
• Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.
• “Sulfonyloxy” refers to the group -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2- alkenyl, -OSO 2 -substituted alkenyl, -OSO 2 -cycloalkyl, -OSO 2 -substituted cylcoalkyl, -OSO 2 - cycloalkenyl, -OSO2-substituted cylcoalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2- heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclic, and -OSO2-substituted heterocyclic, wherein alkyl, substituted alkyl
• “Sulfate” or “sulfate ester” refers the group -O-SO 2 -OH, -O-SO 2 -O-alkyl, -O-SO 2 -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- SO 2 -O-aryl, -O-SO 2 -O-substituted aryl, -O-SO 2 -O-heteroaryl, -O-SO 2 -O-substituted heteroaryl, - O-SO2-O-heterocyclic, and -O-O-SO 2
• 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.
• thioheteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.
• heterocyclooxy refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.
• substituted when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.
• Each M + may independently be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 )4; or an alkaline earth ion, such as [Ca 2+ ]0.5, [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions).
• an alkali ion such as K + , Na + , Li +
• an ammonium ion such as + N(R 60 )4
• -NR 80 R 80 is meant to include -NH 2 , -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N- morpholinyl.
• substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O-M + , -OR 70 , -SR 70 , -S – M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 – M + , -SO 3 R 70 , -OSO 2 R 70 , -OSO 3 – M + , -OSO 3 R 70 , -PO 3 -2 (M + ) 2 , -P(O)(OR 70 )O – M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)
• 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)2R 70 , -S(O)2O-M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2 O-M + , -OS(O)2OR 70 , -P(O)(O-)2(M + )2, -P(O)(OR 70 )O-M + , -P(O)(OR 70 ), -C(O)R 70 ,
• a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
• polymers arrived at by defining substituents with further substituents to themselves e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.
• the maximum number of such substitutions is three.
• serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.
• 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)-0-C(0)-.
• any of the groups disclosed herein which contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
• the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
• pharmaceutically acceptable salt means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
• “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.
• salt thereof means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like.
• the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient.
• salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
• solvent refers to a complex formed by combination of solvent molecules with molecules or ions of the solute.
• the solvent can be an organic compound, an inorganic compound, or a mixture of both.
• Some examples of solvents include, but are not limited to, methanol, N,N-d ⁇ methyl formamidc, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
• Stereoisomers refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
• pyrazoles imidazoles, benzimidazoles, triazoles, and tetrazoles.
• “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 non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
• the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, proteins which contain at least one N-terminal methionine residue (e.g., to facilitate production in a recombinant host cell); immunologically tagged proteins; and the like.
• a polypeptide is an antibody.
• “Native amino acid sequence” or “parent amino acid sequence” are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include at least one modified amino acid residue.
• 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, lie 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, Val 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, disaccharides.
• carbohydrate derivative includes compounds where one or more functional groups of a carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemistry) or absent (e.g., eliminated or replaced by H).
• a variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates.
• glycoside refers to a sugar molecule or group bound to a moiety via a glycosidic bond.
• the moiety that the glycoside is bound to can be a cleavable linker as described herein.
• a glycosidic bond can link the glycoside to the other moiety through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O- glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl).
• glycosides can be cleaved from the moiety they are attached to, such as by chemically-mediated hydrolysis or enzymatically- mediated hydrolysis.
• antibody is used in the broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), and the like.
• An antibody is capable of binding a target antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
• a target antigen can have one or more binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) formed by one or more variable regions of an antibody.
• CDRs complementarity determining regions
• natural antibody refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism.
• Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies.
• the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies.
• humanized antibody or “humanized immunoglobulin” refers to a non human (e.g., mouse or rabbit) antibody containing one or more amino acids (in a framework region, a constant region or a CDR, for example) that have been substituted with a correspondingly positioned amino acid from a human antibody.
• humanized antibodies produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody.
• Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.
• framework substitutions are identified by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No.
• a subject rabbit antibody may be humanized according to the methods set forth in US20040086979 and US20050033031. Accordingly, the antibodies described above may be humanized using methods that are well known in the art.
• chimeric antibodies refer to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species.
• variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3.
• An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although domains from other mammalian species may be used.
• An immunoglobulin polypeptide immunoglobulin light or heavy chain variable region is composed of a framework region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”.
• the extent of the framework region and CDRs have been defined (see, “Sequences of Proteins of Immunological Interest,” E. Rabat et ah, U.S. Department of Health and Human Services, 1991).
• the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs.
• the CDRs are primarily responsible for binding to an epitope of an antigen.
• a “parent Ig polypeptide” is a polypeptide comprising an amino acid sequence which lacks an aldehyde-tagged constant region as described herein.
• the parent polypeptide may comprise a native sequence constant region, or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions).
• 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.
• the present disclosure provides antibody-drug conjugate structures, that include a branched HIPS linker.
• the disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the same.
• conjugate e.g., an antibody-drug conjugate (ADC).
• ADC antibody-drug conjugate
• conjugate is meant a polypeptide (e.g., an antibody) covalently attached to two or more other moieties (e.g., a drugs or active agents).
• an antibody-drug conjugate according to the present disclosure includes two or more drugs or active agents covalently attached to an antibody.
• the polypeptide (e.g., 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.
• the conjugate is a polypeptide conjugate, which includes a polypeptide (e.g., an antibody) conjugated to two or more other moieties.
• the two or more moieties conjugated to the polypeptide can each independently be any of a variety of moieties of interest such as, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilization of the polypeptide to a membrane or a surface.
• the conjugate is a drug conjugate, where a polypeptide is an antibody, thus providing an antibody-drug conjugate (ADC).
• ADC antibody-drug conjugate
• the conjugate can be a drug conjugate, where a polypeptide is conjugated to two or more drugs or active agents.
• Various types of drugs or active agents may be used in the conjugates and are described in more detail below.
• Moieties of interest can be conjugated to the polypeptide (e.g., antibody) at any desired site of the polypeptide.
• the present disclosure provides, for example, a polypeptide having moieties conjugated at two or more sites on the polypeptide, such as a site at or near the C-terminus of the polypeptide, a position at or near the N-terminus of the polypeptide, and a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). Combinations of the above 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 polypeptide at the a- carbon of an amino acid residue.
• a conjugate includes a polypeptide where the side chain of an amino acid residue in the polypeptide 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 polypeptide where the a-carbon of an amino acid residue in the polypeptide 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 polypeptide 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 polypeptide at multiple sites in the polypeptide. In some embodiments, two moieties may be conjugated to a single amino acid residue of the polypeptide.
• two moieties may be conjugated to the same amino acid residue of the polypeptide.
• two moieties are conjugated to a first amino acid residue of the polypeptide and two other moieties are conjugated to a second amino acid residue of the polypeptide.
• a polypeptide 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 polypeptide are each conjugated to a pair of moieties (i.e., two moieties), where each pair of moieties is conjugated to the polypeptide through a branched linker as described herein.
• 1 amino acid residue in the polypeptide 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 polypeptide are each conjugated to a pair of moieties through a branched linker as described herein.
• the one or more amino acid residues of the polypeptide 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 polypeptide.
• the conjugate may include moieties of interest conjugated to an unnatural amino acid residue of the polypeptide.
• the moieties of interest may be conjugated to the polypeptide at a single natural or unnatural amino acid residue as described above.
• One or more natural or unnatural amino acid residues in the polypeptide 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 polypeptide may each be conjugated to two moieties through a branched linker, such that multiple sites in the polypeptide are conjugated to the moieties of interest.
• a polypeptide 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
• detectable labels may be conjugated to the polypeptide.
• combinations of different payloads may be conjugated to the poypeptide.
• embodiments of the present disclosure include, but are not limited to, the following: a conjugate of a polypeptide and two or more drugs; a conjugate of a polypeptide and two or more active agents; a conjugate of a polypeptide and two or more detectable labels; and combinations thereof.
• the polypeptide (e.g., antibody) and the moieties of interest are conjugated through a conjugation moiety.
• the polypeptide and the moieties of interest may each be bound (e.g., covalently bonded) to the conjugation moiety, thus indirectly binding the polypeptide 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 polypeptide 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-Ao- Pictet-Spengler (HIPS) conjugation moiety and an aza-hydrazino-Ao-Pictet-Spengler (azaHIPS) conjugation moiety, respectively.
• HIPS hydrazino-Ao- Pictet-Spengler
• azaHIPS aza-hydrazino-Ao-Pictet-Spengler
• each R independently includes a moiety of interest (e.g., drug or active agent) that is conjugated to the polypeptide (e.g., conjugated to the polypeptide 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 polypeptide that includes a 2-formylglycine residue (fGly) is reacted with the conjugation moiety to produce a polypeptide conjugate, thus attaching the two or more drugs or active agents to the polypeptide through the conjugation moiety.
• fGly 2-formylglycine residue
• 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.
• R 21 and R 22 are each independently selected from any of the substituents described for R’ and R” above.
• Other 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 of different payloads may be conjugated to the poypeptide 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”, “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 labels 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 polypeptide may be conjugated to two or more moieties of interest, where one or more amino acids of the polypeptide are modified before conjugation to the moieties of interest. Modification of one or more amino acids of the polypeptide may produce a polypeptide that contains one or more reactive groups suitable for conjugation to the moieties of interest.
• the polypeptide 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 polypeptide 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) 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 (i.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).
• a “converted” sulfatase motif i.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.
• 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:
• L(C/S)TPSR L(C/S)TPSR.
• 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 polypeptide 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 polypeptide may be contacted with a reactive partner under conditions suitable to provide for conjugation of two or more drugs to the polypeptide.
• 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 polypeptide (e.g., an antibody) having at least one amino acid residue that has been attached to two or more moieties of interest (e.g., drugs or active agents).
• an amino acid residue of the polypeptide 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 polypeptide e.g., antibody
• an amino acid residue of the polypeptide 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 polypeptide through the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
• the term fGly refers to the amino acid residue of the polypeptide (e.g., antibody) that is coupled to the moieties of interest (e.g., drugs or active agents).
• the conjugate includes a polypeptide (e.g., an 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 polypeptide (e.g., an 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.
• 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
• W 1 is a first drug
• W 2 is a second drug
• W 3 is a polypeptide.
• 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 .
• Combinations of various Z 1 , Z 2 , Z 3 and Z 4 are possible. For example, in some instances, Z 1 is C-L B -W 2 , Z 2 is CR 4 , Z 3 is CR 4 , and 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 C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or 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 C 2-3 substituted alkenyl.
• R 1 is alkynyl or substituted alkynyl, such as C 2- 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 C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C 6 aryl or C 6 substituted aryl.
• R 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 C6 heteroaryl or C 6 substituted heteroaryl.
• R 1 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
• R 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 C 3-5 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.
• R 2 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R 2 is methyl. In certain embodiments, R 2 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl. In certain embodiments, R 2 is alkynyl or substituted alkynyl. In certain embodiments, R 2 is alkoxy or substituted alkoxy.
• 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 C6 aryl or C6 substituted aryl.
• R 2 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
• R 2 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 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 C 3-6 substituted heterocyclyl, or a C 3-5 heterocyclyl or C 3-5 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 C1-4 alkyl or C1-4 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 C 2-6 alkenyl or C 2-6 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 C6 aryl or C6 substituted aryl.
• R 3 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl.
• R 3 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
• R 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 C 3-5 heterocyclyl or C 3-5 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 C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 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 C 2-3 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 C 5 substituted aryl, or a C 6 aryl or C 6 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 C6 heteroaryl or C 6 substituted heteroaryl.
• R 4 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a 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).
• W 3 is a polypeptide (e.g., an antibody).
• W 3 comprises one or more fGly’ residues as described herein.
• the polypeptide is attached to the rest of the conjugate through an fGly’ residue as described herein. Examples of polypeptides and 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 polypeptide (e.g., an 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 polypeptide, such as an antibody.
• L A is attached to W 3 through a conjugation moiety, and thus W 3 is indirectly bonded to the linker L A through the hydrazinyl- indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
• W 3 is a polypeptide (e.g., an antibody), and thus L A is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the polypeptide (antibody), e.g., the linker L A is indirectly bonded to the polypeptide (antibody) through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
• L A is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the polypeptide (antibody), e.g., the linker L A is indirectly bonded to the polypeptide (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, methoxypoly ethylene 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.
• 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.
• the sum of a, b, c, d, e and f is 0 to 6. In certain embodiments, the sum of a, b, c, d, e and f is 0. 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.
• the sum of a, b, c, d, e and f is 5. In certain embodiments, the sum of a, b, c, d, e and f is 6. In certain embodiments, a, b, c, d, e and f are each 1. In certain embodiments, a, b, c, d and e are each 1 and f is 0. In certain embodiments, a, b, c and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e and f are each 0. In certain embodiments, a and b are each 1 and c, d, e and f are each 0. In certain embodiments, a and b are each 1 and c, 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.
• 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 comprise one or more groups independently selected from a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, and a diamine (e.g., a linking group that includes an alkylene diamine).
• L 1 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. In some embodiments, L 1 comprises a modified polyethylene glycol. In some embodiments, L 1 comprises an amino acid residue. In some embodiments, L 1 comprises an alkyl group or a substituted alkyl. In some embodiments, L 1 comprises an aryl group or a substituted aryl group. In some embodiments, 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: -(L 1 ) a - is -(T 1 -V 1 ) a -; -(L 2 )b- is -(T 2 -V 2 )b-; -(L 3 )c- is -(T 3 -V 3 )c-; -(L 4 ) d - is -(T 4 -V 4 ) d -; -(L 5 )e- is -(T 5 -V 5 )e-; and -(L 6 )f- is -(T 6 -V 6 )f-, wherein T 1 , T 2 , T 3 , T 4 , T 5 and T 6 , if present, are
• the sum of a, b, c, d, e and f is 0 to 6. In certain embodiments, the sum of a, b, c, d, e and f is 0. 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.
• the sum of a, b, c, d, e and f is 5. In certain embodiments, the sum of a, b, c, d, e and f is 6. In certain embodiments, a, b, c, d, e and f are each 1. In certain embodiments, a, b, c, d and e are each 1 and f is 0. In certain embodiments, a, b, c and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e and f are each 0. In certain embodiments, a and b are each 1 and c, d, e and f are each 0. In certain embodiments, a 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.
• 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 polypeptide (e.g., an 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 polypeptide, such as an antibody.
• L B is attached to W 3 through a conjugation moiety, and thus W 3 is indirectly bonded to the second linker L B through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
• W 3 is a polypeptide (e.g., an antibody), and thus L B is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the polypeptide (antibody), e.g., the linker L B is indirectly bonded to the polypeptide (antibody) through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
• L B is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the polypeptide (antibody), e.g., the linker L B is indirectly bonded to the polypeptide (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.
• 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 )l-(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, l and m are each independently 0 or 1.
• the sum of g, h, i, j, k, l and m is 0 to 7. In certain embodiments, the sum of g, h, i, j, k, l and m is 0. In certain embodiments, the sum of g, h, i, j, k, l and m is 1. In certain embodiments, the sum of g, h, i, j, k, l and m is 2. In certain embodiments, the sum of g, h, i, j, k, l and m is 3. In certain embodiments, the sum of g, h, i, j, k, l and m is 4.
• the sum of g, h, i, j, k, l and m is 5. In certain embodiments, the sum of g, h, i, j, k, l and m is 6. In certain embodiments, the sum of g, h, i, j, k, l and m is 7. In certain embodiments, g, h, i, j, k, l and m are each 1. In certain embodiments, g, h, i, j, k and l are each 1 and m is 0. In certain embodiments, g, h, i, j and k are each 1 and l and m are each 0.
• g, h, i and j are each 1 and k, l and m are each 0. In certain embodiments, g, h, and i are each 1 and j, k, l and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, l and m are each 0. In certain embodiments, g is 1 and h, i, j, k, l and m are each 0. In certain embodiments, g, h, i, j, k, l 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 .
• 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 ) l -(L 13 ) m -, where: -(L 7 )g- is -(T 7 -V 7 )g-; -(L 8 )h- is -(T 8 -V 8 )h-; -(L 9 ) i - is -(T 9 -V 9 ) i -; -(L 10 ) j - is -(T 10 -V 10 ) j -; -(L 11 )k- is -(T 11 -V 11 )k-; -(L 12 )l- is -(T 12 -V 12 )l-; and -(L 13 ) m - is -(T 13 -V 13 -,
• the sum of g, h, i, j, k, l and m is 0 to 7. In certain embodiments, the sum of g, h, i, j, k, l and m is 0. In certain embodiments, the sum of g, h, i, j, k, l and m is 1. In certain embodiments, the sum of g, h, i, j, k, l and m is 2. In certain embodiments, the sum of g, h, i, j, k, l and m is 3. In certain embodiments, the sum of g, h, i, j, k, l and m is 4.
• the sum of g, h, i, j, k, l and m is 5. In certain embodiments, the sum of g, h, i, j, k, l and m is 6. In certain embodiments, the sum of g, h, i, j, k, l and m is 7. In certain embodiments, g, h, i, j, k, l and m are each 1. In certain embodiments, g, h, i, j, k and l are each 1 and m is 0. In certain embodiments, g, h, i, j and k are each 1 and l and m are each 0.
• g, h, i and j are each 1 and k, l and m are each 0. In certain embodiments, g, h, and i are each 1 and j, k, l and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, l and m are each 0. In certain embodiments, g is 1 and h, i, j, k, l and m are each 0. In certain embodiments, g, h, i, j, k, l 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.
• V 13 if present, is attached to the second drug or active agent.
• 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 (C1-C12)alkyl, a substituted (C1-C12)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),
• 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 (C 1 -C 12 )alkyl or a substituted (C 1 -C 12 )alkyl.
• (C1-C12)alkyl is a straight chain or branched alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms.
• (C 1 - C12)alkyl may be an alkyl or substituted alkyl, such as C1-C12 alkyl, or C1-C10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl.
• (C1-C12)alkyl is a C2-alkyl.
• (C1-C12)alkyl may be an alkylene or substituted alkylene, such as C 1 -C 12 alkylene, or C 1 -C 10 alkylene, or C 1 -C 6 alkylene, or C 1 -C 3 alkylene.
• (C 1 -C 12 )alkyl is a C 1 -alkylene (e.g., CH 2 ).
• (C1-C12)alkyl is a C2-alkylene (e.g., CH2CH2).
• (C1-C12)alkyl is a C3-alkylene (e.g., CH2CH2CH2).
• substituted (C 1 -C 12 )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 (C 1 -C 12 )alkyl may be a substituted alkyl, such as substituted C1-C12 alkyl, or substituted C1-C10 alkyl, or substituted C1-C6 alkyl, or substituted C1-C3 alkyl.
• substituted (C1-C12)alkyl is a substituted C2-alkyl.
• substituted (C 1 -C 12 )alkyl may be a substituted alkylene, such as substituted C 1 -C 12 alkylene, or substituted C 1 -C 10 alkylene, or substituted C 1 -C 6 alkylene, or substituted C 1 -C 3 alkylene.
• substituted (C1-C12)alkyl is a substituted C1-alkylene (e.g., C1-alkylene substituted with -SO3H).
• substituted (C1-C12)alkyl is a substituted C2-alkylene.
• substituted (C 1 -C 12 )alkyl is a substituted C 3 -alkylene.
• substituted (C 1 - C12)alkyl may include C1-C12 alkylene (e.g., C3-alkylene or C5-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) 7 ), or may include C 1 -C 12 alkylene (e.g., C3-alkylene) substituted with a -CONHCH2CH2SO3H group, or may include C1-C12 alkylene (e.g., C5-alkylene) substituted with a -NHCOCH2SO3H group.
• 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-Cn/alkyl, a substituted (Ci-Cn/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).
• 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
• the tether group includes a cycloalkyl or substituted cycloalkyl.
• 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 .
• 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 ) includes an ethylene diamine (EDA) moiety, e.g., an EDA containing tether group.
• EDA ethylene diamine
• W includes one or more EDA moieties, such as where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5 or 6).
• the linked ethylene diamine (EDA) 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, or 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,
• 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 instances, n is 2. In some instances, n is 3. In some instances, n is 6. In some instances, 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
• p is 1. In certain embodiments, 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, lie 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, Val 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 CM 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
• PAB para- aminobenz
• 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: [00398] In some embodiments, 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 alkyl or CM substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 14 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl. In certain embodiments, R 14 is alkynyl or substituted alkynyl. In certain embodiments, R 14 is alkoxy or substituted alkoxy. In certain embodiments, R 14 is amino or substituted amino.
• 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. In certain embodiments, R 14 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a Ce aryl or Ce substituted aryl.
• R 14 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a Ce heteroaryl or Ce substituted heteroaryl.
• R 14 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
• R 14 is heterocyclyl or substituted heterocyclyl, such as C 3-8 heterocyclyl or C 3-8 substituted heterocyclyl, such as a C 3-6 heterocyclyl or C 3-6 substituted heterocyclyl, or a C 3-5 heterocyclyl or C 3-5 substituted heterocyclyl.
• 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 an 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 glycoside or glycoside derivative can be selected from the following structures: [00 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)- , -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, where q is an integer from 1 to 6.
• q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.
• each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
• R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 15 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or 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 C6 aryl or C6 substituted aryl.
• R 15 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl.
• R 15 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
• R 15 is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C 3-5 heterocyclyl or C 3-5 substituted heterocyclyl.
• each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, 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 -(T 1 - V 1 )a-(T 2 -V 2 )b-(T 3 -V 3 )c-(T 4 -V 4 )d-(T 5 -V 5 )e-(T 6 -V 6 )f-, where a, b, c, d, e and f are each independently 0 or 1.
• T 1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl
• T 2 , T 3 , T 4 , T 5 and T 6 are each independently selected from (C1-C12)alkyl, substituted (C1- C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR 13 OH)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
• 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 (C1-C12)alkyl and V 1 is -CO-; T 2 is AA and V 2 is absent; T 3 is PABC and V 3 is absent; and d, e and f are each 0; or wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-; T 2 is (PEG)n and V 2 is -CO-; T 3 is AA and V 3 is absent; T 4 is PABC and V 4 is absent; and e and f are each 0; or wherein: T 1 is (C1-C12)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
• 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 )l-(T 13 -V 13 )m-, where g, h, i, j, k, l and m are each independently 0 or 1.
• T 7 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl
• T 8 , T 9 , T 10 , T 11 , T 12 and T 13 are each independently selected from (C1-C12)alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, - (CR 13 OH)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
• 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 11 , 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 an 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 and V 7 , V 8 , V 9 , V 10 ,V 11 , V 12 and V 13 are selected from the following: wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)alkyl and V 8 is -CO-; T 9 is AA and V 9 is absent; T 10 is PABC and V 10 is absent; and k, l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)alkyl and V 8 is -CONH-; T 9 is (PEG) n and V 9 is -CO-; T 10 is AA and V 10 is absent; and T 11 is PABC and V 11 is absent; and l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)al
• 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 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.
• the 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 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 (i.e., cleavage of the first cleavable moiety is still needed in order to cleave the cleavable linker).
• the cleavable moieties included in the cleavable linker may each be an enzymatically cleavable moiety.
• the first cleavable moiety can be a first enzymatically cleavable moiety and the second cleavable moiety can be a second enzymatically cleavable moiety.
• An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more portions as described above through the enzymatic action of an enzyme.
• the 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.
• 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 (i.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 i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage
• 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.
• the second cleavable moiety is a galactoside.
• the second cleavable moiety is a glucoside.
• the second cleavable moiety is a mannoside.
• the second cleavable moiety is a fucoside.
• the second cleavable moiety is O-GlcNAc.
• 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.
• conjugates according to the present disclosure include, but are not limited to, the following structures:
• conjugates according to the present disclosure include, but are not limited to, the following structures:
• the present disclosure provides compounds useful for producing the conjugates described herein.
• the compound can be attached to two or more drugs or active agents and may also include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety useful for conjugation of the drugs or active agents to a polypeptide (e.g., an antibody).
• a polypeptide e.g., an antibody
• the conjugation moiety in the compound may be conjugated to a polypeptide (e.g., antibody), thus indirectly binding the drugs or active agents and the polypeptide (antibody) together.
• the compound is a compound of formula (II): wherein:
• 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 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
• W 1 is a first drug
• W 2 is a second drug.
• the substituents Z 1 , Z 2 , Z 3 , Z 4 , R 2 , R 3 , R 4 , L A , L B , W 1 , and W 2 are as described above in relation to the conjugates of formula (I).
• the T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 and V 6 , and T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , T 13 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 and V 13 substituents are as described above in relation to the conjugates of formula (I).
• 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 (C1-C12)alkyl and V 1 is -CO-; T 2 is AA and V 2 is absent; T 3 is PABC and V 3 is absent; and d, e and f are each 0; or wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-; T 2 is (PEG)n and V 2 is -CO-; T 3 is AA and V 3 is absent; T 4 is PABC and V 4 is absent; and e and f are each 0; or wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-; T 2 is an amino acid analog and V 2 is -NH-; T 3 is (PEG)n and V
• 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 11 , V 12 and V 13 are selected from the following: wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)alkyl and V 8 is -CO-; T 9 is AA and V 9 is absent; T 10 is PABC and V 10 is absent; and k, l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C 1 -C 12 )alkyl and V 8 is -CONH-; T 9 is (PEG)n and V 9 is -CO-; T 10 is AA and V 10 is absent; and T 11 is PABC and V 11 is absent; and l and m are each 0; or wherein: T 7 is absent and V 7 is -NHCO-; T 8 is (C1-C12)
• Compounds of formula (II) can be used in conjugation reactions described herein, where two or more drugs or active agents attached to a hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety is conjugated to a polypeptide (e.g., antibody) to form an antibody-drug conjugate.
• a polypeptide e.g., antibody
• Examples of compounds according to the present disclosure include, but are not limited to, the following structures: , , ⁇
• a subject conjugate can comprise as substituent W 3 a polypeptide (e.g., an antibody).
• the amino acid sequence of the polypeptide (antibody) can be modified to include a 2-formylglycine (fGly) residue.
• amino acids may be referred to by their standard name, their standard three letter abbreviation and/or their standard one letter abbreviation, such as: Alanine or Ala or A; Cysteine or Cys or C; Aspartic acid or Asp or D; Glutamic acid or Glu or E; Phenylalanine or Phe or F; Glycine or Gly or G; Histidine or His or H; Isoleucine or lie 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 Val or V; Tryptophan or Trp or W; and Tyrosine or Tyr or Y.
• the amino acid sequence of a polypeptide or an antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to a 2-formylglycine (fGly) residue by action of a formylglycine generating enzyme (FGE) either in vivo (e.g., at the time of translation of an aldehyde tag- containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system).
• FGE formylglycine generating enzyme
• Such sulfatase motifs may also be referred to herein as an FGE-modification site.
• a minimal sulfatase motif of an aldehyde tag is usually 5 or 6 amino acid residues in length, usually no more than 6 amino acid residues in length.
• Sulfatase motifs provided in an Ig polypeptide are at least 5 or 6 amino acid residues, and can be, for example, from 5 to 16, 6- 16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acid residues in length.
• polypeptides of interest include those where one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide for a sequence of a sulfatase motif in the polypeptide.
• amino acid residues such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19
• the polypeptide includes a modification (insertion, addition, deletion, and/or substitution/replacement) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide.
• a modification insertion, addition, deletion, and/or substitution/replacement of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide.
• an amino acid sequence native to the polypeptide e.g., antibody
• the total number of modifications of residues can be reduced, e.g., by site-specification modification (insertion, addition, deletion, substitution/replacement) of amino acid residues flanking the native amino acid residues to provide a sequence of the desired sulfatase motif.
• the extent of modification of the native amino acid sequence of the target antibody is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target antibody may minimize the impact such modifications may have upon antibody function and/or structure.
• aldehyde tags of particular interest are those comprising at least a minimal sulfatase motif (also referred to a “consensus sulfatase motif”)
• aldehyde tags can thus comprise a minimal sulfatase motif of 5 or 6 residues, or can be longer and comprise a minimal sulfatase motif which can be flanked at the N- and/or C- terminal sides of the motif by additional amino acid residues.
• Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.
• An aldehyde tag can be present at or near the C-terminus of an Ig heavy chain; e.g., an aldehyde tag can be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C- terminus of a native, wild-type Ig heavy chain.
• An aldehyde tag can be present within a CH1 domain of an Ig heavy chain.
• An aldehyde tag can be present within a CH2 domain of an Ig heavy chain.
• An aldehyde tag can be present within a CH3 domain of an Ig heavy chain.
• An aldehyde tag can be present in an Ig light chain constant region, e.g., in a kappa light chain constant region or a lambda light chain constant region.
• the sulfatase motif used may be described by the formula: X 1 Z 10 X 2 Z 20 X 3 Z 30 (I’) where Z 10 is cysteine or serine (which can also be represented by (C/S)); Z 20 is either a proline or alanine residue (which can also be represented by (P/A)); Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 is present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar,
• Z 10 is cysteine or
• the amino acid sequence of an antibody heavy and/or light chain can be modified to provide a sequence of at least 5 amino acids of the formula X 1 Z 10 X 2 Z 20 X 3 Z 30 , where Z 10 is cysteine or serine; Z 20 is a proline or alanine residue; Z 30 is an aliphatic amino acid or a basic amino acid; X 1 is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the polypeptide, X 1 is present; X 2 and X 3 are each independently any amino acid.
• the sulfatase motif is generally selected so as to be capable of conversion by a selected FGE, e.g., an FGE present in a host cell in which the aldehyde tagged polypeptide is expressed or an FGE which is to be contacted with the aldehyde tagged polypeptide in a cell-free in vitro method.
• FGE e.g., an FGE present in a host cell in which the aldehyde tagged polypeptide is expressed or an FGE which is to be contacted with the aldehyde tagged polypeptide in a cell-free in vitro method.
• the sulfatase motif can be of the formula: X 1 CX 2 PX 3 Z 30 (I”) where X 1 may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 is present;
• X 2 and X 3 independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic
• sulfatase motifs include LCTPSR (SEQ ID NO://), MCTPSR (SEQ ID NO://), VCTPSR (SEQ ID NO://), LCSPSR (SEQ ID NO://), LCAPSR (SEQ ID NO://), LCVPSR (SEQ ID NO://), LCGPSR (SEQ ID NO://), ICTPAR (SEQ ID NO://), LCTPSK (SEQ ID NO://), MCTPSK (SEQ ID NO://), VCTPSK (SEQ ID NO://), LCSPSK (SEQ ID NO://), LCAPSK (SEQ ID NO://), LCVPSK (SEQ ID NO://), LCGPSK (SEQ ID NO://), LCTPSA (SEQ ID NO://), ICTPAA (SEQ ID NO://), MCTPSA (SEQ ID NO://), VCTPSA (SEQ ID NO://), LCAPSA (SEQ ID NO://), LCVPSA (SEQ ID NO://), LCVPSA (SEQ ID NO://), LCVPSA (
• the fGly-containing sulfatase motif can be of the formula: X 1 (fGly)X 2 Z 20 X 3 Z 30 (I’’’) where fGly is the formylglycine residue; Z 20 is either a proline or alanine residue (which can also be represented by (P/A)); Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may
• the polypeptide containing the fGly residue may be conjugated to a drug or active agent by reaction of the fGly with a reactive moiety (e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above) of a linker attached to the drug or active agent to produce an fGly’-containing sulfatase motif.
• a reactive moiety e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above
• the term fGly refers to the amino acid residue of the sulfatase motif that is coupled to the drug or active agent through a linker (e.g., a branched linker) as described herein.
• the fGly’-containing sulfatase motif can be of the formula: X 1 (fGly’)X 2 Z 20 X 3 Z 30 (II) where fGly’ is the amino acid residue coupled to the drug or active agent through a linker (e.g., a branched linker) as described herein;
• Z 20 is either a proline or alanine residue (which can also be represented by (P/A));
• Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), is
• the amino acid sequence of an antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an fGly residue by action of an FGE either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag- containing protein with an FGE in a cell-free system).
• a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an fGly residue by action of an FGE either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag- containing protein with an FGE in a cell-free system).
• the antibody used to generate a conjugate of the present disclosure include at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CHI, a CH2, a CH3, and a CH4 domain), or an Ig light chain constant region.
• an Ig constant region e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CHI, a CH2, a CH3, and a CH4 domain)
• target Ig polypeptides e.g., target antibodies.
• the site in an antibody into which a sulfatase motif is introduced can be any convenient site.
• the extent of modification of the native amino acid sequence of the target polypeptide is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), and/or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target antibody may minimize the impact such modifications may have upon antibody function and/or structure.
• An antibody heavy chain constant region can include Ig constant regions of any heavy chain isotype, non-naturahy occurring Ig heavy chain constant regions (including consensus Ig heavy chain constant regions).
• An Ig constant region amino acid sequence can be modified to include an aldehyde tag, where the aldehyde tag is present in or adjacent a solvent- accessible loop region of the Ig constant region.
• An Ig constant region amino acid sequence can be modified by insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, or more than 16 amino acids, to provide an amino acid sequence of a sulfatase motif as described above.
• an aldehyde-tagged antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least a CHI domain; at least a CHI and a CH2 domain; a CHI, a CH2, and a CH3 domain; or a CHI, a CH2, a CH3, and a CH4 domain).
• an aldehyde-tagged Ig heavy chain constant region e.g., at least a CHI domain; at least a CHI and a CH2 domain; a CHI, a CH2, and a CH3 domain; or a CHI, a CH2, a CH3, and a CH4 domain.
• the aldehyde- tagged Ig heavy chain constant region can include heavy chain constant region sequences of an IgA, IgM, IgD, IgE, IgGl, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypic variant of same, e.g., human heavy chain constant region sequences or mouse heavy chain constant region sequences, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an fGly-modified Ig polypeptide. Allotypic variants of Ig heavy chains are known in the art.
• an aldehyde-tagged antibody comprises an aldehyde-tagged Ig light chain constant region.
• the aldehyde-tagged Ig light chain constant region can include constant region sequences of a kappa light chain, a lambda light chain, e.g., human kappa or lambda light chain constant regions, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an fGly-modified antibody.
• Exemplary constant regions include human gamma 1 and gamma 3 regions. With the exception of the sulfatase motif, a constant region may have a wild-type amino acid sequence, or it may have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90% or at least 95% identical) to a wild type amino acid sequence.
• the sulfatase motif is at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain.
• an isolated aldehyde- tagged antibody can comprise a heavy chain constant region amino acid sequence modified to include a sulfatase motif as described above, where the sulfatase motif is in or adjacent a surface- accessible loop region of the antibody heavy chain constant region.
• a sulfatase motif can be provided within or adjacent one or more of these amino acid sequences of such modification sites of an Ig heavy chain.
• an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif adjacent and N-terminal and/or adjacent and C- terminal to these modification sites.
• an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modifications sites.
• an Ig heavy chain polypeptide amino acid sequence may be modified to include two motifs, which may be adjacent to one another, or which may be separated by one, two, three, four or more (e.g., from about 1 to about 25, from about 25 to about 50, or from about 50 to about 100, or more, amino acids.
• a native amino acid sequence provides for one or more amino acid residues of a sulfatase motif sequence
• selected amino acid residues of the modification sites of an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) so as to provide a sulfatase motif at the modification site.
• An antibody used in an antibody-drug conjugate of the present disclosure can have any of a variety of antigen-binding specificities, including but not limited to, e.g., an antigen present on a cancer cell; an antigen present on an autoimmune cell; an antigen present on a pathogenic microorganism; an antigen present on a virus-infected cell (e.g., a human immunodeficiency virus-infected cell); an antigen present on a diseased cell; and the like.
• an antibody conjugate can bind an antigen, where the antigen is present on the surface of the cell.
• An antibody conjugate of the present disclosure can bind antigen with a suitable binding affinity, e.g., from 5 x 10 6 M to 10 7 M, from 10 7 M to 5 x 10 7 M, from 5 x 10 7 M to 10 8 M, from 10 8 M to 5 x 10 8 M, from 5 x 10 8 M to 10 9 M, or a binding affinity greater than 10 9 M.
• a suitable binding affinity e.g., from 5 x 10 6 M to 10 7 M, from 10 7 M to 5 x 10 7 M, from 5 x 10 7 M to 10 8 M, from 10 8 M to 5 x 10 8 M, from 5 x 10 8 M to 10 9 M, or a binding affinity greater than 10 9 M.
• a subject antibody conjugate can bind an antigen present on a cancer cell (e.g., a tumor- specific antigen; an antigen that is over-expressed on a cancer cell; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).
• a subject antibody conjugate can be specific for an antigen on a cancer cell, where the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).
• a subject antibody conjugate can bind an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by the vims; where the antigen is expressed on a cell type that is infected by a vims; etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor.
• a subject antibody conjugate can bind an antigen present on a cell infected with a vims, and the conjugated moiety can be a dmg, such as a viral fusion inhibitor.
• a conjugate or a compound of the present disclosure can include as substituents W 1 and W 2 a dmg or active agent.
• Any of a number of drugs are suitable for use, or can be modified to be rendered suitable for use, as a reactive partner to conjugate to an antibody. Examples of drugs include small molecule drugs and peptide dmgs.
• “Small molecule dmg” as used herein refers to a compound, e.g., an organic compound, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa.
• a small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom.
• the drug or active agent can be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as a camptothecine, or an analog or derivative thereof, or a pharmaceutically active camptothecine moiety and/or a portion thereof.
• a topoisomerase inhibitor (e.g., camptothecine, or analog or derivative thereof) conjugated to the polypeptide can be any of a variety of topoisomerase inhibitors, for example camptothecine or camptothecine moieties such as, but not limited to, camptothecine and analogs and derivatives thereof as described herein.
• drugs that find use in the conjugates and compounds described herein include, but are not limited to, a topoisomerase inhibitor, for example camptothecine or a camptothecine derivative, such as SN-38, Belotecan, Exatecan, 9-aminocamptothecin (9-AC), topotecan, i es-Me-topotecan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/US2022/012325, the disclosure of which is incorporated herein by reference.
• a topoisomerase inhibitor for example camptothecine or a camptothecine derivative, such as SN-38, Belotecan, Exatecan, 9-aminocamptothecin (9-AC), topotecan, i es-Me-topotecan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/US2022/0123
• the drug or active agent can be a maytansine.
• Maytansine “maytansine moiety”, “maytansine active agent moiety” and “maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and/or portions thereof.
• a maytansine conjugated to the polypeptide can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein (e.g., deacylmaytansine).
• the drug or active agent can be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and/or a portion thereof.
• An auristatin conjugated to the polypeptide can be any of a variety of auristatin moieties such as, but not limited to, an auristatin and analogs and derivatives thereof as described herein.
• Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to an auristatin or an auristatin derivative, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), derivatives thereof, and the like.
• the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and/or a portion thereof.
• a duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin moieties such as, but not limited to, a duocarmycin and analogs and derivatives thereof as described herein.
• Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to a duocarmycin or a duocarmycin derivative, such as duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof, and the like.
• the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.
• the drug is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulatory agent, a toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.
• a cytotoxin can include any compound that leads to cell death (e.g., necrosis or apoptosis) or a decrease in cell viability.
• Kinase inhibitors can include, but are not limited to, Adavosertib, Afatinib, Axitinib, Bosutinib, Cetuximab, Cobimetinib, Crizotinib, Cabozantinib, Dacomitinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ruxolitinib, Sorafenib, Sunitinib, Tucatinib, Vandetanib, Vemurafenib, and the like.
• selective estrogen receptor modulators include, but are not limited to, Endoxifen, Tamoxifen, Afimoxifene, Toremifene, and the like.
• Immunostimulatory agents can include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, and the like.
• TLR agonists include, but are not limited to, imiquimod, resiquimod, and the like.
• Oligonucleotide dugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, volanesorsen, inotersen, tofersen, tominersen, and the like.
• Aptamer drugs include, but are not limited to, pegaptanib, AS 1411, REG1,
• Cytokines include, but are not limited to, Albinterferon Alfa-2B, Aldesleukin, ALT-801, Anakinra, Ancestim, Avotermin, Balugrastim, Bempegaldesleukin, Binetrakin, Cintredekin Besudotox, CTCE-0214, Darbepoetin alfa, Denileukin diftitox, Dulanermin, Edodekin alfa, Emfilermin, Epoetin delta, Erythropoietin, Human interleukin-2, Interferon alfa, Interferon alfa-2c, Interferon alfa-nl, Interferon alfa-n3, Interferon alfacon-1, Interferon beta- la, Interferon beta- lb, Interferon gamma- lb, Interferon Kappa, Interleukin- 1 alpha, Interleukin- 10, Interleukin-7, Leno
• Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.
• Peptide drug refers to amino-acid containing polymeric compounds, and is meant to encompass naturally-occurring and non-naturally-occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics.
• the peptide drugs may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source).
• Peptide drugs can range in molecular weight, and can be from 200 Da to 10 kDa or greater in molecular weight.
• Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; anti- angiogenic peptides; peptides that activate B cells; peptides that activate T cells; anti- viral peptides; peptides that inhibit viral fusion; peptides that increase production of one or more lymphocyte populations; anti-microbial peptides; growth factors; growth hormone-releasing factors; vasoactive peptides; anti inflammatory peptides; peptides that regulate glucose metabolism; an anti-thrombotic peptide; an anti-nociceptive peptide; a vasodilator peptide; a platelet aggregation inhibitor; an analgesic; and the like.
• Additional examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to Tubulysin M, Calicheamicin, a STAT3 inhibitor, alpha- Amanitin, an aurora kinase inhibitor, belotecan, and an anthracy cline.
• 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 produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent.
• Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents.
• Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.
• Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. 6,323,315.
• dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure.
• Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad.
• duocarmycins and active analogs and derivatives thereof e.g., including the synthetic analogues, KW-2189 and CB 1-TMl
• benzodiazepines and active analogs and derivatives thereof e.g., pyrrolobenzodiazepine (PBD).
• Agents that act to reduce cellular proliferation are known in the art and widely used.
• Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CytoxanTM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
• alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazene
• Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6- mercaptopurine (6-MP), pentostatin, 5 -fluorouracil (5-FU), methotrexate, lO-propargyl-5,8- dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.
• CYTOSAR-U cytarabine
• cytosine arabinoside including, but not limited to, fluorouracil (5-FU), floxuridine (FudR), 6-thiogu
• 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, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g.
• anthracycline daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epimbicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.
• phenoxizone biscyclopeptides e.g. dactinomycin
• basic glycopeptides e.g.
• anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
• Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
• Hormone modulators and steroids include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.
• adrenocorticosteroids e.g. prednisone, dexamethasone, etc.
• estrogens and pregestins e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.
• adrenocortical suppressants e.g.
• chemotherapeutic agents include metal complexes, e.g. cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, e.g. N-methylhydrazine; epidophyllo toxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc.
• metal complexes e.g. cisplatin (cis-DDP), carboplatin, etc.
• ureas e.g. hydroxyurea
• hydrazines e.g. N-methylhydrazine
• epidophyllo toxin e.g. hydroxyurea
• hydrazines e.g. N-methylhydrazine
• epidophyllo toxin e.g. hydroxyurea
• hydrazines e.g. N-
• mycophenolic acid mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4- morpholinyl)propoxy )quinazoline) ; etc .
• Taxanes are suitable for use.
• “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug.
• “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOLTM, TAXOTERETM (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S.
• Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., TaxotereTM docetaxel, as noted above) and 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 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-g; (8) colony- stimulating factors; and (9) inhibitors of angiogenesis.
• RTK tyrosine kinase
• tumor-associated antigen antagonists such as antibodies that bind specifically to a tumor antigen
• apoptosis receptor agonists such as antibodies that bind specifically to a tumor antigen
• interleukin-2 interleukin-2
• IFN-a IFN-a
• IFN-g IFN-g
• colony- stimulating factors and (9) inhibitors of angiogenesis.
• examples of drugs include small molecule drugs, such as
• the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell
• the antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agent.
• a cancer chemotherapeutic agent such as a microtubule affecting agent.
• the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid.
• Embodiments of the present disclosure include conjugates where an antibody is conjugated to two or more drug moieties, such as 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug moieties.
• drug moieties such as 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug
• the drug moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein.
• the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2.
• DAR drug-to-antibody ratio
• the conjugates have an average DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugates have an average DAR of 1 to 10. In certain embodiments, the conjugates have an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugates have an average DAR of 5 to 10 (e.g., 8). By average is meant the arithmetic mean.
• the two drugs or active agents attached to the branched linker are the same drug or active agent.
• a first branch of a branched linker may be attached to a drug or an active agent and a second branch of the branched linker may be attached to the same drug or the same active agent as the first branch.
• the two drugs or active agents attached to the branched linker are different drugs or active agents.
• a first branch of a branched linker may be attached to a first drug or a first active agent and a second branch of the branched linker may be attached to a second drug or a second active agent different from the first drug or the first active agent attached to the first branch.
• the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect.
• the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both payloads act, thereby increasing overall potency of the ADC.
• the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately,
• the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).
• the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways.
• 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 two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an auristatin (e.g., MMAE) as described herein.
• the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an iRGD peptide as described herein.
• the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and an iRGD peptide as described herein.
• the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein.
• the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein.
• a kinase inhibitor e.g., Sorafenib, Lapatinib, Gefitinib, and the like
• the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein.
• the two different drugs or active agents are attached to the branched linker
• the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a selective estrogen receptor modulator (e.g.,
• Drugs to be conjugated to a polypeptide may be modified to incorporate a reactive partner for reaction with the polypeptide.
• the drug is a peptide drug
• the reactive moiety e.g., aminooxy or hydrazide can be positioned at an N-terminal region, the N-terminus, a C- terminal region, the C-terminus, or at a position internal to the peptide.
• an example of a method involves synthesizing a peptide drug having an aminooxy group.
• the peptide is synthesized from a Boc-protected precursor.
• An amino group of a peptide can react with a compound comprising a carboxylic acid group and oxy-N-Boc group.
• the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidin-l-yloxy)propanoic acid.
• Other variations on the compound comprising a carboxylic acid group and oxy-N-protecting group can include different number of carbons in the alkylene linker and substituents on the alkylene linker.
• the reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and oxy-N-protecting group occurs through standard peptide coupling chemistry.
• peptide coupling reagents examples include, but not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1- benzotriazole diethylphosphate-l-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (l-(3- dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidinium hexafluorophosphosphate), DPP A (diphenylphosphorazidate), B OP (benzotriazol- 1 -yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (0-benzotriazol-l-yl-yl-
• HOBt and DIC can be used as peptide coupling reagents.
• Deprotection to expose the amino-oxy functionality is performed on the peptide comprising an N-protecting group.
• Deprotection of the N-oxysuccinimide group occurs according to standard deprotection conditions for a cyclic amide group. Deprotecting conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions include a hydrazine reagent, amino reagent, or sodium borohydride. Deprotection of a Boc protecting group can occur with TFA.
• reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine.
• the product and intermediates can be purified by conventional means, such as HPLC purification.
• HPLC purification HPLC purification.
• pH and steric hindrance i.e., the accessibility of the amino acid residue to reaction with a reactive partner of interest
• Modifying reaction conditions to provide for optimal conjugation conditions is well within the skill of the ordinary artisan, and is routine in the art. Where conjugation is conducted with a polypeptide present in or on a living cell, the conditions are selected so as to be physiologically compatible.
• the pH can be dropped temporarily for a time sufficient to allow for the reaction to occur but within a period tolerated by the cell (e.g., from about 30 min to 1 hour).
• Physiological conditions for conducting modification of polypeptides on a cell surface can be similar to those used in a ketone-azide reaction in modification of cells bearing cell-surface azides (see, e.g., U.S. 6,570,040).
• Small molecule compounds containing, or modified to contain, an oc-nucleophilic group that serves as a reactive partner with a compound or conjugate disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the present disclosure.
• General methods are known in the art for chemical synthetic schemes and conditions useful for synthesizing a compound of interest (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).
• conjugates of the present disclosure can be formulated in a variety of different ways.
• the conjugate is formulated in a manner compatible with the drug, the antibody, the condition to be treated, and the route of administration to be used.
• a pharmaceutical composition that includes any of the conjugates of the present disclosure and a pharmaceutically-acceptable excipient.
• the conjugate e.g., antibody-drug conjugate
• the conjugate is provided as a liquid injectable (such as in those embodiments where they are administered intravenously or directly into a tissue)
• the conjugate can be provided as a ready-to- use dosage form, or as a reconstitutable storage- stable powder or liquid composed of pharmaceutically acceptable carriers and excipients.
• conjugates can be provided in a pharmaceutical composition comprising a therapeutically effective amount of a conjugate and a pharmaceutically acceptable carrier (e.g., saline).
• a pharmaceutically acceptable carrier e.g., saline
• the pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like).
• the formulations are suitable for administration to a mammal, such as those that are suitable for administration to a human.
• the antibody-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug (i.e., the drug prior to conjugation to the antibody).
• an effective amount e.g., a therapeutically effective amount
• antibody-drug conjugates of the present disclosure can include a cleavable linker, such as an enzymatically cleavable linker that includes a first enzymatically cleavable moiety and a second enzymatically cleavable moiety.
• the cleavable linker can be cleaved under appropriate conditions to separate or release the drug from the antibody at a desired target site of action for the drug.
• the second cleavable linker which protects the first cleavable linker from cleavage, may be cleaved in order to allow the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker into two or more portions, thus releasing the drug from the antibody-drug conjugate at a desired site of action.
• the first cleavable moiety can be an enzymatically cleavable moiety.
• the enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated).
• a first enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein.
• the second cleavable moiety can be an enzymatically cleavable moiety.
• the enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated).
• a second enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein.
• the first enzyme and the second enzyme are different enzymes.
• the first enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated).
• the first enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate.
• the antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the first enzyme.
• the first enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated.
• the first enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated.
• the first enzyme is present in an overabundance at the desired site of action due to localization of the first enzyme at a particular area or location.
• the first enzyme may be associated with a certain structure within the desired site of action, such as lysosomes. In some cases, the first enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject. In some embodiments, the lysosomes that include the first enzyme, are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug. In certain embodiments, the first enzyme is an esterase.
• the second enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated).
• the second enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate.
• the antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the second enzyme.
• the second enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated.
• the second enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated.
• the second enzyme is present in an overabundance at the desired site of action due to localization of the second enzyme at a particular area or location.
• the second enzyme may be associated with a certain structure within the desired site of action, such as lysosomes.
• the second enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject.
• the lysosomes that include the second enzyme are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug.
• the second enzyme is a glucuronidase, a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like.
• Any suitable enzymes can be used for cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein.
• Other enzymes may also be suitable for use in cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein, such as but not limited to, enzymes from other vertebrates (e.g., primates, mice, rats, cats, pigs, quails, goats, dogs, etc.).
• the antibody-drug conjugate is substantially stable under standard conditions.
• substantially stable is meant that the cleavable linker of the antibody- drug conjugate does not undergo a significant amount of cleavage in the absence of a first enzyme and a second enzyme as described above.
• the second cleavable moiety can protect the first cleavable moiety from being cleaved, and as such the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of a second enzyme as described above.
• the cleavable linker of the antibody-drug conjugate may be substantially stable such that 25% or less of the antibody-drug conjugate is cleaved in the absence of the first enzyme and/or second enzyme, such as 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less.
• the antibody-drug conjugate is substantially stable such that the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the first enzyme and/or second enzyme, but can be cleaved when in the presence of the first enzyme and the second enzyme.
• the antibody-drug conjugate can be substantially stable after administration to a subject.
• the antibody-drug conjugate is substantially stable after administration to a subject, and then, when the antibody-drug conjugate is in the presence of the second enzyme at a desired site of action, the second cleavable moiety can be cleaved from the cleavable linker, thus exposing the first cleavable moiety to subsequent cleavage by the first enzyme, which in turn releases the drug at the desired site of action.
• the antibody- drug conjugate after administration to a subject is stable for an extended period of time in the absence of the first enzyme and/or second enzyme, such as 1 hr or more, or 2 hrs or more, or 3 hrs or more, or 4 hrs or more, or 5 hrs or more, or 6 hrs or more, or 7 hrs or more, or 8 hrs or more, or 9 hrs or more, or 10 hrs or more, or 15 hrs or more, or 20 hrs or more, or 24 hrs (1 day) or more, or 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days (1 week) or more.
• the first enzyme and/or second enzyme such as 1 hr or more, or 2 hrs or more, or 3 hrs or more, or 4 hrs or more, or 5 hrs or more, or 6 hrs or more, or 7 days (1 week) or more.
• the antibody-drug conjugate is stable at a range pH values for an extended period of time in the absence of the first enzyme and/or second enzyme, such as at a pH ranging from 2 to 10, or from 3 to 9, or from 4 to 8, or from 5 to 8, or from 6 to 8, or from 7 to 8.
• the antibody-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug.
• treatment is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g.
• treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition.
• treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease; and/or (iii) relief, that is, causing the regression of clinical symptoms.
• the subject to be treated can be one that is in need of therapy, where the subject to be treated is one amenable to treatment using the parent drug. Accordingly, a variety of subjects may be amenable to treatment using the antibody-drug conjugates disclosed herein. Generally, such subjects are “mammals”, with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees and monkeys).
• domestic pets e.g., dogs and cats
• livestock e.g., cows, pigs, goats, horses, and the like
• rodents e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease
• the amount of antibody-drug conjugate administered can be initially determined based on guidance of a dose and/or dosage regimen of the parent drug.
• the antibody- drug conjugates can provide for targeted delivery and/or enhanced serum half-life of the bound drug, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen.
• the antibody-drug conjugates can provide for reduced dose and/or reduced administration in a dosage regimen relative to the parent drug prior to being conjugated in an antibody-drug conjugate of the present disclosure.
• the antibody-drug conjugates can provide for controlled stoichiometry of drug delivery, dosages of antibody-drug conjugates can be calculated based on the number of drug molecules provided on a per antibody-drug conjugate basis.
• an antibody-drug conjugate is administered.
• the frequency of administration of an antibody-drug conjugate can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc.
• an antibody-drug conjugate is administered once per month, twice per month, three times per month, every other week, once per week (qwk), twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily (qd/od), twice a day (bds/bid), or three times a day (tds/tid), etc.
• the present disclosure provides methods that include delivering a conjugate of the present disclosure to an individual having a cancer.
• the methods are useful for treating a wide variety of cancers, including, but not limited to breast, ovarian, colon, lung, stomach, and pancreatic cancer.
• the term “treating” includes one or more (e.g., each) of: reducing growth of a solid tumor, inhibiting replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with a cancer.
• Carcinomas that can be treated using a subject method include, but are not limited to, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, cervical carcinoma, uterine carcinoma, testicular carcinoma, and epithelial carcinoma, etc.
• methods of treating cancer in a subject including administering to the subject a therapeutically effective amount of a conjugate of the present disclosure, where the administering is effective to treat cancer in the subject.
• the method of treating cancer includes administering to the subject a therapeutically effective amount of pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to treat cancer in the subject.
• Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
• Compounds as described herein can be purified by any purification protocol known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins.
• the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J.
• the subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods.
• a variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.
• Synthetic reagents were purchased from Sigma- Aldrich, Acros, AK Scientific, or other commercial sources and were used without purification.
• Anhydrous solvents were obtained from commercial sources in sealed bottles.
• Compounds 5, 12, 29 and 74 were obtained commercially from Shanghai Medicilon and used without purification.
• Cytotoxins belotecan 16 and MMAE 13 were obtained from commercial sources and used as received. In all cases, solvent was removed under reduced pressure with a Buchi Rotovapor R-l 14 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed with a Biotage Isolera chromatography system.
• Preparative HPLC purifications were performed using Waters preparative HPLC unit equipped with Phenomenex Kinetex 5 mhi EVO C18 150 x 21.2 mm column.
• Low-resolution mass spectra were acquired on Agilent Technology 6120 Quadmpole LC/MS, equipped with Agilent 1260 Infinity HPLC system, G1314 variable wavelength detector, and Agilent Poroshell 120 SB Cl 8, 4.6 mm x 50 mm column at room temperature using 10-100% gradient of water and acetonitrile containing 0.1% formic acid.
• HPLCs were monitored at 254 or 205 nm.
• a solution of nitro compound 6 (116 mg, 0.2 mmol) in 1 mL of THF was combined with a solution of ammonium chloride (85 mg, 1.6 mmol) in 1 mL of water, and 0.5 mL of methanol at ambient temperature.
• the resulting mixture was treated with zinc powder (104 mg, 1.6 mmol) in several small portions.
• reaction mixture was treated with PNP carbonate 12 (350 mg, 0.34 mmol) at room temperature and stirred overnight, then treated directly with 100 ⁇ L of piperidine (1 mmol) at 0 °C. After one hour, reaction mixture was purified by reversed-phase chromatography (C18 column, 0-60% v/v gradient of CH 3 CN/H 2 O with 0.05% TFA) to obtain 260 mg (0.19 mmol, 63% yield) of compound 19 as a white powder.
• reaction mixture was treated directly with piperidine (40 ⁇ L, 0.4 mmol), stirred for 20 minutes, and purified by reversed-phase HPLC (C18 column, 10-70% v/v gradient of CH 3 CN/H 2 O with 0.05% TFA). Fractions containing the desired product were lyophilized to give 10 mg (7 ⁇ mol, 32% yield) of compound 21 as a white powder.
• reaction mixture was stirred for 1 hour, then directly treated with piperidine (40 ⁇ L, 0.4 mmol). After 30 minutes, reaction n mixture was purified by reversed-phase HPLC (C18 column, 10-70% v/v gradient of CH3CN/H2O with 0.05% TFA) to give 18 mg (11 ⁇ mol, 68% yield) of compound 24 as a colorless solid.
• MMAE construct 25 [00554] To a mixture of compound 24 (4 mg, 2.4 ⁇ mol), DIPEA (1.6 ⁇ L, 10 ⁇ mol), and HOAt (0.3 mg, 2.4 ⁇ mol) in 1 mL DMF were added bis-PFP ester 11 (1.1 mg, 1.2 ⁇ mol) in four portions over 5 minutes. The resulting mixture was stirred for 1 hour and concentrated under vacuum. The residue was dissolved in 0.5 mL of methanol, cooled down to 0 °C, and treated with 0.75 mL of 1M aqueous LiOH solution.
• reaction mixture was stirred at room temperature and was monitored by LC-MS. After starting material was consumed, the solution was concentrated under vacuum to remove DMF. The residue was dissolved in 1 mL of methanol and slowly treated with 1.5 mL of 1M aqueous LiOH solution at 0 °C. Reaction mixture was stirred for 15 minutes, then warmed up to room temperature and stirring continued for 1 hours, until hydrolysis was judged complete by LCMS analysis. Reaction mixture was quenched by addition of 1 mL of 1M HCl, followed by 1 mL of 0.5M pH 4.7 acetate buffer, concentrated under vacuum, and purified by reversed-phase HPLC (C18, 0-75% v/v CH 3 CN- H2O with 0.05% TFA).
• Reaction mixture was stirred for 1 h until coupling was found complete by LCMS analysis. Solvent was removed under reduced pressure; the residue was dissolved in MeOH (1 mL) and treated with 1M aqueous LiOH solution (1mL) at 0 °C. Reaction mixture was allowed to slowly warm up to room temperature, stirred for additional 1 h, and quenched with pH 4.7 acetate buffer (1mL). Solids were filtered off, and the clear filtrate was purified by reversed-phase prep HPLC (C18 column, 0-75% acetonitrile-water with 0.05% TFA). Pure fractions were combined and lyophilized to give 30 mg (24 ⁇ mol, 89% yield) of compound 72 as a yellow solid.
• Reaction mixture was allowed to stand for 1 h until reaction was judged complete by LCMS analysis, and treated with piperidine (49 ⁇ L, 0.49 mmol) at room temperature.
• Reaction mixture was directly purified by reversed-phase prep HPLC (C18 column, 0-70% acetonitrile-water with 0.05% TFA). Pure fractions were collected and lyophilized to give 24 mg of compound 73 as a yellow solid (8.5 ⁇ mol, 70% yield).
• Reaction mixture was allowed to stir at 0 °C for 1 h, slowly warmed to room temperature, and quenched by adding aqueous HCl (1.0 M) to pH 4.
• the mixture was purified by reversed-phase prep HPLC (C18 column, 0-50% acetonitrile-water/0.05% TFA) to give 0.020 g of compound 78 (0.021 mmol, 70% yield) as an off-white solid.
• reaction mixture was transferred to a separatory funnel, diluted with water (30 mL), and extracted with EtOAc (2x30 mL). Organic layer was washed with water and brine, dried over sodium sulfate. Solvents were removed in vacuum to give 0.50 g of crude product 82 as a colorless oil, which was used further without purification.
• reaction mixture was stirred for 45 min, then directly treated with piperidine (50 ⁇ L). After 30 min, reaction mixture was quenched with aqueous 0.05% TFA (1 mL) and purified by reversed-phase prep HPLC (C18 column, 0-50% acetonitrile-water/0.05% TFA). Fractions containing the desired product were combined and lyophilized to yield 38 mg of amine 85 as a pale-yellow solid (28 ⁇ mol, 90% yield). LRMS (ESI): m/z 1351.6 [M+H] + , Calcd for C65H90N8O23 m/z: 1351.6.
• reaction mixture was stirred for 30 min and then treated directly with piperidine (50 ⁇ L). After 20 min, reaction mixture was purified by reversed-phase prep HPLC (C18 column, 0-50% acetonitrile-water/0.05% TFA). Pure fractions containing product were combined and lyophilized to yield 15 mg of compound 86 (5 ⁇ mol, 69% yield) as a pale- yellow solid.
• reaction mixture was stirred for 1 h and then directly treated with piperidine (8 ⁇ L, 160 ⁇ mol) at room temperature. After 20 minutes, reaction mixture was purified by reversed-phase prep HPLC (C18, 0-70% v/v MeCN-H2O with 0.05% TFA). Lyophilized pure fractions gave 2.8 mg of compound 92 (1 ⁇ mol, 26 % yield) as a yellow powder.
• Reaction mixture was stirred for 30 min, then piperidine (52 uL, 0.52 mmol) was added, and stirring continued for 20 minutes.
• Reaction mixture was purified directly by reversed-phase chromatography (C18, 0-100% v/v MeCN-H2O with 0.05% TFA). Lyophilized pure fractions gave 22 mg of compound 98 (16 ⁇ mol, 62 % yield) as a yellow powder.
• reaction mixture was allowed to stand at room temperature for 1 h and then treated with piperidine (22 ⁇ L, 0.22 mmol). After 20 minutes, reaction mixture was purified by reversed-phase prep HPLC (C18, 0-50% v/v MeCN-H 2 O/10 mM ammonium formate). Pure fractions containing product were combined and lyophilized to give 7 mg of compound 103 (2.8 ⁇ mol, 56% yield) as a tan powder.
• Reaction mixture was allowed to stir for 1 h, then piperidine (50 ⁇ L) was added to directly to the mixture and stirring continued for 30 mins. Reaction mixture was quenched by adding 2 mL of aqueous 0.05% TFA solution and purified by reversed-phase prep HPLC (C18, 0-50% v/v MeCN-H2O with 0.05% TFA). Pure fractions were lyophilized to give 13 mg of compound 109 (9 ⁇ mol, 90% yield) as a pale-yellow solid.
• Reaction mixture was allowed to stir for 30 mins, then piperidine (50 ⁇ L) was added directly to the mixture and stirring continued for 30 min.
• Reaction mixture was purified by reversed-phase prep HPLC (C18, 0-50% v/v MeCN-H2O with 0.05% TFA). Lyophilization of pure fractions gave 5 mg of compound 110 (1.6 ⁇ mol, 36% yield) as a pale-yellow solid.
• Reaction mixture was directly purified by reversed phase HPLC using C18 column (H2O/CH3CN with 0.05% TFA, 90:10 to 0:100 v/v). Fractions containing the desired compound were pooled and lyophilized to yield compound 120 (280 mg, 0.57 mmol,
• reaction mixture was stirred for 20 minutes and combined with amine 17 (55 mg, 58 ⁇ mol) in 1 mL of DMF. Reaction mixture was stirred for 30 minutes, then piperidine (115 ⁇ L, 1.2 mmol) was added to the mixture at room temperature. After 20 minutes, reaction mixture was directly purified by reversed phase prep HPLC (C18, 0-50% v/v MeCN-H2O with 0.05% TFA). Lyophilization of pure fractions afforded 34 mg (23 ⁇ mol, 40% yield) of compound 130 as a yellow powder.
• Reaction mixture was stirred for 30 minutes, monitored by LCMS analysis. After reaction was judged complete, piperidine (40 ⁇ L) was added to the mixture, and stirring continued for 20 minutes. Reaction mixture was then purified by reversed-phase prep HPLC (C18, 0-50% v/v MeCN-H 2 O with 0.05% TFA). Pure fractions were lyophilized to afford 14.6 mg of compound 141 as a yellow powder (11 ⁇ mol, 55% yield). LRMS (ESI): m/z 1321.5 [M+H] + , Calcd for C63H84N8O23 m/z 1321.6.
• reaction mixture was stirred for one hour, then compound 145 (25 mg, 27 ⁇ mol) was added to the mixture, and stirring continued for 1 h, until coupling was judged complete by LCMS analysis.
• reaction mixture was treated with triethylamine (0.4 mL) and stirred at room temperature for 5 h.
• Reaction mixture was purified by reversed phase prep HPLC (Cl 8, 0-70% v/v MeCN- H2O with 0.05% TFA). Pure fractions were collected and lyophilized to obtain compound 146 as a yellow solid (26 mg, 18 pmol, 67% yield).
• reaction mixture was treated with PNP-carbonate 12 (116 mg, 115 ⁇ mol) and stirred at room temperature overnight until all starting materials were consumed as judged by HPLC analysis. Reaction mixture was then diluted with water (10 mL), the resulting precipitate was collected and dissolved in THF (3 mL). The THF solution was then treated with aq. LiOH (1 mL, 1M) in at 0 °C, stirred for 30 min, warmed up to room temperature, and stirred for 1 h. Reaction mixture was purified by reversed phase prep HPLC (C18, 0-70% v/v MeCN-H2O with 0.05% TFA).
• reaction mixture was directly treated with piperidine (62 ⁇ L, 0.63 mmol) at room temperature, stirred for 20 minutes, and purified by reversed-phase prep HPLC (C18, 0-70% v/v MeCN-H2O with 0.05% TFA). Pure fractions were collected and lyophilized to afford compound 150 as a yellow solid (26 mg, 18 ⁇ mol, 55% yield).
• reaction mixture was allowed to warm to room temperature and stirred overnight.
• the mixture was briefly purified by passing through a silica gel pad (0-6% MeOH-DCM as an eluent) to give crude compound 167, which was dissolved in EtOAc (2 mL) and combined with Pd/C (10 wt %, 20 mg) and triethylamine (20 ⁇ L, 220 ⁇ mol).
• Reaction flask was then evacuated and filled with hydrogen gas from a balloon, in three repeating cycles. Reaction mixture was vigorously stirred for 48 h at room temperature with H 2 balloon attached, then filtered through a pad of celite.
• Reaction mixture was stirred for 1 h, then DMF (0.5 mL) and piperidine (50 ⁇ L) were added to the mixture. After 30 minutes, the reaction was semi-purified by silica gel chromatography with a gradient of 0 to 5% MeOH in DCM to give crude compound 170. Next, a solution of 170 in 1 mL of acetonitrile was treated with Fmoc-Val-OPFP 171 (62 mg, 120 ⁇ mol) and DIPEA (22 ⁇ L, 120 ⁇ mol) at room temperature.
• reaction mixture was purified by silica gel chromatography (MeOH-DCM 0-5% gradient) to yield compound 172 (70 mg, 51 ⁇ mol, 83% yield) as a yellow solid.
• reaction mixture was concentrated and then reconstituted in formic acid (1 mL) at room temperature. After 30 minutes, formic acid was removed in vacuum, and the residue was reconstituted in DMF (1 mL) and piperidine (50 ⁇ L). After stirring for 15 minutes at room temperature, the reaction mixture was directly purified by reversed phase HPLC using C18 column (H 2 O/CH 3 CN with 0.05% TFA, 90:10 to 35:65 v/v). Fractions containing the desired compound were pooled and lyophilized to yield compound 175 (0.7 mg, 0.2 ⁇ mol, 33% yield) as a yellow powder.

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